warmstart colorimetric lamp 2  (New England Biolabs)


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    Structured Review

    New England Biolabs warmstart colorimetric lamp 2
    Production of molecular biology reagents. (A) Purified Br512 Bst DNA polymerase visualized in a polyacrylamide gel stained with coomassie blue. (B) <t>Colorimetric</t> <t>LAMP</t> assay using the Br512 Bst DNA polymerase produced in vitro in both fresh conditions (top panel) and using rehydrated samples (bottom panel) after a 2 week storage at room temperature. Cell-free reactions based on PEP and MDX were prepared using the low-cost drying system and protected with sucrose (120 and 15 mM, respectively). A synthetic dsDNA fragment from actin B gene ( Homo sapiens ) was used as a target in the following amounts: 0, 0.025, 0.25, 2.5, 250, and 2500 pmoles. Primers used in this assay are described in Table S11 . Negative reactions were pink-colored, and positive reactions changed to yellow. (C, D) A PCR product encoding the Bsa I restriction endonuclease (2043 bp) was amplified using a single PCR with four oligonucleotides. An inner set of core primers provided a template for secondary amplification by longer oligonucleotides. The resulting product had extended terminal sequences that helped protect the coding region from exonuclease degradation. (E) Testing of Bsa I by restriction endonuclease digestion of luxpGEX plasmid. Digestion was performed using Bsa I produced by cell-free technology. Plasmid DNA samples were treated with (1) FastDigest Eco31I (Thermo Scientific, FD0293) (Isoschizomer: Bsa I), (2) Bsa I in cell extract, and (3) Bsa I in cell extract: 100% glycerol (1:1). Expected size of bands after digestion: 6440 and 4433 bp.
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    Images

    1) Product Images from "Constructing Cell-Free Expression Systems for Low-Cost Access"

    Article Title: Constructing Cell-Free Expression Systems for Low-Cost Access

    Journal: ACS Synthetic Biology

    doi: 10.1021/acssynbio.1c00342

    Production of molecular biology reagents. (A) Purified Br512 Bst DNA polymerase visualized in a polyacrylamide gel stained with coomassie blue. (B) Colorimetric LAMP assay using the Br512 Bst DNA polymerase produced in vitro in both fresh conditions (top panel) and using rehydrated samples (bottom panel) after a 2 week storage at room temperature. Cell-free reactions based on PEP and MDX were prepared using the low-cost drying system and protected with sucrose (120 and 15 mM, respectively). A synthetic dsDNA fragment from actin B gene ( Homo sapiens ) was used as a target in the following amounts: 0, 0.025, 0.25, 2.5, 250, and 2500 pmoles. Primers used in this assay are described in Table S11 . Negative reactions were pink-colored, and positive reactions changed to yellow. (C, D) A PCR product encoding the Bsa I restriction endonuclease (2043 bp) was amplified using a single PCR with four oligonucleotides. An inner set of core primers provided a template for secondary amplification by longer oligonucleotides. The resulting product had extended terminal sequences that helped protect the coding region from exonuclease degradation. (E) Testing of Bsa I by restriction endonuclease digestion of luxpGEX plasmid. Digestion was performed using Bsa I produced by cell-free technology. Plasmid DNA samples were treated with (1) FastDigest Eco31I (Thermo Scientific, FD0293) (Isoschizomer: Bsa I), (2) Bsa I in cell extract, and (3) Bsa I in cell extract: 100% glycerol (1:1). Expected size of bands after digestion: 6440 and 4433 bp.
    Figure Legend Snippet: Production of molecular biology reagents. (A) Purified Br512 Bst DNA polymerase visualized in a polyacrylamide gel stained with coomassie blue. (B) Colorimetric LAMP assay using the Br512 Bst DNA polymerase produced in vitro in both fresh conditions (top panel) and using rehydrated samples (bottom panel) after a 2 week storage at room temperature. Cell-free reactions based on PEP and MDX were prepared using the low-cost drying system and protected with sucrose (120 and 15 mM, respectively). A synthetic dsDNA fragment from actin B gene ( Homo sapiens ) was used as a target in the following amounts: 0, 0.025, 0.25, 2.5, 250, and 2500 pmoles. Primers used in this assay are described in Table S11 . Negative reactions were pink-colored, and positive reactions changed to yellow. (C, D) A PCR product encoding the Bsa I restriction endonuclease (2043 bp) was amplified using a single PCR with four oligonucleotides. An inner set of core primers provided a template for secondary amplification by longer oligonucleotides. The resulting product had extended terminal sequences that helped protect the coding region from exonuclease degradation. (E) Testing of Bsa I by restriction endonuclease digestion of luxpGEX plasmid. Digestion was performed using Bsa I produced by cell-free technology. Plasmid DNA samples were treated with (1) FastDigest Eco31I (Thermo Scientific, FD0293) (Isoschizomer: Bsa I), (2) Bsa I in cell extract, and (3) Bsa I in cell extract: 100% glycerol (1:1). Expected size of bands after digestion: 6440 and 4433 bp.

    Techniques Used: Purification, Staining, Lamp Assay, Produced, In Vitro, Polymerase Chain Reaction, Amplification, Plasmid Preparation

    2) Product Images from "Development and Validation of a Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Glaesserella (Haemophilus) parasuis"

    Article Title: Development and Validation of a Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Glaesserella (Haemophilus) parasuis

    Journal: Microorganisms

    doi: 10.3390/microorganisms9010041

    Results of the WarmStart Colorimetric LAMP 2X Master Mix assay for detection of LAMP amplicons with the naked eye. Samples 1–8: serial dilutions of DNA from strain G. ( H.) parasuis DSM 21448 starting at concentrations of 10 ng/µL up to 1 fg/µL. Sample 9: DNA from Actinobacillus minor CCUG 38923 T . Sample 10: no template control.
    Figure Legend Snippet: Results of the WarmStart Colorimetric LAMP 2X Master Mix assay for detection of LAMP amplicons with the naked eye. Samples 1–8: serial dilutions of DNA from strain G. ( H.) parasuis DSM 21448 starting at concentrations of 10 ng/µL up to 1 fg/µL. Sample 9: DNA from Actinobacillus minor CCUG 38923 T . Sample 10: no template control.

    Techniques Used:

    3) Product Images from "Toward a next-generation diagnostic tool: A review on emerging isothermal nucleic acid amplification techniques for the detection of SARS-CoV-2 and other infectious viruses"

    Article Title: Toward a next-generation diagnostic tool: A review on emerging isothermal nucleic acid amplification techniques for the detection of SARS-CoV-2 and other infectious viruses

    Journal: Analytica Chimica Acta

    doi: 10.1016/j.aca.2021.339338

    RT-LAMP assay to detect SARS-CoV-2. The LAMP primer mix contain three pairs of target-specific primers: two forward primers, two backward primers, and two loop primers. The LAMP reaction mix contained DNA polymerase, reverse transcriptase, isothermal buffer, and signal reporter. A simple heating block can be used to amplify the target region of the viral RNA using colorimetric detection. The image on the right shows the time-dependent color change, adapted from Dao Thi et al., [ 125 ]. Reprinted with permission from AAAS. Created with BioRender.com . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: RT-LAMP assay to detect SARS-CoV-2. The LAMP primer mix contain three pairs of target-specific primers: two forward primers, two backward primers, and two loop primers. The LAMP reaction mix contained DNA polymerase, reverse transcriptase, isothermal buffer, and signal reporter. A simple heating block can be used to amplify the target region of the viral RNA using colorimetric detection. The image on the right shows the time-dependent color change, adapted from Dao Thi et al., [ 125 ]. Reprinted with permission from AAAS. Created with BioRender.com . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: RT Lamp Assay, Blocking Assay

    Smartphone-based handheld POC instrument. A) Simple workflow of SARS-CoV-2 sample preparation by brief heat treatment and transfer of the thermally lysed sample to a syringe. Another syringe was loaded with LAMP reaction mix. B) Photograph of the microfluidic chip integrated POC tool. C) Disposable microfluidic cartridge. Figure adapted with permission from Ganguli et al., [ 83 ].
    Figure Legend Snippet: Smartphone-based handheld POC instrument. A) Simple workflow of SARS-CoV-2 sample preparation by brief heat treatment and transfer of the thermally lysed sample to a syringe. Another syringe was loaded with LAMP reaction mix. B) Photograph of the microfluidic chip integrated POC tool. C) Disposable microfluidic cartridge. Figure adapted with permission from Ganguli et al., [ 83 ].

    Techniques Used: Sample Prep, Chromatin Immunoprecipitation

    4) Product Images from "Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern"

    Article Title: Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2021.713713

    Colorimetric RT-LAMP for COVID-19 diagnosis validation using 100 clinical samples. Clinical samples were collected from symptomatic and hospitalized patients by nasopharyngeal swabs in partnership with CT-Vacinas/UFMG, Belo Horizonte, Brazil. Samples were obtained from different parts including Brazilian Southeast and Northeast regions. The reaction was performed at 65°C during 30 min using WarmStart ® colorimetric LAMP master mix (NEB #M1800) in 20 μL final volume. The RT-LAMP reaction targeted SARS-CoV-2 N gene. Yellow content indicates positive reaction, whereas the pink pattern reveals nonreagent samples. Amplicons were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification. Latter pattern confirmed specific SARS-CoV-2 amplification that matches with yellow output tubes, which is not observed in pink nonreagent tests. +C, positive control using RNA extracted from laboratory-Vero E6 cultured inactivated SARS-CoV-2; NTC, nontemplate control. Clinimetric parameters from these samples are presented in Supplementary Figure S1 .
    Figure Legend Snippet: Colorimetric RT-LAMP for COVID-19 diagnosis validation using 100 clinical samples. Clinical samples were collected from symptomatic and hospitalized patients by nasopharyngeal swabs in partnership with CT-Vacinas/UFMG, Belo Horizonte, Brazil. Samples were obtained from different parts including Brazilian Southeast and Northeast regions. The reaction was performed at 65°C during 30 min using WarmStart ® colorimetric LAMP master mix (NEB #M1800) in 20 μL final volume. The RT-LAMP reaction targeted SARS-CoV-2 N gene. Yellow content indicates positive reaction, whereas the pink pattern reveals nonreagent samples. Amplicons were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification. Latter pattern confirmed specific SARS-CoV-2 amplification that matches with yellow output tubes, which is not observed in pink nonreagent tests. +C, positive control using RNA extracted from laboratory-Vero E6 cultured inactivated SARS-CoV-2; NTC, nontemplate control. Clinimetric parameters from these samples are presented in Supplementary Figure S1 .

    Techniques Used: Agarose Gel Electrophoresis, Staining, Amplification, Positive Control, Cell Culture

    Colorimetric RT-LAMP allows the detection of SARS-CoV-2 VOCs and VOIs. RT-LAMP reaction was performed at 65°C for 30 min, using the WarmStart ® colorimetric LAMP 2× master mix (NEB #M1804), using multiplex N 2/ E 1 primer sets. The amplicons were migrated in agarose gel at 2% to confirm amplification, as indicated by the characteristic ladder highlighted by GelRed ® staining. NTC, nontemplate control; CS, clinical sample; and +C, positive control. The top panel shows a schematic representation of SARS-CoV-2 spike protein (upper) and where the main mutations are highlighted and represented in SARS-CoV-2 virions (right hand side) present in VOC gamma (B.1), delta (B.1.167.2), and VOI zeta (P.2). The VOCs alpha (B.1.1.7) and beta (B.1.3.51), first reported in the United Kingdom and South Africa, respectively, are also represented. K417N: lysine-to-asparagine substitution at position 417 of spike protein at the receptor biding domain (RBD); V445A: valine-to-alanine substitution at position 445 and so on. L, leucine; Q, glutamine; E, glutamic acid; Y, tyrosine; T, threonine; P, proline; H, histidine; D, aspartic acid; S, serine; F, phenylalanine. del, deletion. Segments of SARS-CoV-2 protein NTD, N-terminal domain; CTD2, C-terminal domain 2 or C terminus of S1 fragment after furin cleavage; FP, fusion peptide; HR1, heptad repeat region 1. SARS-CoV-2 variants were previously sequenced. Variants of interest B.1.1.371 and B.1.1.374 were first reported in Saudi Arabia and Finland, respectively, ( https://cov-lineages.org/ ). Created with biorender.com .
    Figure Legend Snippet: Colorimetric RT-LAMP allows the detection of SARS-CoV-2 VOCs and VOIs. RT-LAMP reaction was performed at 65°C for 30 min, using the WarmStart ® colorimetric LAMP 2× master mix (NEB #M1804), using multiplex N 2/ E 1 primer sets. The amplicons were migrated in agarose gel at 2% to confirm amplification, as indicated by the characteristic ladder highlighted by GelRed ® staining. NTC, nontemplate control; CS, clinical sample; and +C, positive control. The top panel shows a schematic representation of SARS-CoV-2 spike protein (upper) and where the main mutations are highlighted and represented in SARS-CoV-2 virions (right hand side) present in VOC gamma (B.1), delta (B.1.167.2), and VOI zeta (P.2). The VOCs alpha (B.1.1.7) and beta (B.1.3.51), first reported in the United Kingdom and South Africa, respectively, are also represented. K417N: lysine-to-asparagine substitution at position 417 of spike protein at the receptor biding domain (RBD); V445A: valine-to-alanine substitution at position 445 and so on. L, leucine; Q, glutamine; E, glutamic acid; Y, tyrosine; T, threonine; P, proline; H, histidine; D, aspartic acid; S, serine; F, phenylalanine. del, deletion. Segments of SARS-CoV-2 protein NTD, N-terminal domain; CTD2, C-terminal domain 2 or C terminus of S1 fragment after furin cleavage; FP, fusion peptide; HR1, heptad repeat region 1. SARS-CoV-2 variants were previously sequenced. Variants of interest B.1.1.371 and B.1.1.374 were first reported in Saudi Arabia and Finland, respectively, ( https://cov-lineages.org/ ). Created with biorender.com .

    Techniques Used: Multiplex Assay, Agarose Gel Electrophoresis, Amplification, Staining, Positive Control

    Analytical sensitivity as revealed by the limit of detection (LoD). RNA was extracted from VTM-nasopharyngeal swab, and the genome viral copies input was calculated based on SARS-CoV-2 E gene-harboring plasmid (Bioclin #K228-1) calibration curve. RT-LAMP reaction was performed at 65°C during 30 min using WarmStart ® colorimetric master LAMP mix (NEB #M1800) in 20 μL final volume (upper panel). Amplicons were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification (bottom panel). cps/μL, viral genome copies per microliter; NTC, nontemplate control; VTM, viral transport medium (Bioclin #G092-1).
    Figure Legend Snippet: Analytical sensitivity as revealed by the limit of detection (LoD). RNA was extracted from VTM-nasopharyngeal swab, and the genome viral copies input was calculated based on SARS-CoV-2 E gene-harboring plasmid (Bioclin #K228-1) calibration curve. RT-LAMP reaction was performed at 65°C during 30 min using WarmStart ® colorimetric master LAMP mix (NEB #M1800) in 20 μL final volume (upper panel). Amplicons were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification (bottom panel). cps/μL, viral genome copies per microliter; NTC, nontemplate control; VTM, viral transport medium (Bioclin #G092-1).

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Staining, Amplification

    Microbial cross-reactivity assay to test SARS-CoV-2 RT-LAMP analytical sensitivity. The test was performed using potentially cross-reacting respiratory viruses or local occurring arboviruses. RT-LAMP reaction was performed at 65°C during 30 min, with additional 10 min, to confirm the absence of cross-reactivity when targeting SARS-CoV-2 E and N genes. The assay was performed using the WarmStart ® colorimetric LAMP 2× master mix (NEB #M1800). Yellow (positive) reaction is observed only when the template is SARS-CoV-2 viral RNA. hRSV, human respiratory syncytial virus; NTC, nontemplate control; M, molecular size marker. RT-LAMP amplification products were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification. DENV3, dengue virus serotype 3; ZIKV, Zika virus; CHIKV, Chikungunya virus; YFV, yellow fever virus; Influenza A (H1N1/H3N2); and influenza B (Yamagata/Victoria).
    Figure Legend Snippet: Microbial cross-reactivity assay to test SARS-CoV-2 RT-LAMP analytical sensitivity. The test was performed using potentially cross-reacting respiratory viruses or local occurring arboviruses. RT-LAMP reaction was performed at 65°C during 30 min, with additional 10 min, to confirm the absence of cross-reactivity when targeting SARS-CoV-2 E and N genes. The assay was performed using the WarmStart ® colorimetric LAMP 2× master mix (NEB #M1800). Yellow (positive) reaction is observed only when the template is SARS-CoV-2 viral RNA. hRSV, human respiratory syncytial virus; NTC, nontemplate control; M, molecular size marker. RT-LAMP amplification products were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification. DENV3, dengue virus serotype 3; ZIKV, Zika virus; CHIKV, Chikungunya virus; YFV, yellow fever virus; Influenza A (H1N1/H3N2); and influenza B (Yamagata/Victoria).

    Techniques Used: Marker, Amplification, Agarose Gel Electrophoresis, Staining

    Colorimetric RT-LAMP for SARS-CoV-2 detection using genes N , E , and RdRp as target. Selected SARS-CoV-2–positive clinical samples by RT-qPCR were classified as low (Ct 18.9 and 21.7), medium (Ct 26.6 and 28.4), and high (Ct 31.6 and 35.2) Ct values for E gene. They were included as input for colorimetric RT-LAMP reaction using primers targeting N , RdRp (A) , and E genes (B) . RT-LAMP SARS-CoV-2 false-negative samples were more frequent when using E and RdRp genes as target (C) . RT-LAMP reaction was performed at 65°C during 30 min, using the WarmStart ® colorimetric LAMP 2× master mix (NEB #M1800). RT-LAMP amplification products were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification. +C, positive control using SARS-CoV-2 RNA extracted from laboratory-cultured inactivated SARS-CoV-2; NTC, nontemplate control.
    Figure Legend Snippet: Colorimetric RT-LAMP for SARS-CoV-2 detection using genes N , E , and RdRp as target. Selected SARS-CoV-2–positive clinical samples by RT-qPCR were classified as low (Ct 18.9 and 21.7), medium (Ct 26.6 and 28.4), and high (Ct 31.6 and 35.2) Ct values for E gene. They were included as input for colorimetric RT-LAMP reaction using primers targeting N , RdRp (A) , and E genes (B) . RT-LAMP SARS-CoV-2 false-negative samples were more frequent when using E and RdRp genes as target (C) . RT-LAMP reaction was performed at 65°C during 30 min, using the WarmStart ® colorimetric LAMP 2× master mix (NEB #M1800). RT-LAMP amplification products were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification. +C, positive control using SARS-CoV-2 RNA extracted from laboratory-cultured inactivated SARS-CoV-2; NTC, nontemplate control.

    Techniques Used: Quantitative RT-PCR, Amplification, Agarose Gel Electrophoresis, Staining, Positive Control, Cell Culture

    Colorimetric RT-LAMP to detect SAR-CoV-2 in RNA extraction–free clinical samples (A) or laboratory-cultured virus (B) . Clinical samples were derived from nasopharyngeal swabs placed on guanidine-containing viral transport medium, diluted 1:10. The RT-PCR Ct values for SARS-CoV-2 based on E gene are as follows: CS134 = 31.8, CS135 = 15.3, CS138 = 18.4, CS139 = 21.7, and CS140 = 24.6. RT-LAMP reaction was performed in 20 μL final volume, incubated at 65°C during 30, 40, or 50 min (inactivated virus) using WarmStart ® colorimetric LAMP master mix (NEB #M1800). Both clinical samples and viruses are RNA extraction–free samples. The amplification products (amplicons) were migrated in agarose gel at 2% to confirm amplification, as indicated by the characteristic ladder highlighted by GelRed ® staining. NTC, nontemplate control; CS, clinical sample; and +C, positive control.
    Figure Legend Snippet: Colorimetric RT-LAMP to detect SAR-CoV-2 in RNA extraction–free clinical samples (A) or laboratory-cultured virus (B) . Clinical samples were derived from nasopharyngeal swabs placed on guanidine-containing viral transport medium, diluted 1:10. The RT-PCR Ct values for SARS-CoV-2 based on E gene are as follows: CS134 = 31.8, CS135 = 15.3, CS138 = 18.4, CS139 = 21.7, and CS140 = 24.6. RT-LAMP reaction was performed in 20 μL final volume, incubated at 65°C during 30, 40, or 50 min (inactivated virus) using WarmStart ® colorimetric LAMP master mix (NEB #M1800). Both clinical samples and viruses are RNA extraction–free samples. The amplification products (amplicons) were migrated in agarose gel at 2% to confirm amplification, as indicated by the characteristic ladder highlighted by GelRed ® staining. NTC, nontemplate control; CS, clinical sample; and +C, positive control.

