New England Biolabs, E2019S, SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit

The SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit utilizes isothermal amplification for use in the analysis of SARS-CoV-2, the novel coronavirus that causes COVID-19. Related Categories Isothermal Amplification & Strand Displacement,, PCR, qPCR & Amplification Technologies Applications Loop-Mediated Isothermal Amplification,, Isothermal Amplification,, DNA Amplification, PCR & qPCR Specification Materials Required but not Supplied Disposable powder-free gloves and any additional PPE required P2/P10, P200, and P1000 aerosol barrier tips Sterile, nuclease-free 1.5 ml microcentrifuge tubes Sterile, nuclease-free 2.0 ml, 5.0 ml, or 15 ml tubes 0.2 ml PCR reaction tube strips with separated tubes and lids (e.g., VWR 20170-004) or attached caps (e.g., VWR 20170-010) or 96-well PCR reaction plates with 8-cap strips Racks for 1.5 ml microcentrifuge tubes and 96-well 0.2 ml PCR reaction tubes Cooler rack for 1.5 microcentrifuge tubes and 96-well 0.2 ml PCR reaction tubes Acceptable surface decontaminants, for example: 10% bleach (1:10 dilution of commercial 5.25-6.0% sodium hypochlorite) Laboratory marking pen Appropriate disposal containers White paper or light background for optimal visualization of colorimetric reaction (e.g., typical printer paper) Micropipettes (2 or 10 μl, 200 μl and 1,000 μl), Multichannel Micropipettes (5-50 μl) -20°C Freezer (frost-free or nonfrost), 4°C Refrigerator Thermocycler, heat block or device that can be set to 65°C PCR Work Station [UV lamp; Laminar flow (Class 100 HEPA filtered)], Vortex Mixer, Tabletop Microcentrifuge FAQ Q: How does the new Omicron variant impact the effectiveness of the SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit? A: The SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit is an isothermal assay that targets regions in the N and E genes of the SARS-CoV-2 viral genome. Primers for both target regions are present in the same reaction, so amplification of either target can generate a positive signal. The new COVID-19 variant, Omicron, contains one point mutation in a region of the E-gene that is targeted by the LAMP primers in this kit. Given the position of this mutation and the overall tolerance of LAMP to point mutations, we do not anticipate a significant impact to assay performance with the Omicron (B.1.1.529) variant. To assess this experimentally, direct testing of IVT RNA representing both the wild-type (Wuhan) and mutant SARS-CoV-2 (Omicron) E­gene templates was conducted in the presence of wild-type N gene RNA, and substantially equivalent results were observed across a 3-log range of inputs. At low input ( 50 copies/rxn), 100% detection of both samples (36/36 replicates tested) was observed. Related application note: Detection of the Omicron variant mutation at position 26,270 in the SARS-CoV-2 E gene using the SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit Q: Can the Colorimetric LAMP 2X Master Mix be used in instruments that utilize fluorescent detection of DNA amplification or real time turbidity measurement? A: The master mix is compatible with fluorescent detection methods when supplemented with DNA intercalating fluorescent dyes and it gives reaction speed similar to conventional LAMP. It is also possible to perform LAMP detection on a real time turbidimeter, although its reaction speed is slightly slower than that of conventional LAMP. Q: How stable are the WarmStart Colorimetric LAMP 2X Master Mixes? A: The Colorimetric LAMP Mixes are stable under standard handling and usage conditions. They contain a low concentration of Tris pH 8 and the pH-sensitive Phenol Red shows bright pink color near this pH. After repeated usage of the same tube, a slight drop of pH is possible but will typically not impact reaction performance provided that the mix is still pink prior to amplification. Occasionally, after multiple uses and extended exposure to air, the pH may drop to below pH 7, and the mix will turn a light orange to yellow color before use. This color change provides a visual indication that the solution is no longer suitable for the colorimetric LAMP reaction. Thus it is recommended to avoid extended exposure to air by closing tube caps and storing the master mix in the vial it was provided in (or in similar vials with O-rings) at -20 °C. If the master mix is pipetted into strip tubes, PCR tubes or similar, we recommend using the material within the same day. Q: The WarmStart LAMP Master Mix has some precipitation in the tube after thawing, is this normal? A: Yes, precipitation of the LAMP Master Mixes after freezing/thawing is normal. It is important to resuspend the mix by thoroughly mixing or vortexing to dissolve all the precipitate material, but after suspension the Master Mixes can be used as normal. Q: What if my colorimetric LAMP mix is orange instead of pink prior to amplification? A: The Colorimetric LAMP Mixes are stable under standard handling and usage conditions. They contain a low concentration of Tris pH 8 and the pH-sensitive Phenol Red shows bright pink color near this pH. After repeated usage of the same tube, a slight drop of pH is possible but will typically not impact reaction performance provided that the mix is still pink prior to amplification. Occasionally, after multiple uses and extended exposure to air, the pH may drop to below pH 7, and the