Colorimetric reverse transcriptional loop-mediated isothermal amplification for rapid detection of SARS-CoV-2

Abstract

Purpose: With the increasing incidence of a novel coronavirus SARS-CoV-2 causing COVID-19 cases, accurate and early detection infection is need of the hour for effective prevention and management. Therefore, the aim of this study was to develop a colorimetric reverse transcriptional loop-mediated isothermal amplification for rapid detection of SARS-CoV-2. Methods & Materials: Inactivated SARS-CoV-2 virus samples were procured from the National Institute of Virology, Pune, India. Various genes were targeted for primer design, such as nucleocapsid, spike, RNA dependent RNA polymerase, and envelop genes region of SARS-CoV-2. In-vitro synthesised viral RNA was used for the standardisation of the RT-LAMP. RT-LAMP products were visualised by the naked eye using hydroxy naphthol blue dye. The sensitivity of RT-LAMP assay was performed by diluting in-vitro synthesised viral RNA at a different concentration such as 5 ng/µl, 25 ng/µl, 50 ng/µl, 200 ng/µl. Additionally, the RNA copy number was estimated and tested with RT-LAMP. In-silico analysis was carried to calculating the percentage of mismatch using various viral sequences, including SARS-CoV-2, other coronaviruses, and other related RNA virus sequences available at GenBank. Results: RT-LAMP assay was standardised using in-vitro synthesised viral RNA. Temperature and time standardisation revealed, all the targets i.e., E, S, N, and RdRp gene regions had an optimum temperature of 63°C and time, 60 min. The sensitivity of all the target genes were ten copies of viral RNA. RT-LAMP amplified products were visualised by the naked eye using hydroxy naphthol blue dye and verified by agarose gel electrophoresis. All the primers used for the RT-LAMP assay showed a zero percent mismatch with SARS-CoV-2 sequences available at GenBank. Conclusion: Colorimetric reverse transcriptional loop-mediated isothermal amplification assay developed in this study could provide a visual and faster alternative to the RT-qPCR assays.