Evaluation of reverse transcriptase-polymerase spiral reaction assay for rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2.

BACKGROUND AND AIM: Existing real-time reverse transcriptase PCR (RT-qPCR) has certain limitations for the point-of-care detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since it requires sophisticated instruments, reagents and skilled laboratory personnel. In this study, we evaluated an assay termed the reverse transcriptase-polymerase spiral reaction (RT-PSR) for rapid and visual detection of SARS-CoV-2. METHODS: The RT-PSR assay was optimized using RdRp gene and evaluated for the detection of SARS-CoV-2. The time of 60min and a temperature of 63°C was optimized for targeting the RNA-dependent RNA polymerase gene of SARS-CoV-2. The sensitivity of the assay was evaluated by diluting the in-vitro transcribed RNA, which amplifies as low as ten copies. RESULTS: The specific primers designed for this assay showed 100% specificity and did not react when tested with other lung infection-causing viruses and bacteria. The optimized assay was validated with 190 clinical samples in two phases, using automated RTPCR based TrueNat test, and the results were comparable. CONCLUSIONS: The RT-PSR assay can be considered for rapid and sensitive detection of SARS-CoV-2, particularly in resource-limited settings. To our knowledge, there is as yet no RT-PSR-based kit developed for SARS-CoV-2.

Deletion in the C-terminal region of the envelope glycoprotein in some of the Indian SARS-CoV-2 genome.

The envelope glycoprotein (E) is the smallest structural component of SARS-CoVs; plays an essential role in the viral replication starting from envelope formation to assembly. The in silico analysis of 2086 whole genome sequences from India performed in this study provides the first observation on the extensive deletion of amino acid residues in the C-terminal region of the envelope glycoprotein in 34 Indian SARS-CoV-2 genomes. These amino acid deletions map to the homopentameric interface and PDZ binding motif (PBM) present in the C-terminal region of E protein as well as immediately after the reverse primer binding region as per Charité protocol in 26 of these genomes, hence, their detection through RT-qPCR may not be hampered and therefore E gene-based RT-qPCR would still detect these isolates. Eight genomes from the State of Odisha had deletion even in the primer binding site. It is possible that the deletions in the C-terminal region of E protein of these genomes are a result of adapting to a newer geographical area and host. The information on the clinical status was available only for 9 out of 34 cases and these were asymptomatic. However, further studies are indispensable to understand the functional consequences of amino acid deletion in the C terminal region of SARS-CoV-2 envelope protein in the viral pathogenesis and host adaptation.