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...

Full description

Bibliographic Details
Main Authors: Yong Yan, Li Zhan, Guoying Zhu, Junyan Zhang, Ping Li, Lixia Chen, Peiyan He, Jianyong Luo, Zhongwen Chen
Format: Article
Language:English
Published: MDPI AG 2022-07-01
Series:Pathogens
Subjects:
Online Access:https://www.mdpi.com/2076-0817/11/8/865
_version_ 1827617682074107904
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
publisher MDPI AG
record_format Article
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
work_keys_str_mv AT yongyan directandrapididentificationofvibriocholeraeserogroupandtoxigenicitybyanovelmultiplexrealtimeassay
AT lizhan directandrapididentificationofvibriocholeraeserogroupandtoxigenicitybyanovelmultiplexrealtimeassay
AT guoyingzhu directandrapididentificationofvibriocholeraeserogroupandtoxigenicitybyanovelmultiplexrealtimeassay
AT junyanzhang directandrapididentificationofvibriocholeraeserogroupandtoxigenicitybyanovelmultiplexrealtimeassay
AT pingli directandrapididentificationofvibriocholeraeserogroupandtoxigenicitybyanovelmultiplexrealtimeassay
AT lixiachen directandrapididentificationofvibriocholeraeserogroupandtoxigenicitybyanovelmultiplexrealtimeassay
AT peiyanhe directandrapididentificationofvibriocholeraeserogroupandtoxigenicitybyanovelmultiplexrealtimeassay
AT jianyongluo directandrapididentificationofvibriocholeraeserogroupandtoxigenicitybyanovelmultiplexrealtimeassay
AT zhongwenchen directandrapididentificationofvibriocholeraeserogroupandtoxigenicitybyanovelmultiplexrealtimeassay