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


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    WarmStart Colorimetric LAMP 2X Master Mix DNA and RNA
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    WarmStart Colorimetric LAMP 2X Master Mix DNA and RNA 500 rxns
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    M1800L
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    Thermostable DNA Polymerases
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    500 rxns
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    New England Biolabs colorimetric warmstart lamp 2x master mix
    WarmStart Colorimetric LAMP 2X Master Mix DNA and RNA
    WarmStart Colorimetric LAMP 2X Master Mix DNA and RNA 500 rxns
    https://www.bioz.com/result/colorimetric warmstart lamp 2x master mix/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    colorimetric warmstart lamp 2x master mix - by Bioz Stars, 2021-05
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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 "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

    3) 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

    4) Product Images from "A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing"

    Article Title: A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing

    Journal: bioRxiv

    doi: 10.1101/2020.06.23.166397

    HNB RT-LAMP enables colorimetric SARS-CoV-2 detection from crude patient samples. A) Schematic illustrating the properties of pH-sensitive (Phenol Red, top) and Mg 2+ concentration sensitive (hydroxynaphthol blue, HNB, bottom) colorimetric readouts for LAMP. Phenol Red interacts with protons (H + ) generated during DNA amplification, which causes a color change from pink/red to yellow (right: the color range of the Phenol Red-containing colorimetric RT-LAMP mastermix (NEB) at relevant pH values). Magnesium pyrophosphate precipitate produced during DNA amplification lowers the free Mg 2+ concentration, causing a color change of the HNB dye from purple to sky-blue (right: the color range of solutions with HNB at relevant Mg 2+ concentrations). B) Influence of QuickExtract on HNB RT-LAMP performance. Shown is the colorimetric HNB readout of RT-LAMP reactions (after 35 minutes; in duplicates) using indicated copy numbers of SARS-CoV-2 RNA standard in water or QuickExtract and the corresponding co-measured end-point fluorescence values (heatmaps are shown below). C) QuickExtract lysis buffer is compatible with HNB colorimetric readout but incompatible with Phenol Red colorimetric readout of RT-LAMP reactions. Shown are RT-LAMP reaction outcomes (upper panel: colorimetric readout after 35 minutes, lower panel: fluorescent end-point values) when using 500 copies of synthetic SARS-CoV-2 RNA standard in indicated sample media diluted 1:1 with water or 2x QuickExtract solution as input. D) HNB RT-LAMP performance on Covid-19 patient samples lysed in QuickExtract solution. Shown is the binary colorimetric HNB readout of RT-LAMP reactions (N gene) using indicated patient samples (sputum (orange), swab (black), gargle (green)) plotted against the corresponding Cq values from RT-qPCR. E) Predictive agreement between HNB RT-LAMP and RT-qPCR assays using patient samples lysed in QuickExtract solution. Samples were grouped according to their RT-qPCR Cq values, and the percentage of detected (black) and not detected (purple) samples (based on HNB RT-LAMP) of the total number of samples per group was plotted. F) Schematic illustrating the serial dilution grid of a Covid-19 positive patient sample (Cq of 28) in QuickExtract. The heatmap (left) indicates Cq values determined by 1-step RT-qPCR (values above Cq 40 are indicated by black crosses). The grid (right) indicates the binary read-out (black: detected; white: not detected) of HNB RT-LAMP as measured by 650 nm absorbance). G) Scatter plot showing HNB RT-LAMP performance (measured by 650 nm absorbance) versus qPCR-determined Cq values on the serial dilution grid shown in F, including no-target controls (NTC) and a Covid-19-negative patient sample (qPCR negative). Horizontal dashed line indicates the maximum absorbance obtained for any negative control (y = 0.602).
    Figure Legend Snippet: HNB RT-LAMP enables colorimetric SARS-CoV-2 detection from crude patient samples. A) Schematic illustrating the properties of pH-sensitive (Phenol Red, top) and Mg 2+ concentration sensitive (hydroxynaphthol blue, HNB, bottom) colorimetric readouts for LAMP. Phenol Red interacts with protons (H + ) generated during DNA amplification, which causes a color change from pink/red to yellow (right: the color range of the Phenol Red-containing colorimetric RT-LAMP mastermix (NEB) at relevant pH values). Magnesium pyrophosphate precipitate produced during DNA amplification lowers the free Mg 2+ concentration, causing a color change of the HNB dye from purple to sky-blue (right: the color range of solutions with HNB at relevant Mg 2+ concentrations). B) Influence of QuickExtract on HNB RT-LAMP performance. Shown is the colorimetric HNB readout of RT-LAMP reactions (after 35 minutes; in duplicates) using indicated copy numbers of SARS-CoV-2 RNA standard in water or QuickExtract and the corresponding co-measured end-point fluorescence values (heatmaps are shown below). C) QuickExtract lysis buffer is compatible with HNB colorimetric readout but incompatible with Phenol Red colorimetric readout of RT-LAMP reactions. Shown are RT-LAMP reaction outcomes (upper panel: colorimetric readout after 35 minutes, lower panel: fluorescent end-point values) when using 500 copies of synthetic SARS-CoV-2 RNA standard in indicated sample media diluted 1:1 with water or 2x QuickExtract solution as input. D) HNB RT-LAMP performance on Covid-19 patient samples lysed in QuickExtract solution. Shown is the binary colorimetric HNB readout of RT-LAMP reactions (N gene) using indicated patient samples (sputum (orange), swab (black), gargle (green)) plotted against the corresponding Cq values from RT-qPCR. E) Predictive agreement between HNB RT-LAMP and RT-qPCR assays using patient samples lysed in QuickExtract solution. Samples were grouped according to their RT-qPCR Cq values, and the percentage of detected (black) and not detected (purple) samples (based on HNB RT-LAMP) of the total number of samples per group was plotted. F) Schematic illustrating the serial dilution grid of a Covid-19 positive patient sample (Cq of 28) in QuickExtract. The heatmap (left) indicates Cq values determined by 1-step RT-qPCR (values above Cq 40 are indicated by black crosses). The grid (right) indicates the binary read-out (black: detected; white: not detected) of HNB RT-LAMP as measured by 650 nm absorbance). G) Scatter plot showing HNB RT-LAMP performance (measured by 650 nm absorbance) versus qPCR-determined Cq values on the serial dilution grid shown in F, including no-target controls (NTC) and a Covid-19-negative patient sample (qPCR negative). Horizontal dashed line indicates the maximum absorbance obtained for any negative control (y = 0.602).

