山羊支原体山羊肺炎亚种重组酶聚合酶扩增技术的建立

Establishment of a Recombinase Polymerase Amplification Method for Detecting Mycoplasma capricolum subsp.capripneumoniae

为检测山羊传染性胸膜肺炎,选择Mccp-arcA假基因作为靶基因,用Primer 3 plus设计筛选生物素标记的特异引物和荧光素标记的探针,将双标记扩增产物结合到核酸检测试纸条,通过优化反应温度和时间,建立了一种检测山羊支原体山羊肺炎亚种的重组酶聚合酶扩增技术,对其灵敏度、重复性、准确性和特异性进行了试验,并初步用于临床样品的检测。结果显示,山羊传染性胸膜肺炎核酸阳性样品能够在核酸检测试纸条上显示阳性条带,阴性样品无条带。试验具有良好的重复性和敏感性(达到10 copies/μL)。用建立的重组酶聚合酶扩增技术与普通PCR方法同时检测疑似山羊传染性胸膜肺炎8份样品,结果均为3份阳性和5份阴性,符合率100%。该检测方法适用于山羊传染性胸膜肺炎的快速诊断,为Mccp的快速检测提供了新的技术。

In order to detect contagious caprine pleuropneumonia,based on the Mccp-arcA pseudogene as target gene,specific primers for biotin labeling and probes lablled by fluorescein were designed and screened using Primer 3 plus software.A recombinase polymerase amplification detection method for Mycoplasma capricolum subsp.capripneumoniae was established by combining the double labeled amplification product with nucleic acid detection strip and optimizing the reaction temperature and time.The sensitivity,repeatability,accuracy and specificity of this method were tested,and it was preliminarily used in the detection of clinical samples.The results showed that the nucleic acid positive samples of contagious caprine pleuropneumonia could show positive bands on the strip,while the negative samples had no bands.It reflects that the test has good repeatability and sensitivity(10 copies/μL).Using the established RPA and general PCR to detect 8 clinical samples of suspected contagious caprine pleuropneumonia,the results of both methods were showed that 3 of the 8 clinical samples were positive,5 of the 8 clinical samples were negative,and the coincidence rate of detection results was 100%.The above results indicated that the RPA detection method established in this study is suitable for rapid diagnosis of contagious caprine pleuropneumonia,and provides a new technology for rapid detection of Mycoplasma capricolum subsp.capripneumoniae.