PMC:7574920 / 9457-11869 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7574920","sourcedb":"PMC","sourceid":"7574920","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7574920","text":"Establishing colorimetric RT-LAMP assay sensitivity using an artificial SARS-CoV-2 RNA template\nTo detect SARS-CoV-2 RNA with RT-LAMP, we used the WarmStart Colorimetric RT-LAMP 2X Master Mix (DNA and RNA) from New England Biolabs. This mix contains two enzymes, an engineered reverse transcriptase (RTx) and a strand-displacing polymerase (Bst 2.0). In addition, the reaction mixture contains oligonucleotide-based aptamers that function as reversible temperature-dependent inhibitors, ensuring that the reaction only runs at an elevated temperature (WarmStart) to avoid nonspecific priming reactions. Several primer sets were recently proposed for RT-LAMP–based detection of SARS-CoV-2 RNA by Zhang et al. (11) and by Yu et al. (10), and these primer sets were subsequently validated with in vitro–translated RNA. We prepared and tested two primer sets for different RNA sections of the SARS-CoV-2 genome, the N-A set targeting the N gene and the 1a-A set targeting open reading frame (ORF) 1a (table S1) (11). Figure 1A shows that the oligonucleotide set for the N gene was capable of detecting 100 IVT RNA molecules in a test reaction with 1 μl of RNA solution, as evidenced by the red-to-yellow color change. The reaction was conducted for up to 1 hour at 65°C. For time points \u003e 30 to 35 min, the negative control frequently became yellowish (Fig. 1A). This was caused by spurious amplification products, which is a well-known problem with RT-LAMP (14). Analysis by gel electrophoresis revealed clearly distinct banding patterns for the correct RT-LAMP reaction products (lanes with ≥100 molecules IVT RNA input) and the spurious reaction products (Fig. 1B).\nFig. 1 Sensitivity of the RT-LAMP assay determined using IVT RNA.\n(A) Defined numbers of in vitro transcribed (IVT) RNA molecules of the SARS-CoV-2 N gene were added to the RT-LAMP reaction and incubated at 65°C. At indicated times, samples were removed from the heating block and cooled on ice to stop the reaction. Photographs were taken using the color scanner function of an office copy machine and show the red to yellow color change in positive samples. (B) The RT-LAMP reaction product (2.5 μl) was analyzed on a 2% agarose gel. The typical band pattern of a successful RT-LAMP reaction was visible in the samples with 100 or more SARS-CoV-2 RNA molecules, i.e., in those samples that showed a color change from red to yellow after 30 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