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

中文关键词: gyrB基因  灿烂弧菌  实时定量PCR  SYBR Green I

英文关键词: gyrB gene  Vibrio splendidus  Real-time quantitative PCR  SYBR Green I

中文摘要:

      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。

英文摘要:

      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.