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

Abstract

Background: The increased transmission of SARS-CoV-2 variants of concern (VOC) which originated in the United Kingdom (B.1.1.7), South Africa (B1.351), Brazil (P.1) and in United States (B.1.427/429) requires a vigorous public health response, including real time strain surveillance on a global scale. Although genome sequencing is the gold standard for identifying these VOCs, it is time consuming and expensive. Here, we describe a simple, rapid and high-throughput reverse-transcriptase PCR (RT-PCR) melting temperature (Tm) screening assay that identifies these three major VOCs.

Methods: RT-PCR primers and four sloppy molecular beacon (SMB) probes were designed to amplify and detect the SARS-CoV-2 N501Y (A23063T) and E484K (G23012A) mutations and their corresponding wild type sequences. After RT-PCR, the VOCs were identified by a characteristic Tm of each SMB. Assay optimization and testing was performed with RNA from SARS-CoV-2 USA WA1/2020 (WT), a B.1.17 and a B.1.351 variant strains. The assay was then validated using clinical samples.

Results: The limit of detection (LOD) for both the WT and variants was 4 and 10 genomic copies/reaction for the 501 and 484 codon assays, respectively. The assay was 100% sensitive and 100% specific for identifying the N501Y and E484K mutations in cultured virus and in clinical samples as confirmed by Sanger sequencing.

Conclusion: We have developed an RT-PCR melt screening test for the three major VOCs which can be used to rapidly screen large numbers of patient samples providing an early warning for the emergence of these variants and a simple way to track their spread.

Keywords: E484K; N501Y; SARS-CoV-2; Variants; melting temperature (Tm); screening test.

Fig. 1.. Analytical limit of detection and Tm values generated by the SMB-501 (A and B) and SMB-484 (C and D) assays tested against SARS-CoV-2 RNA.

A and C- SARS-CoV-2 wildtype (WT) RNA; B- B.1.1.7 mutant (MT) RNA and D- B.351 mutant (MT) RNA at the indicated number of genomic equivalents (GEs).

Fig. 2.. The effect of target concentration on the melt peak height generated by the SMB-501 (A and B) and SMB-484 (C and D) assays tested against SARS-CoV-2 RNA.

A and C- SARS-CoV-2 wildtype (WT) RNA; B- B.1.1.7 mutant (MT) RNA and D- B.351 mutant (MT) RNA at the indicated number of genomic equivalents (GEs).

Fig. 3.. Sloppy molecular beacon (SMB) Tm profile of positive clinical nasopharyngeal (NP) samples tested using the SMB-501 and SMB-484 Tm assay.

Tm signatures consisting of the Tm values for both the WT probe (blue) and MT probe (orange) are shown for the SMB-501 assay (A) and the SMB-484 assay (B). Ref WT indicates the Tm profile of the reference, WT SARS-CoV-2 strain and Ref MT indicates the TM profile of the reference MT SARS-CoV-2 B.1.1.7 strain (A) and B.351 (B). SMBP1 – SMBP46 indicate that Tm profiles of the 46 COVID positive clinical samples tested in this study with SMB-501 assay and the 26 COVID positive samples tested by SMB-484 assay. Error bars show +/− one standard deviation.

Fig.4.. Prevalence of N501Y variant strains among the tested sample set over time.

The proportion of N501Y and E484K variants is shown for samples obtained during the periods of October-November (n=9 tested with SMB-501/n=3 tested with SMB-484), January (n=16/n=12), February (n=15/n=9) and the first week of March (n=6/n=2). No samples from December were tested in our study.