Multiplex SARS-CoV-2 Genotyping Reverse Transcriptase PCR for Population-Level Variant Screening and Epidemiologic Surveillance

Abstract

Emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with concerning phenotypic mutations is of public health interest. Genomic surveillance is an important tool for pandemic response, but many laboratories do not have the resources to support population-level sequencing. We hypothesized that a nucleic acid amplification test (NAAT) to genotype mutations in the viral spike protein could facilitate high-throughput variant surveillance. We designed and analytically validated a one-step multiplex allele-specific reverse transcriptase polymerase chain reaction (RT-qPCR) to detect three non-synonymous spike protein mutations (L452R, E484K, N501Y). Assay specificity was validated with next-generation whole-genome sequencing. We then screened a large cohort of SARS-CoV-2 positive specimens from our San Francisco Bay Area population. Between December 1, 2020 and March 1, 2021, we screened 4,049 unique infections by genotyping RT-qPCR, with an assay failure rate of 2.8%. We detected 1,567 L452R mutations (38.7%), 34 N501Y mutations (0.84%), 22 E484K mutations (0.54%), and 3 (0.07%) E484K+N501Y mutations. The assay had perfect (100%) concordance with whole-genome sequencing in a validation subset of 229 specimens, and detected B.1.1.7, B.1.351, B.1.427, B.1.429, B.1.526, and P.2 variants, among others. The assay revealed rapid emergence of L452R in our population, with a prevalence of 24.8% in December 2020 that increased to 62.5% in March 2021. We developed and clinically implemented a genotyping RT-qPCR to conduct high-throughput SARS-CoV-2 variant screening. This approach can be adapted for emerging mutations and immediately implemented in laboratories already performing NAAT worldwide using existing equipment, personnel, and extracted nucleic acid.

Precision Response to the Rise of the SARS-CoV-2 B.1.1.7 Variant of Concern by Combining Novel PCR Assays and Genome Sequencing for Rapid Variant Detection and Surveillance

“…Other groups have also implemented rRT-PCR assays to detect mutations associated with VOCs. Wang et al describe the design and use of assays targeting N501Y, E484K, and L452R, which are all found in different VOCs; this approach allowed them to detect the rise of SARS-CoV-2 strains carrying L452R in the San Francisco Bay Area ( 25 ). Very similar to the strategy implemented in our study, another group used commercially available assays to test for the H69/V70 deletion and N501Y in 35,208 SARS-CoV-2-positive samples in France to detect the rise of B.1.1.7 there ( 26 ).…”

RT-qPCR assays for SARS-CoV-2 variants of concern in wastewater reveals compromised vaccination-induced immunity

“…High transmission rates and lower vaccine efficacy towards these variants ( Bernal et al, 2021 ; Hoffmann et al, 2021 ; Z. Wang et al, 2021 ) resulted in a greater threat of infectivity and morbidity compared to the original SARS-CoV-2 virus. Therefore, quick detection and quantification of variants of concern, are vital for initiating response and for proper policy-making for pandemic containment.…”

“…Such an assay can help assess a variant's frequency within a population and contribute to policy guidelines. Indeed a number of publications targeting variants of concern detection using RT-qPCR ( Barreto et al, 2020 ; Bivins et al, 2021 ; Gand et al, 2021 ; Vega-Magaña et al, 2021 ; Vogels et al, 2021 ; H. Wang et al, 2021 ; Yaniv et al, 2021 ) as well as available commercial kits (e.g., TaqPath ThermoScientific, GT molecular, PerkinElmer) have emerged for individual assessments.…”

SARS-CoV-2 variants of concern, Gamma (P.1) and Delta (B.1.617), sensitive detection and quantification in wastewater employing direct RT-qPCR

“…Such an assay can help assess a variant's frequency within the population and contribute to policy guidelines. Indeed a number of publications targeting variants of concern detection using RT-qPCR [13][14][15][16][17][18] as well as available commercial kits (TaqPath ThermoScientific, GT molecular, PerkinElmer, etc. ), have emerged.…”