Development of multiplex S-gene-targeted RT-PCR for rapid identification of SARS-CoV-2 variants by extended S-gene target failure

•Rapid screening assay of SARS-CoV-2 variants is required for clinical laboratories.•Four targets, 3 S-gene and 1N-gene regions were detected by multiplex RT-PCR.•The VOC were estimated by the extended S-gene target failure patterns.•The VOC results of eSGTF and S-gene sequencing had a high concordance rate.•Multiplex RT-PCR and eSGTF patterns enable high-throughput screening of VOCs. Tracking SARS-CoV-2 variants of concern (VOC) by genomic sequencing is time-consuming. The rapid screening of VOCs is necessary for clinical laboratories. In this study, we developed a rapid screening method based on multiplex RT-PCR by extended S-gene target failure (eSGTF), a false negative result caused by S-gene mutations. Three S-gene target (SGT) regions (SGT1, codons 65–72; SGT2, codons 152–159; and SGT3, codons 370–377) and an N-gene region (for internal control) were detected in single-tube. Four types of VOC (Alpha, Delta, Omicron BA.1, and Omicron BA.2) are classified by positive/negative patterns of 3 S-gene regions (eSGTF pattern). The eSGTF patterns of VOCs were as follows (SGT1, SGT2, SGT3; P, positive; N, negative): Alpha, NPP; Delta, PNP; Omicron BA.1, NPN pattern; and Omicron BA.2, PPN. As compared with the S-gene sequencing, eSGTF patterns were identical to the specific VOCs (concordance rate = 96.7%, N = 206/213). Seven samples with discordant results had a minor mutation in the probe binding region. The epidemics of VOCs estimated by eSGTF patterns were similar to those in Japan. Multiplex RT-PCR and eSGTF patterns enable high-throughput screening of VOCs. It will be useful for the rapid determination of VOCs in clinical laboratories.