基于lolB基因的SYBR GreenⅠ实时定量PCR检测病原霍乱弧菌方法的建立

Development of a SYBR GreenⅠreal-time PCR for detection of Vibrio cholerae based on lolB gene

中文关键词: 霍乱弧菌  lolB基因  SYBR GreenⅠReal-time PCR

英文关键词: Vibrio cholerae  lolB gene  SYBR GreenⅠReal-time PCR

中文摘要:

      基于霍乱弧菌lolB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检测霍乱弧菌的方法。常规PCR检测仅霍乱弧菌扩增出大小为519bp的目的片段，其他3种病原弧菌均为阴性；SYBR GreenⅠ实时定量PCR，在Tm为86.5~87 ℃，扩增产物的熔解曲线只出现1个单特异峰，无引物二聚体，表明该引物具有较好的特异性。SYBR GreenⅠ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；所制作的标准曲线在2.59×108~2.59×100拷贝数之间有较好的线性关系，相关系数为0.993，能对霍乱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4~5 h，较传统方法敏感、操作简单、耗时短，是霍乱弧菌引起的水产动物疾病的快速诊断及流行病调查的有效方法。

英文摘要:

      The lolB gene has been shown to code an outermembrane lipoprotein, and serve as a reliable molecular marker for the detection of all V.cholerae serogroup and biotypes. A pair of specific primers based on lolB gene of V.cholerae was designed, and a real-time PCR using SYBR Green I for V.cholerae detection was established. A 519bp gene fragment was amplified from chromosomal DNA of V.cholerae, and no positive reaction was detected in 3 other pathogenic Vibrio species using conventional PCR. The melting curve analysis of SYBR GreenⅠreal-time PCR showed one specific peak with melting temperature（Tm）of 86.5~87 ℃, and no primer-dimers peak present. The results indicated that the PCR primers have good specificity. Amplification curves of SYBR GreenⅠreal-time PCR revealed the geometric phase and plateau phase of PCR. Analysis of standard curves revealed excellent correlation between the quantity of bacteria (2.59×108 to 2.59×100) and PCR threshold cycle (Ct),with the correlation rate of 0.993. The results indicated that the SYBR GreenⅠreal-time PCR could be used as an effective assay for quantification of pathogenic V.cholerae. The assay could be completed within 4~5 h from extraction of nucleic acids to analysis of results. The SYBR GreenⅠreal-time PCR was a simple, rapid, specific and sensitive method for the diagnosis of aquatic animal diseases and investigation of epidemics caused by V.cholerae.