conventional pcr warmstart lamp kit  (New England Biolabs)


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    Structured Review

    New England Biolabs conventional pcr warmstart lamp kit
    Conventional <t>PCR</t> to distinguish S. frugiperda and S. exigua . (A) PCR with <t>LAMP</t> external primer set FAW_F3 and FAW_B3 produced a 781 bp product from S. frugiperda DNA only. (B) PCR with primer FAW_F3 and alternative reverse primer FAW_UR produed a 501 bp product from S. frugiperda DNA only. (C) PCR with primers BAW_DF and FAW_UR produced a 454 bp product from S. exigua DNA only. Abbreviations are NC (non-template control), FAW (fall armyworm S. frugiperda ), BAW (beet armyworm S. exigua ), TCW (tobacco cutworm S. litura ), ACL (African cotton leafworm S. littoralis ), RAW (rice armyworm Mythimna separata ), and CBW (cotton bollworm Helicoverpa armigera ).
    Conventional Pcr Warmstart Lamp Kit, 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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    Images

    1) Product Images from "Development of a simple and accurate molecular tool for Spodoptera frugiperda species identification using LAMP"

    Article Title: Development of a simple and accurate molecular tool for Spodoptera frugiperda species identification using LAMP

    Journal: bioRxiv

    doi: 10.1101/2020.04.07.029678

    Conventional PCR to distinguish S. frugiperda and S. exigua . (A) PCR with LAMP external primer set FAW_F3 and FAW_B3 produced a 781 bp product from S. frugiperda DNA only. (B) PCR with primer FAW_F3 and alternative reverse primer FAW_UR produed a 501 bp product from S. frugiperda DNA only. (C) PCR with primers BAW_DF and FAW_UR produced a 454 bp product from S. exigua DNA only. Abbreviations are NC (non-template control), FAW (fall armyworm S. frugiperda ), BAW (beet armyworm S. exigua ), TCW (tobacco cutworm S. litura ), ACL (African cotton leafworm S. littoralis ), RAW (rice armyworm Mythimna separata ), and CBW (cotton bollworm Helicoverpa armigera ).
    Figure Legend Snippet: Conventional PCR to distinguish S. frugiperda and S. exigua . (A) PCR with LAMP external primer set FAW_F3 and FAW_B3 produced a 781 bp product from S. frugiperda DNA only. (B) PCR with primer FAW_F3 and alternative reverse primer FAW_UR produed a 501 bp product from S. frugiperda DNA only. (C) PCR with primers BAW_DF and FAW_UR produced a 454 bp product from S. exigua DNA only. Abbreviations are NC (non-template control), FAW (fall armyworm S. frugiperda ), BAW (beet armyworm S. exigua ), TCW (tobacco cutworm S. litura ), ACL (African cotton leafworm S. littoralis ), RAW (rice armyworm Mythimna separata ), and CBW (cotton bollworm Helicoverpa armigera ).

    Techniques Used: Polymerase Chain Reaction, Produced

    2) 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

    3) Product Images from "An integrated one-step assay combining thermal lysis and loop-mediated isothermal DNA amplification (LAMP) in 30 min from E. coli and M. smegmatis cells on a paper substrate"

    Article Title: An integrated one-step assay combining thermal lysis and loop-mediated isothermal DNA amplification (LAMP) in 30 min from E. coli and M. smegmatis cells on a paper substrate

    Journal: bioRxiv

    doi: 10.1101/594374

    Panel A shows the schematic of the one-step lysis and LAMP protocol. The LAMP reaction mix is spotted on a pre-treated 5 mm paper disc. The paper disc is put in a polyethylene pouch, which is then heat sealed. The pouch is then subjected to a temperature of 60 °C for LAMP. PicoGreen is spotted on the paper disc after amplification and allowed to bind to the dsDNA for 5 min. Fluorescence intensity (Ex./Em.: 480 nm/ 520 nm) is measured using the microplate reader. Panel B shows the schematic diagram of the biochemistry of the reaction. The thermal lysis causes release of the genomic DNA which serves as the template for subsequent DNA amplification. PicoGreen is added to the mixture which binds to the dsDNA molecules. The recorded fluorescence intensities were then analysed to confirm the efficacy of the one-step protocol.
    Figure Legend Snippet: Panel A shows the schematic of the one-step lysis and LAMP protocol. The LAMP reaction mix is spotted on a pre-treated 5 mm paper disc. The paper disc is put in a polyethylene pouch, which is then heat sealed. The pouch is then subjected to a temperature of 60 °C for LAMP. PicoGreen is spotted on the paper disc after amplification and allowed to bind to the dsDNA for 5 min. Fluorescence intensity (Ex./Em.: 480 nm/ 520 nm) is measured using the microplate reader. Panel B shows the schematic diagram of the biochemistry of the reaction. The thermal lysis causes release of the genomic DNA which serves as the template for subsequent DNA amplification. PicoGreen is added to the mixture which binds to the dsDNA molecules. The recorded fluorescence intensities were then analysed to confirm the efficacy of the one-step protocol.

