Journal
JOURNAL OF CLINICAL MICROBIOLOGY
Volume 59, Issue 12, Pages -Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/JCM.01446-21
Keywords
COVID-19; diagnostics; molecular epidemiology; variants
Categories
Funding
- National Institute of Biomedical Imaging and Bioengineering grant [U54 EB027690 02S1]
- National Institutes of Health [UL1 TR002378]
- Doris Duke Charitable Foundation Clinical Scientist Development Award [2019089]
- Centers for Disease Control and Prevention [75D30121C10084]
- Emory Woodruff Health Sciences Center COVID-19 CURE Award
- National Institute of Allergy and Infectious Diseases [T32AI074492]
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A new multiplex real-time reverse transcription-PCR assay was developed to accurately identify SARS-CoV-2 mutations in the spike receptor binding domain, and can be quickly modified to detect new mutations that emerge.
To provide an accessible and inexpensive method to surveil for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutations, we developed a multiplex real-time reverse transcription-PCR (rRT-PCR) assay, the Spike single-nucleotide polymorphism (SNP) assay, to detect specific mutations in the spike receptor binding domain. A single primer pair was designed to amplify a 348-bp region of spike, and probes were initially designed to detect K417, E484K, and N501Y. The assay was evaluated using characterized variant sample pools and residual nasopharyngeal samples. Variant calls were confirmed by SARS-CoV-2 genome sequencing in a subset of samples. Subsequently, a fourth probe was designed to detect L452R. The lower limit of 95% detection was 2.46 to 2.48 log10 genome equivalents (GE)/ml for the three initial targets (similar to 1 to 2 GE/reaction). Among 253 residual nasopharyngeal swabs with detectable SARS-CoV-2 RNA, the Spike SNP assay was positive in 238 (94.1%) samples. All 220 samples with threshold cycle (C-T) values of <30 for the SARS-CoV-2 N2 target were detected, whereas 18/33 samples with N2 C-T values of >= 30 were detected. Spike SNP results were confirmed by sequencing in 50/50 samples (100%). Addition of the 452R probe did not affect performance for the original targets. The Spike SNP assay accurately identifies SARS-CoV-2 mutations in the receptor binding domain, and it can be quickly modified to detect new mutations that emerge.
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