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目的 建立鉴定布鲁杆菌属及种的膜基因芯片技术方法.方法 试验菌株羊种标准株16M,牛种标准株544A,猪种标准株1330S;临床标本羊种81190,牛种80176、104M,猪种80177、19971、19972,共9个样本,均为中国疾病预防控制中心鼠布基地菌种库保存.提取各菌种DNA,进行PCR扩增,并以耶尔森菌O∶9为对照.设计布鲁杆菌鞭毛蛋白基因flhA保守片段中两个位点(379、576)的单个核苷酸多态性探针,在探针中引物入生物素,并将探针固定在膜上,制成膜芯片,与目的片段进行杂交,用生物素显色技术检测目的片段.结果 9个样本的扩增产物长度均为357 bp,与预期结果一致.与布鲁杆菌有相同抗原的耶尔森菌O∶9无特异性PCR产物产生.在鞭毛蛋白基因序列379和576两个核苷酸位点上,猪种为379G和576C,牛种为379T和576T,羊种为379T和576C,与所设计探针序列完全相同.经膜基因芯片实验验证,猪种菌株在379G、576C及F位点(公共探针)显色;牛种菌株在379T、576T及F位点显色;羊种菌株在379T、576C及F位点显色.结论 膜基因芯片可用于布鲁杆菌属和种的鉴定,且是一种优越的鉴定方法.“,”Objective To establish an accurate testing method for detection of Brucella and Brucella Abortus,Brucella Melitensis,Brucella suis.Methods The strains include the standard Brucella melitensis 16M,Brucella abortus 544A,the standard Brucella suis 1330s,and the clinical specimens Brucella melitensis 81190,Brucella abortus 80176,104M,Brucella suis 80177,19971,19972.All 9 strains were saved at the Base for Prevention and Control of Plague and Brucellosis,the Chinese Center for Disease Prevention and Control.DNA was extracted and used to amplify by PCR,Yersinia pestis O ∶ 9 served as control.The single nucleotide polymorphisms probes were designed on the basis of twosites (379,576) in conservative segment from Blucella flhA,and made into gene chip.According to part sequence of flhA,primers were labeled by biotin,target gene was amplified by PCR,and hybridized with the designed gene chip,and finally,biotin was used for identification.Results The length of all 9 strains were all 357 bp,consistent with expected results.The Yersinia pestis O ∶ 9 that had the same antigens with the Brucella showed no specific PCR products.At the 379 and 576 points of gene,the Brucella suis was 379G,576C,the Brucella Abortus was 379T,576T,the Brucella Melitensis was 379T,576C,all these results were identical to the designed probe sequence.Experimental verification of membrane gene chip,the Brucella suis showed color at the points of 379G,576C and F,the Brucella Abortus showed color at the points of 379T,576T and F,the Brucella Melitensis showed color at the points of 379T,576C and F.Conclusion Brucella gene chip can be used to identify Brucella rapidly.