生物技术通报 ›› 2018, Vol. 34 ›› Issue (11): 205-209.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0365

• 研究报告 • 上一篇    下一篇

猪PEDV与PDCoV二重RT-PCR检测方法的建立

孔维欢1,2, 王晶晶1,2, 万鹏程2, 石国庆2   

  1. 1. 石河子大学动物科技学院,石河子 832000;
    2. 新疆农垦科学院畜牧兽医研究所,石河子 832000
  • 收稿日期:2018-04-17 出版日期:2018-11-26 发布日期:2018-11-28
  • 作者简介:孔维欢,男,硕士研究生,研究方向:动物遗传育种与繁殖;E-mail:919282632@qq.com
  • 基金资助:
    国家重点研发计划项目(2017YFD0501904,2017-2020)

Establishment of a Duplex RT-PCR Detection Method for Porcine PEDV and PDCOV

KONG Wei-huan1,2, Wang Jing-jing1,2, WAN Peng-cheng2, SHI Guo-qing2   

  1. 1. College of Animal Science and Technology,Shihezi University,Shihezi 832000;
    2. Animal Husbandry and Veterinary Institute,Xinjiang Academy of Agricultural and Reclamation Science,Shihezi 832000
  • Received:2018-04-17 Published:2018-11-26 Online:2018-11-28

摘要: 旨在建立能同时检测出猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)和猪丁型冠状病毒(Porcine delta corona virus,PDCOV)的二重RT-PCR检测方法。根据GenBank已收录发表的PEDV和PDCOV基因序列设计2对特异性引物,首先运用RT-PCR反应技术,通过PEDV和PDCOV病毒的目的基因的单项扩增,对反应条件进行优化。然后通过特异性试验、敏感性试验以及临床样品的检测验证确定二重RT-PCR方法的建立。结果显示,目的基因PEDV-M扩增的片段大小是750 bp,PDCOV-N扩增的片段大小是372 bp;能够同时检测出PEDV和PDCOV目的基因,却检测不到PRV、CSFV、PPV三种病毒;体系优化扩增PEDV-PDCOV混合核酸浓度下限为100 pg/μL;能够初步诊断出临床疑似病毒感染的病例。结果表明,成功的建立PEDV-PDCOV二重RT-PCR检测方法,此方法敏感性高、特异性强,是一种能够快速有效的检测出猪PEDV-PDCOV单病毒感染或多病毒混合感染的临床诊断方法。

关键词: PEDV, PDCOV, RT-PCR, 临床诊断

Abstract: The aim of this study is to establish a double RT-PCR assay for simultaneously detecting porcine epidemic diarrhea virus(PEDV)and porcine delta corona virus(PDCOV). Two pairs of specific primers were designed according to the sequences of PEDV and PDCOV genes published in GenBank. Firstly,the RT-PCR reaction technique was used to optimize the reaction conditions through the single amplification of the target genes of PEDV and PDCOV viruses. Then,the establishment of double RT-PCR method was confirmed by specificity test,sensitivity test and clinical sample detection. Results showed as:The size of the amplified target gene PEDV-M was 750 bp,and 372 bp for PDCOV-N fragment;PEDV and PDCOV genes were detected simultaneously,but 3 viruses of PRV,CSFV and PPV were not detected. The minimum concentration limit of mixed nucleic acid concentration for optimizing and amplifying PEDV-PDCOV was 100 pg/μL,thus it can be used to identify the clinical suspected virus infection. The results showed that a double RT-PCR detection method for PEDV-PDCOV virus is established successfully,this method is highly sensitive and specific and a rapid and effective method for the clinical detection of PEDV-PDCOV single virus infection or multi-virus mixed infection in pigs.

Key words: PEDV, PDCOV, RT-PCR, clinical diagnosis