    Techniques Used: Lysis, Amplification, Fluorescence

    Estimation of the minimum reaction time for E. coli (panel A) and M. smegmatis (panel B) using gel electrophoresis. Lane 1: DNA ladder (10 bp to 300 bp). Lane 2: NMC indicates negative control without any mastermix in the reaction mixture. Lane 3: NTC indicates negative control without any bacterial cells in the reaction mixture. Lane 4: LAMP for the recommended duration of 60 min (positive control). Lanes 5-10: LAMP performed for 5 min, 10 min, 15 min, 20 min, 30 min and 45 min respectively. The minimum reaction times for E. coli and M. smegmatis are 10 min and 45 min respectively.
    Figure Legend Snippet: Estimation of the minimum reaction time for E. coli (panel A) and M. smegmatis (panel B) using gel electrophoresis. Lane 1: DNA ladder (10 bp to 300 bp). Lane 2: NMC indicates negative control without any mastermix in the reaction mixture. Lane 3: NTC indicates negative control without any bacterial cells in the reaction mixture. Lane 4: LAMP for the recommended duration of 60 min (positive control). Lanes 5-10: LAMP performed for 5 min, 10 min, 15 min, 20 min, 30 min and 45 min respectively. The minimum reaction times for E. coli and M. smegmatis are 10 min and 45 min respectively.

    Techniques Used: Nucleic Acid Electrophoresis, Negative Control, Positive Control

    Detection sensitivity of our integrated assay with E. coli (panel A) and M. smegmatis (panel B) using gel electrophoresis. Lane 1: 10 bp to 300 bp DNA ladder. Lane 2: No master-mix control (NMC). Lane 3: No template control (NTC). Lane 4: LAMP of purified genomic DNA, used as a positive control. Lanes 5 – 10 in panel A: Reactions using different cell concentrations (10 7 CFU/mL, 10 6 CFU/mL, 10 4 CFU/mL, 10 2 CFU/mL, 10 0 CFU/mL and 10 -2 CFU/mL respectively). Lanes 5 – 10 in panel B: Reactions using different cell concentrations (10 8 CFU/mL, 10 7 CFU/mL, 10 5 CFU/mL, 10 3 CFU/mL, 10 1 CFU/mL and 10 -1 CFU/mL respectively). Gel electrophoresis can detect 1000 CFU/mL of M smegmatis and 100 CFU/mL of E. coli .
    Figure Legend Snippet: Detection sensitivity of our integrated assay with E. coli (panel A) and M. smegmatis (panel B) using gel electrophoresis. Lane 1: 10 bp to 300 bp DNA ladder. Lane 2: No master-mix control (NMC). Lane 3: No template control (NTC). Lane 4: LAMP of purified genomic DNA, used as a positive control. Lanes 5 – 10 in panel A: Reactions using different cell concentrations (10 7 CFU/mL, 10 6 CFU/mL, 10 4 CFU/mL, 10 2 CFU/mL, 10 0 CFU/mL and 10 -2 CFU/mL respectively). Lanes 5 – 10 in panel B: Reactions using different cell concentrations (10 8 CFU/mL, 10 7 CFU/mL, 10 5 CFU/mL, 10 3 CFU/mL, 10 1 CFU/mL and 10 -1 CFU/mL respectively). Gel electrophoresis can detect 1000 CFU/mL of M smegmatis and 100 CFU/mL of E. coli .

    Techniques Used: Nucleic Acid Electrophoresis, Purification, Positive Control

    4) Product Images from "COV-ID: A LAMP sequencing approach for high-throughput co-detection of SARS-CoV-2 and influenza virus in human saliva"

    Article Title: COV-ID: A LAMP sequencing approach for high-throughput co-detection of SARS-CoV-2 and influenza virus in human saliva

