Development of a Novel Multiplex PCR Method for the Rapid Detection of SARS-CoV-2, Influenza A Virus, and Influenza B Virus

作者：Liang Ma, Haoyan Zhu, Yongwei Jiang, Xiaomu Kong, Peng Gao, Yi Liu, Meimei Zhao, Guo‐Xiong Deng, Yongtong Cao · 发表于：International Journal of Analytical Chemistry · 年份：2024 · DOI：10.1155/2024/4950391 · 被引用次数：8 · 研究领域：SARS-CoV-2 detection and testing、SARS-CoV-2 and COVID-19 Research、Influenza Virus Research Studies

Objective. A sensitive and specific multiplex fluorescence rapid detection method was established for simultaneous detection of SARS-CoV-2, influenza A virus, and influenza B virus in a self-made device within 30 min, with a minimum detection limit of 200 copies/mL. Methods. Based on the genome sequences of SARS-CoV-2, influenza A virus (FluA), and influenza B virus (FluB) with reference to the Chinese Center for Disease Control and Prevention and related literature, specific primers were designed, and a multiplex fluorescent PCR system was established. The simultaneous and rapid detection of SARS-CoV-2, FluA, and FluB was achieved by optimizing the concentrations of Taq DNA polymerase as well as primers, probes, and Mg2+. The minimum detection limits of the nucleic acid rapid detection system for SARS-CoV-2, FluA, and FluB were evaluated. Results. By optimizing the amplification system, the N enzyme with the best amplification performance was selected, and the optimal concentration of Mg2+ in the multiamplification system was 3 mmol/L; the final concentrations of SARS-CoV-2 NP probe and primer were 0.15 μmol/L and 0.2 μmol/L, respectively; the final concentrations of SARS-CoV-2 ORF probe and primer were both 0.15 μmol/L; the final concentrations of FluA probe and primer were 0.2 μmol/L and 0.3 μmol/L, respectively; the final concentrations of FluB probe and primer were 0.15 μmol/L and 0.25 μmol/L, respectively. Conclusion. A multiplex real-time quantitative fluorescence RT-P...