Understanding the Use of Real Time Reverse Transcriptase-Polymerase Chain Reaction (rRT-PCR) For Covid-19 Diagnosis

Abstract

Background: Adequate know ledge of real tim e Reverse Transcriptase -Polymerase Chain Re action (rRT-PCR) is critical for accurate implementation of the assay, interpretation of results and report ing. This mini-review describes the principles, procedures, and level of development of rRT-PCR assays for the control of the COVID-19 pandemic. Methods: A narrative review w as carried out to describe the principles of rRT -PCR, provide an update on the landscape of rRT-PCR protocols and elucidate the process control involved in preanalytical,analytical and post-analytical stages of COVID-19 testing . Review Findings: The rRT-PCR is currently considered to be the acceptable standard for confirming COVID-19 diagnosis based on SARS-CoV-2 RNA detection via conversion to cDNA and amplification of target genes in real time using sequence specific TaqMan® probes. Available evidence indicates that dif ferent rRT-PCR protocols varying in number and type of target genes within SARS-CoV-2 genome are currently available for validation and emergency use approval (EUA) in pandemic countries. A total of 1 – 3 target genes, comprising the ORF1a, ORF1b, RNA dependent RNA polymerase (RdRp), Nucleoplasid protein gene (N), Spike glycoprotein gene (S) and Envelope protein gene (E) are detected by these proto cols. Conclusion: rRT-PCR remains the most sensitive method for confirming, monitoring and managing COVID-19 disease in the ongoing pandemic in all affected countries. The need for validation of every rRT PCR protocol prior to deployment for COVID-19 testing and research into the development of alternative testing protocols are strongly recommended.