Taqman-探针荧光定量PCR鉴定副溶血弧菌方法的建立

Establishment of a Method for the Identification of Vibrio Parahaemolyticus by Taqman-Probe Fluorescence Quantitative PCR Analysis

目的建立一种Taqman荧光定量PCR鉴定副溶血弧菌的方法。方法以副溶血弧菌标准株(ATCC-VPJS421)和其它常见致病菌标准株为研究对象,通过Gene Bank获取toxR基因的序列,采用生物信息学软件设计特异性PCR引物及Taqman探针,在SLAN 96P荧光定量PCR仪进行扩增检测,评价该检测方法的特异度和灵敏度。结果 (1)设计的引物能够扩增出特异性条带;(2)扩增体系中0.5μl探针的扩增效果优于1.0μl探针;(3)Taqman-探针荧光定量PCR检测方法对副溶血弧菌toxR基因的检测灵敏度为10-1 mg/L;(4)在检测粪肠球菌、金黄色葡萄球菌、腐生葡萄球菌、霍氏肠杆菌、铜绿假单胞菌、大肠埃希菌、溶藻弧菌、创伤弧菌、梅氏弧菌和弗尼斯弧菌等10种常见致病菌时未出现阳性扩增,特异度为100%。结论成功建立Taqman探针荧光定量PCR鉴定副溶血弧菌的方法,该方法特异度、灵敏度均较好,适用于副溶血弧菌的快速检测,具有良好的应用价值。

Objective To establish a method for the identification of Vibrio parahaemolyticus based on Taqman-fluorescence probe quantitative PCR method targeting toxR gene.Methods Taking the standard strain of Vibrio parahaemolyticus（VPJS421）and other ommon pathogenic bacteria＇standard strain as the research object,using the bio-software to design specific PCR primers and Taqman probe of Vibrio parahaemolyticus toxR gene and detected by fluorescence quantitative PCR instrument.Results（1）The designed primers could amplify specific bands.（2）The amplification efficiency of the 0.5μl probe in the amplification system was better than that of the 1.0μl probe.（3）The detection sensitivity of toxR gene of Vibrio parahaemolyticus by Taqman fluorescence quantitative PCR was 10-1 mg/L.（4）The detection method did not show positive amplification in detection of Enterococcus faecalis,Staphylococcus aureus,Saprophytic staphylococcus,Enterobacter hormaechei,Pseudomonas aeruginosa,Escherichia coli,Vibrio alginolyticus,Vibrio vulnficus,Vibrio metschnikovii and Vibrio furnissii 10 other common pathogenic bacteria.The specificity was 100%.Conlusion The fluorescence quantitative PCR method for the identification of Vibrio parahaemolyticus was successfully established.The method was sensitivty and specificity,and it is suitable for rapid detection of Vibrio parahaemolyticus and has a good application value.