重组酶介导的等温核酸扩增（RAA）检测布氏杆菌方法的建立

Abstract[Objective]The common gene fragments of all species and genera of Brucella were screened,and theprimers and probes suitable for amplification of target genes were designed.The method ofdetection of Brucella was established and optimized by using recombinant enzyme-mediatedisothermal nucleic acid amplification technology.[Methods](1)The multi sequence gene fragments of Brucella were found in the DNA sequencedatabase,and the specific gene sequences were compared and analyzed.The gene sequencessuitable for general detection were selected.According to the selected gene sequences,primers and probes were designed by using the design software;(2)Method of extracting nucleic acid;(3)To establish a new method for detection of Brucella by recombinant enzyme mediatedisothermal nucleic acid amplification (RAA);(4)The sensitivity,specificity and repeatability of the new method for detection ofBrucella were tested.Results]In this study,a new method for the detection of Brucella was successfully established byusing recombinant enzyme mediated isothermal nucleic acid amplification.The wholeamplification was carried out at 39 C for 20 minutes.The sensitivity of this method is as lowas 100 copies of recombinant plasmids containing Brucella,and the specificity of this methodis high,and there is no non-specific amplification with the common bacteria and viruses onthe existing Brucella host.The primers used in this method are made from all kinds ofBrucella.Under the test of existing samples,they can be detected positive and have highrepeatability.[Conclusions]In this study,the recombinant enzyme-mediated isothermal nucleic acid amplificationtechnology for detection of Brucella is rapid,simple,highly sensitive,specific andreproducible.It is suitable for the field testing of animal husbandry or the rapid testing ofsamples before the research in schools and laboratories.Due to the epidemic situation andV