Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Evidence-Based Evaluation of PCR Diagnostics for SARS-Cov-2 and The Omicron Variants by Sanger Sequencing

Version 1 : Received: 11 April 2022 / Approved: 12 April 2022 / Online: 12 April 2022 (03:57:29 CEST)

How to cite: Lee, S.H. Evidence-Based Evaluation of PCR Diagnostics for SARS-Cov-2 and The Omicron Variants by Sanger Sequencing. Preprints 2022, 2022040091 (doi: 10.20944/preprints202204.0091.v1). Lee, S.H. Evidence-Based Evaluation of PCR Diagnostics for SARS-Cov-2 and The Omicron Variants by Sanger Sequencing. Preprints 2022, 2022040091 (doi: 10.20944/preprints202204.0091.v1).

Abstract

Both SARS-CoV-2 and SARS-CoV 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 policy-makers. This article presented Sanger sequencing evidence to show that the RT-PCR diagnostic protocol established in 2003 for SARS-CoV can in fact detect SARS-CoV-2 accurately due to the well-known nonspecific PCR amplification of DNAs with similar 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 21 (42%) were false-positive. Although the other 29 positive isolates were categorized as Omicron variant by partial sequencing of the N gene, and the RBD and the NTD of the S gene, 9 (31%) showed focal to complete sequencing failure in the S gene segments due to multi-allelic SNPs. During the course of the study, an Omicron variant isolate containing a BA.1 NTD and a BA.2 RBD in its S gene was also detected. Routine partial S gene sequencing of all PCR-positive samples can timely discover multi-allelic SNPs and viral recombination in the circulating variants for investigation of their impacts on vaccine efficacies, therapeutics and diagnostics.

Keywords

SARS-CoV-2; SARS-CoV; RT-PCR; Sanger sequencing; RT-qPCR; receptor-binding domain (RBD); N-terminal domain (NTD); Omicron; multi-allelic SNPs; false-positive

Subject

MEDICINE & PHARMACOLOGY, General Medical Research

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