猪流行性腹泻病毒和猪圆环病毒4型双重SYBR GreenⅠ实时荧光定量PCR方法的建立  

Development of a SYBR GreenⅠ-based real-time PCR assay for detection of porcine epidemic diarrhea virus and porcine circovirus 4

作　　者：李洪炫 滑宇鹏 彭琳颖 高家俊 陈琳琳[1] 李法昊 金钺[1] 陈红英[1] LI Hongxuan;HUA Yupeng;PENG Linying;GAO Jiajun;CHEN Linlin;LI Fahao;JIN Yue;CHEN Hongying(College of Animal Science and Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China)

机构地区：[1]河南农业大学、动物医学院,河南郑州450046

出　　处：《中国兽医学报》2023年第1期23-28,共6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人才支持计划资助项目(21HASTIT039)。

摘　　要：分别靶向猪流行性腹泻病毒(PEDV)M基因和猪圆环病毒4型(PCV4)ORF2基因,合成了2对特异性引物,建立了同时检测PEDV和PCV4的双重SYBR GreenⅠ荧光PCR检测方法。结果显示,PEDV和PCV4在一个反应体系中出现明显不同的熔解峰(Tm),PEDV的Tm值为84℃,PCV4的Tm值为79℃,而对其他常见的非靶标猪病毒没有显示特定的熔解峰,且对于PEDV和PCV4的最低检测极限分别为55.2,44.1拷贝/μL。利用本研究建立的双重荧光PCR方法对30份猪临床样品进行检测,结果显示,PEDV和PCV4的检出率分别为40%(12/30)和60%(18/30),PEDV和PCV4混合感染的检出率为33%(10/30),相较于常规PCR方法更加灵敏。该方法能够同时对PEDV和PCV4进行检测,极大地提高了检测效率和准确度,为PEDV和PCV4的诊断与防控提供技术支持。Two pairs of primers were synthesized according to the M gene of PEDV and the ORF2 gene of PCV4.A SYBR GreenⅠ-based quantitative real-time PCR(qPCR)assay for detection of PEDV and PCV4 was established after optimizing the amplification conditions.The results showed that PEDV and PCV4 had distinct different melting temperatures(Tm)in one reaction system.The Tmvalue of PEDV was 84℃,and the Tmvalue of PCV4 was 79℃,while no specific melting peak was shown for other non-targeted porcine common viruses.The detection limits were 55.2 copies/μL of PEDV and 44.1 copies/μL of PCV4,respectively.Thirty porcine clinical samples were screened by a duplex SYBR Green I-based qPCR developed in this study.The results of qPCR showed that the positive rates of PEDV and PCV4 were 40%(12/30),60%(18/30).The positive rate of co-infection with PEDV and PCV4 was 33%(10/30).This method can detect PEDV and PCV4 at the same time,greatly improving the detection efficiency and accuracy,and providing technical support for the diagnosis,prevention and control of PEDV and PCV4.

关 键 词：PEDV PCV4 荧光定量PCR 

分 类 号：S852.65[农业科学—基础兽医学]