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 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.

Highly Sensitive Lineage Discrimination of SARS-CoV-2 Variants through Allele-Specific Probe PCR

“…A potential weakness is that the method does require at least two independent AS-PCRs to perform lineage identification for individual samples, although an approach using pooled samples, as described in the study, could be used for local lineage prevalence estimation. Babiker et al constructed a multiplex ASP-PCR based on SNP identification from dropout of signal for probes targeting S:K417 and the S:E484K and S:N501Y mutations, although these are uninformative for variant identification (Table S3) ( 25 ). The Vogels et al multiplex RT-qPCR method discriminates Alpha, Beta, Gamma, and others by targeting the deletions ORF1a:3675–3677del and S:69/70del ( 26 ).…”

Highly-Sensitive Lineage Discrimination of SARS-CoV-2 Variants through Allele-Specific Probe Polymerase Chain Reaction

“…Babiker, et al (2021) constructed a multiplex, single reaction ASP-PCR based on SNP identification from dropout of signal for probes targeting S:K417 and the S:E484K and S:N501Y mutations, although these are likely uninformative for variant identification (see Supplementary Table 3). 20 The Vogels et al (2021) multiplex RT-qPCR method discriminates Alpha, Beta, Gamma, and other by targeting the deletions ORF1a:3675-3677del and S:69/70del. 21 Lastly, Harper et al (2021) designed a genotyping panel for identifying 19 SNPS based off of PACE chemistry and allele-specific forward primers.…”

Advances in the Rapid Diagnostic of Viral Respiratory Tract Infections

“…These methods, known as nucleic acid amplification techniques (NAAT), inactivate the virions during preliminary purification steps and can simultaneously detect multiple viruses (Lu et al, 2021). However, the presence of nucleic acid does not always mean active infection (Babiker et al, 2021), and significant issues stem during results interpretation: a weak signal explaining either the end of an infection or a recent evolving one and undetectable virus in the upper respiratory tract samples in the respiratory tract infections (Crozier et al, 2021). Furthermore, since respiratory viruses are prone to antigenic drift due to genetic point mutations and reassortment, it is fundamental to identify these changes within the viral genomes.…”