Department of Biotechnology, Pukyong National University, Busan 608-737, Republic of Korea.
Mol Cell Probes. 2013 Oct-Dec;27(5-6):171-5. doi: 10.1016/j.mcp.2013.04.001. Epub 2013 May 6.
Vibrio parahaemolyticus is a significant cause of human gastrointestinal disorders worldwide, transmitted primarily by ingestion of raw or undercooked contaminated seafood. In this study, a multiplex PCR assay for the detection and differentiation of V. parahaemolyticus strains was developed using primer sets for a species-specific marker, groEL, and two virulence markers, tdh and trh. Multiplex PCR conditions were standardised, and extracted genomic DNA of 70 V. parahaemolyticus strains was used for identification. The sensitivity and efficacy of this method were validated using artificially inoculated shellfish and seawater. The expected sizes of amplicons were 510 bp, 382 bp, and 171 bp for groEL, tdh and trh, respectively. PCR products were sufficiently different in size, and the detection limits of the multiplex PCR for groEL, tdh and trh were each 200 pg DNA. Specific detection and differentiation of virulent from non-virulent strains in shellfish homogenates and seawater was also possible after artificial inoculation with various V. parahaemolyticus strains. This newly developed multiplex PCR is a rapid assay for detection and differentiation of pathogenic V. parahaemolyticus strains, and could be used to prevent disease outbreaks and protect public health by helping the seafood industry maintain a safe shellfish supply.
副溶血性弧菌是一种重要的人类胃肠道疾病病原体，主要通过摄入生的或未煮熟的受污染海鲜传播。在这项研究中，我们使用groEL 种特异性标记物和两个毒力标记物 tdh 和 trh 的引物组，开发了一种用于检测和区分副溶血性弧菌菌株的多重 PCR 检测方法。对多重 PCR 条件进行了标准化，并使用 70 株副溶血性弧菌的提取基因组 DNA 进行了鉴定。使用人工接种贝类和海水验证了该方法的灵敏度和功效。groEL、tdh 和 trh 的扩增子预期大小分别为 510 bp、382 bp 和 171 bp。PCR 产物在大小上有足够的差异，groEL、tdh 和 trh 的多重 PCR 检测限分别为 200 pg DNA。在人工接种各种副溶血性弧菌菌株后，也可以在贝类匀浆和海水中对毒力菌株和非毒力菌株进行特异性检测和区分。这种新开发的多重 PCR 是一种快速检测和区分致病性副溶血性弧菌菌株的方法，通过帮助海鲜行业保持安全的贝类供应，可以预防疾病爆发和保护公众健康。
