Journal: Journal of Medical Virology

Article Title: A pyrosequencing protocol for rapid identification of SARS‐CoV‐2 variants

doi: 10.1002/jmv.27770

Figure Lengend Snippet: Schematic representation of the location of forward and reverse PCR primers (arrows) used to amplify four regions of the S gene of SARS‐CoV‐2. PCR 1 and PCR 2 target sequences corresponding to the N‐terminal domain (NTD) of the S protein and overlap by 11 bases. PCR 2 resides within the receptor‐binding domain (RBD), and PCR 4 overlies the junction between the S1 and S2 subunits (S1/S2). Common S gene mutations reported in SARS‐CoV‐2 variants, detectable by pyrosequencing the PCR products, using sequencing primers or forward PCR primers as listed in Table , are indicated in the respective boxes. Forty‐two mutations detected in specimens analyzed in the current study appear in bold type. Other rare, novel mutations may be detected within targeted sequences but are not listed.

Article Snippet: Several commercial SARS‐CoV‐2 genotyping assays are available, including the MassARRAY® SARS‐CoV‐2 Panel (Agena Biosciences), which uses RT‐PCR followed by single‐base extension of mutation‐specific probes and mass spectrometry, the TaqMan SARS‐CoV‐2 Mutation Panel (Thermo Fisher Scientific), which uses RT‐PCR and TaqMan SNP technology, the AllplexTM SARS‐CoV‐2 Variants I and II Assays (SeeGene), which use RT‐PCR and a Tagging Oligonucleotide Cleavage and Extension (TOCETM) technology, and CoviDetectTM Variants and CoviDetectTM 4‐plex (PentaBase), which uses real‐time RT‐PCR and DNA melt curve analysis.

Techniques: Binding Assay, Sequencing