两株布鲁菌MLST新序列型(ST)的鉴定

目的 对北京地区分离自布病患者血液的2株布鲁菌进行多位点序列分型(Multilocus sequence typing,MLST)及其遗传学特征进行研究,为布病的预防和控制提供科学依据。方法 应用布鲁菌属特异性PCR(BCSP31-PCR)和种/型特异性PCR(AMOS-PCR)对分离的2株布鲁菌进行鉴定,采用MLST技术对2株布鲁菌分离株的19个管家基因、1个外膜蛋白基因及1个基因间区的序列进行测定,与MLST数据库中的等位基因序列进行比对,确定菌株的等位基因谱及9位点和21位点序列型(sequence type,ST)。采用聚类分析法研究2株菌ST型与各种布鲁菌已知ST型的遗传进化关系。结果 2株分离株经BCSP31-PCR鉴定为布鲁菌属细菌,AMOS-PCR鉴定为非羊种、非牛种1、2、4型、非猪种1型、非绵羊附睾种布鲁菌;MLST分析显示,2株菌的等位基因编号(gap/aroA/glk/dnaK/gyrB/trpE/cobQ/int-hyp/omp25/prpE/caiA/csdB/soxA/leuA/ mviM/fumC/fbaA/ddlA/putA/mutL/acnA)分别为2、1、2、2、1、3、1、4、1、13、2、2、2、2、1、1、3、7、6、2、3和2、1、2、2、1、3、1、4、29、13、2、2、2、2、1、1、3、7、6、2、3,比对MLST数据库,结果显示这2株细菌的等位基因编号组合未见报道,为新ST型。聚类分析显示这2种新的ST型与牛种布鲁菌ST型处于同一分支。结论 北京地区分离的2株布鲁菌为新ST型牛种布鲁菌,已被MLST数据库确认,分别命名为ST74和ST75(9位点序列型)与ST108和ST109(21位点序列型)。与同属牛种布鲁菌的ST型遗传关系最近,该结果为北京地区布病的预防和控制提供了科学依据。

Identification of two new sequence type of Brucella isolates by multilocus sequence typing

We defined the sequence type (ST) of 2 Brucella strains isolated from patients’ blood in Beijing by multilocus sequence typing (MLST) and investigate their genetic characteristics to provide scientific basis for the control and prevention of brucellosis. The 2 isolates were identified using Brucella genus specific BCSP31-PCR and species-specific AMOS-PCR. Nineteen house-keeping genes, one out membrane protein gene and one gene-inter-region sequence of the strains were further analyzed using MLST. The sequences were compared with the existing allele sequences on the MLST website to determine the allelic profile and sequence type and to determine the 9-loci ST and 21-loci ST. Clustering analysis was used to investigate the genetic relationship between the 2 new STs and other known STs of Brucella. The 2 isolates were identified as Brucella with BCSP31-PCR. AMOS-PCR results suggested that the isolates were not B. melitensis, not type 1, 2 and 4 B. abortus, not type 1 B. suis, and not B. ovis. MLST analysis revealed that 2 novel allele combinations (gap/aroA/glk/dnaK/gyrB/trpE/cobQ/int-hyp/omp25/prpE/caiA/csdB/soxA/leuA/mviM/fumC/fbaA/ddlA/putA/mutL/acnA) 2, 1, 2, 2, 1, 3, 1, 4, 1, 13, 2, 2, 2, 2, 1, 1, 3, 7, 6, 2, 3 and 2, 1, 2, 2, 1, 3, 1, 4, 29, 13, 2, 2, 2, 2, 1, 1, 3, 7, 6, 2, 3 were found in the isolates but not reported before. Clustering analysis revealed that the 2 novel STs located at the same branches as the STs of B. abortus. The 2 isolates from Beijing were identified as 2 novel STs by this study which were confirmed by the MLST website,and were assigned as ST74 and ST75 (9-loci ST), ST108 and ST109 (21-loci ST). The 2 STs were most genetically closed to the STs belonging to B. abortus. The results provide scientific basis for the control and prevention of brucellosis in Beijing.