    Techniques Used: RNA Extraction, Cell Culture, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Incubation, Amplification, Agarose Gel Electrophoresis, Staining, Positive Control

    5) Product Images from "A diagnostic LAMP assay for rapid identification of an invasive plant pest, fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae)"

    Article Title: A diagnostic LAMP assay for rapid identification of an invasive plant pest, fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae)

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-04496-x

    Detection sensitivity of FAW gBlock dsDNA amplicons (upper), evaluating amount of FAW DNA with gBlock DNA (lower). ( a ) Amplification profile with gBlock templates ranging from 10 8 to 10 copies at ten-fold dilution. ( b ) Anneal derivative of gBlock LAMP amplicons, with an anneal derivative of 81 °C. ( c ) Amplification profile of four-fold dilution of FAW DNA (VAITC 10726) and gBlock DNA (10 5 copies, pink). ( d ) Anneal derivative of LAMP amplicons showing two peaks, 78.5 °C for FAW DNA dilutions and 81 °C for gBlock DNA (pink).
    Figure Legend Snippet: Detection sensitivity of FAW gBlock dsDNA amplicons (upper), evaluating amount of FAW DNA with gBlock DNA (lower). ( a ) Amplification profile with gBlock templates ranging from 10 8 to 10 copies at ten-fold dilution. ( b ) Anneal derivative of gBlock LAMP amplicons, with an anneal derivative of 81 °C. ( c ) Amplification profile of four-fold dilution of FAW DNA (VAITC 10726) and gBlock DNA (10 5 copies, pink). ( d ) Anneal derivative of LAMP amplicons showing two peaks, 78.5 °C for FAW DNA dilutions and 81 °C for gBlock DNA (pink).

    Techniques Used: Amplification

    Mitochondrial COI DNA sequence (3′ region) alignment showing FAW LAMP primers. Sequence of FAW (grey shading, from Kim et al . 37 ) and other closely related Spodoptera species 20 , 55 obtained from GenBank. Reverse primers are underlined; FIP (5′-3′) is made by combining F1 (reverse compliment) and F2; BIP (5′-3′) is made by combining B1 and B2 (reverse compliment).
    Figure Legend Snippet: Mitochondrial COI DNA sequence (3′ region) alignment showing FAW LAMP primers. Sequence of FAW (grey shading, from Kim et al . 37 ) and other closely related Spodoptera species 20 , 55 obtained from GenBank. Reverse primers are underlined; FIP (5′-3′) is made by combining F1 (reverse compliment) and F2; BIP (5′-3′) is made by combining B1 and B2 (reverse compliment).

    Techniques Used: Sequencing

    Optimised LAMP assay performed on FAW larva and adult moth laboratory DNA extracts. ( a ) Amplification profile, with 7 positive samples amplifying in approx. 10 min and negative sample (dark blue) showing a flat line. ( b ) Anneal derivative of LAMP amplicons, with an anneal derivative of 78.5 °C.
    Figure Legend Snippet: Optimised LAMP assay performed on FAW larva and adult moth laboratory DNA extracts. ( a ) Amplification profile, with 7 positive samples amplifying in approx. 10 min and negative sample (dark blue) showing a flat line. ( b ) Anneal derivative of LAMP amplicons, with an anneal derivative of 78.5 °C.

    Techniques Used: Lamp Assay, Amplification

    Time-series of FAW LAMP (new assay primers) using colorimetric master mix. Ninety minutes total amplification time shown in increments of 15 min. Samples: (1) Spodoptera frugiperda , (2) Spodoptera litura (PNG), (3) Spodoptera exigua, (4) Helicoverpa armigera conferta, (5) Mythimna convecta, (6) Leucania loreyi, (7) no-template negative control and (8) FAW gBlock DNA dilution 10 6 . The colour change from pink to yellow in tube 1 and 8 indicates positive samples. Negative samples did not change colour.
    Figure Legend Snippet: Time-series of FAW LAMP (new assay primers) using colorimetric master mix. Ninety minutes total amplification time shown in increments of 15 min. Samples: (1) Spodoptera frugiperda , (2) Spodoptera litura (PNG), (3) Spodoptera exigua, (4) Helicoverpa armigera conferta, (5) Mythimna convecta, (6) Leucania loreyi, (7) no-template negative control and (8) FAW gBlock DNA dilution 10 6 . The colour change from pink to yellow in tube 1 and 8 indicates positive samples. Negative samples did not change colour.

    Techniques Used: Amplification, Negative Control

    Maximum Likelihood tree (5′-COI DNA sequences) of samples used for testing FAW LAMP assay. Bootstrap values indicated on nodes. AgVic, Agricultural Victoria; CSIRO, Commonwealth Scientific and Industrial Research Organisation; QDAF, Department of Agriculture and Fisheries Queensland; Vic, Victoria Australia; Qld, Queensland Australia; PNG, Papua New Guinea.
    Figure Legend Snippet: Maximum Likelihood tree (5′-COI DNA sequences) of samples used for testing FAW LAMP assay. Bootstrap values indicated on nodes. AgVic, Agricultural Victoria; CSIRO, Commonwealth Scientific and Industrial Research Organisation; QDAF, Department of Agriculture and Fisheries Queensland; Vic, Victoria Australia; Qld, Queensland Australia; PNG, Papua New Guinea.

    Techniques Used: Lamp Assay

    DNA sensitivity test of FAW LAMP and FAW real-time PCR assays. ( a,b ) A four-fold DNA dilution series of two biological replicates of FAW larvae (VAITC 10707 and 10726) DNA amount ranging from 40.0 ng/µL to 2.441 × 10 –3 ng/µL. ( a ) FAW LAMP assay amplification time, sensitive to all 8 DNA dilutions tested. ( b ) Real-time PCR Cq values sensitive to all 8 dilutions tested. ( c,d ) A four-fold DNA dilution series of two biological replicates of FAW adult moth (VAITC 10728 and 10729) DNA amount ranging from 1.0 ng/µL to 6.1 × 10 –5 ng/µL. ( c ) FAW LAMP assay amplification time, sensitive to only 5 out of 8 DNA dilutions tested. ( d ) Real-time PCR Cq values sensitive to only 5 out of 8 DNA dilutions tested. Black and white circles represent biological replicate DNA samples.
    Figure Legend Snippet: DNA sensitivity test of FAW LAMP and FAW real-time PCR assays. ( a,b ) A four-fold DNA dilution series of two biological replicates of FAW larvae (VAITC 10707 and 10726) DNA amount ranging from 40.0 ng/µL to 2.441 × 10 –3 ng/µL. ( a ) FAW LAMP assay amplification time, sensitive to all 8 DNA dilutions tested. ( b ) Real-time PCR Cq values sensitive to all 8 dilutions tested. ( c,d ) A four-fold DNA dilution series of two biological replicates of FAW adult moth (VAITC 10728 and 10729) DNA amount ranging from 1.0 ng/µL to 6.1 × 10 –5 ng/µL. ( c ) FAW LAMP assay amplification time, sensitive to only 5 out of 8 DNA dilutions tested. ( d ) Real-time PCR Cq values sensitive to only 5 out of 8 DNA dilutions tested. Black and white circles represent biological replicate DNA samples.

    Techniques Used: Real-time Polymerase Chain Reaction, Lamp Assay, Amplification

    6) Product Images from "Fully 3D Printed Integrated Reactor Array for Point-of-Care Molecular Diagnostics"

    Article Title: Fully 3D Printed Integrated Reactor Array for Point-of-Care Molecular Diagnostics

    Journal: Biosensors & bioelectronics

    doi: 10.1016/j.bios.2018.03.009

    Colorimetric and fluorescence based detection for NAATs in 3D printed reactor array. A) Representative photographs of colorimetric LAMP assay for detection of N. meningitidis with 0, 50, 500 and 5000 CFU/reaction on the same chip, alongside LAMP fluorescence based image at given time interval. B) LAMP amplification curves for P. falciparum with 0, 0.1 1, 10, 100, 1000 pg per reaction. C) Calibration curve for P. falciparum as function of log target concentration, n=3. D) LAMP amplification curves for N. meningitidis with 0, 50, 500, 5000 CFU per reaction. E) Calibration curve for N. meningitidis as function of log target concentration, n=3. WarmStart ® LAMP master mix was used.
    Figure Legend Snippet: Colorimetric and fluorescence based detection for NAATs in 3D printed reactor array. A) Representative photographs of colorimetric LAMP assay for detection of N. meningitidis with 0, 50, 500 and 5000 CFU/reaction on the same chip, alongside LAMP fluorescence based image at given time interval. B) LAMP amplification curves for P. falciparum with 0, 0.1 1, 10, 100, 1000 pg per reaction. C) Calibration curve for P. falciparum as function of log target concentration, n=3. D) LAMP amplification curves for N. meningitidis with 0, 50, 500, 5000 CFU per reaction. E) Calibration curve for N. meningitidis as function of log target concentration, n=3. WarmStart ® LAMP master mix was used.

    Techniques Used: Fluorescence, Lamp Assay, Chromatin Immunoprecipitation, Amplification, Concentration Assay

    7) Product Images from "Real-time fluorometric and end-point colorimetric isothermal assays for detection of equine pathogens C. psittaci and equine herpes virus 1: validation, comparison and application at the point of care"

    Article Title: Real-time fluorometric and end-point colorimetric isothermal assays for detection of equine pathogens C. psittaci and equine herpes virus 1: validation, comparison and application at the point of care

    Journal: BMC Veterinary Research

    doi: 10.1186/s12917-021-02986-8

    Colorimetric and SYBR Green LAMP assays testing of the same clinical swab suspension samples. cLAMP assay (top row) and sgLAMP assay (bottom row) testing for presence of C. psittaci (A), and EHV-1 (B). In (C) C. psittaci results of rapidly processed placental and tissue swabs suspensions using cLAMP, and (D) test results of C. psittaci cLAMP yielding ambiguous result (orange colour, top), unambiguous negative (pink colour, middle) and unambiguous positive (yellow colour, bottom). Tested swab samples IDs are (from left): F1 tissues, F2 tissues1 and placenta1, F105 placenta, F4 nasal, M3 cervix, M11 vagina, and S79 penile (Table S5). Positive and negative tests ate denoted with P and N, respectively. Sample M3 in EHV-1 cLAMP, and sample S79 in both C. psittaci and EHV-1 cLAMPs yielded ambiguous test results (orange colour, indicated by orange stars). The images were taken with iPhone SE
    Figure Legend Snippet: Colorimetric and SYBR Green LAMP assays testing of the same clinical swab suspension samples. cLAMP assay (top row) and sgLAMP assay (bottom row) testing for presence of C. psittaci (A), and EHV-1 (B). In (C) C. psittaci results of rapidly processed placental and tissue swabs suspensions using cLAMP, and (D) test results of C. psittaci cLAMP yielding ambiguous result (orange colour, top), unambiguous negative (pink colour, middle) and unambiguous positive (yellow colour, bottom). Tested swab samples IDs are (from left): F1 tissues, F2 tissues1 and placenta1, F105 placenta, F4 nasal, M3 cervix, M11 vagina, and S79 penile (Table S5). Positive and negative tests ate denoted with P and N, respectively. Sample M3 in EHV-1 cLAMP, and sample S79 in both C. psittaci and EHV-1 cLAMPs yielded ambiguous test results (orange colour, indicated by orange stars). The images were taken with iPhone SE

    Techniques Used: SYBR Green Assay

    Real-time and colorimetric C. psittaci and EHV-1 LAMP assays. The real-time fluorometer Genie III displays of (A) a C. psittaci and EHV-1 amplification curves within 30 min amplification time, and (B) High-resolution melts (HRM). Positive rtLAMP result is denoted with time to amplify (mm:ss) and HRM (°C), whilst negative result does not record either. End-point detection using C. psittaci (top row) and EHV-1 (bottom row) (C) cLAMP and (D) sgLAMP assays. cLAMP reaction resulting in yellow colour after amplification denote a positive result, and while pink colour represents a negative test result. sgLAMP reaction showing a bright yellow-green colour denote a positive test result and orange colour depict a negative result. The images were taken with iPhone SE
    Figure Legend Snippet: Real-time and colorimetric C. psittaci and EHV-1 LAMP assays. The real-time fluorometer Genie III displays of (A) a C. psittaci and EHV-1 amplification curves within 30 min amplification time, and (B) High-resolution melts (HRM). Positive rtLAMP result is denoted with time to amplify (mm:ss) and HRM (°C), whilst negative result does not record either. End-point detection using C. psittaci (top row) and EHV-1 (bottom row) (C) cLAMP and (D) sgLAMP assays. cLAMP reaction resulting in yellow colour after amplification denote a positive result, and while pink colour represents a negative test result. sgLAMP reaction showing a bright yellow-green colour denote a positive test result and orange colour depict a negative result. The images were taken with iPhone SE

    Techniques Used: Amplification

    8) Product Images from "Multicenter international assessment of a SARS-CoV-2 RT-LAMP test for point of care clinical application"

    Article Title: Multicenter international assessment of a SARS-CoV-2 RT-LAMP test for point of care clinical application

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0268340

    Effect of primer multiplexing and supplements. (A) Evaluation of RT-LAMP performance with the indicated primer multiplexing. RT-LAMP reactions were carried out with 15 copies of SARS-CoV-2 RNA at the optimized concentration for each primer set (see Fig 2A in bold). (B) Evaluation of RT-LAMP performance with 40mM GuHCl and/or 0.5M betaine. Reactions were performed with multiplexed Gene E1 and ORF1a primers. (C) LoD assessment of the best RT-LAMP condition with the indicated copy numbers of SARS-CoV-2 RNA. (D) Fluorescent readouts and color changes of the reactions in (C) at 60 minutes. Each condition was evaluated with 10 replicates. NTC, no template control; TTR, time to results; RFU, relative fluorescent units; Error bars represent mean ± standard deviations.
    Figure Legend Snippet: Effect of primer multiplexing and supplements. (A) Evaluation of RT-LAMP performance with the indicated primer multiplexing. RT-LAMP reactions were carried out with 15 copies of SARS-CoV-2 RNA at the optimized concentration for each primer set (see Fig 2A in bold). (B) Evaluation of RT-LAMP performance with 40mM GuHCl and/or 0.5M betaine. Reactions were performed with multiplexed Gene E1 and ORF1a primers. (C) LoD assessment of the best RT-LAMP condition with the indicated copy numbers of SARS-CoV-2 RNA. (D) Fluorescent readouts and color changes of the reactions in (C) at 60 minutes. Each condition was evaluated with 10 replicates. NTC, no template control; TTR, time to results; RFU, relative fluorescent units; Error bars represent mean ± standard deviations.

    Techniques Used: Multiplexing, Concentration Assay

    Screening primer performance at a low copy number of SARS-CoV-2 RNA. (A) A schematic showing a DNA template amplified by LAMP and the primers targeted to the regions in the template. (B) Location of the 7 target regions for the 14 primer sets in the SARS-CoV-2 genome (NC_045512.2, [ 34 ]). The indicated target region is that amplified by the outer F3 and B3 primers. (C) Matrix of test conditions. Each primer set was tested with the indicated primer molar ratio (black), and primer concentrations (blue). A total of 16 conditions were tested for each of the 14 primer sets, with 4 replicates per condition. Other reaction reagents are indicated in red. (D) Screening results for primer Gene E1. Top panel: For the indicated primer mixes (X-axis), red and blue bars indicate TTR using 30 copies of positive control SARS-CoV-2 RNA (TTR PC ) or no template (TTR NTC ), respectively. Red and blue circles indicate sensitivity and specificity, respectively. Bottom left graph shows an example of the fluorescent signal obtained with STYO 9 dye over the 60 minute reaction period for PC (red) or NTC (blue–undetected) using the indicated Gene E1 primer mix. Green line: Threshold to designate TTR. Bottom right panel shows an example of the phenol red colour at 60 minutes. (E and F) Screening results of primer ORF1a (E) and human ACTB (F); format as in (D). (G) Summary of the best two primer concentrations for the top performing four primer sets with adequate performance based on sensitivity, specificity and TTR. NTC, no template control; PC, positive control (30 copies of SARS-CoV-2 RNA); Sensitivity, the percentage of PC replicates with amplifications; Specificity, the percentage of NTC replicates without amplifications; RFU, relative fluorescence units; TTR, time to results (minutes), the time point that the RFU curve crossing the fluorescent threshold; Error bars represent mean ± standard deviations.
    Figure Legend Snippet: Screening primer performance at a low copy number of SARS-CoV-2 RNA. (A) A schematic showing a DNA template amplified by LAMP and the primers targeted to the regions in the template. (B) Location of the 7 target regions for the 14 primer sets in the SARS-CoV-2 genome (NC_045512.2, [ 34 ]). The indicated target region is that amplified by the outer F3 and B3 primers. (C) Matrix of test conditions. Each primer set was tested with the indicated primer molar ratio (black), and primer concentrations (blue). A total of 16 conditions were tested for each of the 14 primer sets, with 4 replicates per condition. Other reaction reagents are indicated in red. (D) Screening results for primer Gene E1. Top panel: For the indicated primer mixes (X-axis), red and blue bars indicate TTR using 30 copies of positive control SARS-CoV-2 RNA (TTR PC ) or no template (TTR NTC ), respectively. Red and blue circles indicate sensitivity and specificity, respectively. Bottom left graph shows an example of the fluorescent signal obtained with STYO 9 dye over the 60 minute reaction period for PC (red) or NTC (blue–undetected) using the indicated Gene E1 primer mix. Green line: Threshold to designate TTR. Bottom right panel shows an example of the phenol red colour at 60 minutes. (E and F) Screening results of primer ORF1a (E) and human ACTB (F); format as in (D). (G) Summary of the best two primer concentrations for the top performing four primer sets with adequate performance based on sensitivity, specificity and TTR. NTC, no template control; PC, positive control (30 copies of SARS-CoV-2 RNA); Sensitivity, the percentage of PC replicates with amplifications; Specificity, the percentage of NTC replicates without amplifications; RFU, relative fluorescence units; TTR, time to results (minutes), the time point that the RFU curve crossing the fluorescent threshold; Error bars represent mean ± standard deviations.

    Techniques Used: Low Copy Number, Amplification, Positive Control, Fluorescence

    Evaluation of the optimized primer concentrations based on limit of detection and specificity. (A) ORF1a, Gene E1, Gene N2 and N-gene primers were assessed at the indicated conditions. Each condition was evaluated with 10 replicates. (B) ACTB primers were evaluated under the indicated conditions. Sensitivity, the percentage of replicates with SARS-CoV-2 RNA or human RNA showing amplifications; Specificity, the percentage of no template controls without amplifications; TTR, time to results (minutes); LoD, limit of detection; Error bars represent mean ± standard deviations.
    Figure Legend Snippet: Evaluation of the optimized primer concentrations based on limit of detection and specificity. (A) ORF1a, Gene E1, Gene N2 and N-gene primers were assessed at the indicated conditions. Each condition was evaluated with 10 replicates. (B) ACTB primers were evaluated under the indicated conditions. Sensitivity, the percentage of replicates with SARS-CoV-2 RNA or human RNA showing amplifications; Specificity, the percentage of no template controls without amplifications; TTR, time to results (minutes); LoD, limit of detection; Error bars represent mean ± standard deviations.

