基于toxR基因的SYBR GreenⅠ实时定量PCR检测病原副溶血弧菌方法的建立

Development of a SYBR GreenⅠreal-time PCR for detection of Vibrio parahaemolyticus based on toxR gene

基于副溶血弧菌toxR基因保守序列设计1对特异性引物,建立了SYBR GreenⅠ实时定量PCR检测副溶血弧菌的方法。常规PCR检测,副溶血弧菌扩增出大小为368bp的目的片段,其他4种病原细菌均为阴性;SYBRGreenⅠ实时定量PCR扩增曲线反映了PCR的指数增长阶段和平台阶段;在Tm为85℃,扩增产物的熔解曲线分析只出现1个单特异峰,无引物二聚体;所制作的标准曲线在2.7×108～2.7×102拷贝数之间有较好的线性关系,相关系数为0.998,能对副溶血弧菌进行准确的定量分析;该方法检测时间从核酸抽提到结果分析仅需4～5h,较传统方法敏感、操作简单,耗时短,是副溶血弧菌引起的水产动物疾病的快速诊断及食品中针对副溶血弧菌安全检测的有效手段。

A pair of specific primers target to toxR gene of V.parahaemolyticus was designed,and a real-time PCR using SYBR Green Ⅰ for V.parahaemolyticus detection was established.The results showed that the PCR primers could amplify 368 bp gene fragment from chromosomal DNA of V.parahaemolyticus,and no positive reaction was detected in 4 other pathogenic Vibrio species using conventional PCR.Amplification curves of SYBR GreenⅠreal time PCR revealed the geometric phase and plateau phase of PCR;the melting curve analysis using SYBR GreenⅠshowed one specific peak with a melting temperature（Tm）of 85℃,and no primer-dimers peak represent.Analysis of standard curves revealed excellent correlation between the quantity of bacteria（2.7×108 to 2.7×102） and PCR threshold cycle（Ct） values,the correlation rate was 0.998（R2=0.998）,indicating that the SYBR GreenⅠreal-time PCR could be used as an effective assay for quantification of pathogenic V.parahaemolyticus.The assay could be completed within 4-5 h from extraction of nucleic acids to analysis of resuts,the SYBR GreenⅠreal-time PCR was a simple,rapid,specific and sensitive method for the diagnose of aquatic animals disease and safety detection of food caused by V.parahaemolyticus.