tetano
Editor, Senior Moderator
J Clin Microbiol
. 2021 May 26;JCM0085921.
doi: 10.1128/JCM.00859-21. Online ahead of print.
Multiplex SARS-CoV-2 Genotyping RT-PCR for Population-Level Variant Screening and Epidemiologic Surveillance
Hannah Wang[SUP] 1 [/SUP], Jacob A Miller[SUP] 2 [/SUP], Michelle Verghese[SUP] 1 [/SUP], Mamdouh Sibai[SUP] 3 [/SUP], Daniel Solis[SUP] 1 [/SUP], Kenji O Mfuh[SUP] 3 [/SUP], Becky Jiang[SUP] 3 [/SUP], Naomi Iwai[SUP] 3 [/SUP], Marilyn Mar[SUP] 3 [/SUP], ChunHong Huang[SUP] 1 [/SUP], Fumiko Yamamoto[SUP] 1 [/SUP], Malaya K Sahoo[SUP] 1 [/SUP], James Zehnder[SUP] 1 [/SUP], Benjamin A Pinsky[SUP] 1 3 4 [/SUP]
Affiliations
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.
. 2021 May 26;JCM0085921.
doi: 10.1128/JCM.00859-21. Online ahead of print.
Multiplex SARS-CoV-2 Genotyping RT-PCR for Population-Level Variant Screening and Epidemiologic Surveillance
Hannah Wang[SUP] 1 [/SUP], Jacob A Miller[SUP] 2 [/SUP], Michelle Verghese[SUP] 1 [/SUP], Mamdouh Sibai[SUP] 3 [/SUP], Daniel Solis[SUP] 1 [/SUP], Kenji O Mfuh[SUP] 3 [/SUP], Becky Jiang[SUP] 3 [/SUP], Naomi Iwai[SUP] 3 [/SUP], Marilyn Mar[SUP] 3 [/SUP], ChunHong Huang[SUP] 1 [/SUP], Fumiko Yamamoto[SUP] 1 [/SUP], Malaya K Sahoo[SUP] 1 [/SUP], James Zehnder[SUP] 1 [/SUP], Benjamin A Pinsky[SUP] 1 3 4 [/SUP]
Affiliations
- PMID: 34037430
- DOI: 10.1128/JCM.00859-21
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.