Bridging the Sequencing Gap: N501Y SNP RT-qPCR Assay Detects First SARS-CoV-2 Beta Variant in the Philippines

Whole genome sequencing (WGS) is used extensively in identifying SARS-CoV-2 variants. However, this method requires stringent sample acceptance criteria, long turn-around time (TAT), expensive processing and maintenance costs, and highly skilled staff. Although sequencing offers comprehensive pathogen insights, a cost-effective tool with faster TAT is beneficial in detecting SARS-CoV-2 variants of concern (VOCs). Here, we used a single nucleotide polymorphism (SNP) RT-qPCR assay to detect the N501Y mutation in banked SARS-CoV-2 RNA extracts (N=452) collected from December 2020 to April 2021. Of the SARS-CoV-2 positives (n=367), 22% carried the N501Y mutation and were classified as probable VOCs. This includes a sample that was confirmed to belong to the Beta lineage and was collected earlier than the first reported Beta variant in the country suggesting an earlier emergence of the variant. Validation experiments for the SNP RT-qPCR assay showed a limit of detection (LOD) of 3.01 copies/μL for both N501 and 501Y targets. A 99.35% concordance with partial S gene Sanger sequencing was observed confirming the presence of the N501Y SNP in 83 samples. In conclusion, the optimized SNP RT-qPCR assay serves as an important complementary or alternative tool for detecting probable SARS-CoV-2 variants, ensuring that samples ineligible for WGS are not overlooked. This effectively resolves sequencing gaps, including stringent sample acceptance criteria, extended TAT, and rigorous data analysis. Therefore, embracing this technology provides a rapid, economical, and dependable solution for managing pathogens of public health concern.