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

When these multiplex assays were seeded with both types of target templates, both Tholoth and NB primer sets led to an increase in OSD fluorescence, measured both in real-time and visually observed at endpoint (Figure 4) . Multiplex assays containing both OSD probes demonstrated an additive effect, with OSD signal being brighter than assays containing only one type of OSD. Having confirmed that both the NB and Tholoth primer sets are able to amplify their respective targets in one-pot individual and multiplex reactions containing N gene and ORF1AB sequences (synthetic or viral, Figures 3, 4) , we tested the multiplex assay containing both Tholoth and NB primers and OSD probes using full length SARS-CoV-2 viral genomic RNA (Figure 5) . Visual observation of endpoint fluorescence revealed a bright signal in multiplex assays containing as few as 10 copies of genomic RNA while reactions containing non-specific human DNA remained dark ( Figure 5 ). Compared to individual LAMP-OSD assays of SARS-CoV-2 viral genomic RNA (Figure 3) , it seems that multiplex LAMP-OSD assay is detecting fewer genomic RNA copies ( Figure 5 ). For instance, while NB and Tholoth LAMP-OSD assays could routinely detect 100 and 1000 genomic RNA copies, respectively, the multiplex LAMP-OSD assay could signal the presence of as few as 10 genomic RNA copies. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.13.039941 doi: bioRxiv preprint in each reaction are indicated below the respective tube. Control reaction received 23 ng of human genomic DNA. Image of endpoint OSD fluorescence is depicted.