    Techniques Used:

    Direct RT-LAMP on raw clinical NP samples without RNA extraction. (A) ROC curves evaluating RT-LAMP performance on 30 positive and 36 negative clinical NP samples with the indicated supplements. 0.5M betaine + 0.25% Igepal CA-630 in green; 0.5M betaine in red; No supplements in pink; 40mM GuHCl + 0.5M betaine in light blue; 40mM GuHCl in black. 1μl of raw samples (without any sample processing) was applied to RT-LAMP reactions, and the reactions were carried out with multiplexing primers for Gene E1 and ORF1a. Significance values were calculated with MedCalc software for ROC curve analysis. TTR* indicates the cutoff providing optimal sensitivity and specificity. (B) Distribution of the RT-LAMP TTRs vs . BGI RT-PCR Ct values with the indicated supplements. Dotted lines indicate cutoffs. (C) Representative fluorescent readouts of RT-LAMP with 0.5M betaine and 0.25% Igepal CA-630. (D) Sensitivity of RT-LAMP at the indicated Ct ranges. Left panel, Clinical NP samples. Right panel, Contrived positives generated by diluting clinical NP positives with negative NP samples.
    Figure Legend Snippet: Direct RT-LAMP on raw clinical NP samples without RNA extraction. (A) ROC curves evaluating RT-LAMP performance on 30 positive and 36 negative clinical NP samples with the indicated supplements. 0.5M betaine + 0.25% Igepal CA-630 in green; 0.5M betaine in red; No supplements in pink; 40mM GuHCl + 0.5M betaine in light blue; 40mM GuHCl in black. 1μl of raw samples (without any sample processing) was applied to RT-LAMP reactions, and the reactions were carried out with multiplexing primers for Gene E1 and ORF1a. Significance values were calculated with MedCalc software for ROC curve analysis. TTR* indicates the cutoff providing optimal sensitivity and specificity. (B) Distribution of the RT-LAMP TTRs vs . BGI RT-PCR Ct values with the indicated supplements. Dotted lines indicate cutoffs. (C) Representative fluorescent readouts of RT-LAMP with 0.5M betaine and 0.25% Igepal CA-630. (D) Sensitivity of RT-LAMP at the indicated Ct ranges. Left panel, Clinical NP samples. Right panel, Contrived positives generated by diluting clinical NP positives with negative NP samples.

    Techniques Used: RNA Extraction, Multiplexing, Software, Reverse Transcription Polymerase Chain Reaction, Generated

    Comparison of RT-LAMP and BGI RT-PCR with extracted RNA from clinical NP samples. (A) Correlation of Ct values with BGI RT-PCR kit vs . other indicated RT-PCR reagents in 30 SARS-CoV-2 positive clinical NP samples. (B) ROC curve evaluating RT-LAMP performance with 30 positive and 36 negative Canadian clinical samples based on the results of BGI RT-PCR kit. TPR: True positive rate; FPR: False positive rate. TTR ≤ 13.2’ was defined as the cut-off to distinguish positive from negative samples with 90% detection sensitivity and 100% specificity. (C) Distribution of RT-LAMP TTRs against BGI RT-PCR Ct values for 30 positive and 36 negative clinical NP samples. BGI RT-PCR and RT-LAMP positives were defined by Ct
    Figure Legend Snippet: Comparison of RT-LAMP and BGI RT-PCR with extracted RNA from clinical NP samples. (A) Correlation of Ct values with BGI RT-PCR kit vs . other indicated RT-PCR reagents in 30 SARS-CoV-2 positive clinical NP samples. (B) ROC curve evaluating RT-LAMP performance with 30 positive and 36 negative Canadian clinical samples based on the results of BGI RT-PCR kit. TPR: True positive rate; FPR: False positive rate. TTR ≤ 13.2’ was defined as the cut-off to distinguish positive from negative samples with 90% detection sensitivity and 100% specificity. (C) Distribution of RT-LAMP TTRs against BGI RT-PCR Ct values for 30 positive and 36 negative clinical NP samples. BGI RT-PCR and RT-LAMP positives were defined by Ct

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    9) Product Images from "Diagnostic utility and validation of a newly developed real time loop mediated isothermal amplification method for the detection of SARS CoV-2 infection"

    Article Title: Diagnostic utility and validation of a newly developed real time loop mediated isothermal amplification method for the detection of SARS CoV-2 infection

    Journal: Journal of Clinical Virology plus

    doi: 10.1016/j.jcvp.2022.100081

    Receiver operating characteristic curve (ROC curve) to study the performance of the newly developed LAMP assay for the detection of SARS CoV-2.
    Figure Legend Snippet: Receiver operating characteristic curve (ROC curve) to study the performance of the newly developed LAMP assay for the detection of SARS CoV-2.

    Techniques Used: Lamp Assay

    Comparison of turnaround time for real time RT-LAMP and real time RT-PCR for the detection of SARS CoV-2.
    Figure Legend Snippet: Comparison of turnaround time for real time RT-LAMP and real time RT-PCR for the detection of SARS CoV-2.

    Techniques Used: Quantitative RT-PCR

    SARS-CoV-2 RNA detected by direct visualization for the presence and absence of turbidity. A - Positive samples showing turbidity . B - Negative samples not showing turbidity.
    Figure Legend Snippet: SARS-CoV-2 RNA detected by direct visualization for the presence and absence of turbidity. A - Positive samples showing turbidity . B - Negative samples not showing turbidity.

    Techniques Used:

    10) Product Images from "A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples"

    Article Title: A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-95799-6

    Limit of detection of the two different RT-LAMP formats and of RT-PCR. ( A ) A known number of copies of in vitro transcribed (IVT) viral RNA (N-gene) were amplified and detected by colorimetric RT-LAMP using the (i) WarmStart Colorimetric LAMP 2 × Master Mix (New England Biolabs) or (ii) the separate components (enzymes purchased individually and an in-house-made colorimetric buffer). The reactions were incubated at 65 °C for 30 min. ( B ) 10 μL of the RT-LAMP reaction were resolved in an agarose gel (2%) electrophoresis. The ladder pattern corresponds to the expected LAMP amplification pattern. ( C ) Limit of detection of ten replicates of the two test formats. ( D ) Standard curve generated by plotting the number of IVT RNA copies (x-axis) vs. the mean of the corresponding RT-PCR threshold cycle (Ct) value (y-axis) of three independent experiments (Original gel images in Fig. S1 ).
    Figure Legend Snippet: Limit of detection of the two different RT-LAMP formats and of RT-PCR. ( A ) A known number of copies of in vitro transcribed (IVT) viral RNA (N-gene) were amplified and detected by colorimetric RT-LAMP using the (i) WarmStart Colorimetric LAMP 2 × Master Mix (New England Biolabs) or (ii) the separate components (enzymes purchased individually and an in-house-made colorimetric buffer). The reactions were incubated at 65 °C for 30 min. ( B ) 10 μL of the RT-LAMP reaction were resolved in an agarose gel (2%) electrophoresis. The ladder pattern corresponds to the expected LAMP amplification pattern. ( C ) Limit of detection of ten replicates of the two test formats. ( D ) Standard curve generated by plotting the number of IVT RNA copies (x-axis) vs. the mean of the corresponding RT-PCR threshold cycle (Ct) value (y-axis) of three independent experiments (Original gel images in Fig. S1 ).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, In Vitro, Amplification, Incubation, Agarose Gel Electrophoresis, Electrophoresis, Generated

    11) Product Images from "Validation of a colorimetric LAMP to detect Loxosceles experimental envenomation"

    Article Title: Validation of a colorimetric LAMP to detect Loxosceles experimental envenomation

    Journal: bioRxiv

    doi: 10.1101/2022.02.09.479769

    Limit of detection of Loxosceles similis DNA with LAMP and PCR. A) LAMP reaction was performed at 71 °C for 60 min using WarmStart ® colorimetric master LAMP mix (NEB #M1800) in 20 μL final volume. Amplicons were resolved in 1.5% agarose gel and stained with SYBR safe (0,009% v/v) (Invitrogen) to confirm DNA amplification. The LoD was established by titrating the L. similis DNA as input, ranging from 10 ng to 0.15 pg. B) PCR amplicons obtained with different L. similis DNA inputs varying from 10 to 0.002 ng. The assay was performed with external primers (F3 and B3) and TaqPlatinum™ enzyme. M: molecular weight standard of 100 bp. NTC: no template control, P: Positive control (10 ng L. similis DNA).
    Figure Legend Snippet: Limit of detection of Loxosceles similis DNA with LAMP and PCR. A) LAMP reaction was performed at 71 °C for 60 min using WarmStart ® colorimetric master LAMP mix (NEB #M1800) in 20 μL final volume. Amplicons were resolved in 1.5% agarose gel and stained with SYBR safe (0,009% v/v) (Invitrogen) to confirm DNA amplification. The LoD was established by titrating the L. similis DNA as input, ranging from 10 ng to 0.15 pg. B) PCR amplicons obtained with different L. similis DNA inputs varying from 10 to 0.002 ng. The assay was performed with external primers (F3 and B3) and TaqPlatinum™ enzyme. M: molecular weight standard of 100 bp. NTC: no template control, P: Positive control (10 ng L. similis DNA).

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Amplification, Molecular Weight, Positive Control

    12) Product Images from "Validation of a rapid, saliva-based, and ultra-sensitive SARS-CoV-2 screening system for pandemic-scale infection surveillance"

    Article Title: Validation of a rapid, saliva-based, and ultra-sensitive SARS-CoV-2 screening system for pandemic-scale infection surveillance

    Journal: Scientific Reports

    doi: 10.1038/s41598-022-08263-4

    The presence of mineral oil markedly reduces the rate of production of false positive RT-LAMP reactions. Two assay chemistries were compared: NEB WarmStart Colorimetric LAMP with UDG (M1804) and Hayat Rapid Colorimetric Fluorometric One Step LAMP SARS-CoV-2 Test Kit, each set up with and without 15 μl mineral oil overlay. Twenty-one identical replicate negative control reactions were set up per condition with a single saliva sample negative for SARS-CoV-2 diluted in VTM and AviSal at a ratio of 1:1:2 and heat inactivated for 10 min at 95 °C. The sample was added to give 5% (NEB) and 3.75% (Hayat) final concentrations of crude saliva in a 25 μl reaction volume. + M.O. with mineral oil overlay; − M.O. without mineral oil. In a typical 30-min reaction runtime, only Hayat chemistry resulted in no false positives and 100% specificity. For the NEB chemistry, false positives were observed after 20 min, even with a mineral oil overlay. Mineral oil overlay markedly reduces the false positive rate.
    Figure Legend Snippet: The presence of mineral oil markedly reduces the rate of production of false positive RT-LAMP reactions. Two assay chemistries were compared: NEB WarmStart Colorimetric LAMP with UDG (M1804) and Hayat Rapid Colorimetric Fluorometric One Step LAMP SARS-CoV-2 Test Kit, each set up with and without 15 μl mineral oil overlay. Twenty-one identical replicate negative control reactions were set up per condition with a single saliva sample negative for SARS-CoV-2 diluted in VTM and AviSal at a ratio of 1:1:2 and heat inactivated for 10 min at 95 °C. The sample was added to give 5% (NEB) and 3.75% (Hayat) final concentrations of crude saliva in a 25 μl reaction volume. + M.O. with mineral oil overlay; − M.O. without mineral oil. In a typical 30-min reaction runtime, only Hayat chemistry resulted in no false positives and 100% specificity. For the NEB chemistry, false positives were observed after 20 min, even with a mineral oil overlay. Mineral oil overlay markedly reduces the false positive rate.

    Techniques Used: Negative Control

    13) Product Images from "Multicenter international assessment of a SARS-CoV-2 RT-LAMP test for point of care clinical application"

    Article Title: Multicenter international assessment of a SARS-CoV-2 RT-LAMP test for point of care clinical application

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0268340

    Effect of primer multiplexing and supplements. (A) Evaluation of RT-LAMP performance with the indicated primer multiplexing. RT-LAMP reactions were carried out with 15 copies of SARS-CoV-2 RNA at the optimized concentration for each primer set (see Fig 2A in bold). (B) Evaluation of RT-LAMP performance with 40mM GuHCl and/or 0.5M betaine. Reactions were performed with multiplexed Gene E1 and ORF1a primers. (C) LoD assessment of the best RT-LAMP condition with the indicated copy numbers of SARS-CoV-2 RNA. (D) Fluorescent readouts and color changes of the reactions in (C) at 60 minutes. Each condition was evaluated with 10 replicates. NTC, no template control; TTR, time to results; RFU, relative fluorescent units; Error bars represent mean ± standard deviations.
    Figure Legend Snippet: Effect of primer multiplexing and supplements. (A) Evaluation of RT-LAMP performance with the indicated primer multiplexing. RT-LAMP reactions were carried out with 15 copies of SARS-CoV-2 RNA at the optimized concentration for each primer set (see Fig 2A in bold). (B) Evaluation of RT-LAMP performance with 40mM GuHCl and/or 0.5M betaine. Reactions were performed with multiplexed Gene E1 and ORF1a primers. (C) LoD assessment of the best RT-LAMP condition with the indicated copy numbers of SARS-CoV-2 RNA. (D) Fluorescent readouts and color changes of the reactions in (C) at 60 minutes. Each condition was evaluated with 10 replicates. NTC, no template control; TTR, time to results; RFU, relative fluorescent units; Error bars represent mean ± standard deviations.

    Techniques Used: Multiplexing, Concentration Assay

    Screening primer performance at a low copy number of SARS-CoV-2 RNA. (A) A schematic showing a DNA template amplified by LAMP and the primers targeted to the regions in the template. (B) Location of the 7 target regions for the 14 primer sets in the SARS-CoV-2 genome (NC_045512.2, [ 34 ]). The indicated target region is that amplified by the outer F3 and B3 primers. (C) Matrix of test conditions. Each primer set was tested with the indicated primer molar ratio (black), and primer concentrations (blue). A total of 16 conditions were tested for each of the 14 primer sets, with 4 replicates per condition. Other reaction reagents are indicated in red. (D) Screening results for primer Gene E1. Top panel: For the indicated primer mixes (X-axis), red and blue bars indicate TTR using 30 copies of positive control SARS-CoV-2 RNA (TTR PC ) or no template (TTR NTC ), respectively. Red and blue circles indicate sensitivity and specificity, respectively. Bottom left graph shows an example of the fluorescent signal obtained with STYO 9 dye over the 60 minute reaction period for PC (red) or NTC (blue–undetected) using the indicated Gene E1 primer mix. Green line: Threshold to designate TTR. Bottom right panel shows an example of the phenol red colour at 60 minutes. (E and F) Screening results of primer ORF1a (E) and human ACTB (F); format as in (D). (G) Summary of the best two primer concentrations for the top performing four primer sets with adequate performance based on sensitivity, specificity and TTR. NTC, no template control; PC, positive control (30 copies of SARS-CoV-2 RNA); Sensitivity, the percentage of PC replicates with amplifications; Specificity, the percentage of NTC replicates without amplifications; RFU, relative fluorescence units; TTR, time to results (minutes), the time point that the RFU curve crossing the fluorescent threshold; Error bars represent mean ± standard deviations.
    Figure Legend Snippet: Screening primer performance at a low copy number of SARS-CoV-2 RNA. (A) A schematic showing a DNA template amplified by LAMP and the primers targeted to the regions in the template. (B) Location of the 7 target regions for the 14 primer sets in the SARS-CoV-2 genome (NC_045512.2, [ 34 ]). The indicated target region is that amplified by the outer F3 and B3 primers. (C) Matrix of test conditions. Each primer set was tested with the indicated primer molar ratio (black), and primer concentrations (blue). A total of 16 conditions were tested for each of the 14 primer sets, with 4 replicates per condition. Other reaction reagents are indicated in red. (D) Screening results for primer Gene E1. Top panel: For the indicated primer mixes (X-axis), red and blue bars indicate TTR using 30 copies of positive control SARS-CoV-2 RNA (TTR PC ) or no template (TTR NTC ), respectively. Red and blue circles indicate sensitivity and specificity, respectively. Bottom left graph shows an example of the fluorescent signal obtained with STYO 9 dye over the 60 minute reaction period for PC (red) or NTC (blue–undetected) using the indicated Gene E1 primer mix. Green line: Threshold to designate TTR. Bottom right panel shows an example of the phenol red colour at 60 minutes. (E and F) Screening results of primer ORF1a (E) and human ACTB (F); format as in (D). (G) Summary of the best two primer concentrations for the top performing four primer sets with adequate performance based on sensitivity, specificity and TTR. NTC, no template control; PC, positive control (30 copies of SARS-CoV-2 RNA); Sensitivity, the percentage of PC replicates with amplifications; Specificity, the percentage of NTC replicates without amplifications; RFU, relative fluorescence units; TTR, time to results (minutes), the time point that the RFU curve crossing the fluorescent threshold; Error bars represent mean ± standard deviations.

    Techniques Used: Low Copy Number, Amplification, Positive Control, Fluorescence

    Evaluation of the optimized primer concentrations based on limit of detection and specificity. (A) ORF1a, Gene E1, Gene N2 and N-gene primers were assessed at the indicated conditions. Each condition was evaluated with 10 replicates. (B) ACTB primers were evaluated under the indicated conditions. Sensitivity, the percentage of replicates with SARS-CoV-2 RNA or human RNA showing amplifications; Specificity, the percentage of no template controls without amplifications; TTR, time to results (minutes); LoD, limit of detection; Error bars represent mean ± standard deviations.
    Figure Legend Snippet: Evaluation of the optimized primer concentrations based on limit of detection and specificity. (A) ORF1a, Gene E1, Gene N2 and N-gene primers were assessed at the indicated conditions. Each condition was evaluated with 10 replicates. (B) ACTB primers were evaluated under the indicated conditions. Sensitivity, the percentage of replicates with SARS-CoV-2 RNA or human RNA showing amplifications; Specificity, the percentage of no template controls without amplifications; TTR, time to results (minutes); LoD, limit of detection; Error bars represent mean ± standard deviations.

    Techniques Used:

    Direct RT-LAMP on raw clinical NP samples without RNA extraction. (A) ROC curves evaluating RT-LAMP performance on 30 positive and 36 negative clinical NP samples with the indicated supplements. 0.5M betaine + 0.25% Igepal CA-630 in green; 0.5M betaine in red; No supplements in pink; 40mM GuHCl + 0.5M betaine in light blue; 40mM GuHCl in black. 1μl of raw samples (without any sample processing) was applied to RT-LAMP reactions, and the reactions were carried out with multiplexing primers for Gene E1 and ORF1a. Significance values were calculated with MedCalc software for ROC curve analysis. TTR* indicates the cutoff providing optimal sensitivity and specificity. (B) Distribution of the RT-LAMP TTRs vs . BGI RT-PCR Ct values with the indicated supplements. Dotted lines indicate cutoffs. (C) Representative fluorescent readouts of RT-LAMP with 0.5M betaine and 0.25% Igepal CA-630. (D) Sensitivity of RT-LAMP at the indicated Ct ranges. Left panel, Clinical NP samples. Right panel, Contrived positives generated by diluting clinical NP positives with negative NP samples.
    Figure Legend Snippet: Direct RT-LAMP on raw clinical NP samples without RNA extraction. (A) ROC curves evaluating RT-LAMP performance on 30 positive and 36 negative clinical NP samples with the indicated supplements. 0.5M betaine + 0.25% Igepal CA-630 in green; 0.5M betaine in red; No supplements in pink; 40mM GuHCl + 0.5M betaine in light blue; 40mM GuHCl in black. 1μl of raw samples (without any sample processing) was applied to RT-LAMP reactions, and the reactions were carried out with multiplexing primers for Gene E1 and ORF1a. Significance values were calculated with MedCalc software for ROC curve analysis. TTR* indicates the cutoff providing optimal sensitivity and specificity. (B) Distribution of the RT-LAMP TTRs vs . BGI RT-PCR Ct values with the indicated supplements. Dotted lines indicate cutoffs. (C) Representative fluorescent readouts of RT-LAMP with 0.5M betaine and 0.25% Igepal CA-630. (D) Sensitivity of RT-LAMP at the indicated Ct ranges. Left panel, Clinical NP samples. Right panel, Contrived positives generated by diluting clinical NP positives with negative NP samples.

