Design of SARS-CoV-2 Variant-Specific PCR Assays Considering Regional and Temporal Characteristics

Monitoring the prevalence of SARS-CoV-2 variants is necessary to make informed public health decisions during the COVID-19 pandemic. PCR assays have received global attention, facilitating a rapid understanding of variant dynamics because they are more accessible and scalable than genome sequencing. However, as PCR assays target only a few mutations, their accuracy could be reduced when these mutations are not exclusive to the target variants. Here we introduce PRIMES, an algorithm that evaluates the sensitivity and specificity of SARS-CoV-2 variant-specific PCR assays across different geographical regions by incorporating sequences deposited in the GISAID database. Using PRIMES, we determined that the accuracy of several PCR assays decreased when applied beyond the geographic scope of the study in which the assays were developed. Subsequently, we used this tool to design Alpha and Delta variant-specific PCR assays for samples from Illinois, USA. In silico analysis using PRIMES determined the sensitivity/specificity to be 0.99/0.99 for the Alpha variant-specific PCR assay and 0.98/1.00 for the Delta variant-specific PCR assay in Illinois, respectively. We applied these two variant-specific PCR assays to six local sewage samples and determined the dominant SARS-CoV-2 variant of either the wild type, the Alpha variant, or the Delta variant. Using next-generation sequencing (NGS) of the spike (S) gene amplicons of the Delta variant-dominant samples, we found six mutations exclusive to the Delta variant (S:T19R, S:D156/157, S:L452R, S:T478K, S:P681R, and S:D950N). The consistency between the variant-specific PCR assays and the NGS results supports the applicability of PRIMES.