鸡马立克氏病毒SYBR GreenⅠ实时荧光定量PCR检测方法的建立

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

【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。

[Objective] To develop a SYBR Green I real-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 Green I real-time flu- orescent quantitative PCR assay for detecting MDV was established with its sensitivity, specifici- ty, repeatability determined. Then we measured the copy number of Marek＇s disease virus be- tween 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 96o//00, 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 fabri- cious of MDV-infected chickens, but there was not any amplification in normal chickens. Mean- while, 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 meth- od, we can detect Marek＇s disease virus quickly and accurately. This study provided a significant way to detect MDV in poultry production.