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

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:

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

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

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

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

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

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

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

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