colorimetric warmstart lamp 2x master mix  (New England Biolabs)


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    Structured Review

    New England Biolabs colorimetric warmstart lamp 2x master mix
    Results of the <t>WarmStart</t> 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.
    Colorimetric Warmstart Lamp 2x Master Mix, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    colorimetric warmstart lamp 2x master mix - by Bioz Stars, 2022-05
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    Images

    1) 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:

    2) 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

    3) 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

    4) 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

    5) 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

    6) 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:

    7) 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

    8) 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

    9) 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

    10) 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

    11) 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

    12) 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

    13) 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

    14) 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

    15) 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

    16) 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

    17) 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

    18) 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

    19) 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

    20) 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

    21) 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

    22) 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 colorimetric warmstart lamp 2x master mix
    Results of the <t>WarmStart</t> 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.
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    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:

    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

    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).

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-021-04496-x

    Figure Lengend 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).

    Article Snippet: We also tested our FAW LAMP assay primers using an alternative colorimetric LAMP master mix (WarmStart Colorimetric LAMP 2 × master mix, DNA & RNA, New England Biolabs Inc.) following published protocols .

    Techniques: 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).

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-021-04496-x

    Figure Lengend 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).

    Article Snippet: We also tested our FAW LAMP assay primers using an alternative colorimetric LAMP master mix (WarmStart Colorimetric LAMP 2 × master mix, DNA & RNA, New England Biolabs Inc.) following published protocols .

    Techniques: 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.

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-021-04496-x

    Figure Lengend 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.

    Article Snippet: We also tested our FAW LAMP assay primers using an alternative colorimetric LAMP master mix (WarmStart Colorimetric LAMP 2 × master mix, DNA & RNA, New England Biolabs Inc.) following published protocols .

    Techniques: 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.

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-021-04496-x

    Figure Lengend 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.

    Article Snippet: We also tested our FAW LAMP assay primers using an alternative colorimetric LAMP master mix (WarmStart Colorimetric LAMP 2 × master mix, DNA & RNA, New England Biolabs Inc.) following published protocols .

    Techniques: 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.

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-021-04496-x

    Figure Lengend 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.

    Article Snippet: We also tested our FAW LAMP assay primers using an alternative colorimetric LAMP master mix (WarmStart Colorimetric LAMP 2 × master mix, DNA & RNA, New England Biolabs Inc.) following published protocols .

    Techniques: 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.

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-021-04496-x

    Figure Lengend 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.

    Article Snippet: We also tested our FAW LAMP assay primers using an alternative colorimetric LAMP master mix (WarmStart Colorimetric LAMP 2 × master mix, DNA & RNA, New England Biolabs Inc.) following published protocols .

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

    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.

    Journal: Biosensors & bioelectronics

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

    doi: 10.1016/j.bios.2018.03.009

    Figure Lengend 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.

    Article Snippet: Bovine serum albumin (BSA), Poly (ethylene glycol) 8000 (PEG), poly(vinyl alcohol) (PVA) are from Sigma-Aldrich, RT-PCR grade water from Ambion, Inc., intercalating Eva Green fluorescent dye from Biotium, Isothermal Master Mix used in LAMP reaction buffer from ISO-001nd, OptiGene, Horsham, UK, WarmStart® colorimetric LAMP 2X master mix from New England Biolabs Inc. N. meningitidis (ATCC 13098) from American Type Culture Collection (ATCC, Rockville, MD).

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