水貂阿留申病毒VP2基因TaqMan荧光定量PCR检测方法的建立与应用  

Establishment and Application of TaqMan Real-time PCR Assay for Detection of VP2 Gene of Aleutian Mink Disease Virus

作　　者：闫文卓 陆涛峰 周洁 赵丽丽[1] 陈洪岩[1] YAN Wenzhuo;LU Taofeng;ZHOU Jie;ZHAO Lili;CHEN Hongyan(State Key Laboratory of Veterinary Biotechnology.Heilongiang Provincial Key Laiboratory of Laboratory Animal and Comparative Medicine,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China;Institute for Laboratory Animal Research,Guizhou University of Traditional Chinese Medicine,Guiyang 550025,China;Shanghai Laboratory Animal Research Center,Shanghai 201203,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所,兽医生物技术国家重点实验室,黑龙江省实验动物与比较医学重点实验室,哈尔滨150069 [2]贵州中医药大学实验动物研究所,贵阳550025 [3]上海实验动物研究中心,上海201203

出　　处：《实验动物与比较医学》2020年第4期289-295,共7页Laboratory Animal and Comparative Medicine

基　　金：国家自然科学基金项目(31700140)。

摘　　要：目的建立一种方便、灵敏的水貂阿留申病毒(AMDV)荧光定量PCR检测方法,从而实现临床样品中AMDV的快速定量检测。方法根据AMDV的结构蛋白VP2的基因保守区域设计一对特异性引物和探针,优化PCR反应条件,建立TaqMan实时荧光定量PCR检测方法,绘制标准曲线,并进行特异性、敏感性和稳定性分析。结果建立的标准曲线在1.0×10^2拷贝/μL至1.0×10^8拷贝/μL之间具有良好的线性关系,相关系数(r^2)大于0.994。该方法检测的灵敏度为10^2拷贝/μL,且与犬细小病毒、犬腺病毒、犬瘟热病毒和水貂病毒性肠炎病毒均无明显交叉反应,特异性良好。重复性试验结果显示,该方法的组内和组间变异系数均小于3%,重复性良好。利用该方法对417份临床组织样品进行检测的结果显示,中国部分地区的AMDV阳性率为81.5%。结论本研究建立了一种重复性、特异性和敏感性良好的AMDV荧光定量PCR检测方法,可用于AMDV的临床检测和流行病学调查。Objective To establish a convenient and sensitive fluorescent quantitative PCR method for the detection of Aleutian mink disease virus(AMDV),aim to implement the rapid quantitative detection of AMDV in clinical samples.Methods According to the conserved region of VP2 gene of AMDV,a pair of specific primers and a probe were designed.The reaction condition was optimized,and the TaqMan real-time fluorescent quantitative PCR detection method was established.The standard curve was mapped,and the specificity,sensitivity and stability of the method were analyzed.Results The correlation coefficient(r^2)of the standard curve was more than 0.994,and it presented a linear relationship in the concentration of the template DNA ranging from 10^2 copies/μL to10^8 copies/μL.The assay was highly specific for amplification from AMDV,but no amplification from CPV,MEV,CDV and CAV.The sensitivity of AMDV detection was 10~2 copies/μL,and the intra-group and inter-group reproducibility tests showed that the variation coefficient of the method was less than 3%.The result of 417 clinical tissue samples tested by this method showed that the positive rate of AMDV was 81.5%in some areas of China.Conclusion A reproducible,specific and sensitive AMDV real-time PCR detection method is established,which can be used for clinical detection and epidemiological investigation of AMDV.

关 键 词：水貂阿留申病 水貂阿留申病毒 实时荧光定量PCR VP2 检测方法 

分 类 号：Q95-33[生物学—动物学]