迟缓爱德华氏菌SYBR Green I实时荧光定量PCR检测方法的建立及其应用

Development of an SYBR Green I real-time PCR assay for detection of Edwardsiella tarda and its application

根据克隆得到的迟缓爱德华氏菌gyrB基因序列设计并合成一对特异性引物,通用性和特异性检测结果显示所设计的引物具有良好的种间特异性和种内通用性,构建含gyrB基因的重组质粒作为标准品,经过反应体系优化后建立了检测迟缓爱德华氏菌的SYBR Green I实时荧光定量PCR检测方法。结果显示,该方法线性关系良好,在T m为63℃时,扩增产物的熔解曲线仅有一个单特异峰,扩增所得标准曲线为y=-3.32x+39.38,相关系数为0.998,扩增效率为1.00,最低能检测到60个拷贝。应用建立的方法对人工感染的大菱鲆病样进行了检测,3个被检样品均呈阳性反应,证明该方法具有较好的适用性。研究表明,所建立的实时荧光定量PCR方法具有特异、敏感、快速、定量的优点,可用于迟缓爱德华氏菌病的快速检测。

According to the sequenced gyrB gene sequence of Edwardsiella tarda, a pair of primers was designed for establishing an SYBR Green I real-time fluorescence quantitative PCR method. A 207 bp gene fragment was amplified from chromosomal DNA of E. tarda from different sources,and no positive reaction was detected in 9 other bacteria species using conventional PCR, which indicated that the primer pair has good inter-species specificity and intra-species commonality. Recombinant plasmid containing gyrB gene of E. tarda was constructed and used to construct the standard curve. The standard curves was y = - 3.32x ＋ 39.38,the correlation coefficient was 0. 998 and the amplification efficiency was 1.00, which indicated that it had a good linear relationship between initial templates and Ct values. The melting curve has only one specific peak when annealing temperature was 63 ℃. The detection limit of the assay was 60 copies per reaction. Turbot samples infected by E. tarda artificially were detected using the real-time PCR assay. All the three samples were positive, which had good agreement with bacteriological analysis by isolation and culture. The results showed that the developed SYBR Green I real-time PCR assay had the advantages of specificity, sensitivity, rapidity and quantification, and would be helpful for E. tarda diagnosis and epidemiology investigation.