    Techniques Used: Concentration Assay, Generated, Amplification, Produced, Fluorescence, Lysis, Quantitative RT-PCR, Serial Dilution, Real-time Polymerase Chain Reaction, Negative Control

    HNB RT-LAMP shows robust performance on QuickExtract-treated patient sample material. A) Comparison between Phenol Red and HNB colorimetric readout of RT-LAMP reactions (in duplicates) on serially diluted synthetic SARS-CoV-2 RNA in water (left) or 1x QuickExtract (right). End-point fluorescence values measured in parallel are shown in heatmaps below. While fluorescent detection indicates successful LAMP in both sample matrices, Phenol Red but not HNB colorimetric readout is inconclusive in QuickExtract buffer (right panel, top rows). B) HNB RT-LAMP performance across a wide range of Covid-19 patients and sample types. Images showing the HNB end-point outcome of RT-LAMP reactions on multiple Covid-19 patient samples (gargle, swab or sputum; samples indicate swabs if not otherwise stated). The respective Cq values of the individual samples (CDC-N1; QuickExtract RT-qPCR or extracted RNA RT-qPCR as indicated) are shown above or below each sample. Colorimetric RT-LAMP using Phenol Red is shown for one sample set (first from top), again with inconclusive outcome. All reactions were performed in duplicates. The HNB color-reaction was read-out at 35 minutes unless indicated otherwise. Summary dotplots for every sample set are shown to the right; samples were classified as detected or not detected based on RT-LAMP outcome and plotted against their respective RT-qPCR determined Cq values.
    Figure Legend Snippet: HNB RT-LAMP shows robust performance on QuickExtract-treated patient sample material. A) Comparison between Phenol Red and HNB colorimetric readout of RT-LAMP reactions (in duplicates) on serially diluted synthetic SARS-CoV-2 RNA in water (left) or 1x QuickExtract (right). End-point fluorescence values measured in parallel are shown in heatmaps below. While fluorescent detection indicates successful LAMP in both sample matrices, Phenol Red but not HNB colorimetric readout is inconclusive in QuickExtract buffer (right panel, top rows). B) HNB RT-LAMP performance across a wide range of Covid-19 patients and sample types. Images showing the HNB end-point outcome of RT-LAMP reactions on multiple Covid-19 patient samples (gargle, swab or sputum; samples indicate swabs if not otherwise stated). The respective Cq values of the individual samples (CDC-N1; QuickExtract RT-qPCR or extracted RNA RT-qPCR as indicated) are shown above or below each sample. Colorimetric RT-LAMP using Phenol Red is shown for one sample set (first from top), again with inconclusive outcome. All reactions were performed in duplicates. The HNB color-reaction was read-out at 35 minutes unless indicated otherwise. Summary dotplots for every sample set are shown to the right; samples were classified as detected or not detected based on RT-LAMP outcome and plotted against their respective RT-qPCR determined Cq values.

