Establishment of Real-time Fluorescent Quantitative PCR for Detecting Marek＇s Disease Virus Based on SYBR Green I | 鸡马立克氏病毒SYBR Green I实时荧光定量PCR检测方法的建立

Chinese. 【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 Green I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 Green I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一0．996），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10。拷贝／vL。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。

English. [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.