基于Omp25基因和环介导等温扩增技术检测布鲁氏菌方法的建立

Establishment of a Detection Method for Brucella Based on Omp25 Gene and Loop-mediated Isothermal Amplification

目的建立灵敏、特异、快速的LAMP检测布鲁氏菌的方法。方法基于布鲁氏菌保守的Omp25序列设计了4套引物,根据文献合成了基于此序列的3套引物,利用7套引物建立了检测方法;应用浊度法和电泳法对4种布鲁氏菌进行检测,确定最佳引物;通过对4种布鲁氏菌和其它17种常见菌同时进行检测评价方法的特异性;通过对不同稀释浓度的4种布鲁氏菌的DNA进行LAMP检测以确定方法的灵敏度。结果本研究新设计的一套引物被确定为最佳引物;用建立的LAMP法对17种常见菌株进行扩增,结果均为阴性,本方法具有很高的特异性;LAMP法检测猪种S2、羊种M5、牛种104 M和布鲁氏菌犬种55226四种布鲁氏菌DNA的灵敏度分别为5×10-4、5×10-5、5×10-3ng/μL和5×10-4ng/μL;检测反应在30 min以内完成。结论本方法具有快速、灵敏、特异的特点,有望成为快速检测布鲁氏菌的有效手段。

Objective Establish a sensitive, specific and rapid loop-mediated isothermal amplification （LAMP） for detection of Brucella species. Method Five sets of primers were designed based on the conservative omp25 sequence in BruceUa species, and three sets of published primers according to the same gene were also used to establish the detection method . Turbidity meter and electrophoresis method were used to screen the best primers for the detection of four species of Brucella. The specificity of the constructed method was evaluated by detecting four species of Brucella and seventeen species of common bacteria. The different dilution of Brucella DNA template was detected with the constructed LAMP to evaluate the sensitivity. Result A new set of primers designed in this study was selected as the best one among seven sets of primers. Seventeen species of other common bacteria were detected by the constructed LAMP, and all the result were negative, indicating that the method had a very high specificity. The detection sensitivity of this LAMP assay was 5×10 -4, 5 × 10 -5, 5 × 10 -3 ng/μL and 5 × 10 -4ng/μL of DNA for the detection of Brucella suis S2, Brucella ovis M5, Brucella abortus 104M and Brucella canis 55226, respectively. The detection reaction was completed in 30 min. Conclusion The LAMP method has rapid, sensitive and specific characteristics, and a potential to develop an effective method for rapid detection of Brucella specieson the scene and in underdeveloped areas.