    Techniques Used: Fluorescence, Quantitative RT-PCR

    bead-LAMP increases sensitivity of RT-LAMP assays. A) Schematic illustrating the bead-LAMP workflow in comparison to the regular RT-LAMP workflow. AMPure XP RNA capture beads were used at 0.6x of the volume of the sample lysate (0.6x beads). B) Performance of bead-LAMP (+ bead enrichment) vs regular RT-LAMP (-bead enrichment) using a synthetic SARS-CoV-2 RNA standard spiked-in at the indicated concentration in the original sample into HeLa cell QuickExtract (QE) lysate. 50 µ l of crude sample in QE, adjusted to 100 µ l final volume with 1x HBSS was used for bead-enrichment. The image shows HNB end-point colorimetric readout of bead-LAMP and RT-LAMP reactions; a magnified view of four wells with beads (top: 5 copies/ µ l is sky-blue; 2 copies/ µ l is purple) and without beads (bottom: both are purple) is shown on the right. All reactions were performed in technical quadruplicates. C) End-point relative fluorescence units (RFUs), with or without prior bead enrichment for reactions shown in B. D) Performance of 1-step RT-qPCR using 2 µ l of the same crude sample preparations as used in B and C. E) Positive detection rates of 1-step RT-qPCR, RT-LAMP and bead-LAMP for reactions shown in B, C and D. F) Performance of bead-LAMP on a Covid-19 positive panel of patient samples in QuickExtract. The images depict the HNB colorimetric end-point readout, and the heatmaps underneath show co-measured end-point relative fluorescence units (RFUs) of RT-LAMP reactions, with or without prior bead enrichment, using eight Covid-19-positive and five negative samples as input (P1-P11, Covid-19 patient sample; CS42 and CS46, healthy controls). 100 µ l of crude sample in QE was used for bead-enrichment. Corresponding Cq values were obtained by measuring 2 µ l of the same QuickExtract (QE) patient samples by 1-step RT-qPCR prior to bead enrichment. G) Bead enrichment increases the sensitivity of RT-LAMP. Patient samples from F) were classified as detected or not detected based on the HNB RT-LAMP assay before (left, open circles) and after (right, filled circles) bead enrichment and plotted against their respective Cq values obtained from QuickExtract (QE) RT-qPCR (Cq values for qPCR negative samples are labelled as not detected, ND). H) Schematic illustrating the pooled testing strategy using bead-LAMP. A single Covid-19 positive patient gargle sample in QuickExtract (Cq ∼28; black) was mixed with different amounts of 95 pooled SARS-CoV-2 negative samples (all in QuickExtract; white) yielding seven sample pools with indicated ratios of positive to negative samples. 40 to 100 µ l of crude sample in QE was used for bead-enrichment depending on the pool sizes. For lysate volumes smaller than 100 µ l, 1x HBSS was added to obtain a final volume of 100 µ l for bead-LAMP. I) Shown is the performance (measured as time to threshold) of bead-LAMP (filled circles) compared to regular RT-LAMP (open circles) on the patient pools defined in H. ND = not detected within 60 minutes of RT-LAMP incubation. J) Images showing the endpoint HNB colorimetric readout (left) and fluorescent readout (endpoint RFU; right) of samples measured in I) with or without prior bead enrichment.
    Figure Legend Snippet: bead-LAMP increases sensitivity of RT-LAMP assays. A) Schematic illustrating the bead-LAMP workflow in comparison to the regular RT-LAMP workflow. AMPure XP RNA capture beads were used at 0.6x of the volume of the sample lysate (0.6x beads). B) Performance of bead-LAMP (+ bead enrichment) vs regular RT-LAMP (-bead enrichment) using a synthetic SARS-CoV-2 RNA standard spiked-in at the indicated concentration in the original sample into HeLa cell QuickExtract (QE) lysate. 50 µ l of crude sample in QE, adjusted to 100 µ l final volume with 1x HBSS was used for bead-enrichment. The image shows HNB end-point colorimetric readout of bead-LAMP and RT-LAMP reactions; a magnified view of four wells with beads (top: 5 copies/ µ l is sky-blue; 2 copies/ µ l is purple) and without beads (bottom: both are purple) is shown on the right. All reactions were performed in technical quadruplicates. C) End-point relative fluorescence units (RFUs), with or without prior bead enrichment for reactions shown in B. D) Performance of 1-step RT-qPCR using 2 µ l of the same crude sample preparations as used in B and C. E) Positive detection rates of 1-step RT-qPCR, RT-LAMP and bead-LAMP for reactions shown in B, C and D. F) Performance of bead-LAMP on a Covid-19 positive panel of patient samples in QuickExtract. The images depict the HNB colorimetric end-point readout, and the heatmaps underneath show co-measured end-point relative fluorescence units (RFUs) of RT-LAMP reactions, with or without prior bead enrichment, using eight Covid-19-positive and five negative samples as input (P1-P11, Covid-19 patient sample; CS42 and CS46, healthy controls). 100 µ l of crude sample in QE was used for bead-enrichment. Corresponding Cq values were obtained by measuring 2 µ l of the same QuickExtract (QE) patient samples by 1-step RT-qPCR prior to bead enrichment. G) Bead enrichment increases the sensitivity of RT-LAMP. Patient samples from F) were classified as detected or not detected based on the HNB RT-LAMP assay before (left, open circles) and after (right, filled circles) bead enrichment and plotted against their respective Cq values obtained from QuickExtract (QE) RT-qPCR (Cq values for qPCR negative samples are labelled as not detected, ND). H) Schematic illustrating the pooled testing strategy using bead-LAMP. A single Covid-19 positive patient gargle sample in QuickExtract (Cq ∼28; black) was mixed with different amounts of 95 pooled SARS-CoV-2 negative samples (all in QuickExtract; white) yielding seven sample pools with indicated ratios of positive to negative samples. 40 to 100 µ l of crude sample in QE was used for bead-enrichment depending on the pool sizes. For lysate volumes smaller than 100 µ l, 1x HBSS was added to obtain a final volume of 100 µ l for bead-LAMP. I) Shown is the performance (measured as time to threshold) of bead-LAMP (filled circles) compared to regular RT-LAMP (open circles) on the patient pools defined in H. ND = not detected within 60 minutes of RT-LAMP incubation. J) Images showing the endpoint HNB colorimetric readout (left) and fluorescent readout (endpoint RFU; right) of samples measured in I) with or without prior bead enrichment.

    Techniques Used: Concentration Assay, Fluorescence, Quantitative RT-PCR, RT Lamp Assay, Real-time Polymerase Chain Reaction, Incubation