    Journal: medRxiv

    doi: 10.1101/2021.04.23.21255523

    Barcoding and PCR amplification of RT-LAMP products (A) Overview of COV-ID. Saliva is collected and inactivated prior to RT-LAMP performed with up to 96 individual sample barcoded primers. LAMP reactions are pooled and further amplified via PCR to introduce Illumina adapter sequences and pool-level dual indexes. A single thermal cycler can amplify 96 or 384 such pools and the resulting “super-pool” can be sequenced overnight to detect multiple amplicons from 9,216 or 36,864 individual patient samples (number of reads in parenthesis assume an output of ∼450M reads from a NextSeq 500). (B) Schematic of the RT-LAMP (step I) of COV-ID. Selected numbered intermediates of RT-LAMP reaction are shown to illustrate how the LAMP barcode, shown in yellow, and the P5 and P7 homology sequences (blue and pink, respectively) are introduced in the final LAMP product. Upon generating the dumb-bell intermediate the reaction proceeds through rapid primed and self-primed extensions to form mixture of various DNA amplicons containing sequences for PCR amplification. A more detailed version of the LAMP phase of COV-ID, including specific sequences, is illustrated in Fig. S1 . (C) Conventional RT-LAMP primers (solid lines) or primers modified for COV-ID (dotted lines) were used for RT-LAMP of SARS-CoV-2. The numbers of inactivated SARS-CoV-2 virions per µL is indicated in the color legend. (D) Schematic of the PCR (step II) of COV-ID. Following RT-LAMP, up to 96 reactions are pooled and purified and Illumina libraries are generated directly by PCR with dual-indexed P5 and P7 adapters in preparation for sequencing. (E) COV-ID primers targeting ACTB mRNA were used for RT-LAMP with HeLa total RNA. LAMP was diluted 1:100, amplified via PCR and resolved on 2% agarose gel.
    Figure Legend Snippet: Barcoding and PCR amplification of RT-LAMP products (A) Overview of COV-ID. Saliva is collected and inactivated prior to RT-LAMP performed with up to 96 individual sample barcoded primers. LAMP reactions are pooled and further amplified via PCR to introduce Illumina adapter sequences and pool-level dual indexes. A single thermal cycler can amplify 96 or 384 such pools and the resulting “super-pool” can be sequenced overnight to detect multiple amplicons from 9,216 or 36,864 individual patient samples (number of reads in parenthesis assume an output of ∼450M reads from a NextSeq 500). (B) Schematic of the RT-LAMP (step I) of COV-ID. Selected numbered intermediates of RT-LAMP reaction are shown to illustrate how the LAMP barcode, shown in yellow, and the P5 and P7 homology sequences (blue and pink, respectively) are introduced in the final LAMP product. Upon generating the dumb-bell intermediate the reaction proceeds through rapid primed and self-primed extensions to form mixture of various DNA amplicons containing sequences for PCR amplification. A more detailed version of the LAMP phase of COV-ID, including specific sequences, is illustrated in Fig. S1 . (C) Conventional RT-LAMP primers (solid lines) or primers modified for COV-ID (dotted lines) were used for RT-LAMP of SARS-CoV-2. The numbers of inactivated SARS-CoV-2 virions per µL is indicated in the color legend. (D) Schematic of the PCR (step II) of COV-ID. Following RT-LAMP, up to 96 reactions are pooled and purified and Illumina libraries are generated directly by PCR with dual-indexed P5 and P7 adapters in preparation for sequencing. (E) COV-ID primers targeting ACTB mRNA were used for RT-LAMP with HeLa total RNA. LAMP was diluted 1:100, amplified via PCR and resolved on 2% agarose gel.

    Techniques Used: Polymerase Chain Reaction, Amplification, Introduce, Modification, Purification, Generated, Sequencing, Agarose Gel Electrophoresis

    5) Product Images from "LAMP-Seq: Population-Scale COVID-19 Diagnostics Using Combinatorial Barcoding"

    Article Title: LAMP-Seq: Population-Scale COVID-19 Diagnostics Using Combinatorial Barcoding

    Journal: bioRxiv

    doi: 10.1101/2020.04.06.025635

    LAMP-Seq: A scalable deep-sequencing based approach for SARS-CoV-2 detection. (A) Schematic outline of a proposed scalable testing procedure involving remote lysis and inactivation of samples, and centralized barcoded RT-LAMP, pooling, and sequencing. (B) Schematic outline of a proposed scalable testing procedure involving remote barcoded RT-LAMP and sample pooling, and centralized sequencing. (C) Schematic of anticipated enzymatic reactions and reaction products. (D) Experimental validation of barcode insertion into FIP primers employed in LAMP reactions. All steps were performed as described in the Methods section, with the exception that plasmid DNA containing the SARS-CoV-2 N-gene (IDT) was used as template instead of a swab sample, no Bst 3.0 or Tris buffer were added, and the reaction was scaled down to a volume of 25 μ l. Samples were run on a 1% agarose gel and visualized using ethidium bromide. (E) Barcoded LAMP reactions templated with either 100 or 10,000 dsDNA molecules were combined after heat inactivation as described for D. Reactions were PCR amplified and sequenced on an Illumina MiSeq sequencer. Relative read counts are shown as mean and standard deviation from two experimental replicates. (F) RT-LAMP reactions with a combination of three barcoded FIP primers, but without Tris or Bst 3.0, were templated with synthetic RNA, and were sequenced using a MiSeq sequencer. Base frequencies are depicted by the size of each letter without applying any read filtering. Increasing phasing noise towards the 3’ end of the amplicon is likely caused by indels in primers. (G) Sensitivity measurement of RT-LAMP reactions as described for D templated with indicated numbers of synthetic RNA molecules. After PCR-amplification, the number of positive reactions was counted on a 1% agarose gel. (H) Likelihood function of the probability of detection for a single RNA molecule.
    Figure Legend Snippet: LAMP-Seq: A scalable deep-sequencing based approach for SARS-CoV-2 detection. (A) Schematic outline of a proposed scalable testing procedure involving remote lysis and inactivation of samples, and centralized barcoded RT-LAMP, pooling, and sequencing. (B) Schematic outline of a proposed scalable testing procedure involving remote barcoded RT-LAMP and sample pooling, and centralized sequencing. (C) Schematic of anticipated enzymatic reactions and reaction products. (D) Experimental validation of barcode insertion into FIP primers employed in LAMP reactions. All steps were performed as described in the Methods section, with the exception that plasmid DNA containing the SARS-CoV-2 N-gene (IDT) was used as template instead of a swab sample, no Bst 3.0 or Tris buffer were added, and the reaction was scaled down to a volume of 25 μ l. Samples were run on a 1% agarose gel and visualized using ethidium bromide. (E) Barcoded LAMP reactions templated with either 100 or 10,000 dsDNA molecules were combined after heat inactivation as described for D. Reactions were PCR amplified and sequenced on an Illumina MiSeq sequencer. Relative read counts are shown as mean and standard deviation from two experimental replicates. (F) RT-LAMP reactions with a combination of three barcoded FIP primers, but without Tris or Bst 3.0, were templated with synthetic RNA, and were sequenced using a MiSeq sequencer. Base frequencies are depicted by the size of each letter without applying any read filtering. Increasing phasing noise towards the 3’ end of the amplicon is likely caused by indels in primers. (G) Sensitivity measurement of RT-LAMP reactions as described for D templated with indicated numbers of synthetic RNA molecules. After PCR-amplification, the number of positive reactions was counted on a 1% agarose gel. (H) Likelihood function of the probability of detection for a single RNA molecule.

