Direct and Rapid Identification of Vibrio Cholerae Serogroup and Toxigenicity by a Novel Multiplex Real-Time Assay
Molecular diagnostic assays for cholera detection have superior sensitivity to conventional assays and are now being accepted as the new standard method, especially the real-time PCR/RT-PCR. However, limited throughput capacity and long detection duration prevent them from detecting more specimens a...
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MDPI AG
2022-07-01
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| Series: | Pathogens |
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| Online Access: | https://www.mdpi.com/2076-0817/11/8/865 |
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| author | Yong Yan Li Zhan Guoying Zhu Junyan Zhang Ping Li Lixia Chen Peiyan He Jianyong Luo Zhongwen Chen |
| author_facet | Yong Yan Li Zhan Guoying Zhu Junyan Zhang Ping Li Lixia Chen Peiyan He Jianyong Luo Zhongwen Chen |
| author_sort | Yong Yan |
| collection | DOAJ |
| description | Molecular diagnostic assays for cholera detection have superior sensitivity to conventional assays and are now being accepted as the new standard method, especially the real-time PCR/RT-PCR. However, limited throughput capacity and long detection duration prevent them from detecting more specimens and more targets in one turnaround time simultaneously. In this study, we utilized nucleic acid extraction-free, direct RT-PCR and high-speed amplification to develop a novel multiplex assay, a quadplex direct one-tube real-time RT-PCR assay, for rapid detection of the serogroup and cholera toxin toxigenicity of <i>Vibrio cholerae</i> targeting the <i>epsM</i>, <i>ctxA</i>, <i>rfb-O1</i>, and <i>rfb-O139</i> genes. Performance of the multiplex assay was evaluated by comparison with the monoplex real-time PCR assay according to the China Cholera Prevention Manual. Detection data from clinical specimens showed that the new assay had good diagnostic sensitivities for <i>epsM</i> (100%, n = 301), <i>ctxA</i> (100%, n = 125), <i>rfb-O1</i> (100%, n = 85), and <i>rfb-O139</i> (97.87%, n = 49). Analysis of the analytical sensitivities with serial dilutions of positive standards showed that the detection limits of the new assay for <i>Vibrio cholerae</i> <i>epsM</i><i>,</i><i>ctxA,</i><i>rfb-O1</i>, and <i>rfb-O139</i> were up to 200, 590, 115, and 1052 copies per mL lower than the monoplex real-time PCR (910, 345, and 1616 copies/mL respectively, for <i>ctxA</i><i>,</i><i>rfb-O1</i>, and <i>rfb-O139</i>). The results indicate that the multiplex assay is a rapid, sensitive, specific, and easy-to-use detection tool for <i>Vibrio cholerae</i>, especially suitable for rapid identification and screening detection of mass specimens. |
| first_indexed | 2024-03-09T09:51:27Z |
| format | Article |
| id | doaj.art-f2fac9c161144824a0924b254740a8f2 |
| institution | Directory Open Access Journal |
| issn | 2076-0817 |
| language | English |
| last_indexed | 2024-03-09T09:51:27Z |
| publishDate | 2022-07-01 |
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| series | Pathogens |
| spelling | doaj.art-f2fac9c161144824a0924b254740a8f22023-12-02T00:08:04ZengMDPI AGPathogens2076-08172022-07-0111886510.3390/pathogens11080865Direct and Rapid Identification of Vibrio Cholerae Serogroup and Toxigenicity by a Novel Multiplex Real-Time AssayYong Yan0Li Zhan1Guoying Zhu2Junyan Zhang3Ping Li4Lixia Chen5Peiyan He6Jianyong Luo7Zhongwen Chen8Jiaxing Key Laboratory of Pathogenic Microbiology, Jiaxing Municipal Center for Disease Control and Prevention, Wenqiao Road 486, Jiaxing 314050, ChinaInstitute of Microbiology, Zhejiang Provincial Center for Disease Control and Prevention, Binsheng Road 3399, Hangzhou 310051, ChinaJiaxing Key Laboratory of Pathogenic Microbiology, Jiaxing Municipal Center for Disease Control and Prevention, Wenqiao Road 486, Jiaxing 314050, ChinaInstitute of Microbiology, Zhejiang Provincial Center for Disease Control and Prevention, Binsheng