    Techniques Used: RNA Extraction, Multiplexing, Software, Reverse Transcription Polymerase Chain Reaction, Generated

    Comparison of RT-LAMP and BGI RT-PCR with extracted RNA from clinical NP samples. (A) Correlation of Ct values with BGI RT-PCR kit vs . other indicated RT-PCR reagents in 30 SARS-CoV-2 positive clinical NP samples. (B) ROC curve evaluating RT-LAMP performance with 30 positive and 36 negative Canadian clinical samples based on the results of BGI RT-PCR kit. TPR: True positive rate; FPR: False positive rate. TTR ≤ 13.2’ was defined as the cut-off to distinguish positive from negative samples with 90% detection sensitivity and 100% specificity. (C) Distribution of RT-LAMP TTRs against BGI RT-PCR Ct values for 30 positive and 36 negative clinical NP samples. BGI RT-PCR and RT-LAMP positives were defined by Ct
    Figure Legend Snippet: Comparison of RT-LAMP and BGI RT-PCR with extracted RNA from clinical NP samples. (A) Correlation of Ct values with BGI RT-PCR kit vs . other indicated RT-PCR reagents in 30 SARS-CoV-2 positive clinical NP samples. (B) ROC curve evaluating RT-LAMP performance with 30 positive and 36 negative Canadian clinical samples based on the results of BGI RT-PCR kit. TPR: True positive rate; FPR: False positive rate. TTR ≤ 13.2’ was defined as the cut-off to distinguish positive from negative samples with 90% detection sensitivity and 100% specificity. (C) Distribution of RT-LAMP TTRs against BGI RT-PCR Ct values for 30 positive and 36 negative clinical NP samples. BGI RT-PCR and RT-LAMP positives were defined by Ct

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    14) Product Images from "Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers"

    Article Title: Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers

    Journal: medRxiv

    doi: 10.1101/2020.07.16.20150250

    Optimized RT-LAMP primer sets for detecting SARS-CoV-2 in human saliva. A) Three RT-LAMP primer sets targeting the SARS-CoV-2 genome (AS1E ( Rabe and Cepko, 2020 ), ORF1e, and CU-N2) were tested with real-time RT-LAMP. Saliva was mixed 1:1 with 2X saliva stabilization solution, heated at 95°C for 10 minutes, and then spiked with in vitro transcribed SARS-CoV-2 RNA at the indicated concentrations. 4 μL of this was added to a master mix containing primers and NEB’s WarmStart LAMP 2x Master Mix in a final reaction volume of 20 μL. Reactions were incubated at 65°C and a fluorescence reading was taken every 30 seconds. EvaGreen was used to monitor amplification products in real-time (X-axis) using a QuantStudio3 quantitative PCR machine. There are 9 lines for each of the three primer sets because three concentrations of spiked in SARS-CoV-2 RNA were each tested in triplicate (0, 400, 800 copies / μL saliva). The saliva samples without SARS-CoV-2 RNA spike in are shown as flat lines. When concentrations are given herein, denominator refers to the raw, pre-diluted saliva sample. The normalized change in fluorescence signal (ΔRn) is shown on the Y-axis. B) Saliva mixed 1:1 with 2X saliva stabilization solution was heated (95°C for 10 minutes) and then spiked with SARS-CoV-2 RNA at the indicated concentrations. Replicates were tested by RT-LAMP with the control RNaseP primer set and three distinct SARS-CoV-2 primer sets (AS1E, ORF1e, and CU-N2). All samples scored positive except those boxed, which are saliva samples that contain no SARS-CoV-2 RNA, as expected.
    Figure Legend Snippet: Optimized RT-LAMP primer sets for detecting SARS-CoV-2 in human saliva. A) Three RT-LAMP primer sets targeting the SARS-CoV-2 genome (AS1E ( Rabe and Cepko, 2020 ), ORF1e, and CU-N2) were tested with real-time RT-LAMP. Saliva was mixed 1:1 with 2X saliva stabilization solution, heated at 95°C for 10 minutes, and then spiked with in vitro transcribed SARS-CoV-2 RNA at the indicated concentrations. 4 μL of this was added to a master mix containing primers and NEB’s WarmStart LAMP 2x Master Mix in a final reaction volume of 20 μL. Reactions were incubated at 65°C and a fluorescence reading was taken every 30 seconds. EvaGreen was used to monitor amplification products in real-time (X-axis) using a QuantStudio3 quantitative PCR machine. There are 9 lines for each of the three primer sets because three concentrations of spiked in SARS-CoV-2 RNA were each tested in triplicate (0, 400, 800 copies / μL saliva). The saliva samples without SARS-CoV-2 RNA spike in are shown as flat lines. When concentrations are given herein, denominator refers to the raw, pre-diluted saliva sample. The normalized change in fluorescence signal (ΔRn) is shown on the Y-axis. B) Saliva mixed 1:1 with 2X saliva stabilization solution was heated (95°C for 10 minutes) and then spiked with SARS-CoV-2 RNA at the indicated concentrations. Replicates were tested by RT-LAMP with the control RNaseP primer set and three distinct SARS-CoV-2 primer sets (AS1E, ORF1e, and CU-N2). All samples scored positive except those boxed, which are saliva samples that contain no SARS-CoV-2 RNA, as expected.

    Techniques Used: In Vitro, Incubation, Fluorescence, Amplification, Real-time Polymerase Chain Reaction

    15) Product Images from "Low saliva pH can yield false positives results in simple RT-LAMP-based SARS-CoV-2 diagnostic tests"

    Article Title: Low saliva pH can yield false positives results in simple RT-LAMP-based SARS-CoV-2 diagnostic tests

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0250202

    Critical value threshold determination for RT-LAMP tests for SARS-CoV-2 detection. A) NEB WarmStart LAMP kit pH is monitored using the pH indicator phenol red. In acid media phenol red has a yellow color and as the pH rises it turns to orange, red and finally pink. Addition of a new complementary nucleotide (dNTP) to a new synthesized DNA chain will form a phosphodiester bond between the α phosphate of the 3’ hydroxide of the pentose acidifying the medium and therefore turning the reaction color from red (basic) to yellow (acidic). B) Representative absorption spectrum from a negative and positive SARS-CoV-2 spiked sample using the NEB kit LAMP. The absorption spectrum for the negative sample is shown in a black line and the positive sample is shown in red line. Measurements were taken at two absorption maximum points, one in yellow (λ = 448 nm) and one in red (570 nm). C) Box plots represent the absorbance values of positive viral RNA-spiked samples and negative samples at 448 and 570 nm (n = 20) D) The quotient of 448/570 nm of negative and positive samples was used to set the crisitcal value threshold at 2. Box plots represent the values between positive and negative SARS-CoV2 spiked samples. Paired t-test of n = 20 **** P
    Figure Legend Snippet: Critical value threshold determination for RT-LAMP tests for SARS-CoV-2 detection. A) NEB WarmStart LAMP kit pH is monitored using the pH indicator phenol red. In acid media phenol red has a yellow color and as the pH rises it turns to orange, red and finally pink. Addition of a new complementary nucleotide (dNTP) to a new synthesized DNA chain will form a phosphodiester bond between the α phosphate of the 3’ hydroxide of the pentose acidifying the medium and therefore turning the reaction color from red (basic) to yellow (acidic). B) Representative absorption spectrum from a negative and positive SARS-CoV-2 spiked sample using the NEB kit LAMP. The absorption spectrum for the negative sample is shown in a black line and the positive sample is shown in red line. Measurements were taken at two absorption maximum points, one in yellow (λ = 448 nm) and one in red (570 nm). C) Box plots represent the absorbance values of positive viral RNA-spiked samples and negative samples at 448 and 570 nm (n = 20) D) The quotient of 448/570 nm of negative and positive samples was used to set the crisitcal value threshold at 2. Box plots represent the values between positive and negative SARS-CoV2 spiked samples. Paired t-test of n = 20 **** P

    Techniques Used: Synthesized

    16) Product Images from "Constructing Cell-Free Expression Systems for Low-Cost Access"

    Article Title: Constructing Cell-Free Expression Systems for Low-Cost Access

    Journal: ACS Synthetic Biology

    doi: 10.1021/acssynbio.1c00342

    Enhancer effect of lactose on gene expression using linear DNA templates in cell-free reactions. CFPS based on (A) PEP or (B) MDX. Details of additives in each cell-free mixture are shown in Table S5 . GamS and Chi6 were added at a final concentration of 2 μM. Except for NTC (no template control), all reactions contained 5 nM DNA (plasmid or linear). Linear DNA templates were amplified with extended 100 bp flanks to protect the template (highlighted with a gray dashed box). Unprotected linear DNA template was amplified with extended 3 bp flanks (denoted as “short flanks”). Black strips are representative images of the fluorescence signal on the plate captured using an imaging system (BioRad GelDoc-Go). Cell-free reactions were incubated at 29 °C for 10 h. All error bars represent standard error over three biological replicates based on three technical measurements.
    Figure Legend Snippet: Enhancer effect of lactose on gene expression using linear DNA templates in cell-free reactions. CFPS based on (A) PEP or (B) MDX. Details of additives in each cell-free mixture are shown in Table S5 . GamS and Chi6 were added at a final concentration of 2 μM. Except for NTC (no template control), all reactions contained 5 nM DNA (plasmid or linear). Linear DNA templates were amplified with extended 100 bp flanks to protect the template (highlighted with a gray dashed box). Unprotected linear DNA template was amplified with extended 3 bp flanks (denoted as “short flanks”). Black strips are representative images of the fluorescence signal on the plate captured using an imaging system (BioRad GelDoc-Go). Cell-free reactions were incubated at 29 °C for 10 h. All error bars represent standard error over three biological replicates based on three technical measurements.

    Techniques Used: Expressing, Concentration Assay, Plasmid Preparation, Amplification, Fluorescence, Imaging, Incubation

    Production of molecular biology reagents. (A) Purified Br512 Bst DNA polymerase visualized in a polyacrylamide gel stained with coomassie blue. (B) Colorimetric LAMP assay using the Br512 Bst DNA polymerase produced in vitro in both fresh conditions (top panel) and using rehydrated samples (bottom panel) after a 2 week storage at room temperature. Cell-free reactions based on PEP and MDX were prepared using the low-cost drying system and protected with sucrose (120 and 15 mM, respectively). A synthetic dsDNA fragment from actin B gene ( Homo sapiens ) was used as a target in the following amounts: 0, 0.025, 0.25, 2.5, 250, and 2500 pmoles. Primers used in this assay are described in Table S11 . Negative reactions were pink-colored, and positive reactions changed to yellow. (C, D) A PCR product encoding the Bsa I restriction endonuclease (2043 bp) was amplified using a single PCR with four oligonucleotides. An inner set of core primers provided a template for secondary amplification by longer oligonucleotides. The resulting product had extended terminal sequences that helped protect the coding region from exonuclease degradation. (E) Testing of Bsa I by restriction endonuclease digestion of luxpGEX plasmid. Digestion was performed using Bsa I produced by cell-free technology. Plasmid DNA samples were treated with (1) FastDigest Eco31I (Thermo Scientific, FD0293) (Isoschizomer: Bsa I), (2) Bsa I in cell extract, and (3) Bsa I in cell extract: 100% glycerol (1:1). Expected size of bands after digestion: 6440 and 4433 bp.
    Figure Legend Snippet: Production of molecular biology reagents. (A) Purified Br512 Bst DNA polymerase visualized in a polyacrylamide gel stained with coomassie blue. (B) Colorimetric LAMP assay using the Br512 Bst DNA polymerase produced in vitro in both fresh conditions (top panel) and using rehydrated samples (bottom panel) after a 2 week storage at room temperature. Cell-free reactions based on PEP and MDX were prepared using the low-cost drying system and protected with sucrose (120 and 15 mM, respectively). A synthetic dsDNA fragment from actin B gene ( Homo sapiens ) was used as a target in the following amounts: 0, 0.025, 0.25, 2.5, 250, and 2500 pmoles. Primers used in this assay are described in Table S11 . Negative reactions were pink-colored, and positive reactions changed to yellow. (C, D) A PCR product encoding the Bsa I restriction endonuclease (2043 bp) was amplified using a single PCR with four oligonucleotides. An inner set of core primers provided a template for secondary amplification by longer oligonucleotides. The resulting product had extended terminal sequences that helped protect the coding region from exonuclease degradation. (E) Testing of Bsa I by restriction endonuclease digestion of luxpGEX plasmid. Digestion was performed using Bsa I produced by cell-free technology. Plasmid DNA samples were treated with (1) FastDigest Eco31I (Thermo Scientific, FD0293) (Isoschizomer: Bsa I), (2) Bsa I in cell extract, and (3) Bsa I in cell extract: 100% glycerol (1:1). Expected size of bands after digestion: 6440 and 4433 bp.

    Techniques Used: Purification, Staining, Lamp Assay, Produced, In Vitro, Polymerase Chain Reaction, Amplification, Plasmid Preparation

    Ultra-low-cost (ULC) cell-free formulation based on PEP. (A) Relative levels of cell-free protein synthesis after the successive removal of reaction components according to Tables S7B and S8B . Every sequential row removes one more reagent in addition to those above it. Cell-free reactions were prepared with PEP or MDX as an energy source. The importance of additional components in the reaction buffers was tested by omission, starting with the most costly and less essential. The activities of the cell-free extracts were measured by sfGFP production and normalized relative to the respective full reaction (PEP/PEP complete reaction or MDX/MDX complete reaction). All measurements were based on three biological and three technical replicates. Relative level of protein synthesis for the ultra-low-cost (ULC) cell-free formulation is highlighted with a black square. (B) Synthesis of fluorescent proteins using the ULC cell-free formulation supplemented with 11.25 mM lactose ( Table S13 ). Reporters: (1) psfGFP, (2) pJL1-eforRed, (3) pJL1-dTomato, (4) pFGC-T7-RibJ-mScarlet, (5) pFGC-T7-RibJ-RRvT, and (6) pFGC-T7-RibJ-mTFP1. (C) Quantification of sfGFP production in ULC-PEP formulation supplemented with 11.25 mM lactose. (D) Regeneration of NTPs, NMPs, and pyruvate during the cell-free reactions based on ULC-PEP formulation, measured by LC-MS at four time points. Samples were prepared as described in Table S13 , replacing the indicated DNA with MQ water. Cell-free extracts were supplemented with 11.25 mM lactose, as indicated. Concentrations of nucleotides and pyruvate were measured by LC-MS.
    Figure Legend Snippet: Ultra-low-cost (ULC) cell-free formulation based on PEP. (A) Relative levels of cell-free protein synthesis after the successive removal of reaction components according to Tables S7B and S8B . Every sequential row removes one more reagent in addition to those above it. Cell-free reactions were prepared with PEP or MDX as an energy source. The importance of additional components in the reaction buffers was tested by omission, starting with the most costly and less essential. The activities of the cell-free extracts were measured by sfGFP production and normalized relative to the respective full reaction (PEP/PEP complete reaction or MDX/MDX complete reaction). All measurements were based on three biological and three technical replicates. Relative level of protein synthesis for the ultra-low-cost (ULC) cell-free formulation is highlighted with a black square. (B) Synthesis of fluorescent proteins using the ULC cell-free formulation supplemented with 11.25 mM lactose ( Table S13 ). Reporters: (1) psfGFP, (2) pJL1-eforRed, (3) pJL1-dTomato, (4) pFGC-T7-RibJ-mScarlet, (5) pFGC-T7-RibJ-RRvT, and (6) pFGC-T7-RibJ-mTFP1. (C) Quantification of sfGFP production in ULC-PEP formulation supplemented with 11.25 mM lactose. (D) Regeneration of NTPs, NMPs, and pyruvate during the cell-free reactions based on ULC-PEP formulation, measured by LC-MS at four time points. Samples were prepared as described in Table S13 , replacing the indicated DNA with MQ water. Cell-free extracts were supplemented with 11.25 mM lactose, as indicated. Concentrations of nucleotides and pyruvate were measured by LC-MS.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy

    Lyoprotectant effects of five sugars individually added to the two cell-free formulations. CFPS based on PEP (A–E) and MDX (F–J) and dehydrated either by high-cost lyophilization or by the low-cost drying method. Samples were dried and stored at room temperature for 1 day and 2 weeks. Cell-free reactions were rehydrated and incubated at 29 °C for 15 h. The final concentrations of additives in the reactions are indicated on the horizontal axes. The percentage of the recovered protein production was calculated relative to that seen in fresh, additive-free reactions with the energy sources PEP or MDX. Error bars represent standard error over three technical measurements. (K) Effects of lactose on protein yields in fresh cell-free reactions based on PEP or MDX as energy sources. Samples were supplemented with lactose, 11.2 mM (PEP mixture; Tables S2 and S5 ) and 13.7 mM (MDX mixture; Tables S1 and S5 ) as indicated. Cell-free reactions for the production of a green fluorescent protein were incubated at 29 °C for 15 h using psfGFP as a DNA template. Yields were calculated relative to fluorescence values seen in PEP-formulated cell-free reactions in the absence of lactose. Error bars represent standard error over three technical measurements.
    Figure Legend Snippet: Lyoprotectant effects of five sugars individually added to the two cell-free formulations. CFPS based on PEP (A–E) and MDX (F–J) and dehydrated either by high-cost lyophilization or by the low-cost drying method. Samples were dried and stored at room temperature for 1 day and 2 weeks. Cell-free reactions were rehydrated and incubated at 29 °C for 15 h. The final concentrations of additives in the reactions are indicated on the horizontal axes. The percentage of the recovered protein production was calculated relative to that seen in fresh, additive-free reactions with the energy sources PEP or MDX. Error bars represent standard error over three technical measurements. (K) Effects of lactose on protein yields in fresh cell-free reactions based on PEP or MDX as energy sources. Samples were supplemented with lactose, 11.2 mM (PEP mixture; Tables S2 and S5 ) and 13.7 mM (MDX mixture; Tables S1 and S5 ) as indicated. Cell-free reactions for the production of a green fluorescent protein were incubated at 29 °C for 15 h using psfGFP as a DNA template. Yields were calculated relative to fluorescence values seen in PEP-formulated cell-free reactions in the absence of lactose. Error bars represent standard error over three technical measurements.

    Techniques Used: Incubation, Fluorescence

    17) Product Images from "Colorimetric isothermal nucleic acid detection of SARS-CoV-2 with dye combination"

    Article Title: Colorimetric isothermal nucleic acid detection of SARS-CoV-2 with dye combination

    Journal: Heliyon

    doi: 10.1016/j.heliyon.2021.e06886

    Colorimetric isothermal nucleic acid detection of SARS-CoV-2 with dye combination. (A) Detection of SARS-CoV-2 with different concentrations of azure II-pheno red combined dye. (B) Sensitivity and accuracy of SARS-CoV-2 detection, WarmStart® Colorimetric LAMP 2X Master Mix (New England Biolabs) as a reference with diluted patient samples (duplicates of 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 and 10 3 copies of RNA as well as non template control (NTC)). (C) Summary of SARS-CoV-2 detection for diluted patient samples, WarmStart® Colorimetric LAMP 2X Master Mix (New England Biolabs) as a reference, color indicates time of detection of amplifying, gray indicates no amplification of no template control.
    Figure Legend Snippet: Colorimetric isothermal nucleic acid detection of SARS-CoV-2 with dye combination. (A) Detection of SARS-CoV-2 with different concentrations of azure II-pheno red combined dye. (B) Sensitivity and accuracy of SARS-CoV-2 detection, WarmStart® Colorimetric LAMP 2X Master Mix (New England Biolabs) as a reference with diluted patient samples (duplicates of 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 and 10 3 copies of RNA as well as non template control (NTC)). (C) Summary of SARS-CoV-2 detection for diluted patient samples, WarmStart® Colorimetric LAMP 2X Master Mix (New England Biolabs) as a reference, color indicates time of detection of amplifying, gray indicates no amplification of no template control.