    Techniques Used: Sequencing, Lysis, Plasmid Preparation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Standard Deviation

    6) Product Images from "Development of a species diagnostic molecular tool for an invasive pest, Mythimna loreyi using LAMP"

    Article Title: Development of a species diagnostic molecular tool for an invasive pest, Mythimna loreyi using LAMP

    Journal: bioRxiv

    doi: 10.1101/2020.10.01.323089

    The sensitivity of the LAMP assay results in three temperature conditions such as 65, 63, and 61 °C for M. loreyi species detected under (A1, B1, and C1) visible light, (A2, B2, and C2) ultraviolet light with Cyber Green and (A3, B3, and C3) gel electrophoresis. The original pink color of the reaction mixture turned yellow in a positive reaction when the product was formed but remained pink in negative reactions. (D) Conventional and multiplex PCR to distinguish M. loreyi . 794bp amplicon amplified only in M. loreyi and conserved partial sequence of ace1 type acetylcholinesterase gene was targeted as an internal reference. Abbreviations are NTC (non-template control), RAW (rice armyworm Mythimna loreyi ), OAW (oriental armyworm M. separata ), TM (Turnip moth Agrotis segetum ), CBW (cotton bollworm Helicoverpa armigera ), BAW (beet armyworm S. exigua ), FAW (fall armyworm S. frugiperda ), and TCW (tobacco cutworm S. litura )
    Figure Legend Snippet: The sensitivity of the LAMP assay results in three temperature conditions such as 65, 63, and 61 °C for M. loreyi species detected under (A1, B1, and C1) visible light, (A2, B2, and C2) ultraviolet light with Cyber Green and (A3, B3, and C3) gel electrophoresis. The original pink color of the reaction mixture turned yellow in a positive reaction when the product was formed but remained pink in negative reactions. (D) Conventional and multiplex PCR to distinguish M. loreyi . 794bp amplicon amplified only in M. loreyi and conserved partial sequence of ace1 type acetylcholinesterase gene was targeted as an internal reference. Abbreviations are NTC (non-template control), RAW (rice armyworm Mythimna loreyi ), OAW (oriental armyworm M. separata ), TM (Turnip moth Agrotis segetum ), CBW (cotton bollworm Helicoverpa armigera ), BAW (beet armyworm S. exigua ), FAW (fall armyworm S. frugiperda ), and TCW (tobacco cutworm S. litura )

    Techniques Used: Lamp Assay, Nucleic Acid Electrophoresis, Multiplex Assay, Polymerase Chain Reaction, Amplification, Sequencing

    7) Product Images from "Development of a Species Diagnostic Molecular Tool for an Invasive Pest, Mythimna loreyi, Using LAMP"

    Article Title: Development of a Species Diagnostic Molecular Tool for an Invasive Pest, Mythimna loreyi, Using LAMP

