Development of a Novel Multiplex PCR Method for the Rapid Detection of SARS-CoV-2, Influenza A Virus, and Influenza B Virus
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 sequ...
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| Format: | Article |
| Language: | English |
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Wiley
2024-01-01
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| Series: | International Journal of Analytical Chemistry |
| Online Access: | http://dx.doi.org/10.1155/2024/4950391 |
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| author | Liang Ma Haoyan Zhu Yongwei Jiang Xiaomu Kong Peng Gao Yi Liu Meimei Zhao Guoxiong Deng Yongtong Cao |
| author_facet | Liang Ma Haoyan Zhu Yongwei Jiang Xiaomu Kong Peng Gao Yi Liu Meimei Zhao Guoxiong Deng Yongtong Cao |
| author_sort | Liang Ma |
| collection | DOAJ |
| description | 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-PCR system for three respiratory viruses of SARS-CoV-2, FluA, and FluB was established with a high amplification efficiency and sensitivity reaching 200 copies/mL for all samples. Combined with the automated microfluidic nucleic acid detection system, the system can achieve rapid detection in 30 minutes. |
| format | Article |
| id | doaj-art-7989cc53501e4c6a8e62603eac584d49 |
| institution | Kabale University |
| issn | 1687-8779 |
| language | English |
| publishDate | 2024-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | International Journal of Analytical Chemistry |
| spelling | doaj-art-7989cc53501e4c6a8e62603eac584d492025-08-20T03:35:48ZengWileyInternational Journal of Analytical Chemistry1687-87792024-01-01202410.1155/2024/4950391Development of a Novel Multiplex PCR Method for the Rapid Detection of SARS-CoV-2, Influenza A Virus, and Influenza B VirusLiang Ma0Haoyan Zhu1Yongwei Jiang2Xiaomu Kong3Peng Gao4Yi Liu5Meimei Zhao6Guoxiong Deng7Yongtong Cao8Department of Clinical LaboratoryDepartment of Clinical LaboratoryDepartment of Clinical LaboratoryDepartment of Clinical LaboratoryDepartment of Clinical LaboratoryDepartment of Clinical LaboratoryDepartment of Clinical LaboratoryDepartment of Clinical LaboratoryDepartment of Clinical LaboratoryObjective. 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-PCR system for three respiratory viruses of SARS-CoV-2, FluA, and FluB was established with a high amplification efficiency and sensitivity reaching 200 copies/mL for all samples. Combined with the automated microfluidic nucleic acid detection system, the system can achieve rapid detection in 30 minutes.http://dx.doi.org/10.1155/2024/4950391 |
| spellingShingle | Liang Ma Haoyan Zhu Yongwei Jiang Xiaomu Kong Peng Gao Yi Liu Meimei Zhao Guoxiong Deng Yongtong Cao Development of a Novel Multiplex PCR Method for the Rapid Detection of SARS-CoV-2, Influenza A Virus, and Influenza B Virus International Journal of Analytical Chemistry |
| title | Development of a Novel Multiplex PCR Method for the Rapid Detection of SARS-CoV-2, Influenza A Virus, and Influenza B Virus |
| title_full | Development of a Novel Multiplex PCR Method for the Rapid Detection of SARS-CoV-2, Influenza A Virus, and Influenza B Virus |
| title_fullStr | Development of a Novel Multiplex PCR Method for the Rapid Detection of SARS-CoV-2, Influenza A Virus, and Influenza B Virus |
| title_full_unstemmed | Development of a Novel Multiplex PCR Method for the Rapid Detection of SARS-CoV-2, Influenza A Virus, and Influenza B Virus |
| title_short | Development of a Novel Multiplex PCR Method for the Rapid Detection of SARS-CoV-2, Influenza A Virus, and Influenza B Virus |
| title_sort | development of a novel multiplex pcr method for the rapid detection of sars cov 2 influenza a virus and influenza b virus |
| url | http://dx.doi.org/10.1155/2024/4950391 |
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