Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan.
Lett Appl Microbiol. 2010 Sep;51(3):293-300. doi: 10.1111/j.1472-765X.2010.02895.x. Epub 2010 Jul 1.
To develop an effective multiplex PCR for simultaneous and rapid detection of Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus, the three most important Vibrio species that can cause devastating health hazards among human.
Species-specific PCR primers were designed based on toxR gene for V. cholerae and V. parahaemolyticus, and vvhA gene for V. vulnificus. The multiplex PCR was validated with 488 Vibrio strains including 322 V. cholerae, 12 V. vulnificus, and 82 V. parahaemolyticus, 20 other Vibrio species and 17 other bacterial species associated with human diseases. It could detect the three target bacteria without any ambiguity even among closely related species. It showed good efficiency in detection of co-existing target species in the same sample. The detection limit of all the target species was ten cells per PCR tube.
Specificity and sensitivity of the multiplex PCR is 100% each and sufficient for simultaneous detection of these potentially pathogenic Vibrio species in clinical and environmental samples.
This simple, rapid and cost-effective method can be applicable in a prediction system to prevent disease outbreak by these Vibrio species and can be considered as an effective tool for both epidemiologist and ecologist.
开发一种有效的多重 PCR 方法，用于同时快速检测霍乱弧菌、创伤弧菌和副溶血性弧菌，这三种最重要的弧菌，它们可对人类健康造成严重危害。
基于霍乱弧菌和副溶血性弧菌的 toxR 基因以及创伤弧菌的 vvhA 基因，设计了种特异性 PCR 引物。该多重 PCR 方法用包括 322 株霍乱弧菌、12 株创伤弧菌、82 株副溶血性弧菌、20 种其他弧菌和 17 种与人类疾病相关的其他细菌在内的 488 株弧菌菌株进行了验证。即使在亲缘关系密切的种中，该方法也能准确、明确地检测到这三种目标细菌。该方法在同一样本中同时检测共存目标种时具有良好的效率。所有目标种的检测限均为每个 PCR 管 10 个细胞。
该多重 PCR 的特异性和灵敏度均为 100%，足以用于临床和环境样本中这些潜在致病性弧菌的同时检测。
这种简单、快速且具有成本效益的方法可应用于预测系统，以预防这些弧菌引起的疾病爆发，并且可以被视为流行病学家和生态学家的有效工具。
