宁夏布鲁氏菌系统鉴定及16SrRNA基因序列分析

Systematic identification and 16SrRNA gene sequence analysis of Brucella in Ningxia

目的鉴定宁夏地区布鲁氏菌历史菌株、近期分离菌株,检测分析16SrRNA基因序列,构建遗传进化树,为宁夏地区布鲁氏菌鉴定、传染源追溯和分子流行病学监测体系提供基础数据。方法利用传统生物学方法和分子生物学方法鉴定具有代表性的20株布鲁氏菌和参考菌株的种和生物型,从GeneBank下载与布鲁氏菌有共同抗原细菌的16SrRNA基因序列,并采用PCR扩增产物直接测序法对布鲁氏菌的16SrRNA扩增、测序,用DNAMAN软件进行序列拼接比对,构建遗传进化树。结果经传统生物学方法和分子生物学方法鉴定20株布鲁氏菌分别为羊种13株,牛种2株,猪种4株,犬种1株。布鲁氏菌历史菌株、近期分离菌株和部分参考菌株16SrRNA经PCR扩增均得到1 412 bp条带,所有菌株和布鲁氏菌有共同抗原细菌的16SrRNA经一致性分析,达到98.54%,进化树显示,选取的具有代表性的分离菌株与参考菌株在同一末端的分支上,遗传距离十分接近。布鲁氏菌与人苍白杆菌在进化树的同一次分支上,遗传距离为0.019,而与霍乱弧菌、大肠杆菌O∶157、耶尔森菌属O∶9的遗传距离较远,遗传距离均大于0.02。结论从遗传进化角度对布鲁氏菌分离株与标准菌株的亲缘关系进行分析,所有种型遗传距离接近,提示16SrRNA为高度保守序列,不宜做遗传进化分析的靶基因,可作为布鲁氏菌属的一个鉴定方法,布鲁氏菌与人苍白杆菌有较近的亲缘关系。

Objective To identify the history Brucella strains,recently isolated strains in Ningxia,and 16SrRNA gene sequence analysis,phylogenetic tree construction,to determine the taxonomic status of the strain,and to provide the basis for the establishment of monitoring and tracking and tracing system for molecular epidemiology of Brucella infection source in Ningxia.Methods 20 representative strains of Brucella species and biotypes were identified by using the method of traditional biological methods and molecular biology methods,and 16SrRNA gene sequences of common bacteria antigen downloaded from the GeneBank standard and reference strains of Brucella,and using PCR 16SrRNA direct sequencing of the PCR products of Brucella were amplified and sequenced,sequence alignments with DNAMAN software phylogenetic tree construction.Results The results showed that identified by traditional biological methods and molecular biology methods in 20 strains of Brucella included 14 strains in sheep,one and two strains in cattle,4 strains in pig and one strain in dog.1412bp bands were obtained by PCR amplification of Brucella 16SrRNA,and all strains 16SrRNA reached 98.54%by consensus analysis in identification of the history Brucella strains,recently isolated strains and some reference strains.Phylogenetic tree showed that the representative isolates were close to the same strain at the same end,and the genetic distance was very close.The genetic distance between Brucella and human pale bacilli on the same branch of phylogenetic tree is 0.019,and the genetic distance of Vibrio cholerae,Escherichia coli O:157 and Jerson O:9 was far greater than 0.02.Conclusion Brucella all kinds of genetic distance,suggesting that 16SrRNA is highly conserved sequence of Brucella,for target gene phylogenetic analysis of Brucella and ochrobactrum anthropi has close relationship.