    Journal: Insects

    doi: 10.3390/insects11110817

    The sensitivity of the LAMP assay results in three temperature conditions, such as 65, 63, and 61 °C, for M. loreyi species detected under ( A1 , B1 , and C1 ) visible light, ( A2 , B2 , and C2 ) ultraviolet light with SYBR Green, and ( A3 , B3 , and C3 ) gel electrophoresis. The original pink color of the reaction mixture turned yellow in a positive reaction when the product was formed but remained pink in negative reactions. ( D ) Conventional and multiplex PCR to distinguish M. loreyi . The 794 bp amplicon was amplified only in M. loreyi, and the conserved partial sequence of ace1 type acetylcholinesterase gene was targeted as an internal reference. Abbreviations are NTC (non-template control), RAW (rice armyworm Mythimna loreyi ), OAW (oriental armyworm Mythimna separata ), TM (Turnip moth Agrotis segetum ), CBW (cotton bollworm Helicoverpa armigera ), BAW (beet armyworm Spodoptera exigua ), FAW (fall armyworm Spodoptera frugiperda ), and TCW (tobacco cutworm Spodoptera litura ).
    Figure Legend Snippet: The sensitivity of the LAMP assay results in three temperature conditions, such as 65, 63, and 61 °C, for M. loreyi species detected under ( A1 , B1 , and C1 ) visible light, ( A2 , B2 , and C2 ) ultraviolet light with SYBR Green, and ( A3 , B3 , and C3 ) gel electrophoresis. The original pink color of the reaction mixture turned yellow in a positive reaction when the product was formed but remained pink in negative reactions. ( D ) Conventional and multiplex PCR to distinguish M. loreyi . The 794 bp amplicon was amplified only in M. loreyi, and the conserved partial sequence of ace1 type acetylcholinesterase gene was targeted as an internal reference. Abbreviations are NTC (non-template control), RAW (rice armyworm Mythimna loreyi ), OAW (oriental armyworm Mythimna separata ), TM (Turnip moth Agrotis segetum ), CBW (cotton bollworm Helicoverpa armigera ), BAW (beet armyworm Spodoptera exigua ), FAW (fall armyworm Spodoptera frugiperda ), and TCW (tobacco cutworm Spodoptera litura ).

    Techniques Used: Lamp Assay, SYBR Green Assay, Nucleic Acid Electrophoresis, Multiplex Assay, Polymerase Chain Reaction, Amplification, Sequencing

    8) Product Images from "Fast, Precise, and Reliable Multiplex Detection of Potato Viruses by Loop-Mediated Isothermal Amplification"

    Article Title: Fast, Precise, and Reliable Multiplex Detection of Potato Viruses by Loop-Mediated Isothermal Amplification

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21228741

    LAMP optimization to determine the limit of detection (LOD). NEB WarmStart qRT-LAMP tests were carried out using ( a ) TRV-PM3 or ( b ) TRV-PM3 plus loop primers. Betaine was added to a final concentration of 800 mM. Template = RNA purified from sample P10 (TRV-positive potato tuber), 1:2 ( a ) or 1:10 ( b ) dilution series. The reaction volume was 10 µL, including 2 µL of the template. Data are mean Ct values plus standard deviations ( n = 3).
    Figure Legend Snippet: LAMP optimization to determine the limit of detection (LOD). NEB WarmStart qRT-LAMP tests were carried out using ( a ) TRV-PM3 or ( b ) TRV-PM3 plus loop primers. Betaine was added to a final concentration of 800 mM. Template = RNA purified from sample P10 (TRV-positive potato tuber), 1:2 ( a ) or 1:10 ( b ) dilution series. The reaction volume was 10 µL, including 2 µL of the template. Data are mean Ct values plus standard deviations ( n = 3).

    Techniques Used: Concentration Assay, Purification

    Initial testing of the tobacco rattle virus (TRV)-LAMP method. The NEB WarmStart qRT-LAMP method was tested with HPLC-purified primer mix 1 (PM1). Template = RNA purified from TRV-infected (positive, green) and uninfected (negative, blue) tuber tissues. The total reaction volume was 10 µL (including 0.5 µL template, 0.5 µL primer mix and 0.1 µL 50× LAMP dye). Positive control (PC, orange) = German Collection of Microorganisms and Cell Cultures (DSMZ) virus isolate TRV PV-0352. Negative non-template control (NC, gray) = milliQ water. For visual clarity, the amplification plots ( a ) show the increase in fluorescence with the number of cycles, and the bar chart ( b ) shows the overall Ct values. RT-LAMP tests were monitored for 1 h (1 min/cycle).
    Figure Legend Snippet: Initial testing of the tobacco rattle virus (TRV)-LAMP method. The NEB WarmStart qRT-LAMP method was tested with HPLC-purified primer mix 1 (PM1). Template = RNA purified from TRV-infected (positive, green) and uninfected (negative, blue) tuber tissues. The total reaction volume was 10 µL (including 0.5 µL template, 0.5 µL primer mix and 0.1 µL 50× LAMP dye). Positive control (PC, orange) = German Collection of Microorganisms and Cell Cultures (DSMZ) virus isolate TRV PV-0352. Negative non-template control (NC, gray) = milliQ water. For visual clarity, the amplification plots ( a ) show the increase in fluorescence with the number of cycles, and the bar chart ( b ) shows the overall Ct values. RT-LAMP tests were monitored for 1 h (1 min/cycle).

