双重PCR快速检测海水贝类中副溶血性弧菌

Rapid detection of Vibrio parahaemolyticus in sea shellfish by duplex PCR

应用双重PCR技术快速检测新鲜海水贝类样品中副溶血性弧菌。根据副溶血性弧菌tdh基因及tl基因设计两对引物,对影响PCR的主要因素进行优化,确定双重PCR最佳反应体系及条件,该方法对于副溶血性弧菌纯培养物的最低检出限为2.5×102 cfu/mL,模拟污染样品的检测限为10cfu/g。对市场随机抽取的77份新鲜红螺样品、63份方形马珂蛤样品、61份菲律宾蛤仔进行检测,8h内快速检出PCR阳性样品分别为15份、8份、5份,采用国家标准方法对PCR结果进行验证,验证结果表明,PCR阴性样品未检出副溶血性弧菌;PCR阳性样品均有副溶血性弧菌检出,春夏两个季节里红螺中副溶血性弧菌的检出率分别为13.3%、25%,方形马珂蛤为6.7%、21.2%,菲律宾蛤仔为0、16.1%。该方法具有较高的灵敏度和准确性,能快速检验海水贝类中副溶血性弧菌。

The duplex PCR was applied to detect Vibrio parahaemolyticus in fresh sea shellfish.The primers were designed based on the tdh gene and tl gene of Vibrio parahaemolyticus.After optimized,the detection limit of the duplex PCR was 2.5×102 cfu/mL and the duplex PCR could detect 10cfu/g of Vibrio parahaemolyticus in the contaminated sea shellfish.The fresh Rapana bezona Linnaeus（77copies）,Mactra veneriformis（63copies）and Ruditapes philippinarum（61copies）were sampled at the market randomly and detected using duplex PCR,and 15,8,and 5copies of PCR positive samples were obtained respectively in 8h.There were no Vibrio parahaemolyticus detected in PCR negative samples while Vibrio parahaemolyticus were detected in all PCR positive samples when tested with the national standard methods.The detection rate of Vibrio parahaemolyticus from 77 copies of fresh Rapana bezona Linnaeus,63 copies of fresh Mactraveneriformis and 61 copies of fresh Ruditapes philippinarum was 13.3%,25%,6.7%,21.2% in the spring and 0,16.1% and in the summer respectively.The method of the duplex PCR could quickly detect the Vibrio parahaemolyticus in fresh sea shellfish with high sensitivity and accuracy.