Duplex RTqPCRs for detection and relative quantification of SARS-CoV-2 variants of concern (VOC)

Protocol Exchange
Rosa M PintóAlbert Bosch

Abstract

During the evolution of the SARS-CoV-2 pandemic, new variants of the virus have emerged and spread worldwide. The increased transmissibility and proclivity of some of these variants to cause more serious disease threatens public health responses against the virus, and they are classified as variants of concern (Variants of Concern, VOCs). While Next-Generation-Sequencing (NGS) is the gold standard to identify VOC, it cannot always be rapidly implemented in some settings to provide information as an early warning tool. Duplex quantitative real time RTqPCR assays offer a sensitive and easy-to-use tool to detect, discriminate, and estimate relative proportions of SARS-CoV-2 variants containing VOC-specific signature mutations from variants lacking it, using allelic discrimination probes. We developed three multiplexed RTqPCR assays that can detect Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2) VOCs by targeting 22281_22289DelCTTTACTTG, 28262_28263InsAACA and 22029_22034DelAGTTCA deletions/insertions in their genomes, respectively. Beta and Delta deletions are mapped to the S gene (residues 241/243 and 157/158, respectively), while Gamma insertion is located between the end of ORF8 and the beginning of N gene. Our specific duple...Continue Reading

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