    Techniques Used: High Performance Liquid Chromatography, Purification, Infection, Positive Control, Amplification, Fluorescence

    TRV LAMP primer mix comparison. NEB WarmStart qRT-LAMP tests with ( a ) HPLC-purified primer mix 1 (TRV-PM1) or ( b ) TRV-PM2 with new forward internal primer (FIP) and backward internal primer (BIP). Template = RNA purified from TRV-infected (positive, green) and uninfected (negative, blue) tuber tissues. We used 1 µL (1:10 dilution) of template together with positive and negative controls in a total volume of 10 µL. Positive control (PC, orange) = DSMZ virus isolate TRV PV-0352. Negative non-template control (NC, gray) = milliQ water. RT-LAMP tests were monitored for 1 h (1 min/cycle).
    Figure Legend Snippet: TRV LAMP primer mix comparison. NEB WarmStart qRT-LAMP tests with ( a ) HPLC-purified primer mix 1 (TRV-PM1) or ( b ) TRV-PM2 with new forward internal primer (FIP) and backward internal primer (BIP). Template = RNA purified from TRV-infected (positive, green) and uninfected (negative, blue) tuber tissues. We used 1 µL (1:10 dilution) of template together with positive and negative controls in a total volume of 10 µL. Positive control (PC, orange) = DSMZ virus isolate TRV PV-0352. Negative non-template control (NC, gray) = milliQ water. RT-LAMP tests were monitored for 1 h (1 min/cycle).

    Techniques Used: High Performance Liquid Chromatography, Purification, Infection, Positive Control

    9) 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

    10) 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

    11) 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

    12) 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

    13) 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

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    New England Biolabs conventional pcr warmstart lamp kit
    Conventional <t>PCR</t> to distinguish S. frugiperda and S. exigua . (A) PCR with <t>LAMP</t> external primer set FAW_F3 and FAW_B3 produced a 781 bp product from S. frugiperda DNA only. (B) PCR with primer FAW_F3 and alternative reverse primer FAW_UR produed a 501 bp product from S. frugiperda DNA only. (C) PCR with primers BAW_DF and FAW_UR produced a 454 bp product from S. exigua DNA only. Abbreviations are NC (non-template control), FAW (fall armyworm S. frugiperda ), BAW (beet armyworm S. exigua ), TCW (tobacco cutworm S. litura ), ACL (African cotton leafworm S. littoralis ), RAW (rice armyworm Mythimna separata ), and CBW (cotton bollworm Helicoverpa armigera ).
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    Conventional PCR to distinguish S. frugiperda and S. exigua . (A) PCR with LAMP external primer set FAW_F3 and FAW_B3 produced a 781 bp product from S. frugiperda DNA only. (B) PCR with primer FAW_F3 and alternative reverse primer FAW_UR produed a 501 bp product from S. frugiperda DNA only. (C) PCR with primers BAW_DF and FAW_UR produced a 454 bp product from S. exigua DNA only. Abbreviations are NC (non-template control), FAW (fall armyworm S. frugiperda ), BAW (beet armyworm S. exigua ), TCW (tobacco cutworm S. litura ), ACL (African cotton leafworm S. littoralis ), RAW (rice armyworm Mythimna separata ), and CBW (cotton bollworm Helicoverpa armigera ).

    Journal: bioRxiv

    Article Title: Development of a simple and accurate molecular tool for Spodoptera frugiperda species identification using LAMP

    doi: 10.1101/2020.04.07.029678

    Figure Lengend Snippet: Conventional PCR to distinguish S. frugiperda and S. exigua . (A) PCR with LAMP external primer set FAW_F3 and FAW_B3 produced a 781 bp product from S. frugiperda DNA only. (B) PCR with primer FAW_F3 and alternative reverse primer FAW_UR produed a 501 bp product from S. frugiperda DNA only. (C) PCR with primers BAW_DF and FAW_UR produced a 454 bp product from S. exigua DNA only. Abbreviations are NC (non-template control), FAW (fall armyworm S. frugiperda ), BAW (beet armyworm S. exigua ), TCW (tobacco cutworm S. litura ), ACL (African cotton leafworm S. littoralis ), RAW (rice armyworm Mythimna separata ), and CBW (cotton bollworm Helicoverpa armigera ).

    Article Snippet: 2.4 LAMP and conventional PCR WarmStart® LAMP Kit (New England Biolabs, Ipswich, MA) used for LAMP assay.

    Techniques: Polymerase Chain Reaction, Produced

    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.

    Journal: medRxiv

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

    doi: 10.1101/2020.07.16.20150250

    Figure Lengend 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.

    Article Snippet: For each reaction, 10 μL WarmStart LAMP 2X Master Mix (NEB #E1700) was combined with 1 μL 20X EvaGreen Dye (Biotium #31000), 2 μL 10X primer mix, and 3 μL DEPC-treated water.

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

    Panel A shows the schematic of the one-step lysis and LAMP protocol. The LAMP reaction mix is spotted on a pre-treated 5 mm paper disc. The paper disc is put in a polyethylene pouch, which is then heat sealed. The pouch is then subjected to a temperature of 60 °C for LAMP. PicoGreen is spotted on the paper disc after amplification and allowed to bind to the dsDNA for 5 min. Fluorescence intensity (Ex./Em.: 480 nm/ 520 nm) is measured using the microplate reader. Panel B shows the schematic diagram of the biochemistry of the reaction. The thermal lysis causes release of the genomic DNA which serves as the template for subsequent DNA amplification. PicoGreen is added to the mixture which binds to the dsDNA molecules. The recorded fluorescence intensities were then analysed to confirm the efficacy of the one-step protocol.

