Development of a real time PCR assay for rapid detection of Vibrio parahaemolyticus from seafood
Development of a real time PCR assay for rapid detection of Vibrio parahaemolyticus from seafood
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开发用于快速检测海鲜中副溶血弧菌的实时 PCR 检测方法
DOI：
10.1007/s13238-012-2017-6
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发表时间：
2012-03-01
期刊：
影响因子：
21.1
通讯作者：
Shi, Xianming
中科院分区：
文献类型：
--
作者：
Liu, Bin;He, Xiaohua;Shi, Xianming
建立了一种新型特异靶点和竞争性内扩增对照（IAC）的海鲜样品中副溶血性弧菌的实时PCR检测方法。使用390种细菌菌株（包括副溶血性弧菌和其他属于弧菌和非弧菌种的菌株）对该检测的特异性进行了评估。实时PCR检测明确区分副溶血性弧菌，纯化基因组DNA的每次PCR检测灵敏度为4.8 fg，计数副溶血性弧菌菌落的每次反应检测灵敏度为1 CFU。避免干扰试验表明，即使在2.1 μg基因组DNA或10(7)CFU背景细菌存在的情况下，仍能准确检测出副溶血性弧菌。此外，IAC用于指示假阴性结果，每次反应IAC低于94拷贝对检测限没有影响。共检测96个海产品样本，其中58个（60.4%）呈阳性，其中3个呈假阴性。因此，实时PCR检测方法可快速检测海产品中副溶血性弧菌污染物。
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.