    Pooled Covid-19 testing strategy using bead-LAMP. A) Performance of bead-LAMP on crude patient samples. The image (top) shows HNB end-point colorimetric readout and the heatmap (bottom) shows co-measured end-point relative fluorescence units (RFUs) of RT-LAMP on serially diluted patient samples in QuickExtract-prepared HeLa cell lysate, with or without prior bead enrichment. Cq values are estimated based on RT-qPCR measurement of the Cq value of the undiluted parental Covid-19 patient sample prior to bead enrichment. All reactions were performed in duplicates. B) RT-qPCR Cq values (CDC-N1) of gargle sample pools used in Fig 4H-J with the indicated fraction of Covid-19 positive gargle sample per pool. For the two large sample pools (pool of 1 in 96 and pool of 1 in 48), the 100 µ l aliquot used for subsequent RT-LAMP and bead-LAMP was measured in addition. C) Readout of a real-time fluorescence RT-LAMP reaction of sample pools with indicated fraction of positive lysate without (left) and with (right) bead enrichment. RFU: relative fluorescent units. D) Schematic illustrating the pooled testing strategy. Eight pools mimicking different total patient sample numbers and different ratios of Covid-19-positive patient samples (0-100%) were generated from one Covid-19-positive QuickExtract patient sample (N1 RT-qPCR with Cq ∼30) mixed at the indicated ratios with QuickExtract HeLa cell lysate at 20 cells/ µ l. E) Shown is the performance (measured as end-point relative fluorescence units (RFU)) of bead-LAMP (filled circles) compared to regular RT-LAMP (open circles) on the patient pools defined in D. ND = not detected within 60 minutes of RT-LAMP incubation. F) Images showing the endpoint HNB colorimetric readout of samples measured in E with or without prior bead enrichment. G) Bead-enrichment makes crude QuickExtract samples compatible with the pH-sensitive Phenol Red based colorimetric readout of RT-LAMP. Images showing the endpoint Phenol Red colorimetric readout (top) and the fluorescent readout (bottom) of two Covid-19 positive pools and one Covid-19 negative pool (qPCR negative) defined in D with (+) or without (-) prior bead enrichment.
    Figure Legend Snippet: Pooled Covid-19 testing strategy using bead-LAMP. A) Performance of bead-LAMP on crude patient samples. The image (top) shows HNB end-point colorimetric readout and the heatmap (bottom) shows co-measured end-point relative fluorescence units (RFUs) of RT-LAMP on serially diluted patient samples in QuickExtract-prepared HeLa cell lysate, with or without prior bead enrichment. Cq values are estimated based on RT-qPCR measurement of the Cq value of the undiluted parental Covid-19 patient sample prior to bead enrichment. All reactions were performed in duplicates. B) RT-qPCR Cq values (CDC-N1) of gargle sample pools used in Fig 4H-J with the indicated fraction of Covid-19 positive gargle sample per pool. For the two large sample pools (pool of 1 in 96 and pool of 1 in 48), the 100 µ l aliquot used for subsequent RT-LAMP and bead-LAMP was measured in addition. C) Readout of a real-time fluorescence RT-LAMP reaction of sample pools with indicated fraction of positive lysate without (left) and with (right) bead enrichment. RFU: relative fluorescent units. D) Schematic illustrating the pooled testing strategy. Eight pools mimicking different total patient sample numbers and different ratios of Covid-19-positive patient samples (0-100%) were generated from one Covid-19-positive QuickExtract patient sample (N1 RT-qPCR with Cq ∼30) mixed at the indicated ratios with QuickExtract HeLa cell lysate at 20 cells/ µ l. E) Shown is the performance (measured as end-point relative fluorescence units (RFU)) of bead-LAMP (filled circles) compared to regular RT-LAMP (open circles) on the patient pools defined in D. ND = not detected within 60 minutes of RT-LAMP incubation. F) Images showing the endpoint HNB colorimetric readout of samples measured in E with or without prior bead enrichment. G) Bead-enrichment makes crude QuickExtract samples compatible with the pH-sensitive Phenol Red based colorimetric readout of RT-LAMP. Images showing the endpoint Phenol Red colorimetric readout (top) and the fluorescent readout (bottom) of two Covid-19 positive pools and one Covid-19 negative pool (qPCR negative) defined in D with (+) or without (-) prior bead enrichment.

    Techniques Used: Fluorescence, Quantitative RT-PCR, Generated, Incubation, Real-time Polymerase Chain Reaction

    5) 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

    Related Articles

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Fully 3D Printed Integrated Reactor Array for Point-of-Care Molecular Diagnostics
    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). .. N. meningitidis was grown in Tryptic Soy Broth (BD, Sparks, MD) and counted on Trypticase™ Soy Agar with 5% Sheep Blood (BD, Sparks, MD).

    Colorimetric Assay:

    Article Title: Simpler, Faster, and Sensitive Zika Virus Assay Using Smartphone Detection of Loop-mediated Isothermal Amplification on Paper Microfluidic Chips
    Article Snippet: For field applications, a simpler type of dye is needed that is affected by neither ambient lighting perturbations nor the presence of other amplification-inhibiting molecules in the sample. .. For this purpose, pH indicator-based colorimetric assay kits could be used (e.g., WarmStart colorimetric LAMP master mix by New England Biolabs), which includes a pH indicator (phenol red) in the master mix reagent, requiring no additional step, no excitation light source, and no closed container (identifiable under ambient light) . ..

    other:

    Article Title: A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing
    Article Snippet: Colorimetric LAMP For HNB colorimetric RT-LAMP detection, reactions were set up as in fluorescent RT-LAMP with the addition of 120 µ M HNB dye solution (20 mM stock in nuclease-free water).

    Infection:

    Article Title: Fast, Precise, and Reliable Multiplex Detection of Potato Viruses by Loop-Mediated Isothermal Amplification
    Article Snippet: The tHDA, CRISDA, and TwistDx-RPA kits did not generate any amplicons, whereas the Agdia-RPA kit generated weak and inconsistent results (data not shown). .. In contrast, the LAMP method (WarmStart LAMP 2× Master Mix from New England Biolabs (NEB), Ipswich, MA, USA) achieved promising initial results, correctly detecting seven of the nine infected tubers. .. The WarmStart LAMP method also generated three false-positive by-products, but these clearly differed from genuine amplicons in terms of cycle threshold (Ct) values, with delays exceeding 10, 20, and 25 min, respectively ( ).

