The SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit utilizes Loop-Mediated Isothermal Amplification (LAMP) to detect SARS-CoV-2 nucleic acid. The kit is available for research use only and includes WarmStart Colorimetric LAMP 2X Master Mix with UDG and a primer mix targeting the N and E regions of the viral genome. Controls are provided to verify assay performance, and include an internal control primer set and a positive control template. Guanidine hydrochloride has been found to increase the speed and sensitivity of the RT-LAMP reaction and is also included.
Figure 1: SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit Components
The SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit includes WarmStart Colorimetric LAMP 2X Master Mix with UDG and a primer mix targeting the N and E regions of the viral genome. Controls are provided to verify assay performance and include an internal control primer set and a positive control template. Guanidine hydrochloride has been found to increase the speed and sensitivity of the RT-LAMP reaction and is also included.
Figure 2: SARS-CoV-2 Genome and SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit Gene Targets
The SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit includes a dual primer mix that targets the N and E gene regions.
WarmStart Colorimetric LAMP 2X Master Mix with UDG is an optimized formulation of Bst 2.0 WarmStart DNA Polymerase and WarmStart RTx in a special low-buffer reaction solution containing a visible pH indicator for rapid and easy detection of LAMP and RT-LAMP reactions. The inclusion of thermolabile UDG and dUTP in the master mix reduces the possibility of carryover contamination between reactions.
This system provides a fast, clear visual detection of amplification based on the production of protons and the subsequent drop in pH that occurs from the extensive DNA polymerase activity in a LAMP reaction. The decrease in pH produces a change in solution color from pink to yellow. The total protocol time is 30 minutes and only requires a simple heating device that can reach 65°C.
A positive detection of the SARS-CoV-2 RNA sequence would be indicated by a yellow color (amplification occurred, protons released, pH-dependent color change from pink to yellow), while a negative result would be indicated by a pink color (no amplification occurred, no protons released, no color change).
Figure 3: SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit Workflow
In the SARS-CoV-2 Colorimetric LAMP Assay Kit, three control reactions are run with each test sample. All reactions should be pink prior to incubation. Using the provided protocol, a Non-Template Control (NTC) reaction will contain all materials of the test sample (master mix, primers, etc) except for the test input nucleic acid itself and serves as a measure of reaction contamination and primer-based mis-amplification. The NTC sample should not amplify and should stay pink throughout the experiment. The Positive Control (PC) will contain master mix, a plasmid that contains the SARS-CoV-2 N-gene (GenBank: MN908947.3) and primers that will amplify this sequence. Amplification should be observed in the PC and the sample should become yellow after incubation. The Internal Control (IC) will contain master mix, test input nucleic acid, and LAMP primers for rActin, an endogenous housekeeping gene. If reagents are active and samples have been handled appropriately, the IC should become yellow after incubation.
Figure 4: The SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit supports on-demand and high-throughput test workflows
The SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit can be used to support different workflows, each of which vary based on the number of recommended control reactions. For low throughput, on-demand testing, each test requires 4 reactions: 3 controls (a NTC, PC, and an IC) and the test sample. Using the kit in this setting would equate to 24 test samples per NEB #E2019S (96 reaction) kit. For high-throughput settings, where plates of samples are analyzed, only 1 NTC and 1 PC need to be run per plate (an IC is recommended per test sample). Using the kit in this setting equate to 47 test samples per NEB #E2019S (96 reaction) kit.
Figure 5: Rapid, simple, color-based detection of SARS-CoV-2 RNA
The SARS-CoV-2 Rapid Colorimetric LAMP Assay was carried out using the indicated controls and either positive sample (human total RNA synthetic SARS-CoV-2 RNA) or negative sample (human total RNA alone). Valid results for Non-Template Control (NTC, pink), Positive Control (PC, yellow) and Internal Control (IC, yellow) are shown. In the positive test sample, isothermal amplification of SARS-CoV-2 RNA leads to a color change from pink to yellow.
Figure 6: Robust Sensitivity and Specificity
The SARS-CoV-2 Rapid Colorimetric LAMP Assay was carried out using either positive samples (human total RNA synthetic SARS-CoV-2 RNA at 5,000, 500 or 50 copies per reaction), negative samples (human total RNA alone) or no template (NTCs) as indicated. A. All positive samples gave a positive result with robust color change from pink to yellow, including detection in all samples at a low input of 50 copies per reaction (n = 20). B. No color change was observed for negative samples (n = 32). C. Summary of results from analogous testing conducted by 3 users in 9 experiments total. At 50 copies per reaction, >95% (277/288) of reactions were positive (yellow). For negative samples, >99% (358/360) of reactions gave no color change (pink) and 2/360 reactions gave a partial color change (orange, as indicated by asterisk for Users 2 and 3).
LAMP has been used in novel workflows by scientists around the world to further COVID-19 research. To access application notes, selected preprints, and to learn more about NEB's COVID-19 Researcher Spotlight series, click here.
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