环介导等温扩增技术快速诊断猪肺炎支原体  

Loop-mediated Isothermal Amplification for Sensitive and Rapid Detection of Mycoplasma hyopneumoniae

作　　者：郭盼盼[1] 黄书林[1] 张跃伟[1] 付萍[1] 李旭妮[1] 李佳禾[1] 吴文学[1] 

机构地区：[1]中国农业大学动物医学院,北京100193

出　　处：《农业生物技术学报》2010年第4期822-826,共5页Journal of Agricultural Biotechnology

基　　金：农业部重点项目(No.2008-G57)资助

摘　　要：利用环介导等温扩增(loop-mediated isothermal amplification,LAMP)方法建立了猪肺炎支原体(Mycoplasma hyopneumoinae)的快速检测方法。该方法以猪肺炎支原体保守性基因16SrRNA为靶序列设计了6条特异引物,在63℃等温条件下,30min即可完成反应。结果显示,LAMP技术优于PCR技术。在敏感性实验中,LAMP方法的能检测出10个包含靶基因片段的重组质粒,敏感性高于PCR方法10倍;特异性实验中,LAMP方法和PCR方法均显示出了高度的特异性;在对127个临床鼻拭子样本的检测中,LAMP方法检测结果是所有样本都为阳性,而PCR检测出了116份阳性。证明本研究所建立的方法,对临床样本的检测的敏感度高于常规的PCR法。LAMP方法具有操作简便、快速、高效、敏感、特异、经济的特点,在研究机构以及基层实验室、现场监测的广泛使用具有一定的潜力。Using the loop-mediated isothermal amplification （LAMP）,a novel method of gene amplification for Mycoplasma hyopneumoniae detection was established.A set of six primers was designed specificity to recognize the 16S rRNA gene,which is a relatively conserved region of M.hyopneumoniae.The LAMP-based assay could be completed within 30 min at 63℃.Results showed that the LAMP-based assay,compared with PCR,had high superiority.The detection limit of the LAMP assay was found to be 10 copies per reaction determined by using a recombined plasmid containing the target sequence,which was 10-fold higher than that of the PCR assay.In addition,both LAMP and PCR were highly specific to M.hyopneumoniae.Furthermore,the LAMP and PCR assay was applied to 127 clinical nasal swab samples collected from pig farms.For the LAMP assay,all the nasal swab specimens tested positive,while for the PCR assay,116 nasal swab specimens tested positive.The data confirmed that LAMP was more sensitive than PCR for the detection of clinical samples.The LAMP assay is easy to perform,extremely rapid,highly sensitive,specificity and cost-effective,therefore it has the great potentiality suitable for diagnosis of M.hyopneumoniae both in well-equipped laboratories and in field situations.

关 键 词：猪肺炎支原体(Mhp) 环介导等温扩增技术(LAMP) PCR 

分 类 号：S858.286.3[农业科学—临床兽医学]