MOST-USDA Joint Research Center for Food Safety and Bor Luh Food Safety Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.
Protein Cell. 2012 Mar;3(3):204-12. doi: 10.1007/s13238-012-2017-6. Epub 2012 Apr 4.
A real time PCR assay for the detection of Vibrio parahaemolyticus in seafood samples was developed using a novel specific target and a competitive internal amplification control (IAC). The specificity of this assay was evaluated using 390 bacterial strains including V. parahaemolyticus, and other strains belonging to Vibrio and non-Vibrio species. The real time PCR assay unambiguously distinguished V. parahaemolyticus with a detection sensitivity of 4.8 fg per PCR with purified genomic DNA or 1 CFU per reaction by counting V. parahaemolyticus colonies. The assays of avoiding interference demonstrated that, even in the presence of 2.1 μg genomic DNA or 10(7) CFU background bacteria, V. parahaemolyticus could still be accurately detected. In addition, the IAC was used to indicate false-negative results, and lower than 94 copies of IAC per reaction had no influence on the detection limit. Ninety-six seafood samples were tested, of which 58 (60.4%) were positive, including 3 false negative results. Consequently, the real time PCR assay is effective for the rapid detection of V. parahaemotyticus contaminants in seafood.
建立了一种实时 PCR 检测方法，用于检测海产品样品中的副溶血性弧菌，该方法使用了一种新的特异性靶标和竞争性内部扩增对照（IAC）。该方法的特异性通过使用 390 株细菌菌株进行评估，包括副溶血性弧菌以及其他属于弧菌和非弧菌属的菌株。实时 PCR 检测方法能够明确地区分副溶血性弧菌，使用纯化基因组 DNA 的检测灵敏度为 4.8 fg/PCR，通过计算副溶血性弧菌的菌落数，反应的检测灵敏度为 1 CFU/反应。避免干扰的检测结果表明，即使存在 2.1 μg 基因组 DNA 或 10(7) CFU 背景细菌，也可以准确地检测到副溶血性弧菌。此外，IAC 可用于指示假阴性结果，反应中 IAC 少于 94 拷贝对检测限没有影响。对 96 个海产品样品进行了检测，其中 58 个（60.4%）为阳性，包括 3 个假阴性结果。因此，该实时 PCR 检测方法可有效快速检测海产品中的副溶血性弧菌污染物。
