Development of a panel of three multiplex allele-specific qRT-PCR assays for quick differentiation of recombinant variants and Omicron subvariants of SARS-CoV-2

Abstract

Quick differentiation of the circulating variants and the emerging recombinant variants of SARS-CoV-2 is essential to monitor their transmission. However, the widely used gene sequencing method is time-consuming and costly when facing the viral recombinant variants, because partial or whole genome sequencing is required. Allele-specific real time RT-PCR (qRT-PCR) represents a quick and cost-effective method in SNP genotyping and has been successfully applied for SARS-CoV-2 variant screening. In the present study, we developed a panel of 3 multiplex allele-specific qRT-PCR assays targeting 12 key differential mutations for quick differentiation of SARS-CoV-2 recombinant variants (XD and XE) and Omicron subvariants (BA.1 and BA.2). Two parallel multiplex qRT-PCR reactions were designed to separately target the protype allele and the mutated allele of the four mutations in each allele-specific qRT-PCR assay. The variation of Cp values (ΔCp) between the two multiplex qRT-PCR reactions was applied for mutation determination. The developed multiplex allele-specific qRT-PCR assays exhibited outstanding analytical sensitivities (with limits of detection [LoDs] of 2.97-27.43 copies per reaction), wide linear detection ranges (107-100 copies per reaction), good amplification efficiencies (82% to 95%), good reproducibility (Coefficient of Variations (CVs) < 5% in both intra-assay and inter-assay tests) and clinical performances (99.5%-100% consistency with Sanger sequencing). The developed multiplex allele-specific qRT-PCR assays in this study provide an alternative tool for quick differentiation of SARS-CoV-2 recombinant variants (XD and XE) and Omicron subvariants (BA.1 and BA.2).

Efficient Tracing of the SARS-CoV-2 Omicron Variants in Santa Barbara County Using a Rapid Quantitative Reverse Transcription PCR Assay

“…Many groups around the world have responded to the emergence of these highly infectious variants by developing several assays to detect viral variants of concern [ 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ]. In this study, we collaborated with SBC public health officers and clinicians to rapidly develop and implement a simple and highly specific RT-qPCR assay to detect Omicron variants in residual diagnostic clinical samples.…”