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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:

    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).

    Journal: International Journal of Molecular Sciences

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

    doi: 10.3390/ijms21228741

    Figure Lengend 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).

    Article Snippet: In contrast, the LAMP method (WarmStart LAMP 2× Master Mix from New England Biolabs (NEB), Ipswich, MA, USA) achieved promising initial results, correctly detecting seven of the nine infected tubers.

    Techniques: 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).

    Journal: International Journal of Molecular Sciences

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

    doi: 10.3390/ijms21228741

    Figure Lengend 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).

    Article Snippet: In contrast, the LAMP method (WarmStart LAMP 2× Master Mix from New England Biolabs (NEB), Ipswich, MA, USA) achieved promising initial results, correctly detecting seven of the nine infected tubers.

    Techniques: 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).

    Journal: International Journal of Molecular Sciences

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

    doi: 10.3390/ijms21228741

    Figure Lengend 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).

    Article Snippet: In contrast, the LAMP method (WarmStart LAMP 2× Master Mix from New England Biolabs (NEB), Ipswich, MA, USA) achieved promising initial results, correctly detecting seven of the nine infected tubers.

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

    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

    HNB RT-LAMP enables colorimetric SARS-CoV-2 detection from crude patient samples. A) Schematic illustrating the properties of pH-sensitive (Phenol Red, top) and Mg 2+ concentration sensitive (hydroxynaphthol blue, HNB, bottom) colorimetric readouts for LAMP. Phenol Red interacts with protons (H + ) generated during DNA amplification, which causes a color change from pink/red to yellow (right: the color range of the Phenol Red-containing colorimetric RT-LAMP mastermix (NEB) at relevant pH values). Magnesium pyrophosphate precipitate produced during DNA amplification lowers the free Mg 2+ concentration, causing a color change of the HNB dye from purple to sky-blue (right: the color range of solutions with HNB at relevant Mg 2+ concentrations). B) Influence of QuickExtract on HNB RT-LAMP performance. Shown is the colorimetric HNB readout of RT-LAMP reactions (after 35 minutes; in duplicates) using indicated copy numbers of SARS-CoV-2 RNA standard in water or QuickExtract and the corresponding co-measured end-point fluorescence values (heatmaps are shown below). C) QuickExtract lysis buffer is compatible with HNB colorimetric readout but incompatible with Phenol Red colorimetric readout of RT-LAMP reactions. Shown are RT-LAMP reaction outcomes (upper panel: colorimetric readout after 35 minutes, lower panel: fluorescent end-point values) when using 500 copies of synthetic SARS-CoV-2 RNA standard in indicated sample media diluted 1:1 with water or 2x QuickExtract solution as input. D) HNB RT-LAMP performance on Covid-19 patient samples lysed in QuickExtract solution. Shown is the binary colorimetric HNB readout of RT-LAMP reactions (N gene) using indicated patient samples (sputum (orange), swab (black), gargle (green)) plotted against the corresponding Cq values from RT-qPCR. E) Predictive agreement between HNB RT-LAMP and RT-qPCR assays using patient samples lysed in QuickExtract solution. Samples were grouped according to their RT-qPCR Cq values, and the percentage of detected (black) and not detected (purple) samples (based on HNB RT-LAMP) of the total number of samples per group was plotted. F) Schematic illustrating the serial dilution grid of a Covid-19 positive patient sample (Cq of 28) in QuickExtract. The heatmap (left) indicates Cq values determined by 1-step RT-qPCR (values above Cq 40 are indicated by black crosses). The grid (right) indicates the binary read-out (black: detected; white: not detected) of HNB RT-LAMP as measured by 650 nm absorbance). G) Scatter plot showing HNB RT-LAMP performance (measured by 650 nm absorbance) versus qPCR-determined Cq values on the serial dilution grid shown in F, including no-target controls (NTC) and a Covid-19-negative patient sample (qPCR negative). Horizontal dashed line indicates the maximum absorbance obtained for any negative control (y = 0.602).

