猪伪狂犬病病毒gG、gE和TK基因多重PCR检测方法的建立及应用  

Establishment and Application of A multiplex PCR Assay for Detection of gG,gE and TK Gene of Porcine Pseudorabies Virus

作　　者：吴晓敏 陈润山 李华明 项维 徐梦然 杨荣荣 雷连成 张付贤 WU Xiao-min;CHEN Run-shan;LI Hua-ming;XIANG Wei;XU Meng-ran;YANG Rong-rong;LEI Lian-cheng;ZHANG Fu-xian(College of Animal Science,Yangtze University,Jingzhou,Hubei,434023,China;Animal Husbandry and Veterinary Service Center of Fang County,Shiyan,Hubei,442100,China;College of Veterinary Medicine,Jilin University,Changchun,Jilin,130062,China)

机构地区：[1]长江大学动物科学学院,湖北荆州434023 [2]湖北省十堰市房县畜牧兽医服务中心,湖北十堰442100 [3]吉林大学动物医学学院,吉林长春130062

出　　处：《动物医学进展》2024年第9期7-12,共6页Progress In Veterinary Medicine

基　　金：国家科技部“十四五”重点研发计划项目(2021YFD1800405);国家自然科学基金项目(32072823)。

摘　　要：建立准确鉴别猪群伪狂犬病病毒(Porcine pseudorabies virus,PRV)野毒株和基因缺失疫苗株高效多重PCR方法,对PRV gG、gE和TK基因保守区域设计合成特异性引物,优化反应体系和条件,建立多重PCR鉴别检测方法,对临床样品进行检测应用。结果成功建立了鉴别PRV gG、gE和TK基因缺失疫苗株和野毒株感染的多重PCR检测方法,该方法对PRV gG、gE和TK基因可进行特异性扩增,对猪圆环病毒2型、猪繁殖与呼吸综合征病毒、猪流行性腹泻病毒和猪副猪嗜血杆菌等相关病原均无扩增,对携带gG、gE和TK混合阳性质粒的最低检测限为2.5×10^(-5) ng/μL,具有良好的重复性。用该方法检测53份猪组织样品,检出PRV野毒感染的样品7份,与国标方法和单一PCR法检测结果符合率为100%。建立的猪伪狂犬病病毒野毒株和基因缺失疫苗株多重PCR鉴别检测方法具有良好的特异性、灵敏性和重复性,在生产中可快速、高效鉴别猪群PRV野毒感染和基因缺失疫苗免疫,可为PRV的快速鉴别检测、流行病学调查和防控提供技术支持。The aim of the study was to rapidly and efficiently diagnose porcine pseudorabies virus(PRV)and to distinguish PRV from vaccine strains.Nine pairs of specific primers were synthesized for genome sequencing of gG、gE and TK genes.The reaction conditions of this multiplex PCR were obtained after optimization,and specificity,sensitivity,reproducibility of multiplex PCR were determination.Results showed that the multiplex PCR could simultaneously detecting gG、gE and TK genes,and the limit was 2.5 × 10^(-5)ng/μL.PCV2,RRSV,PEDV,and Hps were not amplified by this method.Among the 53 samples from porcine,7 samples were positive based on the multiplex PCR for gE genes,which indicated that these 7 samples were infected by wild-type virus.These positive samples were the same samples based on single PCR.The concordance of the results based on two methods was 100%.In conclusion,the multiplex PCR could be used for the detection and epidemic logical investigation for pseudorabies virus.

关 键 词：猪伪狂犬病病毒 gG基因 GE基因 TK基因 多重PCR 

分 类 号：S852.65[农业科学—基础兽医学]