基于QIAxcel毛细管凝胶电泳分析系统的7种猪病毒检测方法的建立与初步应用  

Establishment and preliminary application of multiplex PCR based on QIAxcel capillary gel electrophoresis to detect seven porcine viral pathogens

作　　者：邬旭龙[1,2] 肖璐 林华 杨泽晓 姚学萍[1] 王印[1] 张鹏飞[1] 姜睿姣[1] WU Xu-long;XIAO Lu;LIN Hua;YANG Ze-xiao;YAO Xue-ping;WANG Yin;ZHANG Peng-fei;JIANG Rui-jiao(College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China;Chengdu Agricultural Science and Technology Vocational College,Chengdu 611134,China;Sichuan Animal Science Academy,Chengdu 610066,China;Chengdu customs district,Chengdu 610041,China)

机构地区：[1]四川农业大学动物医学院,四川成都611130 [2]成都农业科技职业学院,四川成都611134 [3]四川省畜牧科学研究院,四川成都610066 [4]成都海关,四川成都6l004l

出　　处：《中国预防兽医学报》2019年第8期824-829,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：“十二五”国家科技支撑计划(2013BAD12B04)

摘　　要：为建立同时检测7种常见猪病毒的毛细管凝胶电泳方法,本研究针对猪伪狂犬病病毒、猪乙型脑炎病毒、猪瘟病毒、猪圆环病毒2型、猪繁殖与呼吸综合征病毒、猪细小病毒及非洲猪瘟病毒保守基因,设计7对特异引物,并在引物的5’端加入一段非同源性标签序列,再设计一对可识别标签序列的通用引物。经反应条件优化,建立了一种基于QIAxcel毛细管凝胶电泳分析系统的7种猪常见病毒多重PCR检测方法。该方法与O型猪口蹄疫病毒(FMDV-O)、猪传染性胃肠炎病毒(TGEV)等均无交叉反应,特异性强;所建立方法对7种病毒质粒标准品的检测下限分别为4.56×10^3拷贝/μL、6.35×10^3拷贝/μL、1.98×10^3拷贝/μL、1.32×10^4拷贝/μL、3.21×10^3拷贝/μL、4.51×10^3拷贝/μL、3.37×10^3拷贝/μL,敏感性较高;重复性试验结果显示各靶条带数值间的变异系数均小于1%,该方法稳定,重复性高。该方法对65份临床样本检测结果与国标或行标单一PCR检测方法相比,二者对单一病原和混合病原检测的一致性分别为94.3%(33/35)、77.7%(14/18)。本研究建立的方法为毛细管凝胶电泳技术在动物疾病病原检测中的应用提供一定的参考。To establish a novel method for detection of 7 porcine viral pathogens simultaneously, seven pairs of primers were designed according to PRV, JEV, CSFV, PCV-2, PRRSV, PPV and ASFV conservative genes regions. Non-homologous tag sequences were introduced into 5’ end of every primer, and a pair of general primers is designed to recognize the tag sequence..After optimizing reaction conditions, a multiplex PCR for simultaneous detection of PRV, JEV, CSFV, PCV-2, PRRSV and PPVwith a QIAxcel capillary gel electrophoresis analyzer was established. Specificity test showed that this method was highly specific,and no cross reaction with other swine viral pathogens, such as O type foot-and-mouth disease virus(FMDV-O), transmissible gastroenteritis of swine(TGEV) and so on. Sensitivity test showed the minimum detection limit for these seven viruses were 4.56 ×10^3 copies/μL, 6.35×10^3 copies/μL, 1.98×10^3 copies/μL, 1.32×10^4 copies/μL, 3.21×10^3 copies/μL, 4.51×10^3 copies/μL, 3.37×10^3 copies/μL, respectively. The results of repeatability test showed that the coefficient of variation of each target bandwas less than1%. 65 clinical samples were detected by using this multiplex PCR method. Compared with the national standard or standard single PCR method, the consistency of single pathogen and mixed pathogen was 94.3%(33≤35) and 77.7%(14≤18), respectively. The method established in this study provides useful references for the application of capillary gel electrophoresis in the detection of animal epidemic diseases.

关 键 词：多重PCR QIAxcel毛细管凝胶电泳(QIAxcel CGE) 猪伪狂犬病病毒 猪乙型脑炎病毒 猪瘟病毒 猪圆环病毒2型 猪繁殖与呼吸综合征病毒 猪细小病毒 非洲猪瘟病毒 

分 类 号：S852.65[农业科学—基础兽医学]