鸡毒支原体 Taq Man实时荧光定量PCR检测方法的建立

Establishment of Taq Man Real-time Fluorescent Quantitative PCR Detection Methods for Mycoplasma gallisepticum

为快速检测鸡毒支原体,根据鸡毒支原体的保守基因设计特异性引物和Taq Man探针,建立鸡毒支原体Taq Man实时荧光定量PCR方法并分析鸡毒支原体培养过程中颜色单位改变法(color change unit,CCU)和荧光定量PCR检测的相关性。结果显示,标准曲线y=-3.646x+44.208,相关系数R^(2)=0.998,最低检测限度为1 copy/μL;与其他菌株等均无交叉反应;组内和组间变异系数均小于3%,表明该方法具有良好的稳定性和可重复性。用建立的实时荧光定量PCR检测方法对26份临床样品进行检测,该方法较普通PCR方法检出率更高。建立的荧光定量PCR与CCU显示鸡毒支原体在对数生长期时,两者具有一定的对应关系。表明建立了鸡毒支原体Taq Man探针实时荧光定量PCR检测方法,可实现对鸡毒支原体的快速检测,为鸡毒支原体感染的防控提供技术基础。

In order to rapidly detect Mycoplasma gallisepticum(MG),specific primers and Taq Man probe were designed according to the conserved gene of Mycoplasma gallisepticum,and a Taq Man real-time fluorescent quantitative PCR method for Mycoplasma gallisepticum was established.The correlation between color change unit(CCU)and fluorescent quantitative PCR detection during Mycoplasma gallisepticum culture was analyzed.The results showed that standard curve:y=-3.646x+44.208,correlation coefficient R^(2)=0.998,the minimum detection limit was 1 copy/μL;no cross reaction with other strains;the coefficients of variation within and between groups were less than 3%,indicating that the method had good stability and repeatability.The established real-time fluorescent quantitative PCR method was used to detect 26 clinical samples.The detection rate of this method was higher than that of ordinary PCR method.The established fluorescent quantitative PCR and CCU showed that there was a certain correspondence between the two in the logarithmic growth phase.This study successfully established a Taq Man real-time fluorescent quantitative PCR detection method for Mycoplasma gallisepticum,which can achieve rapid diagnosis of Mycoplasma gallisepticum and provide a technical basis for the prevention and control of Mycoplasma gallisepticum.