Road 3399, Hangzhou 310051, ChinaJiaxing Key Laboratory of Pathogenic Microbiology, Jiaxing Municipal Center for Disease Control and Prevention, Wenqiao Road 486, Jiaxing 314050, ChinaJiaxing Key Laboratory of Pathogenic Microbiology, Jiaxing Municipal Center for Disease Control and Prevention, Wenqiao Road 486, Jiaxing 314050, ChinaJiaxing Key Laboratory of Pathogenic Microbiology, Jiaxing Municipal Center for Disease Control and Prevention, Wenqiao Road 486, Jiaxing 314050, ChinaJiaxing Key Laboratory of Pathogenic Microbiology, Jiaxing Municipal Center for Disease Control and Prevention, Wenqiao Road 486, Jiaxing 314050, ChinaJiaxing Key Laboratory of Pathogenic Microbiology, Jiaxing Municipal Center for Disease Control and Prevention, Wenqiao Road 486, Jiaxing 314050, ChinaMolecular diagnostic assays for cholera detection have superior sensitivity to conventional assays and are now being accepted as the new standard method, especially the real-time PCR/RT-PCR. However, limited throughput capacity and long detection duration prevent them from detecting more specimens and more targets in one turnaround time simultaneously. In this study, we utilized nucleic acid extraction-free, direct RT-PCR and high-speed amplification to develop a novel multiplex assay, a quadplex direct one-tube real-time RT-PCR assay, for rapid detection of the serogroup and cholera toxin toxigenicity of <i>Vibrio cholerae</i> targeting the <i>epsM</i>, <i>ctxA</i>, <i>rfb-O1</i>, and <i>rfb-O139</i> genes. Performance of the multiplex assay was evaluated by comparison with the monoplex real-time PCR assay according to the China Cholera Prevention Manual. Detection data from clinical specimens showed that the new assay had good diagnostic sensitivities for <i>epsM</i> (100%, n = 301), <i>ctxA</i> (100%, n = 125), <i>rfb-O1</i> (100%, n = 85), and <i>rfb-O139</i> (97.87%, n = 49). Analysis of the analytical sensitivities with serial dilutions of positive standards showed that the detection limits of the new assay for <i>Vibrio cholerae</i> <i>epsM</i><i>,</i><i>ctxA,</i><i>rfb-O1</i>, and <i>rfb-O139</i> were up to 200, 590, 115, and 1052 copies per mL lower than the monoplex real-time PCR (910, 345, and 1616 copies/mL respectively, for <i>ctxA</i><i>,</i><i>rfb-O1</i>, and <i>rfb-O139</i>). The results indicate that the multiplex assay is a rapid, sensitive, specific, and easy-to-use detection tool for <i>Vibrio cholerae</i>, especially suitable for rapid identification and screening detection of mass specimens.https://www.mdpi.com/2076-0817/11/8/865<i>Vibrio cholerae</i>cholera toxindirect PCRmultiplex real-time PCRmolecular diagnosis |
| spellingShingle | Yong Yan Li Zhan Guoying Zhu Junyan Zhang Ping Li Lixia Chen Peiyan He Jianyong Luo Zhongwen Chen Direct and Rapid Identification of Vibrio Cholerae Serogroup and Toxigenicity by a Novel Multiplex Real-Time Assay Pathogens <i>Vibrio cholerae</i> cholera toxin direct PCR multiplex real-time PCR molecular diagnosis |
| title | Direct and Rapid Identification of Vibrio Cholerae Serogroup and Toxigenicity by a Novel Multiplex Real-Time Assay |
| title_full | Direct and Rapid Identification of Vibrio Cholerae Serogroup and Toxigenicity by a Novel Multiplex Real-Time Assay |
| title_fullStr | Direct and Rapid Identification of Vibrio Cholerae Serogroup and Toxigenicity by a Novel Multiplex Real-Time Assay |
| title_full_unstemmed | Direct and Rapid Identification of Vibrio Cholerae Serogroup and Toxigenicity by a Novel Multiplex Real-Time Assay |
| title_short | Direct and Rapid Identification of Vibrio Cholerae Serogroup and Toxigenicity by a Novel Multiplex Real-Time Assay |
| title_sort | direct and rapid identification of vibrio cholerae serogroup and toxigenicity by a novel multiplex real time assay |
| topic | <i>Vibrio cholerae</i> cholera toxin direct PCR multiplex real-time PCR molecular diagnosis |
| url | https://www.mdpi.com/2076-0817/11/8/865 |
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