基于gyrB基因的SYBR Green I实时定量PCR检测病原灿烂弧菌  

Development of a real-time PCR method for the detection of Vibrio splendidus based on gyrB gene

作　　者：于永翔[1,2] 王印庚[2] 刘智超[1,2] 廖梅杰[2] 张正[2] 荣小军[2] 李彬[2] 战文斌[1] 

机构地区：[1]中国海洋大学水产学院,青岛266100 [2]农业部海洋渔业可持续发展重点实验室,中国水产科学研究院黄海水产研究所,青岛266071

出　　处：《渔业科学进展》2014年第3期134-142,共9页Progress in Fishery Sciences

基　　金：十二五863项目(2012AA10A412-4);科研院所技术开发研究专项项目(2011EG34219);国家科技支撑计划课题(2012BAD17B03);国家自然科学基金项目(30901120;31202016);青岛市战略性新兴产业培育计划项目(13-4-1-65-hy)共同资助

摘　　要：灿烂弧菌Vibrio splendidus是多数海水养殖动物的主要致病菌,对养殖业危害较大。本研究根据灿烂弧菌gyrB基因的保守序列设计特异性引物,建立了SYBR Green I实时定量PCR检测灿烂弧菌的方法。构建含gyrB基因的重组质粒作为标准品,进行SYBR Green I实时定量PCR,在Tm为62℃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=-3.338x+37.67,相关系数为0.999,扩增效率为0.99,最低能检测到20个拷贝。实验结果表明,该检测技术具有较高的特异性、敏感性和重复性,对灿烂弧菌病的快速诊断和流行病学调查有重要意义。Vibrio splendidus is a deleterious pathogenic bacterium for most marine animals, and it causes great losses in aquaculture industry. To develop a quantitative detection method of V. splendidus is important because its pathogenicity is closely related to the population density. To develop a rapid SYBR Green I real-time fluorescence quantitative PCR method, a pair of specific primers were designed according to V. splendidus gyrB gene to determine the specificity and sensitivity. The real-time PCR amplification conditions were optimized. Recombinant plasmid containing gyrB gene of V. splendidus was constructed and used to establish the standard curve. The detection limit and reproducibility were calculated. A 251 bp fragment was amplified from chromosomal DNA, but no positive reaction was detected in 9 other bacteria species using conventional PCR, which indicated that the primer pair has good intra-species specificity and inter-species commonafity. The standard curve was y = -3. 338x ＋ 37.67; the correlation coefficient was 0. 999 and the amplification efficiency was 0.99, indicating a good linear relationship between initial templates and CT values. The melting curve had only one specific peak when annealing temperature was 62℃. The detection limit of the assay was 20 copies per reaction. The results indicated that the established SYBR Green I real-time fluorescence quantitative PCR method for V. splendidus had high specificity, sensitivity and repeatability, which may help V. splendidus diagnosis and epidemiology.

关 键 词：gyrB基因 灿烂弧菌 实时定量PCR SYBR Green I 

分 类 号：S963.7[农业科学—水产养殖]