《中国畜牧兽医》 ›› 2018, Vol. 45 ›› Issue (7): 1804-1812.doi: 10.16431/j.cnki.1671-7236.2018.07.010

• 生物技术 • 上一篇    下一篇

伪狂犬病病毒野毒株与gE基因缺失疫苗株TaqMan实时荧光定量PCR鉴别方法的建立

兰德松1,2,3,4, 顾贵波3,4, 侯振中1,2   

  1. 1. 东北农业大学动物医学院, 哈尔滨 150030;
    2. 农业部动物疫病病原生物学重点实验室, 哈尔滨 150030;
    3. 辽宁省动物疫病预防控制中心, 沈阳 110164;
    4. 辽宁省动物医学研究院, 沈阳 110164
  • 收稿日期:2017-12-29 出版日期:2018-07-20 发布日期:2018-07-20
  • 通讯作者: 侯振中 E-mail:houzz_1963@163.com
  • 作者简介:兰德松(1982-),男,湖北洪湖人,博士,高级兽医师,研究方向:动物疫病防诊治技术,E-mail:landesong@163.com
  • 基金资助:

    辽宁省"百千万人才工程"资助项目(2016921040)

Development of a TaqMan Real-time PCR for Differentiation of Wild-type Strain from gE-deleted Vaccine Strain of Pseudorabies Virus

LAN Desong1,2,3,4, GU Guibo3,4, HOU Zhengzhong1,2   

  1. 1. College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China;
    2. Key Laboratory of Animal Pathogen Biology, Ministry of Agriculture, Harbin 150030, China;
    3. Liaoning Provincial Center for Animal Disease Prevention and Control, Shenyang 110164, China;
    4. Liaoning Animal Medical Research Institute, Shenyang 110164, China
  • Received:2017-12-29 Online:2018-07-20 Published:2018-07-20

摘要:

为实现对伪狂犬病病毒(pseudorabies virus,PRV)野毒株与gE基因缺失疫苗株的快速、敏感、特异的鉴别诊断,本试验针对PRV gDgE基因设计了2套特异性引物和TaqMan探针,建立了PRV野毒株与gE基因缺失疫苗株的TaqMan实时荧光定量PCR鉴别方法,对引物和探针浓度、退火温度等进行了优化,对方法进行敏感性、特异性、重复性试验,并进行临床样品检测。结果显示,建立的针对gDgE基因的TaqMan实时荧光定量PCR方法线性相关系数(R2)分别为0.996和0.980,均呈良好的线性关系;检测限分别为39.4和12.1拷贝/μL;与圆环病毒2型、猪瘟病毒、猪繁殖与呼吸综合征病毒均无交叉反应;重复性试验结果显示,针对gD基因的批内和批间变异系数分别为1.43%~1.86%、1.10%~2.07%,针对gE基因的批内和批间变异系数分别为0.98%~1.41%、1.12%~1.86%。应用建立的TaqMan实时荧光定量PCR与普通PCR分别对11份临床疑似感染样品进行检测,阳性率分别为36.4%和27.3%。结果表明,该方法敏感性高、特异性强、重复性好,可作为伪狂犬病病毒野毒株与gE基因缺失疫苗株的早期鉴别诊断和定量检测的有效手段。

关键词: 伪狂犬病病毒(PRV); 野毒株; gE基因缺失疫苗株; TaqMan实时荧光定量PCR; 鉴别

Abstract:

In order to establish a TaqMan Real-time PCR for rapid,sensitive and specific distinguishing the wild strain and gE-deleted vaccine strain of pseudorabies virus (PRV),two sets of primers and TaqMan probes were designed for gD and gE genes of PRV,respectively,then a set of 2 novel Real-time PCR were developed for quantitative detection and differentiation of wild-type strain from gE-deleted vaccine strain of PRV.The concentration of primers and probes,the annealing temperature in TaqMan Real-time PCR were optimized,the sensitivity,specificity and reproducibility of the assays were determined,and were applied to the detection of clinical samples.The R2 value of the TaqMan Real-time PCR standard curve were 0.996 and 0.980,respectively,both showed good linear response.The detection limit of the assays were 39.4 and 12.1 copies/μL,respectively.The assays were highly specific for PRV,without cross-reaction with other common swine viral pathogens,such as porcine circovirus type 2 (PCV2),classical swine fever virus (CSFV),porcine reproductive and respiratory syndrome virus (PRRSV).Intra-batch and inter-batch reproducibility tests showed that the coefficient of variation (CV) were 1.43% to 1.86%,1.10% to 2.07% (gD gene) and 0.98% to 1.41%,1.12% to 1.86% (gE gene),respectively.Applying the TaqMan Real-time PCR and the conventional PCR to detect 11 PR suspected clinical samples,they got 36.4% and 27.3% positivity,respectively.All these results indicated that the TaqMan Real-time PCR were both sensitive,specific and reproducible,and could be used as a useful tool for quantitative detection and differentiation of wild-type strain from gE-deleted vaccine strain of PRV.

Key words: pseudorabies virus (PRV); wild strain; gE-deleted vaccine strain; TaqMan Real-time PCR; differentiation

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