One-pot Detection of COVID-19 with Real-time Reverse-transcription Loop-mediated Isothermal Amplification (RT-LAMP) Assay and Visual RT-LAMP Assay

Deguo Wang

The one-pot real-time RT-LAMP assay and one-pot visual RT-LAMP assay for detection of COVID-19 were established in the study, and the detection limit was ≥ 6 copies. Although the specificity of the established RT-LAMP assays had been not determined, they were still considered to be highly specific for following reason. Upon alignment in DNA Data Bank of Japan, the target sequence of established RT-LAMP assays had 100% identities (233/233) with that of 35 COVID-19 strains, and had 88% identities (207/233) with that of 52 congeneric SARS Coronavirus strains, among which there were 12 bp on the RT-LAMP primers, it was reported that 3 bp primer-template mismatches extend the detection time from 21 min to 47 min 10 , therefore, the established RT-LAMP assays were theoretically highly specific. The Bst DNA/RNA Polymerase 3.0 (New England Biolabs, Inc., MA, USA) had been used in the study. The enzyme has faborable perforance of both amplification and reverse transcription activity, so it can use either DNA or RNA as template 11, 12 , no reverse transcriptase is needed, and the estalished assays can be directly used for detection of the RNA virus. The nucleic acid extraction had been omitted in the established one-pot real-time or visual RT-LAMP assays, which had greatly reduced the infection risk of the operators. Furthermore, it was recommended the visual RT-LAMP assay over the real-time RT-LAMP assay, one of four Negative Controls in above sensitivity determination of the real-time RT-LAMP assay had amplification, while all four Negative Controls of the visual RT-LAMP assay had no amplification, because the aerosol that may leak was washed by water in water bath, the false positive rate caused by aerosol of the real-time RT-LAMP assay was higher than that of the visual RT-LAMP assay which sunk the reaction tube in water.