    Techniques Used: Amplification

    18) Product Images from "A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples"

    Article Title: A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-95799-6

    Limit of detection of the two different RT-LAMP formats and of RT-PCR. ( A ) A known number of copies of in vitro transcribed (IVT) viral RNA (N-gene) were amplified and detected by colorimetric RT-LAMP using the (i) WarmStart Colorimetric LAMP 2 × Master Mix (New England Biolabs) or (ii) the separate components (enzymes purchased individually and an in-house-made colorimetric buffer). The reactions were incubated at 65 °C for 30 min. ( B ) 10 μL of the RT-LAMP reaction were resolved in an agarose gel (2%) electrophoresis. The ladder pattern corresponds to the expected LAMP amplification pattern. ( C ) Limit of detection of ten replicates of the two test formats. ( D ) Standard curve generated by plotting the number of IVT RNA copies (x-axis) vs. the mean of the corresponding RT-PCR threshold cycle (Ct) value (y-axis) of three independent experiments (Original gel images in Fig. S1 ).
    Figure Legend Snippet: Limit of detection of the two different RT-LAMP formats and of RT-PCR. ( A ) A known number of copies of in vitro transcribed (IVT) viral RNA (N-gene) were amplified and detected by colorimetric RT-LAMP using the (i) WarmStart Colorimetric LAMP 2 × Master Mix (New England Biolabs) or (ii) the separate components (enzymes purchased individually and an in-house-made colorimetric buffer). The reactions were incubated at 65 °C for 30 min. ( B ) 10 μL of the RT-LAMP reaction were resolved in an agarose gel (2%) electrophoresis. The ladder pattern corresponds to the expected LAMP amplification pattern. ( C ) Limit of detection of ten replicates of the two test formats. ( D ) Standard curve generated by plotting the number of IVT RNA copies (x-axis) vs. the mean of the corresponding RT-PCR threshold cycle (Ct) value (y-axis) of three independent experiments (Original gel images in Fig. S1 ).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, In Vitro, Amplification, Incubation, Agarose Gel Electrophoresis, Electrophoresis, Generated

    19) Product Images from "End-to-end system for rapid and sensitive early-detection of SARS-CoV-2 for resource-poor and field-test environments using a $51 lab-in-a-backpack"

    Article Title: End-to-end system for rapid and sensitive early-detection of SARS-CoV-2 for resource-poor and field-test environments using a $51 lab-in-a-backpack

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0259886

    CentriDrive and reagents for LAMP assay. (a) The CentriDrive. (b) Circuit diagram of the CentriDrive. 1. Switch. 2. Power input. 3. LCD. 4. Breadboard. 5. Geekcreit Nano. 6. Infrared sensor. 7. ESC. 8. Potentiometer. 9. Motor. (c) List of all reagents for LAMP assay. Chemical A, Lysis/Inactivation buffer. Chemical B.1, NaI powder. Chemical B.2, 1 N HCl. Chemical B.3, Triton X-100. Chemical C, Silica binding suspension. Chemical D, 80% ethanol solution. Chemical E.1, PBS buffer. Chemical E.2, Primer solution. Chemical E.3, WarmStart ® Colorimetric LAMP 2X Master Mix. Chemical E.4, SARS-CoV-2 solution. *The Arduino nano is shown in the figure due to limitations of the drawing software, but the Geekcreit Nano was used in the final test.
    Figure Legend Snippet: CentriDrive and reagents for LAMP assay. (a) The CentriDrive. (b) Circuit diagram of the CentriDrive. 1. Switch. 2. Power input. 3. LCD. 4. Breadboard. 5. Geekcreit Nano. 6. Infrared sensor. 7. ESC. 8. Potentiometer. 9. Motor. (c) List of all reagents for LAMP assay. Chemical A, Lysis/Inactivation buffer. Chemical B.1, NaI powder. Chemical B.2, 1 N HCl. Chemical B.3, Triton X-100. Chemical C, Silica binding suspension. Chemical D, 80% ethanol solution. Chemical E.1, PBS buffer. Chemical E.2, Primer solution. Chemical E.3, WarmStart ® Colorimetric LAMP 2X Master Mix. Chemical E.4, SARS-CoV-2 solution. *The Arduino nano is shown in the figure due to limitations of the drawing software, but the Geekcreit Nano was used in the final test.

    Techniques Used: Lamp Assay, Lysis, Binding Assay, Software

    20) Product Images from "Clinical validation of colorimetric RT-LAMP, a fast, highly sensitive and specific COVID-19 molecular diagnostic tool that is robust to detect SARS-CoV-2 variants of concern"

    Article Title: Clinical validation of colorimetric RT-LAMP, a fast, highly sensitive and specific COVID-19 molecular diagnostic tool that is robust to detect SARS-CoV-2 variants of concern

    Journal: medRxiv

    doi: 10.1101/2021.05.26.21257488

    Microbial cross-reactivity assay to test SARS-CoV-2 RT-LAMP analytical sensitivity. The test was performed using potentially cross-reacting respiratory viruses (A) or local occurring arboviruses (B). RT-LAMP reaction was performed at 65 °C during 30 min, with additional 10 min, to confirm the absence of cross-reactivity when using SARS-CoV-2 N gene as target. The assay was performed using the WarmStart ® colorimetric LAMP 2x master mix (NEB #M1800). Yellow (positive) reaction is only observed when the template is SARS-CoV-2 viral RNA. hRSV: human respiratory syncytial virus; NTC: non-template control; M: molecular size marker. RT-LAMP amplification products were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification. DENV3: Dengue virus serotype 3; ZIKV: Zika virus; CHIKV: Chikungunya virus; Influenza A (H1N1/H3N2); Influenza B (Yamagata/Victoria).
    Figure Legend Snippet: Microbial cross-reactivity assay to test SARS-CoV-2 RT-LAMP analytical sensitivity. The test was performed using potentially cross-reacting respiratory viruses (A) or local occurring arboviruses (B). RT-LAMP reaction was performed at 65 °C during 30 min, with additional 10 min, to confirm the absence of cross-reactivity when using SARS-CoV-2 N gene as target. The assay was performed using the WarmStart ® colorimetric LAMP 2x master mix (NEB #M1800). Yellow (positive) reaction is only observed when the template is SARS-CoV-2 viral RNA. hRSV: human respiratory syncytial virus; NTC: non-template control; M: molecular size marker. RT-LAMP amplification products were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification. DENV3: Dengue virus serotype 3; ZIKV: Zika virus; CHIKV: Chikungunya virus; Influenza A (H1N1/H3N2); Influenza B (Yamagata/Victoria).

    Techniques Used: Marker, Amplification, Agarose Gel Electrophoresis, Staining

    Analytical sensitivity as revealed by the limit of detection (LoD). RNA was extracted from VTM-nasopharyngeal swab and the genome viral copies input was calculated based on SARS-CoV-2 E gene-harboring plasmid (Bioclin #K228-1) calibration curve. RT-LAMP reaction was performed at 65 °C during 30 min using WarmStart ® colorimetric master LAMP mix (NEB #M1800) in 20 μL final volume (upper panel). Amplicons were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification (bottom panel). cps/µL: viral genome copies per microliter; NTC: non-template control; VTM: viral transport medium (Bioclin #G092-1).
    Figure Legend Snippet: Analytical sensitivity as revealed by the limit of detection (LoD). RNA was extracted from VTM-nasopharyngeal swab and the genome viral copies input was calculated based on SARS-CoV-2 E gene-harboring plasmid (Bioclin #K228-1) calibration curve. RT-LAMP reaction was performed at 65 °C during 30 min using WarmStart ® colorimetric master LAMP mix (NEB #M1800) in 20 μL final volume (upper panel). Amplicons were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification (bottom panel). cps/µL: viral genome copies per microliter; NTC: non-template control; VTM: viral transport medium (Bioclin #G092-1).

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Staining, Amplification

    Colorimetric RT-LAMP to detect SAR-CoV-2 in RNA extraction-free clinical samples (A) or laboratory-cultured virus (B). Clinical samples were derived from nasopharyngeal swabs placed on guanidine-containing viral transport medium, diluted 1:10. The RT-PCR Ct values for SARS-CoV-2 based on E gene are: CS134 = 31.8; CS135 = 15.3; CS138 = 18.4; CS139 = 21.7 and CS140 = 24.6. RT-LAMP reaction was performed in 20 μL final volume, incubated at 65 °C during 30, 40 or 50 min (inactivated virus) using WarmStart ® colorimetric LAMP master mix (NEB #M1800). Both, clinical samples or viruses, are RNA extraction-free samples. The amplification products (amplicons) were migrated in agarose gel at 2% to confirm amplification, as indicated by the characteristic ladder highlighted by GelRed ® staining. NTC: non-template control; CS: clinical sample; +C: positive control.
    Figure Legend Snippet: Colorimetric RT-LAMP to detect SAR-CoV-2 in RNA extraction-free clinical samples (A) or laboratory-cultured virus (B). Clinical samples were derived from nasopharyngeal swabs placed on guanidine-containing viral transport medium, diluted 1:10. The RT-PCR Ct values for SARS-CoV-2 based on E gene are: CS134 = 31.8; CS135 = 15.3; CS138 = 18.4; CS139 = 21.7 and CS140 = 24.6. RT-LAMP reaction was performed in 20 μL final volume, incubated at 65 °C during 30, 40 or 50 min (inactivated virus) using WarmStart ® colorimetric LAMP master mix (NEB #M1800). Both, clinical samples or viruses, are RNA extraction-free samples. The amplification products (amplicons) were migrated in agarose gel at 2% to confirm amplification, as indicated by the characteristic ladder highlighted by GelRed ® staining. NTC: non-template control; CS: clinical sample; +C: positive control.

    Techniques Used: RNA Extraction, Cell Culture, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Incubation, Amplification, Agarose Gel Electrophoresis, Staining, Positive Control

    Colorimetric RT-LAMP allows the detection of SARS-CoV-2 variants of concern (VOC) and interest (VOI). RT-LAMP reaction was performed at 65 °C for 30 min, using the WarmStart ® colorimetric LAMP 2x master mix (NEB #M1804), using either SARS-CoV-2 N gene set1 primers (upper left panel) or multiplex N2/E1 primer sets (down left panel). The amplicons were migrated in agarose gel at 2% to confirm amplification, as indicated by the characteristic ladder highlighted by GelRed ® staining. NTC: non-template control; CS: clinical sample; +C: positive control. The right hand panel shows a schematic representation of SARS-CoV-2 spike protein (upper) and highlights as representative SARS-CoV-2 virions, the main marker mutations present in Brazilian VOC P.1, VOI P.2 and N.10 as well as the VOC B.1.1.7 and B1.3.51 firstly reported in the United Kingdom and South Africa, respectively. K417N: lysine to asparagine substitution at position 417 of spike protein at the receptor biding domain (RBD); V445A: valine to alanine substitution at position 445 and so on. L: Leucine; Q: glutamine; E: glutamic acid; Y: tyrosine; T: threonine; P: proline; H: histidine; D: aspartic acid; S: serine; F: phenylalanine. del: deletion. Segments of SARS-CoV-2 protein NTD: N-terminal domain; CTD2: C-terminal domain 2 or C terminus of S1 fragment after furin cleavage; FP: fusion peptide; HR1: heptad repeat region 1. SARS-CoV-2 variants were previously sequenced. Variants of interest B.1.1.371 and B.1.1.374 were first reported in Saudi Arabia and Finland, respectively ( https://cov-lineages.org/ ).
    Figure Legend Snippet: Colorimetric RT-LAMP allows the detection of SARS-CoV-2 variants of concern (VOC) and interest (VOI). RT-LAMP reaction was performed at 65 °C for 30 min, using the WarmStart ® colorimetric LAMP 2x master mix (NEB #M1804), using either SARS-CoV-2 N gene set1 primers (upper left panel) or multiplex N2/E1 primer sets (down left panel). The amplicons were migrated in agarose gel at 2% to confirm amplification, as indicated by the characteristic ladder highlighted by GelRed ® staining. NTC: non-template control; CS: clinical sample; +C: positive control. The right hand panel shows a schematic representation of SARS-CoV-2 spike protein (upper) and highlights as representative SARS-CoV-2 virions, the main marker mutations present in Brazilian VOC P.1, VOI P.2 and N.10 as well as the VOC B.1.1.7 and B1.3.51 firstly reported in the United Kingdom and South Africa, respectively. K417N: lysine to asparagine substitution at position 417 of spike protein at the receptor biding domain (RBD); V445A: valine to alanine substitution at position 445 and so on. L: Leucine; Q: glutamine; E: glutamic acid; Y: tyrosine; T: threonine; P: proline; H: histidine; D: aspartic acid; S: serine; F: phenylalanine. del: deletion. Segments of SARS-CoV-2 protein NTD: N-terminal domain; CTD2: C-terminal domain 2 or C terminus of S1 fragment after furin cleavage; FP: fusion peptide; HR1: heptad repeat region 1. SARS-CoV-2 variants were previously sequenced. Variants of interest B.1.1.371 and B.1.1.374 were first reported in Saudi Arabia and Finland, respectively ( https://cov-lineages.org/ ).

    Techniques Used: Multiplex Assay, Agarose Gel Electrophoresis, Amplification, Staining, Positive Control, Marker

    Colorimetric RT-LAMP for COVID-19 diagnosis validation using one hundred clinical samples. Clinical samples were collected from symptomatic patients by nasopharyngeal swabs in partnership with CT-Vacinas/UFMG, Belo Horizonte, Brazil. Samples were obtained from different parts including Brazilian Southeast and Northeast regions. The reaction was performed at 65 °C during 30 min using WarmStart ® colorimetric LAMP master mix (NEB #M1800) in 20 μL final volume. The RT-LAMP reaction targeted SARS-CoV-2 N gene. Yellow content indicate positive reaction while pink pattern reveal non-reagent samples. Amplicons were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification. Latter pattern confirmed specific SARS-CoV-2 amplification that matches with yellow output tubes which is not observed in pink non-reagent tests. +C: positive control using RNA extracted from laboratory-Vero E6 cultured inactivated SARS-CoV-2. NTC: non-template control.
    Figure Legend Snippet: Colorimetric RT-LAMP for COVID-19 diagnosis validation using one hundred clinical samples. Clinical samples were collected from symptomatic patients by nasopharyngeal swabs in partnership with CT-Vacinas/UFMG, Belo Horizonte, Brazil. Samples were obtained from different parts including Brazilian Southeast and Northeast regions. The reaction was performed at 65 °C during 30 min using WarmStart ® colorimetric LAMP master mix (NEB #M1800) in 20 μL final volume. The RT-LAMP reaction targeted SARS-CoV-2 N gene. Yellow content indicate positive reaction while pink pattern reveal non-reagent samples. Amplicons were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification. Latter pattern confirmed specific SARS-CoV-2 amplification that matches with yellow output tubes which is not observed in pink non-reagent tests. +C: positive control using RNA extracted from laboratory-Vero E6 cultured inactivated SARS-CoV-2. NTC: non-template control.

    Techniques Used: Agarose Gel Electrophoresis, Staining, Amplification, Positive Control, Cell Culture

    Colorimetric RT-LAMP for SARS-CoV-2 detection using genes N, E and RdRp as target. Selected SARS-CoV-2 positive clinical samples by RT-qPCR were classified as low (Ct 18.9 and 21.7); medium (Ct 26.6 and 28.4) and high (Ct 31.6 and 35.2) Ct values for E gene. They were included as input for colorimetric RT-LAMP reaction using primers targeting N, RdRp (A) and E genes (B). RT-LAMP SARS-CoV-2 false negative samples are more frequent when using E and RdRp genes as target (C). RT-LAMP reaction was performed at 65 °C during 30 min, using the WarmStart ® colorimetric LAMP 2x master mix (NEB #M1800). RT-LAMP amplification products were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification. +C: positive control using SARS-CoV-2 RNA extracted from laboratory-cultured inactivated SARS-CoV-2. NTC: non-template control.
    Figure Legend Snippet: Colorimetric RT-LAMP for SARS-CoV-2 detection using genes N, E and RdRp as target. Selected SARS-CoV-2 positive clinical samples by RT-qPCR were classified as low (Ct 18.9 and 21.7); medium (Ct 26.6 and 28.4) and high (Ct 31.6 and 35.2) Ct values for E gene. They were included as input for colorimetric RT-LAMP reaction using primers targeting N, RdRp (A) and E genes (B). RT-LAMP SARS-CoV-2 false negative samples are more frequent when using E and RdRp genes as target (C). RT-LAMP reaction was performed at 65 °C during 30 min, using the WarmStart ® colorimetric LAMP 2x master mix (NEB #M1800). RT-LAMP amplification products were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification. +C: positive control using SARS-CoV-2 RNA extracted from laboratory-cultured inactivated SARS-CoV-2. NTC: non-template control.

    Techniques Used: Quantitative RT-PCR, Amplification, Agarose Gel Electrophoresis, Staining, Positive Control, Cell Culture

    21) Product Images from "Multicenter international assessment of a SARS-CoV-2 RT-LAMP test for point of care clinical application"

    Article Title: Multicenter international assessment of a SARS-CoV-2 RT-LAMP test for point of care clinical application

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0268340

    Effect of primer multiplexing and supplements. (A) Evaluation of RT-LAMP performance with the indicated primer multiplexing. RT-LAMP reactions were carried out with 15 copies of SARS-CoV-2 RNA at the optimized concentration for each primer set (see Fig 2A in bold). (B) Evaluation of RT-LAMP performance with 40mM GuHCl and/or 0.5M betaine. Reactions were performed with multiplexed Gene E1 and ORF1a primers. (C) LoD assessment of the best RT-LAMP condition with the indicated copy numbers of SARS-CoV-2 RNA. (D) Fluorescent readouts and color changes of the reactions in (C) at 60 minutes. Each condition was evaluated with 10 replicates. NTC, no template control; TTR, time to results; RFU, relative fluorescent units; Error bars represent mean ± standard deviations.
    Figure Legend Snippet: Effect of primer multiplexing and supplements. (A) Evaluation of RT-LAMP performance with the indicated primer multiplexing. RT-LAMP reactions were carried out with 15 copies of SARS-CoV-2 RNA at the optimized concentration for each primer set (see Fig 2A in bold). (B) Evaluation of RT-LAMP performance with 40mM GuHCl and/or 0.5M betaine. Reactions were performed with multiplexed Gene E1 and ORF1a primers. (C) LoD assessment of the best RT-LAMP condition with the indicated copy numbers of SARS-CoV-2 RNA. (D) Fluorescent readouts and color changes of the reactions in (C) at 60 minutes. Each condition was evaluated with 10 replicates. NTC, no template control; TTR, time to results; RFU, relative fluorescent units; Error bars represent mean ± standard deviations.

    Techniques Used: Multiplexing, Concentration Assay

    Screening primer performance at a low copy number of SARS-CoV-2 RNA. (A) A schematic showing a DNA template amplified by LAMP and the primers targeted to the regions in the template. (B) Location of the 7 target regions for the 14 primer sets in the SARS-CoV-2 genome (NC_045512.2, [ 34 ]). The indicated target region is that amplified by the outer F3 and B3 primers. (C) Matrix of test conditions. Each primer set was tested with the indicated primer molar ratio (black), and primer concentrations (blue). A total of 16 conditions were tested for each of the 14 primer sets, with 4 replicates per condition. Other reaction reagents are indicated in red. (D) Screening results for primer Gene E1. Top panel: For the indicated primer mixes (X-axis), red and blue bars indicate TTR using 30 copies of positive control SARS-CoV-2 RNA (TTR PC ) or no template (TTR NTC ), respectively. Red and blue circles indicate sensitivity and specificity, respectively. Bottom left graph shows an example of the fluorescent signal obtained with STYO 9 dye over the 60 minute reaction period for PC (red) or NTC (blue–undetected) using the indicated Gene E1 primer mix. Green line: Threshold to designate TTR. Bottom right panel shows an example of the phenol red colour at 60 minutes. (E and F) Screening results of primer ORF1a (E) and human ACTB (F); format as in (D). (G) Summary of the best two primer concentrations for the top performing four primer sets with adequate performance based on sensitivity, specificity and TTR. NTC, no template control; PC, positive control (30 copies of SARS-CoV-2 RNA); Sensitivity, the percentage of PC replicates with amplifications; Specificity, the percentage of NTC replicates without amplifications; RFU, relative fluorescence units; TTR, time to results (minutes), the time point that the RFU curve crossing the fluorescent threshold; Error bars represent mean ± standard deviations.
    Figure Legend Snippet: Screening primer performance at a low copy number of SARS-CoV-2 RNA. (A) A schematic showing a DNA template amplified by LAMP and the primers targeted to the regions in the template. (B) Location of the 7 target regions for the 14 primer sets in the SARS-CoV-2 genome (NC_045512.2, [ 34 ]). The indicated target region is that amplified by the outer F3 and B3 primers. (C) Matrix of test conditions. Each primer set was tested with the indicated primer molar ratio (black), and primer concentrations (blue). A total of 16 conditions were tested for each of the 14 primer sets, with 4 replicates per condition. Other reaction reagents are indicated in red. (D) Screening results for primer Gene E1. Top panel: For the indicated primer mixes (X-axis), red and blue bars indicate TTR using 30 copies of positive control SARS-CoV-2 RNA (TTR PC ) or no template (TTR NTC ), respectively. Red and blue circles indicate sensitivity and specificity, respectively. Bottom left graph shows an example of the fluorescent signal obtained with STYO 9 dye over the 60 minute reaction period for PC (red) or NTC (blue–undetected) using the indicated Gene E1 primer mix. Green line: Threshold to designate TTR. Bottom right panel shows an example of the phenol red colour at 60 minutes. (E and F) Screening results of primer ORF1a (E) and human ACTB (F); format as in (D). (G) Summary of the best two primer concentrations for the top performing four primer sets with adequate performance based on sensitivity, specificity and TTR. NTC, no template control; PC, positive control (30 copies of SARS-CoV-2 RNA); Sensitivity, the percentage of PC replicates with amplifications; Specificity, the percentage of NTC replicates without amplifications; RFU, relative fluorescence units; TTR, time to results (minutes), the time point that the RFU curve crossing the fluorescent threshold; Error bars represent mean ± standard deviations.

