猪圆环病毒2型和4型双重PCR检测方法的建立  

Development of a duplex PCR for detection of porcine circovirus type 2 and 4

作　　者：徐通 侯承尧 田润博 崔建涛[1] 赵丽[2] 陈红英[1] XU Tong;HOU Chengyao;TIAN Runbo;CUI Jiantao;ZHAO Li;CHEN Hongying(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450002,China;College of Veterinary Medicine,Henan University of Animal Husbandry and Economy,Zhengzhou 450046,China)

机构地区：[1]河南农业大学动物医学院,河南郑州450002 [2]河南牧业经济学院动物医药学院,河南郑州450046

出　　处：《中国兽医学报》2021年第8期1490-1494,共5页Chinese Journal of Veterinary Science

基　　金：中原千人计划资助项目(204200510015);河南省高校科技创新团队支持计划资助项目(19IRTSTHN007);河南牧业经济学院博士启动资金资助项目(2020HNUAHEDF010)。

摘　　要：旨在建立一种能同时快速检测猪圆环病毒2型(PCV2)和4型(PCV4)的双重PCR方法。参考GenBank中PCV2和PCV4基因组序列,分别在PCV2 Rep和PCV4 Cap基因高度保守区设计2对特异性引物,经双重PCR条件优化,建立同时快速检测PCV2/4的双重PCR方法。该方法能同时扩增出264 bp(PCV2)和391 bp(PCV4) 2条特异性片段,而对其他8种常见猪病原扩增均为阴性;PCV2/4的最低检测量分别为61.1,54.9拷贝/μL。应用该方法对河南及山西省内60份临床样品进行检测,检测结果表明PCV2的检出率为51.67%(31/60),PCV4的检出率为18.33%(11/60),混合感染检出率为16.67%(10/57),单一PCR及双重PCR检测结果符合率100%。本研究建立的双重PCR检测方法具有敏感性高、特异性强、重复性好等优点,适合对PCV2和PCV4鉴别诊断和检测。To develop a rapid duplex PCR assay for simultaneous detection of PCV2 and PCV4,two pairs of specific primers were designed and synthesized according to the conserved regions of the Rep gene of PCV2 and Cap gene of PCV4.The duplex PCR detecting PCV2 and PCV4 simultaneously was successfully established by optimizing amplification conditions.The specific fragments of PCV2 and PCV4 were simultaneously amplified by the duplex PCR from samples containing PCV2 and PCV4,but no PCR products were amplified from other common pathogens.The detection limits of the assay were 61.1 copies/μL for PCV2 and 54.9 copies/μL for PCV4.A total of 60 clinical samples of porcine from Henan province and Shanxi province were collected and detected by the single PCR assay and duplex PCR assay.The detection results showed that the positive rate of PCV2 was 51.67%(31/60),the positive rate of PCV4 was 18.33%(11/60),the positive rate of PCV2 and PCV4 co-infection was 16.67%(10/57)and the coincidence between single PCR and duplex PCR was 100%.The results suggested that the duplex PCR established in this study is characterized by convenience,high sensitivity,stability and specificity,which provides effective support for differentiating the clinical samples with PCV2 and PCV4 co-infection.

关 键 词：猪圆环病毒2型 猪圆环病毒4型 双重PCR 

分 类 号：S852.65[农业科学—基础兽医学]