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Single-Amplicon Multiplex Real-Time Reverse Transcription-PCR with Tiled Probes To Detect SARS-CoV-2 spike Mutations Associated with Variants of Concern
- Source :
- Journal of Clinical Microbiology
- Publication Year :
- 2021
- Publisher :
- American Society for Microbiology, 2021.
-
Abstract
- 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 (∼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 (CT) values of
- Subjects :
- Microbiology (medical)
variants
Mutation
SARS-CoV-2
COVID-19
Reverse Transcription
Amplicon
Biology
Real-Time Polymerase Chain Reaction
medicine.disease_cause
molecular epidemiology
Molecular biology
DNA sequencing
Reverse transcriptase
Real-time polymerase chain reaction
Virology
diagnostics
medicine
Humans
RNA, Viral
SNP
Multiplex
Primer (molecular biology)
Subjects
Details
- ISSN :
- 1098660X and 00951137
- Volume :
- 59
- Database :
- OpenAIRE
- Journal :
- Journal of Clinical Microbiology
- Accession number :
- edsair.doi.dedup.....d53fdeac64a0baf1bbbd9195ce57ef4e