食品中副溶血弧菌荧光定量PCR方法快速检测
Rapid detection of Vibrio parahaemolyticus in food by fluorescence quantitative PCR
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摘要:目的 利用荧光定量PCR技术, 建立食品中副溶血弧菌污染的快速、准确的定量检测方法.方法 根据副溶血弧菌gyrase基因序列设计合成副溶血弧菌FQ-PCR诊断试剂盒, 研究其灵敏度、特异性, 并应用于模拟食品样本检测.结果 在31株不同菌株的检测中, 8株副溶血弧菌结果均为阳性, 而其他23株弧菌科及其他菌属的菌株均为阴性; 纯菌条件下定量检测低限10 cfu/ml; 对模拟样本直接进行, 检测低限为103cfu/g, 经培养增菌后, 检测低限则达到2 cfu/g.结论 该方法特异性强、敏感性高, 操作简便快速, 检验周期仅需36 h, 扩增与检测在封闭单管进行, 可避免常规PCR方法易污染缺陷, 具有很好的推广应用前景.Abstract:Objective To constructa rapid and accurate detection method of Viborior ahaemoly ticus with fulo rescence quantitative PCR(FQ-PCR)in food.Methods Adiagnodstic kitwas developed according to the gyrase gene in Vibrio Parahaemolyticus.The sensitivity and specialty of method was assessed, and the method was applied to detectartificial contaminated food samples.Results The results of all eightstrains of Vibrio par ahemolyticus were positive, and the results of the other 23 strains respective belong to Vibrionaceae and other genus were negative.The quantitative detection limitof the mehtod was 10 cfu/ml in pure clutured broth, and the detection limitof the method was 103 cfu/g and 2 cfu/g in non-cultured and o ver night-cultured ar tificial contaminated food samples respectively.Conclusion FQ-PCR method has high sensitivity and specialty.The method is handy and rapid, and the whole process can finish within 36 hr.Compared with the conventional PCR method.FQ-PCR methods is insusceptible to operation contamination for the whole flow of PCR and detection shutin arreaction tube.FQ-PCR method has potential applied value in screening of food borne patho gen.
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