鸭圆环病毒实时荧光定量PCR检测方法的建立及初步应用  

Establishment of Real-time PCR Detection Method of Duck Circovirus and Preliminary Application

作　　者：刘玲凤[1] 黎振标 司兴奎[1] 

机构地区：[1]佛山科学技术学院生命科学与工程学院,广东佛山528231

出　　处：《中国家禽》2017年第7期15-19,共5页China Poultry

基　　金：广东省高等学校高层次人才项目(粤财教[2011]431号-132)

摘　　要：为实现对鸭圆环病毒(DuCV)的快速检测,分析比对Gen Bank中登录的DuCV全基因组序列,在其保守区设计并合成一对特异性引物,将扩增的212 bp片段克隆入pMD-18T载体中,获得pMD18DuCV重组质粒,通过SYBR GreenⅠ荧光掺入法分别对系列稀释pMD18DuCV中DuCV特异性片段进行扩增,通过pMD18DuCV浓度对数与Ct值制作了标准曲线并推导出直线回归方程,二者相关系数(R^2)为0.9968,建立了DuCV实时荧光定量PCR检测方法,重复性和灵敏性检测结果表明重复性良好,检测下限为67.2拷贝/μL;将建立的DuCV实时荧光定量PCR检测方法分别对鸭肝炎病毒(DHV)、大肠杆菌、新城疫病毒(NDV)、H9亚型禽流感病毒、小鹅瘟病毒(GPV)核酸或cDNA进行特异性检测,结果表明特异性良好。初步应用结果表明,建立的DuCV实时荧光定量PCR方法检出率为26.4%(62/235),明显优于常规PCR检测结果15.3%(36/235)。To establish the rapid detection method for duck circovirus （DuCV）,whole genome sequences registered in GenBank were analyzed and compared,and a pair of specific primers for amplification of 212 bp fragment of conserved region were designed and synthesized,and the recombinant plasmid nominated by pMD18DuCV was obtained in which the specific fragment of DuCV was cloned into pMD-18T vector. With the serially diluted pMD18DuCV plasmids were amplified by SYBR Green I fluorescence incorporation, a standard curve and deduced linear regression equation was obtained by the logarithm of pMD18DuCV concentration and Ct values with the correlation coefficient （R2） value 0.9968,the reproducibility and sensitivity were detected respectively. The resuhs showed that the detection limit was 67.2 copies/μL with excellent reproducibility. The specificity of this method was detected with nucleotide of cDNA of duck hepatitis virus （DHV） ,E. coli,Newcastle disease virus （NDV）,H9 subtype avian influenza virus,goose parvovirus （GPV）,respectively,and the results indicated the specificity was satisfied. Preliminary applied results showed that the detection rate of DuCV real-time PCR method was 26.4% （62/235）,which was more sensitive compaired with 15.3% （36/235）detection rate of conventional PCR method.

关 键 词：鸭圆环病毒 实时荧光定量PCR SYBR GreenⅠ 

分 类 号：S855.3[农业科学—临床兽医学]