    Techniques Used: Low Copy Number, Amplification, Positive Control, Fluorescence

    Evaluation of the optimized primer concentrations based on limit of detection and specificity. (A) ORF1a, Gene E1, Gene N2 and N-gene primers were assessed at the indicated conditions. Each condition was evaluated with 10 replicates. (B) ACTB primers were evaluated under the indicated conditions. Sensitivity, the percentage of replicates with SARS-CoV-2 RNA or human RNA showing amplifications; Specificity, the percentage of no template controls without amplifications; TTR, time to results (minutes); LoD, limit of detection; Error bars represent mean ± standard deviations.
    Figure Legend Snippet: Evaluation of the optimized primer concentrations based on limit of detection and specificity. (A) ORF1a, Gene E1, Gene N2 and N-gene primers were assessed at the indicated conditions. Each condition was evaluated with 10 replicates. (B) ACTB primers were evaluated under the indicated conditions. Sensitivity, the percentage of replicates with SARS-CoV-2 RNA or human RNA showing amplifications; Specificity, the percentage of no template controls without amplifications; TTR, time to results (minutes); LoD, limit of detection; Error bars represent mean ± standard deviations.

    Techniques Used:

    Direct RT-LAMP on raw clinical NP samples without RNA extraction. (A) ROC curves evaluating RT-LAMP performance on 30 positive and 36 negative clinical NP samples with the indicated supplements. 0.5M betaine + 0.25% Igepal CA-630 in green; 0.5M betaine in red; No supplements in pink; 40mM GuHCl + 0.5M betaine in light blue; 40mM GuHCl in black. 1μl of raw samples (without any sample processing) was applied to RT-LAMP reactions, and the reactions were carried out with multiplexing primers for Gene E1 and ORF1a. Significance values were calculated with MedCalc software for ROC curve analysis. TTR* indicates the cutoff providing optimal sensitivity and specificity. (B) Distribution of the RT-LAMP TTRs vs . BGI RT-PCR Ct values with the indicated supplements. Dotted lines indicate cutoffs. (C) Representative fluorescent readouts of RT-LAMP with 0.5M betaine and 0.25% Igepal CA-630. (D) Sensitivity of RT-LAMP at the indicated Ct ranges. Left panel, Clinical NP samples. Right panel, Contrived positives generated by diluting clinical NP positives with negative NP samples.
    Figure Legend Snippet: Direct RT-LAMP on raw clinical NP samples without RNA extraction. (A) ROC curves evaluating RT-LAMP performance on 30 positive and 36 negative clinical NP samples with the indicated supplements. 0.5M betaine + 0.25% Igepal CA-630 in green; 0.5M betaine in red; No supplements in pink; 40mM GuHCl + 0.5M betaine in light blue; 40mM GuHCl in black. 1μl of raw samples (without any sample processing) was applied to RT-LAMP reactions, and the reactions were carried out with multiplexing primers for Gene E1 and ORF1a. Significance values were calculated with MedCalc software for ROC curve analysis. TTR* indicates the cutoff providing optimal sensitivity and specificity. (B) Distribution of the RT-LAMP TTRs vs . BGI RT-PCR Ct values with the indicated supplements. Dotted lines indicate cutoffs. (C) Representative fluorescent readouts of RT-LAMP with 0.5M betaine and 0.25% Igepal CA-630. (D) Sensitivity of RT-LAMP at the indicated Ct ranges. Left panel, Clinical NP samples. Right panel, Contrived positives generated by diluting clinical NP positives with negative NP samples.

    Techniques Used: RNA Extraction, Multiplexing, Software, Reverse Transcription Polymerase Chain Reaction, Generated

    Comparison of RT-LAMP and BGI RT-PCR with extracted RNA from clinical NP samples. (A) Correlation of Ct values with BGI RT-PCR kit vs . other indicated RT-PCR reagents in 30 SARS-CoV-2 positive clinical NP samples. (B) ROC curve evaluating RT-LAMP performance with 30 positive and 36 negative Canadian clinical samples based on the results of BGI RT-PCR kit. TPR: True positive rate; FPR: False positive rate. TTR ≤ 13.2’ was defined as the cut-off to distinguish positive from negative samples with 90% detection sensitivity and 100% specificity. (C) Distribution of RT-LAMP TTRs against BGI RT-PCR Ct values for 30 positive and 36 negative clinical NP samples. BGI RT-PCR and RT-LAMP positives were defined by Ct
    Figure Legend Snippet: Comparison of RT-LAMP and BGI RT-PCR with extracted RNA from clinical NP samples. (A) Correlation of Ct values with BGI RT-PCR kit vs . other indicated RT-PCR reagents in 30 SARS-CoV-2 positive clinical NP samples. (B) ROC curve evaluating RT-LAMP performance with 30 positive and 36 negative Canadian clinical samples based on the results of BGI RT-PCR kit. TPR: True positive rate; FPR: False positive rate. TTR ≤ 13.2’ was defined as the cut-off to distinguish positive from negative samples with 90% detection sensitivity and 100% specificity. (C) Distribution of RT-LAMP TTRs against BGI RT-PCR Ct values for 30 positive and 36 negative clinical NP samples. BGI RT-PCR and RT-LAMP positives were defined by Ct

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    22) Product Images from "Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers"

    Article Title: Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers

    Journal: medRxiv

    doi: 10.1101/2020.07.16.20150250

    Optimized RT-LAMP primer sets for detecting SARS-CoV-2 in human saliva. A) Three RT-LAMP primer sets targeting the SARS-CoV-2 genome (AS1E ( Rabe and Cepko, 2020 ), ORF1e, and CU-N2) were tested with real-time RT-LAMP. Saliva was mixed 1:1 with 2X saliva stabilization solution, heated at 95°C for 10 minutes, and then spiked with in vitro transcribed SARS-CoV-2 RNA at the indicated concentrations. 4 μL of this was added to a master mix containing primers and NEB’s WarmStart LAMP 2x Master Mix in a final reaction volume of 20 μL. Reactions were incubated at 65°C and a fluorescence reading was taken every 30 seconds. EvaGreen was used to monitor amplification products in real-time (X-axis) using a QuantStudio3 quantitative PCR machine. There are 9 lines for each of the three primer sets because three concentrations of spiked in SARS-CoV-2 RNA were each tested in triplicate (0, 400, 800 copies / μL saliva). The saliva samples without SARS-CoV-2 RNA spike in are shown as flat lines. When concentrations are given herein, denominator refers to the raw, pre-diluted saliva sample. The normalized change in fluorescence signal (ΔRn) is shown on the Y-axis. B) Saliva mixed 1:1 with 2X saliva stabilization solution was heated (95°C for 10 minutes) and then spiked with SARS-CoV-2 RNA at the indicated concentrations. Replicates were tested by RT-LAMP with the control RNaseP primer set and three distinct SARS-CoV-2 primer sets (AS1E, ORF1e, and CU-N2). All samples scored positive except those boxed, which are saliva samples that contain no SARS-CoV-2 RNA, as expected.
    Figure Legend Snippet: Optimized RT-LAMP primer sets for detecting SARS-CoV-2 in human saliva. A) Three RT-LAMP primer sets targeting the SARS-CoV-2 genome (AS1E ( Rabe and Cepko, 2020 ), ORF1e, and CU-N2) were tested with real-time RT-LAMP. Saliva was mixed 1:1 with 2X saliva stabilization solution, heated at 95°C for 10 minutes, and then spiked with in vitro transcribed SARS-CoV-2 RNA at the indicated concentrations. 4 μL of this was added to a master mix containing primers and NEB’s WarmStart LAMP 2x Master Mix in a final reaction volume of 20 μL. Reactions were incubated at 65°C and a fluorescence reading was taken every 30 seconds. EvaGreen was used to monitor amplification products in real-time (X-axis) using a QuantStudio3 quantitative PCR machine. There are 9 lines for each of the three primer sets because three concentrations of spiked in SARS-CoV-2 RNA were each tested in triplicate (0, 400, 800 copies / μL saliva). The saliva samples without SARS-CoV-2 RNA spike in are shown as flat lines. When concentrations are given herein, denominator refers to the raw, pre-diluted saliva sample. The normalized change in fluorescence signal (ΔRn) is shown on the Y-axis. B) Saliva mixed 1:1 with 2X saliva stabilization solution was heated (95°C for 10 minutes) and then spiked with SARS-CoV-2 RNA at the indicated concentrations. Replicates were tested by RT-LAMP with the control RNaseP primer set and three distinct SARS-CoV-2 primer sets (AS1E, ORF1e, and CU-N2). All samples scored positive except those boxed, which are saliva samples that contain no SARS-CoV-2 RNA, as expected.

    Techniques Used: In Vitro, Incubation, Fluorescence, Amplification, Real-time Polymerase Chain Reaction

    23) Product Images from "Low saliva pH can yield false positives results in simple RT-LAMP-based SARS-CoV-2 diagnostic tests"

    Article Title: Low saliva pH can yield false positives results in simple RT-LAMP-based SARS-CoV-2 diagnostic tests

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0250202

    Critical value threshold determination for RT-LAMP tests for SARS-CoV-2 detection. A) NEB WarmStart LAMP kit pH is monitored using the pH indicator phenol red. In acid media phenol red has a yellow color and as the pH rises it turns to orange, red and finally pink. Addition of a new complementary nucleotide (dNTP) to a new synthesized DNA chain will form a phosphodiester bond between the α phosphate of the 3’ hydroxide of the pentose acidifying the medium and therefore turning the reaction color from red (basic) to yellow (acidic). B) Representative absorption spectrum from a negative and positive SARS-CoV-2 spiked sample using the NEB kit LAMP. The absorption spectrum for the negative sample is shown in a black line and the positive sample is shown in red line. Measurements were taken at two absorption maximum points, one in yellow (λ = 448 nm) and one in red (570 nm). C) Box plots represent the absorbance values of positive viral RNA-spiked samples and negative samples at 448 and 570 nm (n = 20) D) The quotient of 448/570 nm of negative and positive samples was used to set the crisitcal value threshold at 2. Box plots represent the values between positive and negative SARS-CoV2 spiked samples. Paired t-test of n = 20 **** P
    Figure Legend Snippet: Critical value threshold determination for RT-LAMP tests for SARS-CoV-2 detection. A) NEB WarmStart LAMP kit pH is monitored using the pH indicator phenol red. In acid media phenol red has a yellow color and as the pH rises it turns to orange, red and finally pink. Addition of a new complementary nucleotide (dNTP) to a new synthesized DNA chain will form a phosphodiester bond between the α phosphate of the 3’ hydroxide of the pentose acidifying the medium and therefore turning the reaction color from red (basic) to yellow (acidic). B) Representative absorption spectrum from a negative and positive SARS-CoV-2 spiked sample using the NEB kit LAMP. The absorption spectrum for the negative sample is shown in a black line and the positive sample is shown in red line. Measurements were taken at two absorption maximum points, one in yellow (λ = 448 nm) and one in red (570 nm). C) Box plots represent the absorbance values of positive viral RNA-spiked samples and negative samples at 448 and 570 nm (n = 20) D) The quotient of 448/570 nm of negative and positive samples was used to set the crisitcal value threshold at 2. Box plots represent the values between positive and negative SARS-CoV2 spiked samples. Paired t-test of n = 20 **** P

    Techniques Used: Synthesized

    24) Product Images from "Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern"

    Article Title: Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2021.713713

    Colorimetric RT-LAMP allows the detection of SARS-CoV-2 VOCs and VOIs. RT-LAMP reaction was performed at 65°C for 30 min, using the WarmStart ® colorimetric LAMP 2× master mix (NEB #M1804), using multiplex N 2/ E 1 primer sets. The amplicons were migrated in agarose gel at 2% to confirm amplification, as indicated by the characteristic ladder highlighted by GelRed ® staining. NTC, nontemplate control; CS, clinical sample; and +C, positive control. The top panel shows a schematic representation of SARS-CoV-2 spike protein (upper) and where the main mutations are highlighted and represented in SARS-CoV-2 virions (right hand side) present in VOC gamma (B.1), delta (B.1.167.2), and VOI zeta (P.2). The VOCs alpha (B.1.1.7) and beta (B.1.3.51), first reported in the United Kingdom and South Africa, respectively, are also represented. K417N: lysine-to-asparagine substitution at position 417 of spike protein at the receptor biding domain (RBD); V445A: valine-to-alanine substitution at position 445 and so on. L, leucine; Q, glutamine; E, glutamic acid; Y, tyrosine; T, threonine; P, proline; H, histidine; D, aspartic acid; S, serine; F, phenylalanine. del, deletion. Segments of SARS-CoV-2 protein NTD, N-terminal domain; CTD2, C-terminal domain 2 or C terminus of S1 fragment after furin cleavage; FP, fusion peptide; HR1, heptad repeat region 1. SARS-CoV-2 variants were previously sequenced. Variants of interest B.1.1.371 and B.1.1.374 were first reported in Saudi Arabia and Finland, respectively, ( https://cov-lineages.org/ ). Created with biorender.com .
    Figure Legend Snippet: Colorimetric RT-LAMP allows the detection of SARS-CoV-2 VOCs and VOIs. RT-LAMP reaction was performed at 65°C for 30 min, using the WarmStart ® colorimetric LAMP 2× master mix (NEB #M1804), using multiplex N 2/ E 1 primer sets. The amplicons were migrated in agarose gel at 2% to confirm amplification, as indicated by the characteristic ladder highlighted by GelRed ® staining. NTC, nontemplate control; CS, clinical sample; and +C, positive control. The top panel shows a schematic representation of SARS-CoV-2 spike protein (upper) and where the main mutations are highlighted and represented in SARS-CoV-2 virions (right hand side) present in VOC gamma (B.1), delta (B.1.167.2), and VOI zeta (P.2). The VOCs alpha (B.1.1.7) and beta (B.1.3.51), first reported in the United Kingdom and South Africa, respectively, are also represented. K417N: lysine-to-asparagine substitution at position 417 of spike protein at the receptor biding domain (RBD); V445A: valine-to-alanine substitution at position 445 and so on. L, leucine; Q, glutamine; E, glutamic acid; Y, tyrosine; T, threonine; P, proline; H, histidine; D, aspartic acid; S, serine; F, phenylalanine. del, deletion. Segments of SARS-CoV-2 protein NTD, N-terminal domain; CTD2, C-terminal domain 2 or C terminus of S1 fragment after furin cleavage; FP, fusion peptide; HR1, heptad repeat region 1. SARS-CoV-2 variants were previously sequenced. Variants of interest B.1.1.371 and B.1.1.374 were first reported in Saudi Arabia and Finland, respectively, ( https://cov-lineages.org/ ). Created with biorender.com .

    Techniques Used: Multiplex Assay, Agarose Gel Electrophoresis, Amplification, Staining, Positive Control

    Microbial cross-reactivity assay to test SARS-CoV-2 RT-LAMP analytical sensitivity. The test was performed using potentially cross-reacting respiratory viruses or local occurring arboviruses. RT-LAMP reaction was performed at 65°C during 30 min, with additional 10 min, to confirm the absence of cross-reactivity when targeting SARS-CoV-2 E and N genes. The assay was performed using the WarmStart ® colorimetric LAMP 2× master mix (NEB #M1800). Yellow (positive) reaction is observed only when the template is SARS-CoV-2 viral RNA. hRSV, human respiratory syncytial virus; NTC, nontemplate control; M, molecular size marker. RT-LAMP amplification products were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification. DENV3, dengue virus serotype 3; ZIKV, Zika virus; CHIKV, Chikungunya virus; YFV, yellow fever virus; Influenza A (H1N1/H3N2); and influenza B (Yamagata/Victoria).
    Figure Legend Snippet: Microbial cross-reactivity assay to test SARS-CoV-2 RT-LAMP analytical sensitivity. The test was performed using potentially cross-reacting respiratory viruses or local occurring arboviruses. RT-LAMP reaction was performed at 65°C during 30 min, with additional 10 min, to confirm the absence of cross-reactivity when targeting SARS-CoV-2 E and N genes. The assay was performed using the WarmStart ® colorimetric LAMP 2× master mix (NEB #M1800). Yellow (positive) reaction is observed only when the template is SARS-CoV-2 viral RNA. hRSV, human respiratory syncytial virus; NTC, nontemplate control; M, molecular size marker. RT-LAMP amplification products were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification. DENV3, dengue virus serotype 3; ZIKV, Zika virus; CHIKV, Chikungunya virus; YFV, yellow fever virus; Influenza A (H1N1/H3N2); and influenza B (Yamagata/Victoria).

    Techniques Used: Marker, Amplification, Agarose Gel Electrophoresis, Staining

    Colorimetric RT-LAMP for SARS-CoV-2 detection using genes N , E , and RdRp as target. Selected SARS-CoV-2–positive clinical samples by RT-qPCR were classified as low (Ct 18.9 and 21.7), medium (Ct 26.6 and 28.4), and high (Ct 31.6 and 35.2) Ct values for E gene. They were included as input for colorimetric RT-LAMP reaction using primers targeting N , RdRp (A) , and E genes (B) . RT-LAMP SARS-CoV-2 false-negative samples were more frequent when using E and RdRp genes as target (C) . RT-LAMP reaction was performed at 65°C during 30 min, using the WarmStart ® colorimetric LAMP 2× master mix (NEB #M1800). RT-LAMP amplification products were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification. +C, positive control using SARS-CoV-2 RNA extracted from laboratory-cultured inactivated SARS-CoV-2; NTC, nontemplate control.
    Figure Legend Snippet: Colorimetric RT-LAMP for SARS-CoV-2 detection using genes N , E , and RdRp as target. Selected SARS-CoV-2–positive clinical samples by RT-qPCR were classified as low (Ct 18.9 and 21.7), medium (Ct 26.6 and 28.4), and high (Ct 31.6 and 35.2) Ct values for E gene. They were included as input for colorimetric RT-LAMP reaction using primers targeting N , RdRp (A) , and E genes (B) . RT-LAMP SARS-CoV-2 false-negative samples were more frequent when using E and RdRp genes as target (C) . RT-LAMP reaction was performed at 65°C during 30 min, using the WarmStart ® colorimetric LAMP 2× master mix (NEB #M1800). RT-LAMP amplification products were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification. +C, positive control using SARS-CoV-2 RNA extracted from laboratory-cultured inactivated SARS-CoV-2; NTC, nontemplate control.

