Cai Tanxi, Jiang Luyan, Yang Chengbo, Huang Kehe
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
FEMS Immunol Med Microbiol. 2006 Mar;46(2):180-6. doi: 10.1111/j.1574-695X.2005.00016.x.
Vibrio parahaemolyticus is recognized as a leading human food-borne pathogen. A TaqMan PCR assay based on the gyrase B gene (gyrB) sequence of V. parahaemolyticus was developed for quantitative detection of V. parahaemolyticus in seafood. The study involving 27 V. parahaemolyticus and 10 strains of other species indicated that the real-time PCR test was highly specific. The sensitivity of the assay was approximately a single CFU per PCR in pure culture and six to eight CFU per PCR in spiked raw oyster, respectively. Real-time PCR values of artificially inoculated oyster homogenates correlated well with plate counts determined using culture methods. A total of 300 seafood samples were analyzed and 78 (26%) of these samples were positive for V. parahaemolyticus using a conventional culture method and 97 (32.3%) using the real-time PCR assay. All culture-positive samples were PCR-positive. However, 19 samples positive by PCR were culture-negative. The results show that retail seafood is commonly contaminated with V. parahaemolyticus in harvest season in eastern China. These data also indicate that real-time PCR can provide sensitive species-specific detection and quantification of V. parahaemolyticus in seafood without prior isolation and characterization of the bacteria by traditional microbiological methods.
副溶血性弧菌被认为是一种主要的食源性人类病原菌。基于副溶血性弧菌gyrase B基因（gyrB）序列开发了一种TaqMan PCR检测方法，用于定量检测海鲜中的副溶血性弧菌。涉及27株副溶血性弧菌和10株其他菌种的研究表明，实时PCR检测具有高度特异性。该检测方法在纯培养中的灵敏度约为每个PCR一个CFU，在加标的生牡蛎中每个PCR为6至8个CFU。人工接种牡蛎匀浆的实时PCR值与使用培养方法测定的平板计数相关性良好。共分析了300份海鲜样品，其中78份（26%）使用传统培养方法检测副溶血性弧菌呈阳性，97份（32.3%）使用实时PCR检测呈阳性。所有培养阳性的样品PCR均为阳性。然而，有19份PCR阳性的样品培养为阴性。结果表明，中国东部收获季节的零售海鲜普遍受到副溶血性弧菌污染。这些数据还表明，实时PCR无需通过传统微生物方法对细菌进行预先分离和鉴定，即可对海鲜中的副溶血性弧菌进行灵敏的种特异性检测和定量。