mix will turn a light orange to yellow color before use. This color change provides a visual indication that the solution is no longer suitable for the colorimetric LAMP reaction. Thus it is recommended to avoid extended exposure to air by closing tube caps and storing the master mix in the vial it was provided in (or in similar vials with O-rings) at -20 °C. If the master mix is pipetted into strip tubes, PCR tubes or similar, we recommend using the material within the same day. Q: Does the WarmStart® Colorimetric LAMP Master Mix with UDG enable carryover prevention/contamination reduction? A: Yes, WarmStart® Colorimetric LAMP Master Mix with UDG is formulated with a mixture of dTTP and dUTP. This ensures both efficient isothermal amplification as well as the incorporation of dU into the reaction products. The mix also contains Antarctic Thermolabile UDG (NEB #M0372). LAMP products containing dU serve as a substrate for uracil DNA glycosylase, allowing carryover contamination prevention. Reactions should be set up at room temperature (typically 22-25°C) prior to isothermal incubation at 65°C to allow dU-containing amplicons to be degraded, or if desired, a 10 minute, 25°C incubation before LAMP can be added to the workflow. Because LAMP can generate large quantities of DNA in very short periods of time, best practices to reduce contamination involve not opening LAMP reactions post amplification. Q: Does pre-heating the thermal block impact LAMP amplification reactions? A: Yes, we have observed that placing LAMP reactions on a pre-heated thermal block at 65 °C (rather than allowing the samples to warm to 65°C as the block heats up) tends to specifically improve detection of low input samples. Q: What regions of the SARS-CoV-2 virus do the LAMP Primers target? How were these primers chosen? A: The SARS-CoV-2 LAMP Primer Mix is a mixture of two primer sets; one set targets the N gene and the second set targets the E gene. The primer sets perform well individually but mixing them improves detection sensitivity. Q: In the SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit, what is the target of the Internal Control Primer Mix (rActin)? A: The Internal Control LAMP Primer Mix targets actin RNA at an exon-exon junction. The primers also include a region that matches an ankyrin sequence in gDNA so low levels of amplification can be observed from mammalian gDNA but this is a minor contribution to overall signal given that rActin RNA is typically present at much higher copy numbers than gDNA. Q: Should I use Guanidine HCl (B2619A) in colorimetric LAMP reactions? A: We recommend colorimetric LAMP reactions contain a final concentration of 40 mM Guanidine HCl and accordingly provide it for addition to the LAMP reaction in the standard SARS-CoV-2 workflow. However, some upstream sample prep workflows may introduce a source of guanidine into the sample which can be carried into the LAMP reaction. If guanidine will be carried into the reaction with the input material, we recommend not exceeding 60 mM in the final reaction and replacing the volume allotted for guanidine HCl with nuclease-free water. Guanidine enhances isothermal amplification, yielding faster detection of SARS-CoV-2 RNA. Colorimetric LAMP was carried out using purified positive samples (10 ng/ul human total RNA + synthetic SARS-CoV-2 RNA at 10,000 copies per reaction). E1 or N2 primer sets from the SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit were used as indicated, and amplification was monitored in real time using a fluorescent dye. For both primer sets, addition of guanidine HCl to a final concentration of 40-60 mM enhanced amplification, resulting in earlier detection and enhanced color change. Q: How are colorimetric LAMP results interpreted in the SARS-CoV-2 Kit? A: Colorimetric LAMP reactions will turn from pink to yellow if amplification occurs. The Internal Control (rActin) and SARS-CoV-2 Positive Control reactions must be yellow and the NTC must be pink for test results to be accurate. Refer to the product manual for more in depth guidelines on interpreting results. Q: After amplification my samples turned orange rather than yellow. Is this acceptable? How do I interpret the results? A: An orange color following incubation at 65°C for 30 minutes suggests amplification occurred but the pH did not decrease sufficiently to turn the reaction yellow. This may be due to inhibition from the input sample, buffering from the input sample or indicates the sample contains a low amount of target nucleic acid. It may be interpreted as positive if all samples were pink prior to amplification and post amplification, both the IC and PC reactions are yellow/orange and the NTC is pink. Q: Can I incubate my colorimetric LAMP Sars-CoV-2 samples longer than 30 minutes at 65°C? How long should I incubate the reaction before checking the results? A: Our recommended incubation time for LAMP reactions is 30 min. With this time, most LAMP reactions containing target nucleic acid will be complete with no amplification in the non-template control. For high copy number samples, incubation times as short as 15 minutes may result in detection. Low input or inhibitor-containing samples may require extended incubation times at 65°C to visualize detection though this may increase the likelihood of false positive results. We typically do not recommend incubation times longer than 40 minutes for this