    Journal: bioRxiv

    Article Title: A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing

    doi: 10.1101/2020.06.23.166397

    Figure Lengend Snippet: HNB RT-LAMP enables colorimetric SARS-CoV-2 detection from crude patient samples. A) Schematic illustrating the properties of pH-sensitive (Phenol Red, top) and Mg 2+ concentration sensitive (hydroxynaphthol blue, HNB, bottom) colorimetric readouts for LAMP. Phenol Red interacts with protons (H + ) generated during DNA amplification, which causes a color change from pink/red to yellow (right: the color range of the Phenol Red-containing colorimetric RT-LAMP mastermix (NEB) at relevant pH values). Magnesium pyrophosphate precipitate produced during DNA amplification lowers the free Mg 2+ concentration, causing a color change of the HNB dye from purple to sky-blue (right: the color range of solutions with HNB at relevant Mg 2+ concentrations). B) Influence of QuickExtract on HNB RT-LAMP performance. Shown is the colorimetric HNB readout of RT-LAMP reactions (after 35 minutes; in duplicates) using indicated copy numbers of SARS-CoV-2 RNA standard in water or QuickExtract and the corresponding co-measured end-point fluorescence values (heatmaps are shown below). C) QuickExtract lysis buffer is compatible with HNB colorimetric readout but incompatible with Phenol Red colorimetric readout of RT-LAMP reactions. Shown are RT-LAMP reaction outcomes (upper panel: colorimetric readout after 35 minutes, lower panel: fluorescent end-point values) when using 500 copies of synthetic SARS-CoV-2 RNA standard in indicated sample media diluted 1:1 with water or 2x QuickExtract solution as input. D) HNB RT-LAMP performance on Covid-19 patient samples lysed in QuickExtract solution. Shown is the binary colorimetric HNB readout of RT-LAMP reactions (N gene) using indicated patient samples (sputum (orange), swab (black), gargle (green)) plotted against the corresponding Cq values from RT-qPCR. E) Predictive agreement between HNB RT-LAMP and RT-qPCR assays using patient samples lysed in QuickExtract solution. Samples were grouped according to their RT-qPCR Cq values, and the percentage of detected (black) and not detected (purple) samples (based on HNB RT-LAMP) of the total number of samples per group was plotted. F) Schematic illustrating the serial dilution grid of a Covid-19 positive patient sample (Cq of 28) in QuickExtract. The heatmap (left) indicates Cq values determined by 1-step RT-qPCR (values above Cq 40 are indicated by black crosses). The grid (right) indicates the binary read-out (black: detected; white: not detected) of HNB RT-LAMP as measured by 650 nm absorbance). G) Scatter plot showing HNB RT-LAMP performance (measured by 650 nm absorbance) versus qPCR-determined Cq values on the serial dilution grid shown in F, including no-target controls (NTC) and a Covid-19-negative patient sample (qPCR negative). Horizontal dashed line indicates the maximum absorbance obtained for any negative control (y = 0.602).

    Article Snippet: Colorimetric LAMP For HNB colorimetric RT-LAMP detection, reactions were set up as in fluorescent RT-LAMP with the addition of 120 µ M HNB dye solution (20 mM stock in nuclease-free water).

    Techniques: Concentration Assay, Generated, Amplification, Produced, Fluorescence, Lysis, Quantitative RT-PCR, Serial Dilution, Real-time Polymerase Chain Reaction, Negative Control

    HNB RT-LAMP shows robust performance on QuickExtract-treated patient sample material. A) Comparison between Phenol Red and HNB colorimetric readout of RT-LAMP reactions (in duplicates) on serially diluted synthetic SARS-CoV-2 RNA in water (left) or 1x QuickExtract (right). End-point fluorescence values measured in parallel are shown in heatmaps below. While fluorescent detection indicates successful LAMP in both sample matrices, Phenol Red but not HNB colorimetric readout is inconclusive in QuickExtract buffer (right panel, top rows). B) HNB RT-LAMP performance across a wide range of Covid-19 patients and sample types. Images showing the HNB end-point outcome of RT-LAMP reactions on multiple Covid-19 patient samples (gargle, swab or sputum; samples indicate swabs if not otherwise stated). The respective Cq values of the individual samples (CDC-N1; QuickExtract RT-qPCR or extracted RNA RT-qPCR as indicated) are shown above or below each sample. Colorimetric RT-LAMP using Phenol Red is shown for one sample set (first from top), again with inconclusive outcome. All reactions were performed in duplicates. The HNB color-reaction was read-out at 35 minutes unless indicated otherwise. Summary dotplots for every sample set are shown to the right; samples were classified as detected or not detected based on RT-LAMP outcome and plotted against their respective RT-qPCR determined Cq values.

    Journal: bioRxiv

    Article Title: A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing

    doi: 10.1101/2020.06.23.166397

    Figure Lengend Snippet: HNB RT-LAMP shows robust performance on QuickExtract-treated patient sample material. A) Comparison between Phenol Red and HNB colorimetric readout of RT-LAMP reactions (in duplicates) on serially diluted synthetic SARS-CoV-2 RNA in water (left) or 1x QuickExtract (right). End-point fluorescence values measured in parallel are shown in heatmaps below. While fluorescent detection indicates successful LAMP in both sample matrices, Phenol Red but not HNB colorimetric readout is inconclusive in QuickExtract buffer (right panel, top rows). B) HNB RT-LAMP performance across a wide range of Covid-19 patients and sample types. Images showing the HNB end-point outcome of RT-LAMP reactions on multiple Covid-19 patient samples (gargle, swab or sputum; samples indicate swabs if not otherwise stated). The respective Cq values of the individual samples (CDC-N1; QuickExtract RT-qPCR or extracted RNA RT-qPCR as indicated) are shown above or below each sample. Colorimetric RT-LAMP using Phenol Red is shown for one sample set (first from top), again with inconclusive outcome. All reactions were performed in duplicates. The HNB color-reaction was read-out at 35 minutes unless indicated otherwise. Summary dotplots for every sample set are shown to the right; samples were classified as detected or not detected based on RT-LAMP outcome and plotted against their respective RT-qPCR determined Cq values.

    Article Snippet: Colorimetric LAMP For HNB colorimetric RT-LAMP detection, reactions were set up as in fluorescent RT-LAMP with the addition of 120 µ M HNB dye solution (20 mM stock in nuclease-free water).

