A Simple Reverse Transcriptase PCR Melting-Temperature Assay To Rapidly Screen for Widely Circulating SARS-CoV-2 Variants

Abstract

The increased transmission of SARS-CoV-2 variants of concern (VOC), which originated in the United Kingdom (B.1.1.7/alpha), South Africa (B1.351/beta), Brazil (P.1/gamma), the United States (B.1.427/429 or epsilon), and India (B.1.617.2/delta), 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 (T_m) screening assay that identifies the first three major VOCs. 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 T_m of each SMB. Assay optimization and testing was performed with RNA from SARS-CoV-2 USA WA1/2020 (wild type [WT]), B.1.1.7, and B.1.351 variant strains. The assay was then validated using clinical samples. The limit of detection 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. We have developed an RT-PCR melt screening test for the major VOCs that 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; melting temperature; screening test; sloppy molecular beacon; variants.

FIG 1

Analytical limit of detection and T_m values generated by the SMB-501 (A and B) and SMB-484 (C and D) assays tested against SARS-CoV-2 RNA in the presence of the nasopharyngeal (NP) matrix. Shown are SARS-CoV-2 wild type (WT) RNA (A and C), B.1.1.7 mutant (MT) RNA (B), and B.351 mutant (MT) RNA (D) at the indicated number of genomic equivalents (GEs).

FIG 2

Sloppy molecular beacon (SMB) T_m profile of positive clinical nasopharyngeal (NP) samples tested using the SMB-501 and SMB-484 T_m assay. T_m signatures consisting of the T_m 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 T_m profile of the reference WT SARS-CoV-2 strain, and Ref MT indicates the T_m profile of the reference MT SARS-CoV-2 B.1.1.7 strain (A) and B.351 (B). SMBP1 to SMBP46 indicate that T_m 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 3

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 to 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.