Single-amplicon, multiplex real-time RT-PCR with tiled probes to detect SARS-CoV-2
spike
mutations associated with variants of concern
To provide an accessible and inexpensive method to surveil for SARS-CoV-2 mutations, we developed a multiplex real-time RT-PCR (the Spike 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 log 10 GE/mL for the three initial targets (∼1-2 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 Ct values < 30 for the SARS-CoV-2 N2 target were detected, whereas 18/33 samples with N2 Ct values ≥ 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 receptor binding domain, and it can be quickly modified to detect new mutations that emerge.