    Journal: bioRxiv

    Article Title: An integrated one-step assay combining thermal lysis and loop-mediated isothermal DNA amplification (LAMP) in 30 min from E. coli and M. smegmatis cells on a paper substrate

    doi: 10.1101/594374

    Figure Lengend Snippet: Panel A shows the schematic of the one-step lysis and LAMP protocol. The LAMP reaction mix is spotted on a pre-treated 5 mm paper disc. The paper disc is put in a polyethylene pouch, which is then heat sealed. The pouch is then subjected to a temperature of 60 °C for LAMP. PicoGreen is spotted on the paper disc after amplification and allowed to bind to the dsDNA for 5 min. Fluorescence intensity (Ex./Em.: 480 nm/ 520 nm) is measured using the microplate reader. Panel B shows the schematic diagram of the biochemistry of the reaction. The thermal lysis causes release of the genomic DNA which serves as the template for subsequent DNA amplification. PicoGreen is added to the mixture which binds to the dsDNA molecules. The recorded fluorescence intensities were then analysed to confirm the efficacy of the one-step protocol.

    Article Snippet: WarmStart LAMP Kit (DNA and RNA) enzyme mix for the LAMP reactions was bought from New England Biolabs (Beverly, USA).

    Techniques: Lysis, Amplification, Fluorescence

    Estimation of the minimum reaction time for E. coli (panel A) and M. smegmatis (panel B) using gel electrophoresis. Lane 1: DNA ladder (10 bp to 300 bp). Lane 2: NMC indicates negative control without any mastermix in the reaction mixture. Lane 3: NTC indicates negative control without any bacterial cells in the reaction mixture. Lane 4: LAMP for the recommended duration of 60 min (positive control). Lanes 5-10: LAMP performed for 5 min, 10 min, 15 min, 20 min, 30 min and 45 min respectively. The minimum reaction times for E. coli and M. smegmatis are 10 min and 45 min respectively.

    Journal: bioRxiv

    Article Title: An integrated one-step assay combining thermal lysis and loop-mediated isothermal DNA amplification (LAMP) in 30 min from E. coli and M. smegmatis cells on a paper substrate

    doi: 10.1101/594374

    Figure Lengend Snippet: Estimation of the minimum reaction time for E. coli (panel A) and M. smegmatis (panel B) using gel electrophoresis. Lane 1: DNA ladder (10 bp to 300 bp). Lane 2: NMC indicates negative control without any mastermix in the reaction mixture. Lane 3: NTC indicates negative control without any bacterial cells in the reaction mixture. Lane 4: LAMP for the recommended duration of 60 min (positive control). Lanes 5-10: LAMP performed for 5 min, 10 min, 15 min, 20 min, 30 min and 45 min respectively. The minimum reaction times for E. coli and M. smegmatis are 10 min and 45 min respectively.

    Article Snippet: WarmStart LAMP Kit (DNA and RNA) enzyme mix for the LAMP reactions was bought from New England Biolabs (Beverly, USA).

    Techniques: Nucleic Acid Electrophoresis, Negative Control, Positive Control

    Detection sensitivity of our integrated assay with E. coli (panel A) and M. smegmatis (panel B) using gel electrophoresis. Lane 1: 10 bp to 300 bp DNA ladder. Lane 2: No master-mix control (NMC). Lane 3: No template control (NTC). Lane 4: LAMP of purified genomic DNA, used as a positive control. Lanes 5 – 10 in panel A: Reactions using different cell concentrations (10 7 CFU/mL, 10 6 CFU/mL, 10 4 CFU/mL, 10 2 CFU/mL, 10 0 CFU/mL and 10 -2 CFU/mL respectively). Lanes 5 – 10 in panel B: Reactions using different cell concentrations (10 8 CFU/mL, 10 7 CFU/mL, 10 5 CFU/mL, 10 3 CFU/mL, 10 1 CFU/mL and 10 -1 CFU/mL respectively). Gel electrophoresis can detect 1000 CFU/mL of M smegmatis and 100 CFU/mL of E. coli .

    Journal: bioRxiv

    Article Title: An integrated one-step assay combining thermal lysis and loop-mediated isothermal DNA amplification (LAMP) in 30 min from E. coli and M. smegmatis cells on a paper substrate

    doi: 10.1101/594374

    Figure Lengend Snippet: Detection sensitivity of our integrated assay with E. coli (panel A) and M. smegmatis (panel B) using gel electrophoresis. Lane 1: 10 bp to 300 bp DNA ladder. Lane 2: No master-mix control (NMC). Lane 3: No template control (NTC). Lane 4: LAMP of purified genomic DNA, used as a positive control. Lanes 5 – 10 in panel A: Reactions using different cell concentrations (10 7 CFU/mL, 10 6 CFU/mL, 10 4 CFU/mL, 10 2 CFU/mL, 10 0 CFU/mL and 10 -2 CFU/mL respectively). Lanes 5 – 10 in panel B: Reactions using different cell concentrations (10 8 CFU/mL, 10 7 CFU/mL, 10 5 CFU/mL, 10 3 CFU/mL, 10 1 CFU/mL and 10 -1 CFU/mL respectively). Gel electrophoresis can detect 1000 CFU/mL of M smegmatis and 100 CFU/mL of E. coli .

