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Multiplex SARS-CoV-2 Genotyping Reverse Transcriptase PCR for Population-Level Variant Screening and Epidemiologic
Hannah Wang1, Jacob A Miller2, Michelle Verghese1
1Department of Pathology, Stanford University School of Medicine, Stanford, California, USA.
Journal of Clinical Microbiology
|May 26, 2021
Summary
A new RT-qPCR assay enables high-throughput surveillance of SARS-CoV-2 variants by genotyping spike protein mutations. This method rapidly detected the emergence of the L452R variant in the San Francisco Bay Area.
Area of Science:
- Virology
- Molecular Biology
- Public Health Genomics
Background:
- Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants pose a significant public health threat.
- Genomic surveillance is crucial for pandemic response, but population-level sequencing is resource-intensive for many laboratories.
- Developing cost-effective, high-throughput methods for variant detection is essential.
Purpose of the Study:
- To develop and validate a nucleic acid amplification test (NAAT) for high-throughput genotyping of key SARS-CoV-2 spike protein mutations.
- To facilitate rapid variant surveillance in resource-limited settings.
- To monitor the prevalence and emergence of specific SARS-CoV-2 variants.
Main Methods:
- Designed and analytically validated a one-step multiplex allele-specific reverse transcriptase quantitative PCR (RT-qPCR) assay.
- Targeted three nonsynonymous spike protein mutations: L452R, E484K, and N501Y.
- Validated assay specificity using next-generation whole-genome sequencing and screened 4,049 SARS-CoV-2 positive specimens.
Main Results:
- The RT-qPCR assay demonstrated high throughput with a 2.8% failure rate.
- Detected high prevalence of L452R mutations (38.7% overall, increasing from 24.8% to 62.5% from Dec 2020 to Mar 2021).
- Achieved 100% concordance with whole-genome sequencing for a validation subset and identified multiple SARS-CoV-2 variants.
Conclusions:
- A validated genotyping RT-qPCR assay enables efficient, high-throughput screening of SARS-CoV-2 variants.
- This approach can be readily implemented in existing NAAT laboratories worldwide.
- The assay facilitates rapid detection of emerging variants, aiding public health response efforts.
