四川农业大学学报 ›› 2013, Vol. 31 ›› Issue (04): 427-432.doi: 10.3969/j.issn.1000-2650.2013.04.013

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鸡马立克氏病毒SYBR GreenⅠ实时荧光定量PCR检测方法的建立

程洋, 冯泽清, 杨辉林, 刘益平, 朱庆   

  1. 四川农业大学动物科技学院, 四川 雅安 625014
  • 收稿日期:2013-11-20 出版日期:2013-12-31 发布日期:2017-02-28
  • 作者简介:程洋,硕士生。
  • 基金资助:
    国家蛋鸡产业体系经费(CARS-41);四川省科技厅育种攻关项目(2011NZ0099-7)

Establishment of Real-time Fluorescent Quantitative PCR for Detecting Marek's Disease Virus Based on SYBR GreenⅠ

CHENG Yang, FENG Ze-qing, YANG Hui-lin, LIU Yi-ping, ZHU Qing   

  1. College of Animal Science and Technology, SiChuan Agricultural University, Yaan 625014, Sichuan, China
  • Received:2013-11-20 Online:2013-12-31 Published:2017-02-28
  • Contact: 朱庆,教授,主要研究方向:家禽遗传育种,E-mail:zhuqing5959@163.com。 E-mail:zhuqing5959@163.com

摘要: [目的] 建立一种能够快速、灵敏地检测鸡马立克氏病病毒(MDV)的SYBR GreenⅠ实时荧光定量检测方法。[方法] 针对马立克氏病毒特异的Meq基因序列设计引物,利用SYBR GreenⅠ染料建立检测马立克氏病毒的实时荧光定量PCR方法,进行敏感性、特异性、重复性试验,并应用该方法检测了罗曼蛋鸡、AA肉鸡、二郎山山地鸡SD02和SD03品系4种鸡只感染组和对照组样本的脾、法氏囊、胸腺等组织中的病毒拷贝数。[结果] 该方法建立的定量标准曲线荧光阈值循环数(Threshold cycle, Ct)与模板拷贝数呈良好线性关系(r=0.996),扩增效率(E)为96%,熔解曲线分析显示其PCR扩增具有良好的特异性,敏感性和重复性试验证明该方法具有较高的灵敏度和稳定性,最低检测浓度为102拷贝/μL。同时运用该方法对试验样本进行检测,结果显示在4个感染组鸡只的肝,脾,肾,胸腺,法氏囊组织中均检测出Meq的拷贝,并计算出了待测样品的病毒拷贝数,相反在未感染组却没有检测出任何Meq的拷贝。试验结果还发现二郎山山地鸡两品系各组织中MDV的拷贝数显著低于AA肉鸡和罗曼蛋鸡各组织。[结论] 建立的检测方法能够快速检测马立克氏病毒,结果准确,成本低廉,可以将其作为生产上监控马立克氏病的一个重要手段。

关键词: 马立克氏病毒, Meq基因, SYBR GreenⅠ实时荧光定量PCR, 检测

Abstract: [Objective] To develop a SYBR GreenⅠreal-time fluorescent quantitative PCR assay for detecting Marek's disease virus (MDV) rapidly and accurately.[Method] Specific primers were designed according to the Meq gene of Marek's disease virus.A SYBR GreenⅠreal-time fluorescent quantitative PCR assay for detecting MDV was established with its sensitivity,specificity,repeatability determined.Then we measured the copy number of Marek's disease virus between the MDV-infection chickens and the normal chickens by using this method.[Results] The results showed good linear relationship between the threshold Cycle (Ct) of the quantitative standard curve and the copy number of template (r=0.996),and the slope and the amplification efficiency (E) of the Meq gene (-3.433 and 96%,respectively).The detection sensitivity was 102 copies/ul.This test had good repetition,samples were detected successfully.It showed that the copy number of MDV had been detected in liver,spleen,kidney,thymus and bursa of fabricious of MDV-infected chickens,but there was not any amplification in normal chickens.Meanwhile,the copy number of MDV in Erlang mountainous chicken's tissues was significantly lower than in AA broilers and Roman layer tissues.[Conclusion] Due to the established detection method,we can detect Marek's disease virus quickly and accurately.This study provided a significant way to detect MDV in poultry production.

Key words: Marek's disease virus, Meq gene, SYBR GreenⅠreal-time fluorescent quantification PCR, detection

中图分类号: 

  • S831.7