鲤科疱疹病毒2型TaqMan荧光定量PCR检测方法的建立  

Establishment of real-time PCR assay for detection of Cyprinid herpesvirus 2

作　　者：徐晔[1] 段宏安[1] 周毅[1] 丁超[1] 

机构地区：[1]连云港出入境检验检疫局,江苏连云港222042

出　　处：《中国预防兽医学报》2014年第12期952-956,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家质检总局公益项目(201310221)

摘　　要：为建立快速和敏感的检测鲤科疱疹病毒2型(Cy HV-2)的方法,本研究根据Cy HV-2 DNA聚合酶基因序列合成引物和Taq Man探针,建立了Cy HV-2荧光定量PCR检测方法。结果显示,以重组质粒为标准品建立的标准曲线在5拷贝/μL^5×108拷贝/μL具有良好的线性关系,相关系数为0.999;其最低检出量为5拷贝/μL,比普通PCR的敏感度高100倍。该方法仅对Cy HV-2的靶基因序列进行扩增,而对锦鲤疱疹病毒、流行性造血器官坏死病病毒、病毒性出血性败血症病毒、传染性胰脏坏死病病毒及鲤春病毒血症病毒核酸扩增结果均为阴性。组内和组间重复试验变异系数的平均值分别为0.5%和0.3%,具有良好的重复性。与常规PCR相比较,该方法具有快速、敏感、特异及高通量检测等优点,适用于对Cy HV-2的快速检测。A real-time PCR method that quantitatively detects Cyprinid herpesvirus 2 （CyHV-2） was developed,in the present study.The standard curve of the method had a linear response from 5 copies/μL to 5×108 copies/μL of target recombinant plasmid.The sensitivity of this assay was approached 5 copies/μL,which was 100 times higher than that of ordinary PCR.The assay had no cross-reaction with other related fish herpesvirus and common fish pathogens,including cyprinid herpesvirus 3,epizootic haematopoietic necrosis virus,viral haemorrhagic septicaemia virus,infectious pancreatic necrosis virus and spring viraemia of carp virus.The coefficient of variability on the inter-and intra-variation were less than 0.5％ and 0.3％,respectively.The assay established in this study had potentially application in clinical detections and survey of CyHV-2.

关 键 词：鲤科疱疹病毒2型 荧光定量PCR 检测 

分 类 号：S852.65[农业科学—基础兽医学]