Leachii支原体PCR检测方法的建立与应用

Development and application of a PCR assay for detection of Mycoplasma leachii

为建立一种leachii支原体的检测方法,本研究根据leachii支原体LPPA外膜蛋白编码基因序列设计一对引物,建立了PCR检测方法。结果显示,该方法仅对leachii支原体扩增出539 bp的目的片段,而与牛支原体、丝状支原体山羊亚种、绵羊肺炎支原体、丝状支原体丝状亚种小菌落型、山羊支原体山羊肺炎亚种等其它支原体无交叉反应,具有良好的特异性;该方法对leachii支原体的最低检测浓度为105 ccu/m L,敏感性较高。采用该方法可以在乳汁和关节液等病料样品中检测到leachii支原体,与分离培养的结果相符。上述结果表明,本研究建立的leachii支原体PCR检测方法具有特异性强、敏感性高的特点,可实现准确、高效检测leachii支原体的目的。

In order to establish a rapid assay for detecting Mycoplasma leachii （M.leachiO, the PCR method was developed with a pair of special primers targeting on the lppA gene. Then, a 539 bp DNA fragment was specifically amplified from M.leachii with a detection limit of 105 ccu/mL by this assay, but no any amplifications detected from other Mycoplasmas （such as Mycoplasma bovis, Mycoplasma mycoides subsp, Capricolum, Mycoplasma ovipneumoniae, Mycoplasma. mycoides subsp. Mycoides small colony type, Mycoplasma capricolum subsp, capripneumoniae）. Using this method, the M.leachii in milk, synovial fluid and other specimen materials could be detected, and these results were consistent with the results of cultivation. These results confirmed that the PCR established in this study was a fast, sensitive, specific and reliable method for detecting M.leachii.