A Simple RT-PCR Melting temperature Assay to Rapidly Screen for Widely Circulating SARS-CoV-2 Variants.

Background: The emergence of more transmissible SARS-CoV-2 variants in the United Kingdom (B.1.1.7), South Africa (B1.351) and Brazil (P.1) requires a vigorous public health response, including real time strain surveillance on a global scale. Although new SARS-CoV-2 variants can be most accurately identified by genomic sequencing, this approach is time consuming and expensive. A simple and more rapid screen for the key SARS-CoV-2 mutations that define variant strains is needed. We developed a simple, rapid and high-throughput reverse-transcriptase PCR (RT-PCR) melting temperature assay that identifies the SARS-CoV-2 N501Y mutation, a key mutation which is present in all three known variant strains of concern.

Methods: RT-PCR primers and two sloppy molecular beacon (SMB) probes were designed to amplify and detect the SARS-CoV-2 N501Y (A23063T) mutation. One SMB was designed with a probe region that was complementary to the wild type sequence (WT) and a second SMB was designed with a probe region that was complementary to the mutant (MT) sequence. Each SMB was labeled with a different fluorophore, and the assay was performed in a single test well. After RT-PCR, WT versus MT SARS-CoV-2 was identified by a characteristic shift in the melting temperature (Tm) of each SMB. Assay optimization and testing was performed with RNA from SARS-CoV-2 USA WA1/2020 (WT) and SARS-CoV-2 hCoV-19/England/204820464/2020 (a B.1.17 variant). The assay was then validated using clinical samples.

Results: The limit of detection (LOD) of the assay for both the WT and the B1.1.7 variant was 4 genomic copies/reaction. The two SMBs produced Tm shifts that were 100% sensitive and 100% specific for identifying the N501Y mutation in cultured virus and in clinical samples as confirmed by Sanger sequencing.

Conclusion: We have developed a rapid screening test for the SARS-CoV-2 N501Y mutation, which is a characteristic of all three SARS-CoV-2 stains of global concern. This assay can be used to rapidly screen large numbers of patient samples for these variants, providing an early warning for the emergence and spread of these strains of concern.