    Techniques: Fluorescence, Quantitative RT-PCR

    bead-LAMP increases sensitivity of RT-LAMP assays. A) Schematic illustrating the bead-LAMP workflow in comparison to the regular RT-LAMP workflow. AMPure XP RNA capture beads were used at 0.6x of the volume of the sample lysate (0.6x beads). B) Performance of bead-LAMP (+ bead enrichment) vs regular RT-LAMP (-bead enrichment) using a synthetic SARS-CoV-2 RNA standard spiked-in at the indicated concentration in the original sample into HeLa cell QuickExtract (QE) lysate. 50 µ l of crude sample in QE, adjusted to 100 µ l final volume with 1x HBSS was used for bead-enrichment. The image shows HNB end-point colorimetric readout of bead-LAMP and RT-LAMP reactions; a magnified view of four wells with beads (top: 5 copies/ µ l is sky-blue; 2 copies/ µ l is purple) and without beads (bottom: both are purple) is shown on the right. All reactions were performed in technical quadruplicates. C) End-point relative fluorescence units (RFUs), with or without prior bead enrichment for reactions shown in B. D) Performance of 1-step RT-qPCR using 2 µ l of the same crude sample preparations as used in B and C. E) Positive detection rates of 1-step RT-qPCR, RT-LAMP and bead-LAMP for reactions shown in B, C and D. F) Performance of bead-LAMP on a Covid-19 positive panel of patient samples in QuickExtract. The images depict the HNB colorimetric end-point readout, and the heatmaps underneath show co-measured end-point relative fluorescence units (RFUs) of RT-LAMP reactions, with or without prior bead enrichment, using eight Covid-19-positive and five negative samples as input (P1-P11, Covid-19 patient sample; CS42 and CS46, healthy controls). 100 µ l of crude sample in QE was used for bead-enrichment. Corresponding Cq values were obtained by measuring 2 µ l of the same QuickExtract (QE) patient samples by 1-step RT-qPCR prior to bead enrichment. G) Bead enrichment increases the sensitivity of RT-LAMP. Patient samples from F) were classified as detected or not detected based on the HNB RT-LAMP assay before (left, open circles) and after (right, filled circles) bead enrichment and plotted against their respective Cq values obtained from QuickExtract (QE) RT-qPCR (Cq values for qPCR negative samples are labelled as not detected, ND). H) Schematic illustrating the pooled testing strategy using bead-LAMP. A single Covid-19 positive patient gargle sample in QuickExtract (Cq ∼28; black) was mixed with different amounts of 95 pooled SARS-CoV-2 negative samples (all in QuickExtract; white) yielding seven sample pools with indicated ratios of positive to negative samples. 40 to 100 µ l of crude sample in QE was used for bead-enrichment depending on the pool sizes. For lysate volumes smaller than 100 µ l, 1x HBSS was added to obtain a final volume of 100 µ l for bead-LAMP. I) Shown is the performance (measured as time to threshold) of bead-LAMP (filled circles) compared to regular RT-LAMP (open circles) on the patient pools defined in H. ND = not detected within 60 minutes of RT-LAMP incubation. J) Images showing the endpoint HNB colorimetric readout (left) and fluorescent readout (endpoint RFU; right) of samples measured in I) with or without prior bead enrichment.

    Journal: bioRxiv

    Article Title: A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing

    doi: 10.1101/2020.06.23.166397

    Figure Lengend Snippet: bead-LAMP increases sensitivity of RT-LAMP assays. A) Schematic illustrating the bead-LAMP workflow in comparison to the regular RT-LAMP workflow. AMPure XP RNA capture beads were used at 0.6x of the volume of the sample lysate (0.6x beads). B) Performance of bead-LAMP (+ bead enrichment) vs regular RT-LAMP (-bead enrichment) using a synthetic SARS-CoV-2 RNA standard spiked-in at the indicated concentration in the original sample into HeLa cell QuickExtract (QE) lysate. 50 µ l of crude sample in QE, adjusted to 100 µ l final volume with 1x HBSS was used for bead-enrichment. The image shows HNB end-point colorimetric readout of bead-LAMP and RT-LAMP reactions; a magnified view of four wells with beads (top: 5 copies/ µ l is sky-blue; 2 copies/ µ l is purple) and without beads (bottom: both are purple) is shown on the right. All reactions were performed in technical quadruplicates. C) End-point relative fluorescence units (RFUs), with or without prior bead enrichment for reactions shown in B. D) Performance of 1-step RT-qPCR using 2 µ l of the same crude sample preparations as used in B and C. E) Positive detection rates of 1-step RT-qPCR, RT-LAMP and bead-LAMP for reactions shown in B, C and D. F) Performance of bead-LAMP on a Covid-19 positive panel of patient samples in QuickExtract. The images depict the HNB colorimetric end-point readout, and the heatmaps underneath show co-measured end-point relative fluorescence units (RFUs) of RT-LAMP reactions, with or without prior bead enrichment, using eight Covid-19-positive and five negative samples as input (P1-P11, Covid-19 patient sample; CS42 and CS46, healthy controls). 100 µ l of crude sample in QE was used for bead-enrichment. Corresponding Cq values were obtained by measuring 2 µ l of the same QuickExtract (QE) patient samples by 1-step RT-qPCR prior to bead enrichment. G) Bead enrichment increases the sensitivity of RT-LAMP. Patient samples from F) were classified as detected or not detected based on the HNB RT-LAMP assay before (left, open circles) and after (right, filled circles) bead enrichment and plotted against their respective Cq values obtained from QuickExtract (QE) RT-qPCR (Cq values for qPCR negative samples are labelled as not detected, ND). H) Schematic illustrating the pooled testing strategy using bead-LAMP. A single Covid-19 positive patient gargle sample in QuickExtract (Cq ∼28; black) was mixed with different amounts of 95 pooled SARS-CoV-2 negative samples (all in QuickExtract; white) yielding seven sample pools with indicated ratios of positive to negative samples. 40 to 100 µ l of crude sample in QE was used for bead-enrichment depending on the pool sizes. For lysate volumes smaller than 100 µ l, 1x HBSS was added to obtain a final volume of 100 µ l for bead-LAMP. I) Shown is the performance (measured as time to threshold) of bead-LAMP (filled circles) compared to regular RT-LAMP (open circles) on the patient pools defined in H. ND = not detected within 60 minutes of RT-LAMP incubation. J) Images showing the endpoint HNB colorimetric readout (left) and fluorescent readout (endpoint RFU; right) of samples measured in I) with or without prior bead enrichment.

