PMC:7574920 / 9553-11121 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":"To 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. 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