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

Virology Journal

Methodology

Yi Liu¹  Xiaomu Kong¹  Liang Ma¹  Haoyan Zhu¹  Peng Gao¹  Guoxiong Deng¹  Yongtong Cao¹  Yongwei Jiang¹  Meimei Zhao¹  Lixia Lu¹ 

[1] Department of Clinical Laboratory, China-Japan Friendship Hospital, No. 2 Yinghua East Street, Chaoyang District, 100029, Beijing, People’s Republic of China;

关键词: Omicron;    Mutation detection;    Asymmetric PCR;    Melting curve analysis;    SARS-CoV-2;   

DOI  :  10.1186/s12985-023-02137-5

 received in 2022-11-03, accepted in 2023-07-22,  发布年份 2023

来源: Springer

【 摘 要 】

BackgroundThe 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.MethodsThe 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 (Tms). 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 sequencing.ResultsAll genotypes were sensitively identified using the developed method with a LOD of 39.1 copies per reaction. The intra- and inter-run coefficients of variation for the Tms were ≤ 0.69% and ≤ 0.84%, with standard deviations ≤ 0.38 °C and ≤ 0.41 °C, respectively. Validation of the assay using known SARS-CoV-2–positive samples demonstrated its ability to correctly identify the targeted mutations and preliminarily characterize the Omicron lineages.ConclusionThe developed assay can provide accurate, reliable, rapid, simple and low-cost detection of the immune-escape mutations located in the spike RBD to detect the Omicron variant and discriminate its lineages, and its use can be easily generalized in clinical laboratories with a fluorescent PCR platform.

【 授权许可】

CC BY   
© BioMed Central Ltd., part of Springer Nature 2023

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