牛支原体的环介导等温扩增快速检测技术研究  

Development of Loop-mediated Isothermal Amplification for Rapid Detection of Mycoplasma bovis

作　　者：侯轩[1] 付萍[1] 张海燕[1] 张跃伟[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》2012年第2期218-224,共7页Journal of Agricultural Biotechnology

基　　金：公益性行业(农业)科研专项经费项目(No.201203056);农业部动物疫情监测与防治项目

摘　　要：牛支原体(Mycoplasma bovis)是感染牛的一种重要的致病性病原体。本研究利用环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术建立了牛支原体的快速检测方法。该方法以牛支原体保守性基因uvrC为靶序列设计了5条特异引物,在58℃等温条件下,60min即可完成反应。LAMP方法能检测出20pg牛支原体DNA,较PCR方法高100倍,用牛鼻支原体(M.bovirhinis)、无乳支原体(M.agalactiae)、精氨酸支原体(M.ariginini)、牛副流感病毒(BPIV)、牛腺病毒(BADV)、牛传染性鼻气管炎病毒(IBRV)作特异性检测,两种方法均有很高的特异性。本研究还将荧光显色剂钙黄绿素(calcein)和Mn2+用于LAMP反应结果的可视化检测。临床鼻拭子样品煮沸后,可直接进行等温扩增,省去了DNA提取步骤。在对167个临床鼻拭子样本的检测中,LAMP方法检测结果阳性率(26.95%)高于PCR方法检测出阳性率(19.16%),这证明本研究所建立的方法,与PCR法比较更适于临床样品的检测。研究结果表明,LAMP方法具有操作简便、快速、高效、敏感、特异、经济等特点,适于基层实验室监测的广泛使用。Mycoplasma bovis is an important pathogen in cattle.In this research we developed a new and rapid test method for Mycoplasma bovis with loop-mediated isothermal amplification（LAMP）.With the 5 specific primers targeting to a total of six distinct sequences on the conservative gene uvrC,the new method could be completed in 60 min under the isothermal condition of 58℃.With the LAMP method,we could detect 20 pg DNA of M.bovis,which was 100 times lower than using the method of PCR.In addition,using M.bovirhinis,M.agalactiae,M.arginini,Bovine parainfluenza virus（BPIV）,Bovine adenoviruses（BADV） and Infectious Bovine rhinotracheitis virus（IBRV） to determine the specificity of LAMP and PCR methods,both the methods of LAMP and PCR displayed the high specificity results.The research had also applied the calcein,a fluorescence visualization reagent,and Mn2＋ in LAMP,so as to visualize the result of the test.Additionally,clinical samples after boiling 10 min which was the best compared with DNA extraction and FTA Card could be directly used for LAMP.In the test for 167 clinical samples of nasal swabs,the positive rate using LAMP（26.95%） was higher than the one using PCR（19.16%）,which proved that LAMP method,in comparison with PCR method,showed a more efficient ability in testing the clinic samples.In conclusion,LAMP method,featuring simplicity,high-efficiency,rapidity,sensitivity,specificity,and more economical,etc.,possesses a potential for base laboratory.

关 键 词：牛支原体(Mycoplasma bovis) 环介导等温扩增(LAMP) PCR 

分 类 号：S858.232.6[农业科学—临床兽医学]