Discrimination of SARS-CoV-2 omicron variant and its lineages by rapid detection of immune-escape mutations in spike protein RBD using asymmetric PCR-based melting curve analysis

作者：Xiaomu Kong, Peng Gao, Yongwei Jiang, Lixia Lu, Meimei Zhao, Yi Liu, Guo‐Xiong Deng, Haoyan Zhu, Yongtong Cao, Liang Ma · 发表于：Virology Journal · 年份：2023 · DOI：10.1186/s12985-023-02137-5 · 被引用次数：9 · 研究领域：SARS-CoV-2 and COVID-19 Research、SARS-CoV-2 detection and testing、Respiratory viral infections research

Abstract Background The SARS-CoV-2 Omicron strain has multiple immune-escape mutations in the spike protein receptor-binding domain (RBD). Rapid detection of these mutations to identify Omicron and its lineages is essential for guiding public health strategies and patient treatments. We developed a two-tube, four-color assay employing asymmetric polymerase chain reaction (PCR)-based melting curve analysis to detect Omicron mutations and discriminate the BA.1, BA.2, BA.4/5, and BA.2.75 lineages. Methods The presented technique involves combinatory analysis of the detection of six fluorescent probes targeting the immune-escape mutations L452R, N460K, E484A, F486V, Q493R, Q498R, and Y505H within one amplicon in the spike RBD and probes targeting the ORF1ab and N genes. After protocol optimization, the analytical performance of the technique was evaluated using plasmid templates. Sensitivity was assessed based on the limit of detection (LOD), and reliability was assessed by calculating the intra- and inter-run precision of melting temperatures (T m s). Specificity was assessed using pseudotyped lentivirus of common human respiratory pathogens and human genomic DNA. The assay was used to analyze 40 SARS-CoV-2–positive clinical samples (including 36 BA.2 and 4 BA.4/5 samples) and pseudotyped lentiviruses of wild-type and BA.1 viral RNA control materials, as well as 20 SARS-CoV-2–negative clinical samples, and its accuracy was evaluated by comparing the results with those of sequenc...