Park Jie Yeun, Jeon Semi, Kim Jun Young, Park Misun, Kim Seonghan
Division of Enteric Bacterial Infections, Korea National Institute of Health, Osong, Korea.
Osong Public Health Res Perspect. 2013 Jun;4(3):133-9. doi: 10.1016/j.phrp.2013.04.004. Epub 2013 Apr 30.
A multiplex real-time polymerase chain reaction (RT-PCR) method was developed for the identification of three Vibrio species: Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus.
Specific primers and probes targeting the hlyA, tlh, and vvhA genes were selected and used for multiplex real-time PCR to confirm the identification of V. cholerae, V. parahaemolyticus, and V. vulnificus, respectively. This method was applied to screen Vibrio species from environmental samples and combining it with a culture-based method, its effectiveness was evaluated in comparison with culture-based methods alone.
Specific PCR fragments were obtained from isolates belonging to the target species, indicating a high specificity of this multiplex real-time PCR. No cross-reactivity with the assay was observed between the tested bacteria. The sensitivity of the multiplex real-time PCR was found to have a lower limit of 10(4) colony-forming units/reaction for all three Vibrio species. The combination strategy raised the isolation ratio of all three Vibrio species 1.26- to 2.75-fold.
This assay provides a rapid, sensitive, and specific technique to detect these three Vibrio species in the environment.
开发一种多重实时聚合酶链反应（RT-PCR）方法，用于鉴定三种弧菌：霍乱弧菌、副溶血性弧菌和创伤弧菌。
选择靶向hlyA、tlh和vvhA基因的特异性引物和探针，用于多重实时PCR，分别确认霍乱弧菌、副溶血性弧菌和创伤弧菌的鉴定。该方法应用于从环境样本中筛选弧菌，并将其与基于培养的方法相结合，与单独的基于培养的方法相比，评估其有效性。
从属于目标物种的分离物中获得了特异性PCR片段，表明该多重实时PCR具有高特异性。在测试细菌之间未观察到与该检测方法的交叉反应。发现多重实时PCR的灵敏度对于所有三种弧菌物种而言下限为10(4) 菌落形成单位/反应。联合策略使所有三种弧菌物种的分离率提高了1.26至2.75倍。
该检测方法提供了一种快速、灵敏和特异的技术，用于检测环境中的这三种弧菌物种。
