引用本文:
【打印本页】   【HTML】   【下载PDF全文】   查看/发表评论  【EndNote】   【RefMan】   【BibTex】
←前一篇|后一篇→ 过刊浏览    高级检索
本文已被:浏览 1630次   下载 657 本文二维码信息
码上扫一扫!
分享到: 微信 更多
基于gyrB基因的SYBR Green I实时定量PCR检测病原灿烂弧菌
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
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
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。
关键词:  gyrB基因  灿烂弧菌  实时定量PCR  SYBR Green I
DOI:10.11758/yykxjz.20140319
分类号:
基金项目: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)和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
Development of a real-time PCR method for the detection of Vibrio splendidus based on gyrB gene
Abstract:
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 commonality. 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.
Key words:  gyrB gene  Vibrio splendidus  Real-time quantitative PCR  SYBR Green I