Rapid identification and tracking of emerging SARS-CoV-2 variants are critical for understanding the transmission dynamics and developing strategies for interrupting the transmission chain. Next-Generation Sequencing (NGS) is an exceptional tool for whole-genome analysis and deciphering new mutations. The technique has been instrumental in identifying the variants of concern (VOC) and tracking this pandemic. However, NGS is complex and expensive for large-scale adoption, and epidemiological monitoring with NGS alone could be unattainable in limited-resource settings. In this study, we explored the application of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) based detection of the variant identified by NGS. We analyzed a total of 78 deidentified samples that screened positive for SARS-CoV-2 from two timeframes, August 2020 and July 2021. All 78 samples were classified into WHO lineages by whole-genome sequencing and then compared with two commercially available RT-qPCR assays for spike protein mutation(s). The data showed good concordance between RT-qPCR and NGS analysis for specific SARS-CoV-2 lineages and characteristic mutations. RT-qPCR assays are quick and cost-effective and thus can be implemented in synergy with NGS for screening NGS-identified mutations of SARS-CoV-2 for clinical and epidemiological interest. Strategic use of NGS and RT-qPCR can offer several COVID-19 epidemiological advantages.


This study suggests that reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a quick and cost-effective alternative to next generation sequencing sequencing (NGS) for screening known mutations of SARS-CoV-2 for clinical and epidemiological interest, especially in developing countries where COVID-19 diagnostic centers are limited by regional sequencing laboratories for screening the mutations in the SARS-CoV-2 clinical samples. The findings in this study depict great potential for RT-qPCR to be an effective strategy offering several epidemiological advantages. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Date of publication

Summer 7-30-2022



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Publisher Citation

Carpenter RE, Tamrakar V, Chahar H, Vine T, Sharma R. Confirming Multiplex RT-qPCR Use in COVID-19 with Next-Generation Sequencing: Strategies for Epidemiological Advantage. Glob Health Epidemiol Genom. 2022 Jul 30;2022:2270965. doi: 10.1155/2022/2270965. PMID: 35950011; PMCID: PMC9339135.