Sin Hang Lee

Both SARS-CoV-2 and SARS-CoV-1 initially appeared in China and spread to other parts of the world. SARS-CoV-2 has generated a COVID-19 pandemic causing more than 6 million human deaths worldwide, while the SARS outbreak quickly ended in six months with a global total of 774 reported deaths. One of the factors contributing to this stunning difference in the outcome between these two outbreaks is the inaccuracy of the RT-qPCR tests for SARS-CoV-2, which generated a large number of false-negative and false-positive test results that have misled patient management and public health policymakers.

This article presents Sanger sequencing evidence to show that the RT-PCR diagnostic protocol established in 2003 for SARS-CoV-1 can in fact detect SARS-CoV-2 accurately due to the well-known ability of the PCR to amplify similar, homeologous sequences. Using nested RT-PCR followed by Sanger sequencing to retest 50 patient samples collected in January 2022 and sold as RT- qPCR positive reference confirmed that 21 (42%) were false-positive. Routine sequencing of the RT- PCR amplicons of the receptor-binding domain (RBD) and N-terminal domain (NTD) of the Spike protein (S) gene is a tool to avoid false positives and to study the effects of amino acid mutations and multi-allelic single nucleotide polymorphisms (SNPs) in the circulating variants for investigation of their impacts on vaccine efficacies, therapeutics and diagnostics.