In-house Reverse transcriptase polymerase chain reaction for detection of SARS-CoV2 with increased Sensitivity

Abstract

Background:With the increasing COVID-19 infection worldwide, economization of the existing RT-PCR based detection assay becomes the need of the hour. Methods: An assessment of optimal PCR conditions for simultaneous amplification for E, S and RdRp gene of SARS-CoV-2 has been made using both fast traditional and multiplex real time PCR using same primer sets. All variables of practical value were studied by amplifying known target-sequences from ten-fold dilutions of archived positive samples of COVID-19. Results: The designed primers for amplification of E, S and RdRp gene of SARS-Cov-2 in single tube Multiplex PCR amplifications have shown efficient amplification of the target region in 37 minutes using thermal cyclers and 169 minutes with HRM based Real time detection using SYBR green master mix, over a wide range of template concentration, and the results were in good concordance with the commercially available detection kits. Conclusion: This fast HRM based Real time multiplex PCR with SYBR green approach offers rapid and sensitive detection of SARS-CoV-2 in a cost effective manner apart from the added advantage of primer pair’s compatibility for use in Traditional multiplex PCR, which offers extended applicability of the assay protocol in resource limited setting.