workflow. Q: My NTC sample(s) turned orange/yellow following incubation at 65°C. What happened? A: If the non-template control turns orange or yellow following incubation, the assay results are invalid and may indicate cross-contamination during reaction set up, potentially from the SARS-CoV-2 Positive Control. If the NTC reaction changes color in repeat testing, replace all reagent stocks and clean workspace with an acceptable surface decontaminate such as 10% bleach (1:10 dilution of commercial 5.25-6.0% sodium hypochlorite). Q: My colorimetric LAMP SARS-CoV-2 Positive Control failed to turn yellow following incubation at 65°C. What happened? A: The positive control reaction should always turn yellow following incubation at 65°C. Failure to change color indicates a reaction setup error. Repeat assay setup. If this continues to happen, please reach out to NEB Tech Support at info@neb.com or through your local NEB contact. Q: What is the SARS-CoV-2 Positive Control included in the kit? A: The SARS-CoV-2 Positive Control (N2117) is a plasmid that contains the SARS-CoV-2 N-gene (GenBank: MN908947.3). The concentration at 12.5X is approximately 1,000,000 copies/uL and each lot is assessed by qPCR. Please note that we do not recommend use of the Positive Control concentration as an absolute copy number for quantitative experiments. Q: How much sample can be added to the colorimetric LAMP reaction as input? A: For best results, we recommend 2 µl of extracted nucleic acid in a 25 µl reaction. Larger or smaller volumes of samples may be tolerated. Ensure the nucleic acid is eluted or prepared in water to avoid carrying over excess buffer to the reaction. Material eluted in TE or similar elution buffer should be kept to less than 5 µl (20% v/v) of the final reaction volume. Samples in transport media (VTM or UTM) should be kept to less than 2 µl (8% v/v). To increase sample volume, use 0.1X Elution Buffer or water for preparation of the nucleic acid. Q: What types of samples/materials are compatible with the SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit? A: For best results, we recommend using extracted nucleic acid. Because colorimetric LAMP reactions change color based on a pH change during amplification, the nucleic acid should be eluted or prepared in water to avoid carrying over excess buffer into the reaction. Material eluted in TE or similar elution buffer should be kept to less than 5 μl (20% v/v) of the final reaction volume. Up to 1 µl (4% v/v) of transport media (UTM or VTM) may be used without impacting the colorimetric reaction. To increase sample volume, use 0.1X Elution Buffer or water for preparation of the nucleic acid. Note that SDS is typically not tolerated well in colorimetric LAMP and guanidine compounds (hydrochloride, isothiocyanate) should be kept to less than 60 mM in the final reaction. Simple and/or crude sample prep methods are being developed in an ongoing basis and some of these are also compatible with colorimetric LAMP. MedRXiv and BioRXiv are good resources for the most up-to-date options regarding sample prep. A selection of preprints describing different sample types and their utility with colorimetric LAMP can be found on the Isothermal Amplification, RT-qPCR, and COVID-19 page. Q: My SARS-CoV-2 sample or Internal Control reaction turned orange/yellow upon sample addition. Does that mean that the sample contains nucleic acid? A: A color change to yellow or orange upon the addition of sample nucleic acid for the internal control or SARS-CoV-2 test sample reactions indicates that the input material is incompatible with the assay and the results are invalid. It should not be interpreted to signify the presence of target nucleic acid. Repeat with a lower sample volume or adjust the sample input pH to ~ 8. If the NTC or PC reactions immediately turn yellow or orange upon setup, repeat assay setup. Q: What instruments are compatible with the SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit? A: Colorimetric LAMP is compatible with a variety of instruments that provide heating to 65°C, including standard thermocylers, qPCR instruments, plate heaters and heat blocks. Hot water or non-instrumented approaches can be used if desired. Performance of the SARS-CoV-2 colorimetric LAMP Kit has been evaluated on the following instruments and performs well under standard conditions unless otherwise noted: ABI 2720 ABI Veritti ABI MiniAmp ABI SimpliAmp ABI 7500 ABI StepOnePlus ABI 7500 ABI QuantStudio 6 Flex Bio-Rad T100 Bio-Rad CFX Connect Bio-Rad CFX96 Bio-Rad Tetrad 2 Bio-Rad C1000 Bio-Rad S1000 Douglas/Agdia Amplifire Axxin T8 Iso (follow recommendations for sample mixing) MiniPCR: Mini16 VWR Digital Heat Block Eppendorf ThermoMixer C Q: Can I monitor the color change of the colorimetric LAMP reactions using a spectrophotometer? A: Yes. The reaction can be monitored in real time or at end point as part of a high-throughput workflow by measuring the absorbance ratio of 432 nm/560 nm. Q: Can the WarmStart Colorimetric LAMP 2X Master Mixes be used in instruments that utilize fluorescence detection? A: The colorimetric master mix is compatible with fluorescent detection methods when supplemented with a DNA intercalating fluorescent dyes (SYTO 9 or similar) and it gives reaction speeds similar to conventional LAMP. However, we recommend using our standard LAMP products (NEB #E1700, E1708) when the primary readout is fluorescence.