Yan Yong, Zhan Li, Zhu Guoying, Zhang Junyan, Li Ping, Chen Lixia, He Peiyan, Luo Jianyong, Chen Zhongwen
Jiaxing Key Laboratory of Pathogenic Microbiology, Jiaxing Municipal Center for Disease Control and Prevention, Wenqiao Road 486, Jiaxing 314050, China.
Institute of Microbiology, Zhejiang Provincial Center for Disease Control and Prevention, Binsheng Road 3399, Hangzhou 310051, China.
Pathogens. 2022 Jul 30;11(8):865. doi: 10.3390/pathogens11080865.
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 Vibrio cholerae targeting the epsM, ctxA, rfb-O1, and rfb-O139 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 epsM (100%, n = 301), ctxA (100%, n = 125), rfb-O1 (100%, n = 85), and rfb-O139 (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 Vibrio cholerae epsM,ctxA,rfb-O1, and rfb-O139 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 ctxA,rfb-O1, and rfb-O139). The results indicate that the multiplex assay is a rapid, sensitive, specific, and easy-to-use detection tool for Vibrio cholerae, especially suitable for rapid identification and screening detection of mass specimens.
用于霍乱检测的分子诊断检测方法比传统检测方法具有更高的灵敏度，目前已被视为新标准方法，尤其是实时PCR/逆转录PCR。然而，通量有限和检测时间长阻碍了它们在一个周转时间内同时检测更多样本和更多靶点。在本研究中，我们利用无需核酸提取的直接逆转录PCR和高速扩增技术，开发了一种新型多重检测方法，即四重直接单管实时逆转录PCR检测方法，用于快速检测霍乱弧菌的血清群和霍乱毒素产毒性，该方法靶向epsM、ctxA、rfb - O1和rfb - O139基因。根据《中国霍乱预防手册》，通过与单重实时PCR检测方法比较，对该多重检测方法的性能进行了评估。临床样本的检测数据表明，新方法对epsM（100%，n = 301）、ctxA（100%，n = 125）、rfb - O1（100%，n = 85）和rfb - O139（97.87%，n = 49）具有良好的诊断敏感性。对阳性标准品进行系列稀释后的分析灵敏度表明，新方法对霍乱弧菌epsM、ctxA、rfb - O1和rfb - O139的检测限分别比单重实时PCR低200、590、115和1052拷贝/mL（ctxA、rfb - O1和rfb - O139的单重实时PCR检测限分别为910、345和1616拷贝/mL）。结果表明，该多重检测方法是一种快速、灵敏且易于使用的霍乱弧菌检测工具，特别适用于大量样本的快速鉴定和筛查检测。
