Sensitive and visual detection of SARS-CoV-2 using polymerase spiral reaction assay

Abstract

Purpose: Testing of SARS-CoV-2 for large populations is crucial for diagnosis, epidemiology, and surveillance of COVID-19. Currently, reverse transcription (RT) qPCR (RT-qPCR) is being performed worldwide and considered as a gold standard. As an alternative nucleic acid amplification test (NAAT)-based method, RT- polymerase spiral reaction (RT-PSR) assay can be a rapid, robust, and cost-effective point-of-care detection of SARS-CoV-2. Methods & Materials: Inactivated SARS-CoV-2 virus samples were provided by the National Institute of Virology, India for this study. Specific primers targeting Nucleocapsid (N) gene, and RNA-dependent RNA polymerase (RdRp) genes of SARS-CoV-2 were designed for RT-PSR assay. Purified RNA was used for the standardisation of the RT-PSR using in vitro synthesised viral RNA. A simple visual detection of SARS-CoV-2 by the naked eye was optimised using hydroxy naphthol blue dye. The sensitivity of the assay was performed by diluting viral RNA at various concentrations. The limit of detection was estimated and tested with RT-PSR. Computational analysis was performed to determine the specificity of the primers by calculating the percentage of mismatch using various novel coronavirus sequences, other coronaviruses, and other related RNA virus sequences. Results: Temperature and time for RT-PSR assay were found to be optimum at 63°C and 60 min, respectively, for both the gene targets. RT-PSR assay amplified even at a concentration of 5 ng/µl and could detect ten copies and one copy of RNA targeting RdRp and N gene, respectively. RT-PSR amplified products were visually detected by the naked eye and further verified by agarose gel electrophoresis. Primers used for the RT-PSR assay showed zero percent mismatch with SARS-CoV-2 sequences and mismatch with other viruses. Conclusion: The RT-PSR assay developed in this study could be considered a good alternative to the RT-qPCR assays.