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Open Access 01-12-2023 | SARS-CoV-2 | Methodology

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

Authors: Xiaomu Kong, Peng Gao, Yongwei Jiang, Lixia Lu, Meimei Zhao, Yi Liu, Guoxiong Deng, Haoyan Zhu, Yongtong Cao, Liang Ma

Published in: Virology Journal | Issue 1/2023

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_ms). 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.

Results

All 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 T_ms 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.

Conclusion

The 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.

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Title: 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
Authors: Xiaomu Kong
Peng Gao
Yongwei Jiang
Lixia Lu
Meimei Zhao
Yi Liu
Guoxiong Deng
Haoyan Zhu
Yongtong Cao
Liang Ma
Publication date: 01-12-2023
Publisher: BioMed Central
Keywords: SARS-CoV-2
Polymerase Chain Reaction
Published in: Virology Journal / Issue 1/2023
Electronic ISSN: 1743-422X
DOI: https://doi.org/10.1186/s12985-023-02137-5

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