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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
于永翔,王印庚,刘智超,廖梅杰,张正,荣小军,李彬,战文斌
作者单位
于永翔 中国海洋大学水产学院青岛 266100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 
王印庚 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 
刘智超 中国海洋大学水产学院青岛 266100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 
廖梅杰 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 
张正 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 
荣小军 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 
李彬 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 
战文斌 中国海洋大学水产学院青岛 266100 
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