    Article Snippet: WarmStart LAMP Kit (DNA and RNA) enzyme mix for the LAMP reactions was bought from New England Biolabs (Beverly, USA).

    Techniques: Nucleic Acid Electrophoresis, Purification, Positive Control

    Barcoding and PCR amplification of RT-LAMP products (A) Overview of COV-ID. Saliva is collected and inactivated prior to RT-LAMP performed with up to 96 individual sample barcoded primers. LAMP reactions are pooled and further amplified via PCR to introduce Illumina adapter sequences and pool-level dual indexes. A single thermal cycler can amplify 96 or 384 such pools and the resulting “super-pool” can be sequenced overnight to detect multiple amplicons from 9,216 or 36,864 individual patient samples (number of reads in parenthesis assume an output of ∼450M reads from a NextSeq 500). (B) Schematic of the RT-LAMP (step I) of COV-ID. Selected numbered intermediates of RT-LAMP reaction are shown to illustrate how the LAMP barcode, shown in yellow, and the P5 and P7 homology sequences (blue and pink, respectively) are introduced in the final LAMP product. Upon generating the dumb-bell intermediate the reaction proceeds through rapid primed and self-primed extensions to form mixture of various DNA amplicons containing sequences for PCR amplification. A more detailed version of the LAMP phase of COV-ID, including specific sequences, is illustrated in Fig. S1 . (C) Conventional RT-LAMP primers (solid lines) or primers modified for COV-ID (dotted lines) were used for RT-LAMP of SARS-CoV-2. The numbers of inactivated SARS-CoV-2 virions per µL is indicated in the color legend. (D) Schematic of the PCR (step II) of COV-ID. Following RT-LAMP, up to 96 reactions are pooled and purified and Illumina libraries are generated directly by PCR with dual-indexed P5 and P7 adapters in preparation for sequencing. (E) COV-ID primers targeting ACTB mRNA were used for RT-LAMP with HeLa total RNA. LAMP was diluted 1:100, amplified via PCR and resolved on 2% agarose gel.

    Journal: medRxiv

    Article Title: COV-ID: A LAMP sequencing approach for high-throughput co-detection of SARS-CoV-2 and influenza virus in human saliva

    doi: 10.1101/2021.04.23.21255523

    Figure Lengend Snippet: Barcoding and PCR amplification of RT-LAMP products (A) Overview of COV-ID. Saliva is collected and inactivated prior to RT-LAMP performed with up to 96 individual sample barcoded primers. LAMP reactions are pooled and further amplified via PCR to introduce Illumina adapter sequences and pool-level dual indexes. A single thermal cycler can amplify 96 or 384 such pools and the resulting “super-pool” can be sequenced overnight to detect multiple amplicons from 9,216 or 36,864 individual patient samples (number of reads in parenthesis assume an output of ∼450M reads from a NextSeq 500). (B) Schematic of the RT-LAMP (step I) of COV-ID. Selected numbered intermediates of RT-LAMP reaction are shown to illustrate how the LAMP barcode, shown in yellow, and the P5 and P7 homology sequences (blue and pink, respectively) are introduced in the final LAMP product. Upon generating the dumb-bell intermediate the reaction proceeds through rapid primed and self-primed extensions to form mixture of various DNA amplicons containing sequences for PCR amplification. A more detailed version of the LAMP phase of COV-ID, including specific sequences, is illustrated in Fig. S1 . (C) Conventional RT-LAMP primers (solid lines) or primers modified for COV-ID (dotted lines) were used for RT-LAMP of SARS-CoV-2. The numbers of inactivated SARS-CoV-2 virions per µL is indicated in the color legend. (D) Schematic of the PCR (step II) of COV-ID. Following RT-LAMP, up to 96 reactions are pooled and purified and Illumina libraries are generated directly by PCR with dual-indexed P5 and P7 adapters in preparation for sequencing. (E) COV-ID primers targeting ACTB mRNA were used for RT-LAMP with HeLa total RNA. LAMP was diluted 1:100, amplified via PCR and resolved on 2% agarose gel.

    Article Snippet: Each 10 μL RT-LAMP reaction mix consisted of 1x Warmstart LAMP 2x Master Mix (NEB Cat. E1700S), 0.7 μM dUTP (Promega Cat. U1191), 1 μM SYTO-9 (Thermo Cat. S34854), 0.1 μL Thermolabile UDG (Enzymatics Cat. G5020L), 1 μL of saliva template and optionally 20 fg of N2 Spike RNA.

    Techniques: Polymerase Chain Reaction, Amplification, Introduce, Modification, Purification, Generated, Sequencing, Agarose Gel Electrophoresis