    Techniques Used: Quantitative RT-PCR, Amplification, Agarose Gel Electrophoresis, Staining, Positive Control, Cell Culture

    25) Product Images from "Colorimetric isothermal nucleic acid detection of SARS-CoV-2 with dye combination"

    Article Title: Colorimetric isothermal nucleic acid detection of SARS-CoV-2 with dye combination

    Journal: Heliyon

    doi: 10.1016/j.heliyon.2021.e06886

    Colorimetric isothermal nucleic acid detection of SARS-CoV-2 with dye combination. (A) Detection of SARS-CoV-2 with different concentrations of azure II-pheno red combined dye. (B) Sensitivity and accuracy of SARS-CoV-2 detection, WarmStart® Colorimetric LAMP 2X Master Mix (New England Biolabs) as a reference with diluted patient samples (duplicates of 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 and 10 3 copies of RNA as well as non template control (NTC)). (C) Summary of SARS-CoV-2 detection for diluted patient samples, WarmStart® Colorimetric LAMP 2X Master Mix (New England Biolabs) as a reference, color indicates time of detection of amplifying, gray indicates no amplification of no template control.
    Figure Legend Snippet: Colorimetric isothermal nucleic acid detection of SARS-CoV-2 with dye combination. (A) Detection of SARS-CoV-2 with different concentrations of azure II-pheno red combined dye. (B) Sensitivity and accuracy of SARS-CoV-2 detection, WarmStart® Colorimetric LAMP 2X Master Mix (New England Biolabs) as a reference with diluted patient samples (duplicates of 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 and 10 3 copies of RNA as well as non template control (NTC)). (C) Summary of SARS-CoV-2 detection for diluted patient samples, WarmStart® Colorimetric LAMP 2X Master Mix (New England Biolabs) as a reference, color indicates time of detection of amplifying, gray indicates no amplification of no template control.

    Techniques Used: Amplification

    26) Product Images from "Colorimetric isothermal nucleic acid detection of SARS-CoV-2 with dye combination"

    Article Title: Colorimetric isothermal nucleic acid detection of SARS-CoV-2 with dye combination

    Journal: Heliyon

    doi: 10.1016/j.heliyon.2021.e06886

    Colorimetric isothermal nucleic acid detection of SARS-CoV-2 with dye combination. (A) Detection of SARS-CoV-2 with different concentrations of azure II-pheno red combined dye. (B) Sensitivity and accuracy of SARS-CoV-2 detection, WarmStart® Colorimetric LAMP 2X Master Mix (New England Biolabs) as a reference with diluted patient samples (duplicates of 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 and 10 3 copies of RNA as well as non template control (NTC)). (C) Summary of SARS-CoV-2 detection for diluted patient samples, WarmStart® Colorimetric LAMP 2X Master Mix (New England Biolabs) as a reference, color indicates time of detection of amplifying, gray indicates no amplification of no template control.
    Figure Legend Snippet: Colorimetric isothermal nucleic acid detection of SARS-CoV-2 with dye combination. (A) Detection of SARS-CoV-2 with different concentrations of azure II-pheno red combined dye. (B) Sensitivity and accuracy of SARS-CoV-2 detection, WarmStart® Colorimetric LAMP 2X Master Mix (New England Biolabs) as a reference with diluted patient samples (duplicates of 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 and 10 3 copies of RNA as well as non template control (NTC)). (C) Summary of SARS-CoV-2 detection for diluted patient samples, WarmStart® Colorimetric LAMP 2X Master Mix (New England Biolabs) as a reference, color indicates time of detection of amplifying, gray indicates no amplification of no template control.

    Techniques Used: Amplification

    27) Product Images from "Application of the colorimetric loop-mediated isothermal amplification (LAMP) technique for genotyping Cre-driver mice"

    Article Title: Application of the colorimetric loop-mediated isothermal amplification (LAMP) technique for genotyping Cre-driver mice

    Journal: The Journal of Veterinary Medical Science

    doi: 10.1292/jvms.21-0658

    Establishment of an effective loop-mediated isothermal amplification (LAMP) primer set for the Cre gene cassette. Genomic DNA isolated from FosCre (f) or C57BL/6 (b) mice was subjected to either polymerase chain reaction (PCR) (A) or colorimetric LAMP reaction (B–C) to identify the presence of the Cre gene. A. An electrophoretic image of the PCR. Glyceraldehyde 3-phosphate dehydrogenase ( Gapdh ) gene is used as an amplification reference. B . A photographic images of the LAMP reaction using primer sets 1, 5, 11, and 15. C . Photographic images of the LAMP reaction using primer set 11 in the presence (+) or absence (−) of loop primers. Asterisks represent changes in liquid color from red to yellow or orange. Similar results were obtained in three independent experiments.
    Figure Legend Snippet: Establishment of an effective loop-mediated isothermal amplification (LAMP) primer set for the Cre gene cassette. Genomic DNA isolated from FosCre (f) or C57BL/6 (b) mice was subjected to either polymerase chain reaction (PCR) (A) or colorimetric LAMP reaction (B–C) to identify the presence of the Cre gene. A. An electrophoretic image of the PCR. Glyceraldehyde 3-phosphate dehydrogenase ( Gapdh ) gene is used as an amplification reference. B . A photographic images of the LAMP reaction using primer sets 1, 5, 11, and 15. C . Photographic images of the LAMP reaction using primer set 11 in the presence (+) or absence (−) of loop primers. Asterisks represent changes in liquid color from red to yellow or orange. Similar results were obtained in three independent experiments.

    Techniques Used: Amplification, Isolation, Mouse Assay, Polymerase Chain Reaction

    Validation of primer set 11L using several Cre -driver mice strains. A . Colorimetric loop-mediated isothermal amplification (LAMP) genotyping for the Cre cassette using primer set 11L was run on genomic DNA of FosCre (f), NesCre (n), MuCre (m), and C57BL/6 mice (b). Photographic images of the tubes of the LAMP product are shown. B . Electrophoretic images of the polymerase chain reaction (PCR) products of the Cre or glyceraldehyde 3-phosphate dehydrogenase gene ( Gapdh ) amplification of the same samples used for Fig. 2A . Similar results were obtained in three independent experiments.
    Figure Legend Snippet: Validation of primer set 11L using several Cre -driver mice strains. A . Colorimetric loop-mediated isothermal amplification (LAMP) genotyping for the Cre cassette using primer set 11L was run on genomic DNA of FosCre (f), NesCre (n), MuCre (m), and C57BL/6 mice (b). Photographic images of the tubes of the LAMP product are shown. B . Electrophoretic images of the polymerase chain reaction (PCR) products of the Cre or glyceraldehyde 3-phosphate dehydrogenase gene ( Gapdh ) amplification of the same samples used for Fig. 2A . Similar results were obtained in three independent experiments.

    Techniques Used: Mouse Assay, Amplification, Genotyping Assay, Polymerase Chain Reaction

    28) Product Images from "Colorimetric isothermal nucleic acid detection of SARS-CoV-2 with dye combination"

    Article Title: Colorimetric isothermal nucleic acid detection of SARS-CoV-2 with dye combination

    Journal: Heliyon

    doi: 10.1016/j.heliyon.2021.e06886

    Colorimetric isothermal nucleic acid detection of SARS-CoV-2 with dye combination. (A) Detection of SARS-CoV-2 with different concentrations of azure II-pheno red combined dye. (B) Sensitivity and accuracy of SARS-CoV-2 detection, WarmStart® Colorimetric LAMP 2X Master Mix (New England Biolabs) as a reference with diluted patient samples (duplicates of 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 and 10 3 copies of RNA as well as non template control (NTC)). (C) Summary of SARS-CoV-2 detection for diluted patient samples, WarmStart® Colorimetric LAMP 2X Master Mix (New England Biolabs) as a reference, color indicates time of detection of amplifying, gray indicates no amplification of no template control.
    Figure Legend Snippet: Colorimetric isothermal nucleic acid detection of SARS-CoV-2 with dye combination. (A) Detection of SARS-CoV-2 with different concentrations of azure II-pheno red combined dye. (B) Sensitivity and accuracy of SARS-CoV-2 detection, WarmStart® Colorimetric LAMP 2X Master Mix (New England Biolabs) as a reference with diluted patient samples (duplicates of 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 and 10 3 copies of RNA as well as non template control (NTC)). (C) Summary of SARS-CoV-2 detection for diluted patient samples, WarmStart® Colorimetric LAMP 2X Master Mix (New England Biolabs) as a reference, color indicates time of detection of amplifying, gray indicates no amplification of no template control.

    Techniques Used: Amplification

    29) Product Images from "A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples"

    Article Title: A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-95799-6

    Limit of detection of the two different RT-LAMP formats and of RT-PCR. ( A ) A known number of copies of in vitro transcribed (IVT) viral RNA (N-gene) were amplified and detected by colorimetric RT-LAMP using the (i) WarmStart Colorimetric LAMP 2 × Master Mix (New England Biolabs) or (ii) the separate components (enzymes purchased individually and an in-house-made colorimetric buffer). The reactions were incubated at 65 °C for 30 min. ( B ) 10 μL of the RT-LAMP reaction were resolved in an agarose gel (2%) electrophoresis. The ladder pattern corresponds to the expected LAMP amplification pattern. ( C ) Limit of detection of ten replicates of the two test formats. ( D ) Standard curve generated by plotting the number of IVT RNA copies (x-axis) vs. the mean of the corresponding RT-PCR threshold cycle (Ct) value (y-axis) of three independent experiments (Original gel images in Fig. S1 ).
    Figure Legend Snippet: Limit of detection of the two different RT-LAMP formats and of RT-PCR. ( A ) A known number of copies of in vitro transcribed (IVT) viral RNA (N-gene) were amplified and detected by colorimetric RT-LAMP using the (i) WarmStart Colorimetric LAMP 2 × Master Mix (New England Biolabs) or (ii) the separate components (enzymes purchased individually and an in-house-made colorimetric buffer). The reactions were incubated at 65 °C for 30 min. ( B ) 10 μL of the RT-LAMP reaction were resolved in an agarose gel (2%) electrophoresis. The ladder pattern corresponds to the expected LAMP amplification pattern. ( C ) Limit of detection of ten replicates of the two test formats. ( D ) Standard curve generated by plotting the number of IVT RNA copies (x-axis) vs. the mean of the corresponding RT-PCR threshold cycle (Ct) value (y-axis) of three independent experiments (Original gel images in Fig. S1 ).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, In Vitro, Amplification, Incubation, Agarose Gel Electrophoresis, Electrophoresis, Generated

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    New England Biolabs warmstart colorimetric lamp 2
    Production of molecular biology reagents. (A) Purified Br512 Bst DNA polymerase visualized in a polyacrylamide gel stained with coomassie blue. (B) <t>Colorimetric</t> <t>LAMP</t> assay using the Br512 Bst DNA polymerase produced in vitro in both fresh conditions (top panel) and using rehydrated samples (bottom panel) after a 2 week storage at room temperature. Cell-free reactions based on PEP and MDX were prepared using the low-cost drying system and protected with sucrose (120 and 15 mM, respectively). A synthetic dsDNA fragment from actin B gene ( Homo sapiens ) was used as a target in the following amounts: 0, 0.025, 0.25, 2.5, 250, and 2500 pmoles. Primers used in this assay are described in Table S11 . Negative reactions were pink-colored, and positive reactions changed to yellow. (C, D) A PCR product encoding the Bsa I restriction endonuclease (2043 bp) was amplified using a single PCR with four oligonucleotides. An inner set of core primers provided a template for secondary amplification by longer oligonucleotides. The resulting product had extended terminal sequences that helped protect the coding region from exonuclease degradation. (E) Testing of Bsa I by restriction endonuclease digestion of luxpGEX plasmid. Digestion was performed using Bsa I produced by cell-free technology. Plasmid DNA samples were treated with (1) FastDigest Eco31I (Thermo Scientific, FD0293) (Isoschizomer: Bsa I), (2) Bsa I in cell extract, and (3) Bsa I in cell extract: 100% glycerol (1:1). Expected size of bands after digestion: 6440 and 4433 bp.
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    Production of molecular biology reagents. (A) Purified Br512 Bst DNA polymerase visualized in a polyacrylamide gel stained with coomassie blue. (B) Colorimetric LAMP assay using the Br512 Bst DNA polymerase produced in vitro in both fresh conditions (top panel) and using rehydrated samples (bottom panel) after a 2 week storage at room temperature. Cell-free reactions based on PEP and MDX were prepared using the low-cost drying system and protected with sucrose (120 and 15 mM, respectively). A synthetic dsDNA fragment from actin B gene ( Homo sapiens ) was used as a target in the following amounts: 0, 0.025, 0.25, 2.5, 250, and 2500 pmoles. Primers used in this assay are described in Table S11 . Negative reactions were pink-colored, and positive reactions changed to yellow. (C, D) A PCR product encoding the Bsa I restriction endonuclease (2043 bp) was amplified using a single PCR with four oligonucleotides. An inner set of core primers provided a template for secondary amplification by longer oligonucleotides. The resulting product had extended terminal sequences that helped protect the coding region from exonuclease degradation. (E) Testing of Bsa I by restriction endonuclease digestion of luxpGEX plasmid. Digestion was performed using Bsa I produced by cell-free technology. Plasmid DNA samples were treated with (1) FastDigest Eco31I (Thermo Scientific, FD0293) (Isoschizomer: Bsa I), (2) Bsa I in cell extract, and (3) Bsa I in cell extract: 100% glycerol (1:1). Expected size of bands after digestion: 6440 and 4433 bp.

    Journal: ACS Synthetic Biology

    Article Title: Constructing Cell-Free Expression Systems for Low-Cost Access

    doi: 10.1021/acssynbio.1c00342

    Figure Lengend Snippet: Production of molecular biology reagents. (A) Purified Br512 Bst DNA polymerase visualized in a polyacrylamide gel stained with coomassie blue. (B) Colorimetric LAMP assay using the Br512 Bst DNA polymerase produced in vitro in both fresh conditions (top panel) and using rehydrated samples (bottom panel) after a 2 week storage at room temperature. Cell-free reactions based on PEP and MDX were prepared using the low-cost drying system and protected with sucrose (120 and 15 mM, respectively). A synthetic dsDNA fragment from actin B gene ( Homo sapiens ) was used as a target in the following amounts: 0, 0.025, 0.25, 2.5, 250, and 2500 pmoles. Primers used in this assay are described in Table S11 . Negative reactions were pink-colored, and positive reactions changed to yellow. (C, D) A PCR product encoding the Bsa I restriction endonuclease (2043 bp) was amplified using a single PCR with four oligonucleotides. An inner set of core primers provided a template for secondary amplification by longer oligonucleotides. The resulting product had extended terminal sequences that helped protect the coding region from exonuclease degradation. (E) Testing of Bsa I by restriction endonuclease digestion of luxpGEX plasmid. Digestion was performed using Bsa I produced by cell-free technology. Plasmid DNA samples were treated with (1) FastDigest Eco31I (Thermo Scientific, FD0293) (Isoschizomer: Bsa I), (2) Bsa I in cell extract, and (3) Bsa I in cell extract: 100% glycerol (1:1). Expected size of bands after digestion: 6440 and 4433 bp.

    Article Snippet: WarmStart Colorimetric LAMP 2× Master Mix (New England Biolabs, M1800S) was used as a control to evaluate the efficiency in the colorimetric LAMP assay.

    Techniques: Purification, Staining, Lamp Assay, Produced, In Vitro, Polymerase Chain Reaction, Amplification, Plasmid Preparation

    Results of the WarmStart Colorimetric LAMP 2X Master Mix assay for detection of LAMP amplicons with the naked eye. Samples 1–8: serial dilutions of DNA from strain G. ( H.) parasuis DSM 21448 starting at concentrations of 10 ng/µL up to 1 fg/µL. Sample 9: DNA from Actinobacillus minor CCUG 38923 T . Sample 10: no template control.

    Journal: Microorganisms

    Article Title: Development and Validation of a Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Glaesserella (Haemophilus) parasuis

    doi: 10.3390/microorganisms9010041

    Figure Lengend Snippet: Results of the WarmStart Colorimetric LAMP 2X Master Mix assay for detection of LAMP amplicons with the naked eye. Samples 1–8: serial dilutions of DNA from strain G. ( H.) parasuis DSM 21448 starting at concentrations of 10 ng/µL up to 1 fg/µL. Sample 9: DNA from Actinobacillus minor CCUG 38923 T . Sample 10: no template control.

    Article Snippet: The isothermal OptiGene Isothermal Master Mix was replaced by the colorimetric WarmStart LAMP 2X Master Mix (New England BioLabs, Frankfurt am Main, Germany).

    Techniques:

    RT-LAMP assay to detect SARS-CoV-2. The LAMP primer mix contain three pairs of target-specific primers: two forward primers, two backward primers, and two loop primers. The LAMP reaction mix contained DNA polymerase, reverse transcriptase, isothermal buffer, and signal reporter. A simple heating block can be used to amplify the target region of the viral RNA using colorimetric detection. The image on the right shows the time-dependent color change, adapted from Dao Thi et al., [ 125 ]. Reprinted with permission from AAAS. Created with BioRender.com . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

    Journal: Analytica Chimica Acta

    Article Title: Toward a next-generation diagnostic tool: A review on emerging isothermal nucleic acid amplification techniques for the detection of SARS-CoV-2 and other infectious viruses

    doi: 10.1016/j.aca.2021.339338

    Figure Lengend Snippet: RT-LAMP assay to detect SARS-CoV-2. The LAMP primer mix contain three pairs of target-specific primers: two forward primers, two backward primers, and two loop primers. The LAMP reaction mix contained DNA polymerase, reverse transcriptase, isothermal buffer, and signal reporter. A simple heating block can be used to amplify the target region of the viral RNA using colorimetric detection. The image on the right shows the time-dependent color change, adapted from Dao Thi et al., [ 125 ]. Reprinted with permission from AAAS. Created with BioRender.com . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

    Article Snippet: The WarmStart LAMP mix developed by New England Biolabs has been widely employed for LAMP-based detection.

    Techniques: RT Lamp Assay, Blocking Assay

    Smartphone-based handheld POC instrument. A) Simple workflow of SARS-CoV-2 sample preparation by brief heat treatment and transfer of the thermally lysed sample to a syringe. Another syringe was loaded with LAMP reaction mix. B) Photograph of the microfluidic chip integrated POC tool. C) Disposable microfluidic cartridge. Figure adapted with permission from Ganguli et al., [ 83 ].

    Journal: Analytica Chimica Acta

    Article Title: Toward a next-generation diagnostic tool: A review on emerging isothermal nucleic acid amplification techniques for the detection of SARS-CoV-2 and other infectious viruses

    doi: 10.1016/j.aca.2021.339338

    Figure Lengend Snippet: Smartphone-based handheld POC instrument. A) Simple workflow of SARS-CoV-2 sample preparation by brief heat treatment and transfer of the thermally lysed sample to a syringe. Another syringe was loaded with LAMP reaction mix. B) Photograph of the microfluidic chip integrated POC tool. C) Disposable microfluidic cartridge. Figure adapted with permission from Ganguli et al., [ 83 ].

    Article Snippet: The WarmStart LAMP mix developed by New England Biolabs has been widely employed for LAMP-based detection.

    Techniques: Sample Prep, Chromatin Immunoprecipitation

    Colorimetric RT-LAMP for COVID-19 diagnosis validation using 100 clinical samples. Clinical samples were collected from symptomatic and hospitalized patients by nasopharyngeal swabs in partnership with CT-Vacinas/UFMG, Belo Horizonte, Brazil. Samples were obtained from different parts including Brazilian Southeast and Northeast regions. The reaction was performed at 65°C during 30 min using WarmStart ® colorimetric LAMP master mix (NEB #M1800) in 20 μL final volume. The RT-LAMP reaction targeted SARS-CoV-2 N gene. Yellow content indicates positive reaction, whereas the pink pattern reveals nonreagent samples. Amplicons were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification. Latter pattern confirmed specific SARS-CoV-2 amplification that matches with yellow output tubes, which is not observed in pink nonreagent tests. +C, positive control using RNA extracted from laboratory-Vero E6 cultured inactivated SARS-CoV-2; NTC, nontemplate control. Clinimetric parameters from these samples are presented in Supplementary Figure S1 .

