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

Multiplex assays designed to detect multiple sequences from an organism are often employed to improve the accuracy of identification. 22, 23 CDC recommended diagnostic protocol for SARS-CoV-2 includes RT-qPCR amplification of at least two different regions of the viral genome. In fact, a recent pre-publication demonstrated a multiplex PCR approach to enhance efficiency of detecting SARS-CoV-2 at low copy numbers. 24 Having determined that the individual LAMP-OSD assays with NB, Tholoth, and Lamb primers could signal the presence of SARS-CoV-2 RNA, we sought to execute these assays in a multiplexed format to create an internally redundant assay for SARS-CoV-2. We chose to multiplex the NB and the Tholoth assays because they target different viral genes: the N gene and the ORF1AB gene, respectively. We first tested the ability of both primer sets to amplify their respective synthetic targets (in vitro RNA transcripts of N gene and ORF1AB gBlock DNA templates) in a multiplex assay format by assembling LAMP-OSD reactions containing equimolar amounts of both Tholoth and NB LAMP primer sets with either only one or both OSD probes.