畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (4): 852-858.doi: 10.11843/j.issn.0366-6964.2018.04.025

• 研究简报 • 上一篇    下一篇

猪丁型冠状病毒与猪流行性腹泻病毒双重实时荧光定量RT-PCR方法的建立和初步应用

罗尚星1, 范京惠1*, 刘宝京1, 师乾凯1, 侯林杉1, 左玉柱1,2*   

  1. 1. 河北农业大学动物医学院, 保定 071001;
    2. 河北农业大学动物科技学院, 保定 071001
  • 收稿日期:2017-08-30 出版日期:2018-04-23 发布日期:2018-04-19
  • 通讯作者: 左玉柱,男,副教授,主要从事动物传染病学的教学、科研和社会服务工作,E-mail:zuoyuzhu@163.com;范京惠,女,博士,副教授,硕导,主要从事兽医微生物与免疫学的教学、科研工作,E-mail:jinghui76@163.com
  • 作者简介:罗尚星(1993-),女,河北张家口人,硕士生,主要从事动物传染病学研究,E-mail:2270989476@qq.com。
  • 基金资助:

    河北农业大学百名青年学术带头人培养计划(0318011);河北省研究生创新能力培养资助项目(CXZZSS2017068);河北省自然科学基金课题(C2015204121)

Establishment and Application of the Real-time Reverse Transcription Quantitative PCR Assay for Porcine Epidemic Diarrhea Virus and Porcine Deltacoronavirus

LUO Shang-xing1, FAN Jing-hui1*, LIU Bao-jing1, SHI Qian-kai1, HOU Lin-shan1, ZUO Yu-zhu1,2*   

  1. 1. College of Veterinary Medicine, Hebei Agricultural University, Baoding 071001, China;
    2. College of Animal Science and Technology, Hebei Agricultural University, Baoding 071001, China
  • Received:2017-08-30 Online:2018-04-23 Published:2018-04-19

摘要:

猪丁型冠状病毒(PDCoV)与猪流行性腹泻病毒(PEDV)均为致病性冠状病毒,可以引起猪呕吐、腹泻脱水和新生仔猪死亡。为了建立一种快速检测PDCoV和PEDV的双重SYBR Green I荧光定量RT-PCR方法,根据GenBank登录的2种病毒基因的保守序列,设计了两对特异性引物,分别扩增其N、M基因保守区,并将其克隆至pMD19-T载体;将10倍梯度稀释的混合重组质粒作为标准品模板,建立了一种检测PDCoV和PEDV的双重的SYBR Green I荧光定量RT-PCR方法,并对其反应的敏感度、特异性和重复性进行了检测。结果表明,本试验所建立的双重荧光定量RT-PCR检测方法对PDCoV和PEDV的最低检测量分别为51和32拷贝·μL-1,且与PBoV、TGEV、PRV、PCV2无交叉反应,特异性较好;对2016年至2017年在河北省收集的130份仔猪腹泻样本检测,结果显示,PDCoV的检出率为16.9%,PEDV的检出率为66.2%,二者同时感染的检出率为2.3%,与普通RT-PCR检测方法相比,敏感性更高。本研究为PDCoV和PEDV的诊断及分子流行病学调查提供了一种快速、定量检测方法。

关键词: 猪丁型冠状病毒, 猪流行性腹泻病毒, 实时定量PCR, 临床检测

Abstract:

Porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhea virus (PEDV) are enterpathogenic coronavirus which cause acute diarrhea, vomiting,dehydration and mortality in neonatal piglets. In order to establish a real-time reverse transcription quantitative PCR (RT-qPCR) assay to detect PDCoV and PEDV, two pairs of specific primers were designed according to the conservative sequence of N gene (PDCoV) and M gene (PEDV) registered in GenBank. The genes of the conservative region were amplified and cloned into pMD19-T vector. Taking Mix 10 times the gradient dilution of recombinant plasmid as a standard template, a RT-qPCR to PDCoV and PEDV was established. The sensitivity, specificity and repeatability were tested. Results showed that the sensitivity of this RT-qPCR assay was 51 and 32 copies·μL-1 for PDCoV and PEDV, respectively. No cross-reaction was detected to PBoV, TGEV, PRV and PCV2. The RT-qPCR assay was applied to the detection of 130 clinical samples collected from Hebei province during 2016-2017. PDCoV positive rate was 16.9%, PEDV positive rate was 66.2%, and co-infection was 2.3%. The sensitivity of fluorescence quantitative PCR was significantly higher than that of ordinary RT-PCR. These results indicated that the detection assay could be applied for rapid and high through-put diagnosis of PDCoV and PEDV.

Key words: porcine deltacoronavirus, porcine epidemic diarrhea virus, real-time quantitative PCR, clinical detection

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