Highly sensitive and specific detection of the SARS-CoV-2 Delta variant by double-mismatch allele-specific PCR

Abstract

The highly transmissible Delta variant of SARS-CoV-2 (B.1.617.2), first identified in India, is currently replacing pre-existing variants in Europe, the USA, and many other parts of the world. It is essential to monitor efficiently its spread to help guide public health policies. Genome sequencing is the gold standard for identification of Delta, but is time-consuming, expensive, and unavailable in many regions. We describe here a rapid and relatively inexpensive alternative to sequencing for specific identification of the Delta variant, by application of double-mismatch allele-specific RT-PCR (DMAS-RT-PCR). The technique exploits forward and reverse allele-specific primers, targeting two spike gene mutations, L452R and T478K, within the same amplicon. The discriminatory power of each primer is enhanced by the presence of an additional mismatch located at the fourth nucleotide from the 3 end. Amplicons are detected in real-time by means of a conventional fluorescently-labelled hydrolysis probe. Specificity was assessed by testing a range of well characterised cell culture-derived viral isolates and clinical samples, most of which had previously been fully sequenced. In all cases the results of viral genotyping by DMAS-RT-PCR were entirely concordant with the results of sequencing, and the assay was shown to discriminate reliably between the Delta variant and other variants of concern (Alpha, B.1.1.7 and Beta, B.1.351), and wild-type SARS-CoV-2. Other respiratory viruses, including influenza A and respiratory syncytial virus, were non-reactive in the assay. The sensitivity of DMAS-RT-PCR matched that of the diagnostic SARS-CoV-2 RT-qPCR screening assay, which targets the E gene. Several samples that could not be sequenced due to insufficient virus could successfully be genotyped by DMAS-RT-PCR. The method we describe would be simple to establish in any laboratory that has the ability to conduct PCR assays and should greatly facilitate monitoring of the spread of the Delta variant throughout the world, and its proportional representation in any SARS-CoV-2-infected population.