用于检测仔猪致病菌的23S rRNA基因芯片的研制  

Development of 23S rRNA gene-based microarray for detection of pathogenic bacteria in piglets

作　　者：伍诚意[1,2] 曹峰子[1,3] 梁之昶[1,4] 章振华[1] 杨兵[1] 周宏专[1,5] 季海峰[1] 陈小玲[1] 

机构地区：[1]北京市农林科学院畜牧兽医研究所,北京100097 [2]江西生物科技职业学院,江西南昌330200 [3]河北农业大学动物科技学院,河北保定071000 [4]北京华大吉比爱生物技术有限公司,北京101300 [5]首都师范大学生命科学院,北京100037

出　　处：《中国兽医科学》2010年第3期258-264,共7页Chinese Veterinary Science

基　　金：北京市科技合同项目(Z0006321000191)

摘　　要：以副猪嗜血杆菌、胸膜肺炎放线杆菌、多杀性巴氏杆菌、大肠杆菌、肠道沙门氏菌、猪链球菌等12种仔猪致病菌为目标细菌,23SrRNA基因为靶基因,从GenBank中下载其23SrDNA全序列,在其变异区设计特异性探针,在保守区设计通用引物。通过序列分析、PCR靶标与探针杂交试验,建立了有13条寡核苷酸探针和2对通用引物的仔猪常见致病菌基因芯片检测方法。以参考菌株粗提基因组DNA的10倍系列稀释模板做PCR,扩增产物与探针杂交,检出芯片的灵敏度为100fg。对33个参考菌株用芯片检测,结果有1例不正确,只鉴定到属;在61个野外分离株中,55株的芯片检测结果与生化或测序结果一致,有6例不一致,符合率为90%。初步研究证明,该检测方法能快速、有效地鉴定多种仔猪常见致病菌,但要区分某些细菌的致病株与非致病株,还要检测毒力/毒素基因。To develop a DNA microarray for rapid detection of pathogenic bacte-ria in piglets,12 bacterial species such as Haemophilus parasuis,Actinobacillus pleuropneumonia,Pasteurella multocida,Escherichia coli,Salmonella enterica and Streptococcus suis were chosen as the targets and 23S rDNA sequences of the bacteria were downloaded from the GenBank.The variable and conserved regions of the sequences were allocated for designing probes and primers.Based on sequence analysis and hybridization tests of PCR amplicons of target species with candidate probes,a DNA microarray equipped with 13 oligonucleotide probes and 2 pair of universal primers was developed.The serial dilutions of crude genomic DNA were amplified and detection limit of the microarray was determined to be approximately 100 fg total DNA.The efficacy of the DNA microarray was assessed with crude DNA from 33 target and non-target reference strains and 61 field isolates identified by 16S rRNA gene sequencing or biochemical tests.The correct identification for species was 32/33（97%） for reference strains and 55/61（90%） for field isolates respectively.The results demonstrated that the developed DNA microarray was a effective and rapid assay for identifica-tion of pathogens in piglets.To discriminate pathogenic from non-pathogenic strains of some bacteria,virulent or toxic genes have to be identified.

关 键 词：仔猪 致病菌 23S RRNA基因 芯片 寡核苷酸探针 

分 类 号：S858.25[农业科学—临床兽医学] Q81[农业科学—兽医学]