牛支原体牛多杀性巴氏杆菌与牛传染性鼻气管炎病毒三重PCR方法的建立与初步应用

Establishment and preliminary application of triple PCR method for Mycoplasma bovis,Pasteurella multocida and infectious bovine rhinotracheitis virus

为建立一种可同时检测牛支原体(MB)、多杀性巴氏杆菌(PM)与牛传染性鼻气管炎病毒(IBRV)的三重PCR检测方法,笔者设计1对特异性引物,引用文献的2对引物,对反应条件与反应体系进行优化,建立了一种三重PCR检测方法,并对其特异性、敏感性和重复性进行了验证。结果显示,最佳退火温度为51.7℃,最佳引物终浓度均为0.5μmol/L;敏感性结果显示,MB、PM、IBRV的最低检出限分别为3.9×10^(-3)ng/μL、6.61×10^(-2)ng/μL与2.97×10^(-2)ng/μL;特异性结果显示该方法只能特异性检测出MB、PM和IBRV。利用建立的三重PCR方法对108份临床病料进行检测,结果与单一PCR一致。结果表明,该方法可用于临床快速检测MB、PM、IBRV。

To estab lish a triple PCR detection method that can simultaneously detect Mycoplasma bovis(MB),Pasteurella multocida(PM) and infectious bovine rhinotracheitis virus(IBRV),design a pair of specific primers and two pairs of primers in cited references.After optimizing the reaction conditions and reaction systems,a triple PCR detection method was established,and its specificity,sensitivity and repeatability were verified.The best annealing temperature was 52℃,and the best primer concentration was 0.5μmol/L,the sensitivity test shows that the minimum detection limits of MB,PM and IBRV of this detection method are respectively 3.9×10^(-3)ng/μL,6.61×10^(-2)ng/μL and 2.97×10^(-2)ng/μL.The specificity test results showed that this method could only detect MB,PM and IBRV specifically.The triple PCR method was used to detect 108 clinical samples,and the results were consistent with those of single PCR.To sum up,this method can be used for rapid clinical detection of MB,PM,IBRV.