H-NS基因作为靶基因的荧光定量PCR快速检测海产品中的副溶血性弧菌
Rapid detection of Vibrio parahaemolyticus in seafood by Real- time PCR targeted to the H- NS gene
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摘要: 目的:建立一种利用SYBR Green I荧光PCR反应快速、准确、便捷检测副溶血性弧菌的方法。方法:根据副溶血性弧菌的H-NS基因的保守区域设计引物,利用副溶血性弧菌的标准菌株以及12株其他种属的常见致病菌对引物的特异性和灵敏度进行评价。基于该基因利用SYBR Green I荧光PCR检测方法建立标准曲线,确定该检测方法的灵敏度。对用不同浓度的菌液污染的灭菌过的蚬子样品进行检测,确定该方法的的可行性。最后用该方法检测30份蚬子样品,并与国标GB4789.7-2013检测结果进行比较。结果:用H-NS基因作为靶基因检测副溶血性弧菌的SYBR Green I荧光PCR检测方法,建立的标准曲线的相关系数为0.998,检测灵敏度为7.3×10 CFU/m L。人工污染蚬子的检出限为6.7×102CFU/m L。对蚬子样品的检测结果为30份样品中有4份含有副溶血性弧菌,与国家标准方法 （GB4789.7-2013）的检测结果具有一致性。结论:表明该方法可以用于副溶血性弧菌的快速检测。Abstract: Objective: In this study,a rapid and accurate method was established to detect Vibrio parahaemolyticus of using SYBR Green I fluorescence PCR reaction.Methods: Primers were designed by using conserved region of the H- NS gene.Twelve non Vibrio parahaemolyticus were amplified by PCR to confirm the specificity.Based on H- NS gene,the standard curve was established and the sensitivity was determined by SYBR Green I fluorescence PCR reaction. Sterile clam samples contaminated by different concentrations of bacteria were detected to determine the feasibility of this method.Then,thirty clam samples were detected,and the results were compared with the GB 4789.7-2013.Results: Correlation coefficient of the standard curve established in this study was 0.998,and the sensitivity was 7.3 × 10 CFU / m L.The detection limit of artificial contamination was 6.7 × 102 CFU / m L.There were four positive results detected in the thirty samples by using SYBR Green I fluorescence PCR method which was consistent with GB 4789.7- 2013.Conclusion: Method of this article could be used in rapid detection of Vibrio parahaemolyticus.
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