布鲁杆菌环介导等温扩增快速检测方法的建立

Detection of Brucella by loop-mediated isothermal amplification

目的建立一种环介导等温扩增（LAMP）快速检测动物布鲁杆菌的方法。方法使用Primer4．0，针对布鲁杆菌外膜蛋白（0MP31）基因保守区设计4条特异引物，在Bst大片段聚合酶的作用下，实现DNA的梯状等温扩增，并在此方法最适扩增条件优化的基础上，对其特异性、灵敏度进行实验，同时与普通PCR灵敏度进行比较，以及进行LAMP可视化实验。结果本研究建立的检测方法对牛种布鲁杆菌（ Brucella abortus ,B.abortus ）544A和104M、羊种布鲁杆菌（ Brucella melitensis ,B.melitensis ）Rev-1和16M、猪种布鲁杆菌（Brucella suis，B.suis）S2和1330S、犬种布鲁杆菌（Brucella canis，B．canis）RM6／66、绵羊附睾种布鲁杆菌（Brucella ovis，B.ovis）63／290、沙林鼠种布鲁杆菌（Brucella neotomae，B．neotomae）5K33检测阳性，而耶尔森氏菌（Yersinia enterocolitica，Y．enterocolitica）0：9、大肠杆菌（Escherichia coli，E．coli）0157：H7和沙门氏菌（Salmonella typhimurium，S．typhimurium）47729检测阴性。LAMP法检出最低DNA浓度为8．5×10^-8mg／L，较普通PCR检测灵敏度高。检测结果既可以通过电泳判定也可以通过可视化判定。结论本研究所建立的布鲁杆菌LAMP检测方法具有特异、灵敏、设备要求简单等特点．适用于基层兽医部门进行布鲁杆菌的快速检测。

Objective A loop-mediated isothermal amplification （LAMP）assay was developed for rapid detection of BruceUa. Methods Using Primer 4.0, we designed four specific primers at brookings coli outer membrane protein （OMP31） gene conservative area, under the action of Bst polymerase larger pieces to realize the step-like isothermal amplification of DNA. In this method, on the basis of optimal amplification conditions, the specificity and sensitivity of this method was compared with conventional PCR, and LAMP visualization experiment was conducted. Results The detection results of Brucella abortus （B.abortus） strain 544, B. abortus strain 104M, Brucella melitensis （B.melitensis ） strain Rev-1, B. melitensis strain 16M, Brneella suis （B.suis ） strain $2, B. suis strainand 1330S, Brucella canis （B.canis ） strain RM6/66, Brucella ovis （B.ovis ） strain 63/290, and Brucella neotomae （ B.neotomae ） strain 5K33 were positive, and Yersinia enterocolitica （ Y.enterocolitica ） 0：9, Escherichia coli（E.coli） O157：H7, and （Salmonella typhimurium,S.typhimurium） 47729 were negative. The minimum detection limit was 8.5 ×10^-8 mg/L. LAMP was more sensitive than PCR. The results can be determined by electrophoresis or through visual judgment. Conclusions LAMP is a simple, specific and sensitive method. LAMP assay is a useful tool for rapid detection of BruceUa.