猪肺炎支原体和猪鼻支原体TaqMan双重荧光PCR检测方法的建立及应用

Establishment and Application of Taq Man Duplex Real-time PCR Detection Method for Mhp and Mhr

为建立一种可快捷鉴别检测猪肺炎支原体(Mhp)和猪鼻支原体(Mhr)的方法,针对Mhp的183基因和Mhr的p37基因保守片段,分别设计合成引物及TaqMan探针,优化反应条件,建立一种可同时检测Mhp和Mhr的TaqMan双重荧光PCR方法,并评价其特异性、敏感性和重复性。结果表明,该方法特异性强,检测其他常见猪呼吸系统病原不发生交叉反应;对标准品pMD18-T-Mhp-183、pMD18-T-Mhr-P37的最低检测限分别达45.2 copies/μL和29.7 copies/μL,比普通PCR检测灵敏度提高10^(3)~10^(4)倍;该方法检测结果的批内与批间变异系数均小于2%。用该方法检测145份广西自治区内临床样品,从中检出82份Mhp和9份Mhr阳性样品。该方法可用于临床样品快速检测及实验室Mhp和Mhr的鉴定,可为Mhp和Mhr在猪群中的早期监测提供技术支持。

To establish a rapid and specific detection method for Mycoplasma pneumoniae(Mhp)and Mycoplasma hyorhinis(Mhr),two pairs of specific primers and two Taq Man probes labeled with different fluorescent groups were synthesized based on gene sequences in the conserved regions of Mhp,Mhr.The Taq Man real-time PCR detection method to Mhp and Mhr was successfully established after optimizing reaction conditions,validating the specificity,sensitivity,reproducibility and finishing the preliminary application in clinical samples.The results showed that this method,with high specificity,did not cross-react with other pathogens related with common pig respiratory diseases;and this method is highly sensitive,with the lowest detection limits of 45.2 copies/μL and 29.7 copies/μL for the standards pMD18-T-Mhp-183 and pMD18-T-Mhr-P37,respectively,and is more sensitive than the common PCR assay.The method was reproducible,and the inter-group and intra-group coefficients of variation were less than 2%.In detected 145 clinical samples,the positive number of Mhp and Mhr were 82 and 9,respectively.In this study,a rapid and specific Taq Man real-time PCR detection method for Mhp and Mhr was successfully established.This method,with high specificity,sensitivity and reproducibility,could be used in rapid detection of clinical samples and the identification of Mhp and Mhr.It provided a new and reliable detection technology for monitoring Mhp and Mhr.