Single-Amplicon Multiplex Real-Time Reverse Transcription-PCR with Tiled Probes To Detect SARS-CoV-2 <i>spike</i> Mutations Associated with Variants of Concern
Ahmed Babiker, Katherine Immergluck, Samuel D. Stampfer, Anuradha Rao, Leda Bassit, Maxwell Su, Vi Nguyen, Victoria Stittleburg, Jessica Ingersoll, Heath L. Bradley, Maud Mavigner, Nils Schoof, Colleen S. Kraft, Ann Chahroudi, Raymond F. Schinazi, Greg S. Martin, Anne Piantadosi, Wilbur A. Lam, Jesse J. Waggoner
Abstract
To provide an accessible and inexpensive method to surveil for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutations, we developed a multiplex real-time reverse transcription-PCR (rRT-PCR) assay, the Spike single-nucleotide polymorphism (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.