基于vhhP2基因的SYBR Green I实时定量PCR检测哈维氏弧菌方法的建立  

Development of a SYBR Green I real-time PCR for detection of Vibrio harveyi based on the vhhP2 gene

作　　者：廖梅杰[1] 张正[1] 荣小军[1] 王印庚[1] 刘智超[1] 栾晶[2] 李彬[1] 王岚[1] 陈贵平[1] 

机构地区：[1]中国水产科学研究院黄海水产研究所、农业部海洋渔业可持续发展重点实验室,山东青岛266071 [2]山东省出入境检验检疫局,山东青岛266001

出　　处：《中国水产科学》2014年第3期611-620,共10页Journal of Fishery Sciences of China

基　　金：国家863计划项目(2012AA10A412-4);国家科技支撑计划项目(2012BAD17B03);国家自然科学基金项目(31202-016);科研院所技术开发研究专项项目(2011EG34219);青岛市战略性新兴产业培育计划项目(13-4-1-65-hy);山东省优秀中青年科学家奖励基金项目(BS2010SW035)

摘　　要：根据哈维氏弧菌(Vibro harveyi)溶血素基因vhhP2的保守序列设计特异性引物,建立了SYBR Green I实时定量PCR检测哈维氏弧菌的方法。构建含vhhP2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60℃时,扩增产物的熔解曲线仅有一个特异峰,扩增所得标准曲线为y=–3.331x+37.48,相关系数为0.998,扩增效率为1,最低可检测到7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,对哈维氏弧菌病的快速诊断和流行病学调查有重要意义。Vibrio harveyi is an important pathogenic bacterium of most marine aquaculture animals that has caused sig-nificant losses within the aquaculture industry. The pathogenicity of V. harveyi is influenced by quorum sensing, mean-ing that population density plays a role in determining the outcome of an infection. Thus, there is a need to develop a method to detect the density of Vibrio harveyi. We designed a pair of specific primers based on the V. harveyi spe-cies-specific vhhP2 gene to establish a SYBR Green I real-time fluorescence quantitative PCR detection method. A 151 bp gene fragment was amplified from the chromosomal DNA of V.harveyi from different sources. The primers did not cross react with nine other bacteria species using conventional PCR, suggesting the primer pair has good intra-species specificity and inter-species commonality. A recombinant plasmid containing the vhhP2 gene of V. harveyi was con-structed and used to construct the standard curve. The standard curve for the Ct values and initial template was repre-sented by the formula：y=-3.331x＋37.48. The correlation coefficient was 0.998 and the amplification efficiency was 1.00, indicating that there was a good linear relationship between initial templates and Ct values. The melting curve had only one specific peak at an annealing temperature of 60℃. The detection limit of the assay was seven copies per reac-tion, which is 10 000 times more sensitive than that of conventional polymerase chain reaction （PCR）. The results of intra-and inter-assay variability tests demonstrated that the method was highly reproducible. Our results suggest that this SYBR Green I real-time PCR assay may be used for the rapid and accurate detection of V. harveyi from infected aquaculture species. This will allow early diagnosis of V. harveyi infection and improve the efficacy of disease preven-tion and surveillance programs.

关 键 词：vhhP2基因 哈维氏弧菌 实时定量PCR SYBR Green I 

分 类 号：S942[农业科学—水产养殖]