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| 基于gyrB基因的SYBR Green I实时定量PCR检测病原灿烂弧菌 |
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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
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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
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| 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 |
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| Development of a real-time PCR method for the detection of Vibrio splendidus based on gyrB gene |
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| 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 |