Identifying SARS-CoV-2 Variants of Concern through saliva-based RT-qPCR by targeting recurrent mutation sites

Abstract

SARS-CoV-2 variants of concern (VOCs) continue to pose a public health threat which necessitates a real-time monitoring strategy to compliment whole genome sequencing. Thus, we investigated the efficacy of competitive probe RT-qPCR assays for six mutation sites identified in SARS-CoV-2 VOCs and, after validating the assays with synthetic RNA, performed these assays on positive saliva samples. When compared with whole genome sequence results, the SΔ69-70 and ORF1aΔ3675-3677 assays demonstrated 93.60% and 68.00% accuracy, respectively. The SNP assays (K417T, E484K, E484Q, L452R) demonstrated 99.20%, 96.40%, 99.60%, and 96.80% accuracies, respectively. Lastly, we screened 345 positive saliva samples from December 7-22, 2021 using Omicron-specific mutation assays and were able to quickly identify rapid spread of Omicron in Upstate South Carolina. Our workflow demonstrates a novel approach for low-cost, real-time population screening of VOCs.

Importance: SARS-CoV-2 variants of concern and their many sublineages can be characterized by mutations present within their genetic sequences. These mutations can provide selective advantages such as increased transmissibility and antibody evasion, which influences public health recommendations such as mask mandates, quarantine requirements, and treatment regimens. Our real-time RT-qPCR workflow allows for strain identification of SARS-CoV-2 positive saliva samples by targeting common mutation sites shared between VOCs and detecting single nucleotides present at the targeted location. This differential diagnostic system can quickly and effectively identify a wide array of SARS-CoV-2 strains, which can provide more informed public health surveillance strategies in the future.

Figure 1.

Allelic discrimination plots of deletion assays ORF1aΔ3675-3677 and SΔ69-70. Synthetic RNA controls from six SARS-CoV-2 type strains was amplified in triplicate at 4□10⁴ genome copies/assay via TaqPath RT-qPCR along with no template controls. The deletion probe from the ORF1aΔ3675-3677 assay produced low intensity fluorescence. Sequenced positive saliva samples (n=125) were loaded in duplicate to determine the detection range of the assay in saliva. Data were plotted by using the absolute fluorescence of each reporter dye probe.

Figure 2.

Allelic discrimination plots of SNP assays for Spike K417T, E484K, E484Q, L452R. Synthetic RNA controls from six SARS-CoV-2 type strains was amplified in triplicate at 4□10⁴ genome copies/assay via TaqPath RT-qPCR along with no template controls. Synthetic RNA strains that failed to amplify on K417T, E484K, E484Q, or L452R assays lacked both alleles. Sequenced positive saliva samples (n=125) were loaded in duplicate to determine the detection range of the assay in saliva. Data were plotted by using the absolute fluorescence of each reporter dye probe.

Figure 3.

Application and interpretation of differential VOC assays. 3A. VOC strain typing by mutation site. Each strain will produce a different combination of results from the six assays. Strains with an alternate allele at the mutation site will produce inconclusive results. 3B. Example strain typing workflow using minimal steps. Saliva samples that are determined positive by routine diagnostic testing are analyzed by various assays that produce differential results for each VOC.