应用双重PCR方法检测羊支原体肺炎病原

Development of a duplex polymerase chain reaction assay for the detection of pathogens for Mycoplasmal pneumonia of sheep and goats

通过对丝状支原体山羊亚种(M.mycoides subsp.capri,Mmc)特异性引物MmcF/MmcR和绵羊肺炎支原体(M.ovipneumontiae,Mo)特异性引物LmF/LmR退火温度、引物浓度比例等条件的选择,建立了一个可以同时检测Mmc和Mo的双重PCR方法。该方法可同时扩增出Mmc 195 bp和Mo 361 bp目的片段,但对其他病原菌不能扩增出任何条带,具有良好的特异性。敏感性试验表明,该方法能够分别检测出0.1ng的Mmc DNA和0.01 ng的Mo DNA,或同时检测出1ng Mmc和1ng Mo混合的DNA。用该双重PCR方法可对实验室保存的4株绵羊肺炎支原体和2株丝状支原体山羊亚种进行准确鉴定,并可从临床病料中检测出相应支原体,表明建立的双重PCR方法可用于Mmc和Mo的快速鉴定、实验室诊断和病原学调查。

Using the primers MmcF/MmcR specific for Mycoplasma mycoides subsp, capri （Mmc） and LmF/LmR specific for M. ovipneumoniae （ Mo）, a duplex polymerase chain reaction （PCR） assay for the detection of Mmc and Mo has been developed in this study. The duplex PCR assay could simultaneously amplify a 195 bp fragment from the Mmc strain PG3 and a 361 bp from the Mo strain Y98, but no DNA fragments were amplified from the other bacteria such as M. capricolum subsp, capripneumoniae, M. hypneumoniae and P. muhocida, etc. The sensitivity test showed that it could detect 0. 1 ng DNA of Mmc and 0. 01 ng DNA of Mo, or the mixed DNA with 1 ng DNA of Mme and 1 ng DNA of Mo. Four Mo isolates and 2 Mmc isolates were accurately identified by the duplex PCR. The specific flagments could be directly amplified from clinical samples, such as pathological lungs and pleural fluid, by using the duplex PCR, respectively. These results demonstrated that the duplex PCR could be applied for the rapid identification of Mmc and Mo, and diagnosis of Mycoplasmal pneumonia of sheep and goats.