鸡毒支原体TaqMan实时荧光定量PCR检测方法的建立及应用

Establishment of a TaqMan real-time PCR assay for detection of Mycoplasma gallisepticum

为建立一种快速、灵敏检测鸡毒支原体(MG)的方法,本研究根据GenBank中登录的MK984197.1,针对PAPV基因的保守序列设计了1对特异性引物探针,将PCR扩增后的PAPV基因克隆至pMD19-T载体,以构建出的重组质粒作为标准阳性质粒标准品,建立鸡毒支原体TaqMan探针荧光定量PCR检测方法,并对该方法的敏感性、特异性和重复性等进行优化与验证。结果显示,Ct值与标准模板在0.53×10^(8)~0.53×10^(3)copies/μL范围内呈良好的线性关系,相关系数(R^(2))为0.998,线性方程的斜率为-3.219,最低检测限度为0.53×10^(2)copies/μL;经组内和组间重复试验,两者变异系数均小于3%,表明该方法具有良好的稳定性和可重复性;与鸡滑液囊支原体(MS)、火鸡支原体(MM)等均无交叉反应,表明该方法具有很好的特异性。利用所建立的MG-TaqMan探针实时荧光定量PCR检测方法对部分地区抽检的60份临床样品进行检测,该方法较普通PCR方法的检出率更高。上述结果表明,本研究成功建立了MG-TaqMan探针实时荧光定量PCR检测方法,可实现对MG的快速灵敏诊断。

In order to establish a rapid and sensitive detection method for Mycoplasma gallisepticum(MG),primers and probes were designed based on the conserved regions of the PAPV gene obtained from Gen Bank(MK984197.1).The PAPV gene amplified by PCR were inserted into the p MD19-T vector as the standard positive plasmid to establish the MG-Taq Man fluorescence quantitative PCR detection method.Furthermore,the sensitivity,specificity and repeatability of the method were optimized and verified.A good linearity of response was observed between the Ctvalues and the standard template in the range of0.53×10^(8)-0.53×10^(3)copies/μL,the correlation coefficient(R^(2))was 0.998,the slope of the linear equation was-3.219,and the minimum detection limit was 0.53×10^(3)copies/u L,the coefficients of variation within a batch and between batches were both lower than 3%.The RT-q PCR assay demonstrated high sensitivity and excellent reproducibility.When MS and MM were used as templates,no positive signals could be detected,indicating that the method had a high specificity for MG.Clinical sample testing revealed the established MG Taq Man probe real-time fluorescence quantitative PCR was more sensitive than using traditional PCR.Above all,the MG Taq Man probe real-time quantitative PCR detection method has been successfully established for the fast and accurate identification of MG.