猪鼻支原体荧光PCR检测方法的建立和应用

Development and application of a real-time PCR for detection of Mycoplasma hyorhinis

为了建立猪鼻支原体(Mycoplasma hyorhinis)快速荧光PCR检测方法,本研究合成了该病原特异性基因P37的重组质粒,根据该基因设计荧光PCR引物和探针,并对设计的引物和探针的特异性、敏感性和重复性进行了检测。结果显示,该方法具有高度的特异性,除了识别猪鼻支原体基因组外,对猪的基因组和猪常见病原的基因组均不发生交叉反应。在敏感性和重复性试验中,该方法对猪鼻支原体的检测限为1 000copies/m L,变异系数小于3%,因此具有优良的敏感性和稳定性。用该方法检测临床样本,阳性检出率为76%(38/50),远高于普通PCR的阳性检出率(40%,20/50)。上述结果表明,本研究建立的荧光PCR方法在特异性、敏感性和稳定性方面均满足猪鼻支原体的临床检测要求,可以用于猪鼻支原体临床样品的检测。

To establish a rapid assay for the detection of Mycoplasma hyorhinis(M.hyorhinis),the recombinant plasmid holding P37 gene of M.hyorhinis was constructed,and a pairs of specific primers and the probe targeting the P37 gene was designed for the real-time PCR assay.The specificity,sensitivity and reproducibility were evaluated in this research.The results showed that the assay has high specificity with only amplification of M.hyorhinis and without reaction with pig genomic DNA and general pathogens.The detection limit of this assay was 1 000 copies/m L and the coefficient of variation was less than 3%,indicating the good sensitivity and reproducibility.Detection of fifty clinical specimens showed 76%(38/50) positive rate with the real-time PCR assay,versus 40%(20/50) with the conventional PCR method.These data indicated that the real-time PCR assay developed in this research has a good specificity,sensitivity and reproducibility for the detection of M.hyorhinis,and is able to be used to detect clinical specimens.