绵羊肺炎支原体RPA检测技术的建立

Establishment of a RPA Method for Detecting Mycoplasma ovipneumoniae

【目的】建立一种快速简便检测绵羊肺炎支原体(Mycoplasma ovipneumoniae,Mo)的方法。【方法】根据Mo膜蛋白P80基因序列,利用Oligo 7软件设计并筛选出特异性扩增引物,通过条件优化建立检测Mo的RPA方法。【结果】该方法可特异性扩增Mo,对其他羊常见病原无特异性扩增,对Mo核酸的检测灵敏度为70fg·μL^-1,与常规PCR敏感性一致。批内和批间结果显示Mo阳性样品均能扩增条带,而阴性对照均无扩增,表明重复性好。对186份临床样品应用建立的RPA方法和常规PCR方法同时进行检测,并对其中40份肺脏样品进行支原体的分离鉴定,结果显示分离鉴定出的13份Mo阳性样品及常规PCR法检测出的95份阳性样品经RPA检测,结果均为阳性。【结论】建立的Mo RPA方法特异性强、重复性好,可作为Mo的快速检测和流行病学调查的一项候选技术。

【Objective】 To develop a rapid detecting method for Mycoplasma ovipneumoniae (Mo).【Method】 Based on the P80 gene sequence,specific primers were designed using Oligo 7 software. Conditions of the recombinase polymerase amplification (RPA) method were optimized for the application.【Result】 The new assay detected P80 gene in Mo specifically,not any other common pathogens of sheep and goats. The detection sensitivity was 70 fg·μL^-1 ,which was the same as provided by conventional PCR. The inter- and intra-batch tests showed that the modified method could amplify the bands on Mo-positive samples,not on Mo-negative specimens,indicating an acceptable repeatability of the methodology. Furthermore,the RPA assay and conventional PCR methods were simultaneously used on 186 clinic samples,as well as the Mycoplasma isolation and identification on 40 lung samples,to show that the RPA assay positively identified all 13 Mo-positive samples detected by Mycoplasma isolation and identification and 95 positive samples detected by the conventional PCR.【Conclusion】The newly developed RPA method had desirable specificity and repeatability on Mo detection. It could be applied for rapid detecting and epidemiological studies on Mo.