Single-Amplicon Multiplex Real-Time Reverse Transcription-PCR with Tiled Probes To Detect SARS-CoV-2 spike Mutations Associated with Variants of Concern

Abstract

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 log₁₀ 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.