《中国畜牧兽医》 ›› 2019, Vol. 46 ›› Issue (5): 1532-1540.doi: 10.16431/j.cnki.1671-7236.2019.05.033

• 临床兽医 • 上一篇    下一篇

多重PCR结合基因芯片技术检测5种猪繁殖障碍性病毒病方法的研究

刘胜利1, 刘玲玲2, 吕玉金2, 吴凤笋2, 李文刚2   

  1. 1. 河南农业大学牧医工程学院, 郑州 450002;
    2. 河南牧业经济学院动物医学院, 郑州 450046
  • 收稿日期:2018-10-08 出版日期:2019-05-20 发布日期:2019-05-20
  • 通讯作者: 李文刚 E-mail:wengang-li@126.com
  • 作者简介:刘胜利(1992-),女,河南濮阳人,硕士,研究方向:动物传染病,E-mail:1179388498@qq.com
  • 基金资助:

    河南牧业经济学院预防兽医学重点学科(MXK2016102);2016年河南省创新型科技团队

Research on Multiplex PCR Combined with DNA Microarray for Detecting Five Kinds of Reproductive Disease Virus

LIU Shengli1, LIU Lingling2, LYU Yujin2, WU Fengsun2, LI Wengang2   

  1. 1. College of Livestock Husbandry and Veterinary Engineering, Henan Agricultural University, Zhengzhou 450002, China;
    2. College of Veterinary Medicine, Henan University of Auimal Husbandry Economy, Zhengzhou 450046, China
  • Received:2018-10-08 Online:2019-05-20 Published:2019-05-20

摘要:

为建立运用多重PCR和基因芯片技术同时检测5种猪繁殖障碍性病毒病的方法。本研究根据GenBank中登录的猪瘟病毒(CSFV)、猪细小病毒(PPV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪日本乙型脑炎病毒(JEV)及猪圆环病毒2型(PCV2)的基因序列设计特异性引物与探针,制备相应的寡核苷酸芯片,检测了5种猪繁殖障碍性疾病病毒的标准毒株,并对16份临床样品进行检测。通过多重PCR扩增出带有荧光标记的5种病毒的特异性基因片段,并与固定有特异性探针的基因芯片杂交。结果显示,本研究建立的多重PCR结合基因芯片检测方法特异性强、稳定性好,灵敏度可达102拷贝/μL。16份临床样品检测结果显示阳性率达87.5%(14/16)。以上结果表明该方法特异性好、灵敏度高,可高效检测以上5种病毒,为其临床诊断及流行病学调查提供了有效的检测方法。

关键词: 多重PCR; 基因芯片; CSFV; PPV; PRRSV; JEV; PCV2

Abstract:

A multiplex PCR combined with DNA microarray method was developed for detection of 5 swine reproductive diseases virus in this study.Five pairs of specific primers and probes were designed according to the published genomic sequences of swine fever virus (CSFV),porcine parvovirus (PPV),porcine reproductive and respiratory syndrome virus (PRRSV),Japanese encephalitis virus (JEV) and porcine circovirus type 2 (PCV2).The corresponding oligonucleotide chips were prepared,16 clinical samples and the standard strains of five reproductive viruses were tested.Specific gene fragments with fluorescent label were amplified by multiplex PCR and hybridized with microarray that immobilized with specific probes.The results showed that the multiplex PCR combined with DNA microarray to detect viral diseases was highly specific and stable,and the sensitivity was up to 102 copies/μL.The test results of 16 clinical samples showed a positive rate of 87.5% (14/16).These data indicated that the sensitive and specific detection method could be applied in these 5 viruses detection for clinical diagnosis and pathogenic epidemiologic investigation.

Key words: multiplex PCR, microarray, CSFV, PPV, PRRSV, JEV, PCV2

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