    Article Snippet: Colorimetric LAMP For HNB colorimetric RT-LAMP detection, reactions were set up as in fluorescent RT-LAMP with the addition of 120 µ M HNB dye solution (20 mM stock in nuclease-free water).

    Techniques: Concentration Assay, Fluorescence, Quantitative RT-PCR, RT Lamp Assay, Real-time Polymerase Chain Reaction, Incubation

    Pooled Covid-19 testing strategy using bead-LAMP. A) Performance of bead-LAMP on crude patient samples. The image (top) shows HNB end-point colorimetric readout and the heatmap (bottom) shows co-measured end-point relative fluorescence units (RFUs) of RT-LAMP on serially diluted patient samples in QuickExtract-prepared HeLa cell lysate, with or without prior bead enrichment. Cq values are estimated based on RT-qPCR measurement of the Cq value of the undiluted parental Covid-19 patient sample prior to bead enrichment. All reactions were performed in duplicates. B) RT-qPCR Cq values (CDC-N1) of gargle sample pools used in Fig 4H-J with the indicated fraction of Covid-19 positive gargle sample per pool. For the two large sample pools (pool of 1 in 96 and pool of 1 in 48), the 100 µ l aliquot used for subsequent RT-LAMP and bead-LAMP was measured in addition. C) Readout of a real-time fluorescence RT-LAMP reaction of sample pools with indicated fraction of positive lysate without (left) and with (right) bead enrichment. RFU: relative fluorescent units. D) Schematic illustrating the pooled testing strategy. Eight pools mimicking different total patient sample numbers and different ratios of Covid-19-positive patient samples (0-100%) were generated from one Covid-19-positive QuickExtract patient sample (N1 RT-qPCR with Cq ∼30) mixed at the indicated ratios with QuickExtract HeLa cell lysate at 20 cells/ µ l. E) Shown is the performance (measured as end-point relative fluorescence units (RFU)) of bead-LAMP (filled circles) compared to regular RT-LAMP (open circles) on the patient pools defined in D. ND = not detected within 60 minutes of RT-LAMP incubation. F) Images showing the endpoint HNB colorimetric readout of samples measured in E with or without prior bead enrichment. G) Bead-enrichment makes crude QuickExtract samples compatible with the pH-sensitive Phenol Red based colorimetric readout of RT-LAMP. Images showing the endpoint Phenol Red colorimetric readout (top) and the fluorescent readout (bottom) of two Covid-19 positive pools and one Covid-19 negative pool (qPCR negative) defined in D with (+) or without (-) prior bead enrichment.

    Journal: bioRxiv

    Article Title: A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing

    doi: 10.1101/2020.06.23.166397

    Figure Lengend Snippet: Pooled Covid-19 testing strategy using bead-LAMP. A) Performance of bead-LAMP on crude patient samples. The image (top) shows HNB end-point colorimetric readout and the heatmap (bottom) shows co-measured end-point relative fluorescence units (RFUs) of RT-LAMP on serially diluted patient samples in QuickExtract-prepared HeLa cell lysate, with or without prior bead enrichment. Cq values are estimated based on RT-qPCR measurement of the Cq value of the undiluted parental Covid-19 patient sample prior to bead enrichment. All reactions were performed in duplicates. B) RT-qPCR Cq values (CDC-N1) of gargle sample pools used in Fig 4H-J with the indicated fraction of Covid-19 positive gargle sample per pool. For the two large sample pools (pool of 1 in 96 and pool of 1 in 48), the 100 µ l aliquot used for subsequent RT-LAMP and bead-LAMP was measured in addition. C) Readout of a real-time fluorescence RT-LAMP reaction of sample pools with indicated fraction of positive lysate without (left) and with (right) bead enrichment. RFU: relative fluorescent units. D) Schematic illustrating the pooled testing strategy. Eight pools mimicking different total patient sample numbers and different ratios of Covid-19-positive patient samples (0-100%) were generated from one Covid-19-positive QuickExtract patient sample (N1 RT-qPCR with Cq ∼30) mixed at the indicated ratios with QuickExtract HeLa cell lysate at 20 cells/ µ l. E) Shown is the performance (measured as end-point relative fluorescence units (RFU)) of bead-LAMP (filled circles) compared to regular RT-LAMP (open circles) on the patient pools defined in D. ND = not detected within 60 minutes of RT-LAMP incubation. F) Images showing the endpoint HNB colorimetric readout of samples measured in E with or without prior bead enrichment. G) Bead-enrichment makes crude QuickExtract samples compatible with the pH-sensitive Phenol Red based colorimetric readout of RT-LAMP. Images showing the endpoint Phenol Red colorimetric readout (top) and the fluorescent readout (bottom) of two Covid-19 positive pools and one Covid-19 negative pool (qPCR negative) defined in D with (+) or without (-) prior bead enrichment.

    Article Snippet: Colorimetric LAMP For HNB colorimetric RT-LAMP detection, reactions were set up as in fluorescent RT-LAMP with the addition of 120 µ M HNB dye solution (20 mM stock in nuclease-free water).

    Techniques: Fluorescence, Quantitative RT-PCR, Generated, Incubation, Real-time Polymerase Chain Reaction