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当前位置: 首页 > 文章 > 鸡马立克氏病毒SYBR GreenⅠ实时荧光定量PCR检测方法的建立 四川农业大学学报 2013,31 (4) 427-432
Position: Home > Articles > Establishment of Real-time Fluorescent Quantitative PCR for Detecting Marek's Disease Virus Based on SYBR GreenⅠ Journal of Sichuan Agricultural University 2013,31 (4) 427-432

鸡马立克氏病毒SYBR GreenⅠ实时荧光定量PCR检测方法的建立

作  者：
程洋;冯泽清;杨辉林;刘益平;朱庆
单  位：
四川农业大学动物科技学院
关键词：
马立克氏病毒;Meq基因;SYBR GreenⅠ实时荧光定量PCR;检测
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。
译  名：
Establishment of Real-time Fluorescent Quantitative PCR for Detecting Marek's Disease Virus Based on SYBR GreenⅠ
作  者：
CHENG Yang;FENG Ze-qing;YANG Hui-lin;LIU Yi-ping;ZHU Qing;College of Animal Science and Technology,Sichuan Agricultural University;
关键词：
Marek's disease virus;;Meq gene;;SYBR GreenⅠreal-time fluorescent quantification PCR;;detection
摘  要：
【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 Meqgene 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(-3.433and 96%,respectively).The detection sensitivity was 102copies/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.

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