水产品中副溶血性弧菌特异性二重PCR检测方法的研究

Specific detection of Vibrio parahaemolyticus in aquatic products by a dulplex PCR

建立快速检测水产品中副溶血性弧菌(Vibrio parahae-molyticus)的二重PCR方法。以副溶血性弧菌特异性基因tlh和toxR为靶基因,选择2对引物,对5株副溶血性弧菌和40株非副溶血性弧菌进行特异性检测;梯度稀释副溶血性弧菌基因组DNA,以不同稀释度DNA作PCR扩增;在鱼肉样品中以不同菌量人工污染,不同增菌时间培养,提取DNA进行PCR扩增;应用该方法对实际样品进行检测。以tlh和toxR为靶基因的两对引物对副溶血性弧菌的检出有很好的特异性。PCR检测的灵敏度在DNA水平上达到28.76pg;人工污染样品,当起始污染量为1CFU/mL时,37℃增菌培养10h即可检出。本试验一共检测了21份水产品样品,有14份检出了副溶血性弧菌。

A rapid dulplex PCR methed was developed to detect Vibrio parahaemolyticus in aquatic products, Two sets of primers that specifically amplify segments of the tlh and toxR genes were designed to detect Vibrio parahaemolyticus, Five Vibrio parahaemolyticus and forty non Vibrio parahaemolyticus were amplified by PCR to confirm the specificity. The Vibrio parahaemolyticus genomic DNA was serially diluted and subjected to PCR amplification to verify sensitivity, Various numbers of bacteria were added into fish, after different time of enrichment, DNA was extracted and amplified by PCR to verify detection limit, This method was applied to detect naturally contaminated samples, This PCR system was specific. The detection limit of the PCR was 28.76 pg with genomic DNA and 1 CFU/mL in artificially contaminated fish after enrichment at 37 ℃ for 10 h. Twenty one aquatic products were detected by this dulplex PCR and fourteen were found to be positive. A rapid, sensitive and specific dulplex PCR methad can be used for detection of Vibrio parahaemolytieus in aquatic products.