    Journal: Frontiers in Microbiology

    Article Title: Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern

    doi: 10.3389/fmicb.2021.713713

    Figure Lengend Snippet: Colorimetric RT-LAMP for COVID-19 diagnosis validation using 100 clinical samples. Clinical samples were collected from symptomatic and hospitalized patients by nasopharyngeal swabs in partnership with CT-Vacinas/UFMG, Belo Horizonte, Brazil. Samples were obtained from different parts including Brazilian Southeast and Northeast regions. The reaction was performed at 65°C during 30 min using WarmStart ® colorimetric LAMP master mix (NEB #M1800) in 20 μL final volume. The RT-LAMP reaction targeted SARS-CoV-2 N gene. Yellow content indicates positive reaction, whereas the pink pattern reveals nonreagent samples. Amplicons were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification. Latter pattern confirmed specific SARS-CoV-2 amplification that matches with yellow output tubes, which is not observed in pink nonreagent tests. +C, positive control using RNA extracted from laboratory-Vero E6 cultured inactivated SARS-CoV-2; NTC, nontemplate control. Clinimetric parameters from these samples are presented in Supplementary Figure S1 .

    Article Snippet: RT-LAMP reactions were performed according to NEB recommendations, containing the following components: 10 μL of WarmStart® Colorimetric LAMP 2× Master Mix [NEB #M1800 or #M1804, the latter contains dUTP UDG (uracil-DNA-glycosylase) to avoid carryover contamination; composition of both are NEB’s proprietary]—ready-to-use mixture of WarmStart® Bst 2.0 DNA polymerase and WarmStart® RTx (reverse transcriptase for one-step transcription/amplification reaction) in presence of a pH sensor that turns from fuchsia (pink) to yellow in presence of increased proton (acid pH) during DNA polymerization on isothermal amplification, 1.6 μmol/L forward inner/backward inner primers (FIP/BIP); 0.2 μmol/L forward and backward outer primers (F3/B3), and 0.4 μmol/L loop forward and loop backward primers (LF/LB); Ultra-pureTM DNAse/RNase-free distilled water (InvitrogenTM #10977015) was added in quantity enough to complete the final volume reaction of 20 μL; isothermal amplification was performed on VeritiTM thermal cycler (Applied Biosystems, Foster City, CA, United States) at 65°C for 30 min. From clinical samples in the first batch, we used as input, 1 μL of RNA extracted from nasopharyngeal swab placed on guanidine-containing VTM, whereas upon optimization, 5 μL source template was considered from the samples in the second group.

    Techniques: Agarose Gel Electrophoresis, Staining, Amplification, Positive Control, Cell Culture

    Colorimetric RT-LAMP allows the detection of SARS-CoV-2 VOCs and VOIs. RT-LAMP reaction was performed at 65°C for 30 min, using the WarmStart ® colorimetric LAMP 2× master mix (NEB #M1804), using multiplex N 2/ E 1 primer sets. The amplicons were migrated in agarose gel at 2% to confirm amplification, as indicated by the characteristic ladder highlighted by GelRed ® staining. NTC, nontemplate control; CS, clinical sample; and +C, positive control. The top panel shows a schematic representation of SARS-CoV-2 spike protein (upper) and where the main mutations are highlighted and represented in SARS-CoV-2 virions (right hand side) present in VOC gamma (B.1), delta (B.1.167.2), and VOI zeta (P.2). The VOCs alpha (B.1.1.7) and beta (B.1.3.51), first reported in the United Kingdom and South Africa, respectively, are also represented. K417N: lysine-to-asparagine substitution at position 417 of spike protein at the receptor biding domain (RBD); V445A: valine-to-alanine substitution at position 445 and so on. L, leucine; Q, glutamine; E, glutamic acid; Y, tyrosine; T, threonine; P, proline; H, histidine; D, aspartic acid; S, serine; F, phenylalanine. del, deletion. Segments of SARS-CoV-2 protein NTD, N-terminal domain; CTD2, C-terminal domain 2 or C terminus of S1 fragment after furin cleavage; FP, fusion peptide; HR1, heptad repeat region 1. SARS-CoV-2 variants were previously sequenced. Variants of interest B.1.1.371 and B.1.1.374 were first reported in Saudi Arabia and Finland, respectively, ( https://cov-lineages.org/ ). Created with biorender.com .

    Journal: Frontiers in Microbiology

    Article Title: Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern

    doi: 10.3389/fmicb.2021.713713

    Figure Lengend Snippet: Colorimetric RT-LAMP allows the detection of SARS-CoV-2 VOCs and VOIs. RT-LAMP reaction was performed at 65°C for 30 min, using the WarmStart ® colorimetric LAMP 2× master mix (NEB #M1804), using multiplex N 2/ E 1 primer sets. The amplicons were migrated in agarose gel at 2% to confirm amplification, as indicated by the characteristic ladder highlighted by GelRed ® staining. NTC, nontemplate control; CS, clinical sample; and +C, positive control. The top panel shows a schematic representation of SARS-CoV-2 spike protein (upper) and where the main mutations are highlighted and represented in SARS-CoV-2 virions (right hand side) present in VOC gamma (B.1), delta (B.1.167.2), and VOI zeta (P.2). The VOCs alpha (B.1.1.7) and beta (B.1.3.51), first reported in the United Kingdom and South Africa, respectively, are also represented. K417N: lysine-to-asparagine substitution at position 417 of spike protein at the receptor biding domain (RBD); V445A: valine-to-alanine substitution at position 445 and so on. L, leucine; Q, glutamine; E, glutamic acid; Y, tyrosine; T, threonine; P, proline; H, histidine; D, aspartic acid; S, serine; F, phenylalanine. del, deletion. Segments of SARS-CoV-2 protein NTD, N-terminal domain; CTD2, C-terminal domain 2 or C terminus of S1 fragment after furin cleavage; FP, fusion peptide; HR1, heptad repeat region 1. SARS-CoV-2 variants were previously sequenced. Variants of interest B.1.1.371 and B.1.1.374 were first reported in Saudi Arabia and Finland, respectively, ( https://cov-lineages.org/ ). Created with biorender.com .

    Article Snippet: RT-LAMP reactions were performed according to NEB recommendations, containing the following components: 10 μL of WarmStart® Colorimetric LAMP 2× Master Mix [NEB #M1800 or #M1804, the latter contains dUTP UDG (uracil-DNA-glycosylase) to avoid carryover contamination; composition of both are NEB’s proprietary]—ready-to-use mixture of WarmStart® Bst 2.0 DNA polymerase and WarmStart® RTx (reverse transcriptase for one-step transcription/amplification reaction) in presence of a pH sensor that turns from fuchsia (pink) to yellow in presence of increased proton (acid pH) during DNA polymerization on isothermal amplification, 1.6 μmol/L forward inner/backward inner primers (FIP/BIP); 0.2 μmol/L forward and backward outer primers (F3/B3), and 0.4 μmol/L loop forward and loop backward primers (LF/LB); Ultra-pureTM DNAse/RNase-free distilled water (InvitrogenTM #10977015) was added in quantity enough to complete the final volume reaction of 20 μL; isothermal amplification was performed on VeritiTM thermal cycler (Applied Biosystems, Foster City, CA, United States) at 65°C for 30 min. From clinical samples in the first batch, we used as input, 1 μL of RNA extracted from nasopharyngeal swab placed on guanidine-containing VTM, whereas upon optimization, 5 μL source template was considered from the samples in the second group.

    Techniques: Multiplex Assay, Agarose Gel Electrophoresis, Amplification, Staining, Positive Control

    Analytical sensitivity as revealed by the limit of detection (LoD). RNA was extracted from VTM-nasopharyngeal swab, and the genome viral copies input was calculated based on SARS-CoV-2 E gene-harboring plasmid (Bioclin #K228-1) calibration curve. RT-LAMP reaction was performed at 65°C during 30 min using WarmStart ® colorimetric master LAMP mix (NEB #M1800) in 20 μL final volume (upper panel). Amplicons were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification (bottom panel). cps/μL, viral genome copies per microliter; NTC, nontemplate control; VTM, viral transport medium (Bioclin #G092-1).

    Journal: Frontiers in Microbiology

    Article Title: Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern

    doi: 10.3389/fmicb.2021.713713

    Figure Lengend Snippet: Analytical sensitivity as revealed by the limit of detection (LoD). RNA was extracted from VTM-nasopharyngeal swab, and the genome viral copies input was calculated based on SARS-CoV-2 E gene-harboring plasmid (Bioclin #K228-1) calibration curve. RT-LAMP reaction was performed at 65°C during 30 min using WarmStart ® colorimetric master LAMP mix (NEB #M1800) in 20 μL final volume (upper panel). Amplicons were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification (bottom panel). cps/μL, viral genome copies per microliter; NTC, nontemplate control; VTM, viral transport medium (Bioclin #G092-1).

    Article Snippet: RT-LAMP reactions were performed according to NEB recommendations, containing the following components: 10 μL of WarmStart® Colorimetric LAMP 2× Master Mix [NEB #M1800 or #M1804, the latter contains dUTP UDG (uracil-DNA-glycosylase) to avoid carryover contamination; composition of both are NEB’s proprietary]—ready-to-use mixture of WarmStart® Bst 2.0 DNA polymerase and WarmStart® RTx (reverse transcriptase for one-step transcription/amplification reaction) in presence of a pH sensor that turns from fuchsia (pink) to yellow in presence of increased proton (acid pH) during DNA polymerization on isothermal amplification, 1.6 μmol/L forward inner/backward inner primers (FIP/BIP); 0.2 μmol/L forward and backward outer primers (F3/B3), and 0.4 μmol/L loop forward and loop backward primers (LF/LB); Ultra-pureTM DNAse/RNase-free distilled water (InvitrogenTM #10977015) was added in quantity enough to complete the final volume reaction of 20 μL; isothermal amplification was performed on VeritiTM thermal cycler (Applied Biosystems, Foster City, CA, United States) at 65°C for 30 min. From clinical samples in the first batch, we used as input, 1 μL of RNA extracted from nasopharyngeal swab placed on guanidine-containing VTM, whereas upon optimization, 5 μL source template was considered from the samples in the second group.

    Techniques: Plasmid Preparation, Agarose Gel Electrophoresis, Staining, Amplification

    Microbial cross-reactivity assay to test SARS-CoV-2 RT-LAMP analytical sensitivity. The test was performed using potentially cross-reacting respiratory viruses or local occurring arboviruses. RT-LAMP reaction was performed at 65°C during 30 min, with additional 10 min, to confirm the absence of cross-reactivity when targeting SARS-CoV-2 E and N genes. The assay was performed using the WarmStart ® colorimetric LAMP 2× master mix (NEB #M1800). Yellow (positive) reaction is observed only when the template is SARS-CoV-2 viral RNA. hRSV, human respiratory syncytial virus; NTC, nontemplate control; M, molecular size marker. RT-LAMP amplification products were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification. DENV3, dengue virus serotype 3; ZIKV, Zika virus; CHIKV, Chikungunya virus; YFV, yellow fever virus; Influenza A (H1N1/H3N2); and influenza B (Yamagata/Victoria).

    Journal: Frontiers in Microbiology

    Article Title: Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern

    doi: 10.3389/fmicb.2021.713713

    Figure Lengend Snippet: Microbial cross-reactivity assay to test SARS-CoV-2 RT-LAMP analytical sensitivity. The test was performed using potentially cross-reacting respiratory viruses or local occurring arboviruses. RT-LAMP reaction was performed at 65°C during 30 min, with additional 10 min, to confirm the absence of cross-reactivity when targeting SARS-CoV-2 E and N genes. The assay was performed using the WarmStart ® colorimetric LAMP 2× master mix (NEB #M1800). Yellow (positive) reaction is observed only when the template is SARS-CoV-2 viral RNA. hRSV, human respiratory syncytial virus; NTC, nontemplate control; M, molecular size marker. RT-LAMP amplification products were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification. DENV3, dengue virus serotype 3; ZIKV, Zika virus; CHIKV, Chikungunya virus; YFV, yellow fever virus; Influenza A (H1N1/H3N2); and influenza B (Yamagata/Victoria).

    Article Snippet: RT-LAMP reactions were performed according to NEB recommendations, containing the following components: 10 μL of WarmStart® Colorimetric LAMP 2× Master Mix [NEB #M1800 or #M1804, the latter contains dUTP UDG (uracil-DNA-glycosylase) to avoid carryover contamination; composition of both are NEB’s proprietary]—ready-to-use mixture of WarmStart® Bst 2.0 DNA polymerase and WarmStart® RTx (reverse transcriptase for one-step transcription/amplification reaction) in presence of a pH sensor that turns from fuchsia (pink) to yellow in presence of increased proton (acid pH) during DNA polymerization on isothermal amplification, 1.6 μmol/L forward inner/backward inner primers (FIP/BIP); 0.2 μmol/L forward and backward outer primers (F3/B3), and 0.4 μmol/L loop forward and loop backward primers (LF/LB); Ultra-pureTM DNAse/RNase-free distilled water (InvitrogenTM #10977015) was added in quantity enough to complete the final volume reaction of 20 μL; isothermal amplification was performed on VeritiTM thermal cycler (Applied Biosystems, Foster City, CA, United States) at 65°C for 30 min. From clinical samples in the first batch, we used as input, 1 μL of RNA extracted from nasopharyngeal swab placed on guanidine-containing VTM, whereas upon optimization, 5 μL source template was considered from the samples in the second group.

    Techniques: Marker, Amplification, Agarose Gel Electrophoresis, Staining

    Colorimetric RT-LAMP for SARS-CoV-2 detection using genes N , E , and RdRp as target. Selected SARS-CoV-2–positive clinical samples by RT-qPCR were classified as low (Ct 18.9 and 21.7), medium (Ct 26.6 and 28.4), and high (Ct 31.6 and 35.2) Ct values for E gene. They were included as input for colorimetric RT-LAMP reaction using primers targeting N , RdRp (A) , and E genes (B) . RT-LAMP SARS-CoV-2 false-negative samples were more frequent when using E and RdRp genes as target (C) . RT-LAMP reaction was performed at 65°C during 30 min, using the WarmStart ® colorimetric LAMP 2× master mix (NEB #M1800). RT-LAMP amplification products were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification. +C, positive control using SARS-CoV-2 RNA extracted from laboratory-cultured inactivated SARS-CoV-2; NTC, nontemplate control.

    Journal: Frontiers in Microbiology

    Article Title: Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern

    doi: 10.3389/fmicb.2021.713713

    Figure Lengend Snippet: Colorimetric RT-LAMP for SARS-CoV-2 detection using genes N , E , and RdRp as target. Selected SARS-CoV-2–positive clinical samples by RT-qPCR were classified as low (Ct 18.9 and 21.7), medium (Ct 26.6 and 28.4), and high (Ct 31.6 and 35.2) Ct values for E gene. They were included as input for colorimetric RT-LAMP reaction using primers targeting N , RdRp (A) , and E genes (B) . RT-LAMP SARS-CoV-2 false-negative samples were more frequent when using E and RdRp genes as target (C) . RT-LAMP reaction was performed at 65°C during 30 min, using the WarmStart ® colorimetric LAMP 2× master mix (NEB #M1800). RT-LAMP amplification products were resolved in 2% agarose gel and stained with GelRed ® (Biotium #41003) to confirm DNA amplification. +C, positive control using SARS-CoV-2 RNA extracted from laboratory-cultured inactivated SARS-CoV-2; NTC, nontemplate control.

    Article Snippet: RT-LAMP reactions were performed according to NEB recommendations, containing the following components: 10 μL of WarmStart® Colorimetric LAMP 2× Master Mix [NEB #M1800 or #M1804, the latter contains dUTP UDG (uracil-DNA-glycosylase) to avoid carryover contamination; composition of both are NEB’s proprietary]—ready-to-use mixture of WarmStart® Bst 2.0 DNA polymerase and WarmStart® RTx (reverse transcriptase for one-step transcription/amplification reaction) in presence of a pH sensor that turns from fuchsia (pink) to yellow in presence of increased proton (acid pH) during DNA polymerization on isothermal amplification, 1.6 μmol/L forward inner/backward inner primers (FIP/BIP); 0.2 μmol/L forward and backward outer primers (F3/B3), and 0.4 μmol/L loop forward and loop backward primers (LF/LB); Ultra-pureTM DNAse/RNase-free distilled water (InvitrogenTM #10977015) was added in quantity enough to complete the final volume reaction of 20 μL; isothermal amplification was performed on VeritiTM thermal cycler (Applied Biosystems, Foster City, CA, United States) at 65°C for 30 min. From clinical samples in the first batch, we used as input, 1 μL of RNA extracted from nasopharyngeal swab placed on guanidine-containing VTM, whereas upon optimization, 5 μL source template was considered from the samples in the second group.

    Techniques: Quantitative RT-PCR, Amplification, Agarose Gel Electrophoresis, Staining, Positive Control, Cell Culture

    Colorimetric RT-LAMP to detect SAR-CoV-2 in RNA extraction–free clinical samples (A) or laboratory-cultured virus (B) . Clinical samples were derived from nasopharyngeal swabs placed on guanidine-containing viral transport medium, diluted 1:10. The RT-PCR Ct values for SARS-CoV-2 based on E gene are as follows: CS134 = 31.8, CS135 = 15.3, CS138 = 18.4, CS139 = 21.7, and CS140 = 24.6. RT-LAMP reaction was performed in 20 μL final volume, incubated at 65°C during 30, 40, or 50 min (inactivated virus) using WarmStart ® colorimetric LAMP master mix (NEB #M1800). Both clinical samples and viruses are RNA extraction–free samples. The amplification products (amplicons) were migrated in agarose gel at 2% to confirm amplification, as indicated by the characteristic ladder highlighted by GelRed ® staining. NTC, nontemplate control; CS, clinical sample; and +C, positive control.

    Journal: Frontiers in Microbiology

    Article Title: Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern

    doi: 10.3389/fmicb.2021.713713

    Figure Lengend Snippet: Colorimetric RT-LAMP to detect SAR-CoV-2 in RNA extraction–free clinical samples (A) or laboratory-cultured virus (B) . Clinical samples were derived from nasopharyngeal swabs placed on guanidine-containing viral transport medium, diluted 1:10. The RT-PCR Ct values for SARS-CoV-2 based on E gene are as follows: CS134 = 31.8, CS135 = 15.3, CS138 = 18.4, CS139 = 21.7, and CS140 = 24.6. RT-LAMP reaction was performed in 20 μL final volume, incubated at 65°C during 30, 40, or 50 min (inactivated virus) using WarmStart ® colorimetric LAMP master mix (NEB #M1800). Both clinical samples and viruses are RNA extraction–free samples. The amplification products (amplicons) were migrated in agarose gel at 2% to confirm amplification, as indicated by the characteristic ladder highlighted by GelRed ® staining. NTC, nontemplate control; CS, clinical sample; and +C, positive control.

    Article Snippet: RT-LAMP reactions were performed according to NEB recommendations, containing the following components: 10 μL of WarmStart® Colorimetric LAMP 2× Master Mix [NEB #M1800 or #M1804, the latter contains dUTP UDG (uracil-DNA-glycosylase) to avoid carryover contamination; composition of both are NEB’s proprietary]—ready-to-use mixture of WarmStart® Bst 2.0 DNA polymerase and WarmStart® RTx (reverse transcriptase for one-step transcription/amplification reaction) in presence of a pH sensor that turns from fuchsia (pink) to yellow in presence of increased proton (acid pH) during DNA polymerization on isothermal amplification, 1.6 μmol/L forward inner/backward inner primers (FIP/BIP); 0.2 μmol/L forward and backward outer primers (F3/B3), and 0.4 μmol/L loop forward and loop backward primers (LF/LB); Ultra-pureTM DNAse/RNase-free distilled water (InvitrogenTM #10977015) was added in quantity enough to complete the final volume reaction of 20 μL; isothermal amplification was performed on VeritiTM thermal cycler (Applied Biosystems, Foster City, CA, United States) at 65°C for 30 min. From clinical samples in the first batch, we used as input, 1 μL of RNA extracted from nasopharyngeal swab placed on guanidine-containing VTM, whereas upon optimization, 5 μL source template was considered from the samples in the second group.

    Techniques: RNA Extraction, Cell Culture, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Incubation, Amplification, Agarose Gel Electrophoresis, Staining, Positive Control