High-surety isothermal amplification and detection of SARS-CoV-2, including with crude enzymes

Sanchita Bhadra; Timothy E Riedel; Simren Lakhotia; Nicholas D Tran; Andrew D Ellington

Following 90 min incubation at 65 °C, visual observation of endpoint OSD fluorescence revealed that the Tholoth LAMP-OSD assay containing 1000 copies of SARS-CoV-2 genomic RNA was brighter compared to assays containing fewer or no SARS-CoV-2 genomic RNA (Figure 7) . Similarly, 6 primer Lamb LAMP-OSD assays containing as few as 100 SARS-CoV-2 genomes were bright compared to controls containing no viral genomes. In contrast, all tubes of NB LAMP-OSD assay demonstrated background fluorescence with no virus-specific increase in OSD signal. These results are consistent with previous reports from NEB, the manufacturer of Bst 2.0 (https://www.neb.com/products/m0374-bst-3-0-dna-polymerase#Product%20Information), that while Bst-LF cannot support all reverse transcription LAMP reactions, for the Tholoth and Lamb primer sets it is suitable for the detection of SARS-CoV-2.