山西省人源羊种布鲁氏菌的MLVA-2 B及rpoB基因分型研究

摘要

Objective To analyze the genotypes of Brucella melitensis strains isolated in Shanxi province by using multiple-locus variable-number tandem-repeat 2B typing ( MLVA-2B) and rpoB typing. Methods Brucella strains were isolated by culturing the blood samples collected from patients with brucello-sis in Shanxi province from 2014 to 2015 and then were identified with conventional methods. MLVA panel 2 ( eight loci) was used to identify the genotypes of the Brucella melitensis strains, and the results were further analyzed by cluster analysis. The rpoB gene was molecularly characterized by PCR amplification and DNA sequencing in those strains. The rpoB nucleotide sequences of all Brucella strains were compared with the published rpoB gene sequence of the Brucella melitensis strain 16M to reveal specific nucleotide variations. Results A total of 41 Brucella strains were isolated in 2014 and 2015 and all of them belonged to Brucella melitensis biovar 3. Those strains were divided into five groups and thirty-one MLVA types based on MLVA panel 2B with a genetic similarity ranging from 64. 78% to 100%. Four mutation sites in rpoB gene were in-vestigated in the 41 Brucella strains and based on that analysis, those strains were grouped as rpoB-2 (629-GTG/985-GTC/1309-CTA,95.12%)andrpoB-2variant(629-GTG/1309-CTA,4. 88%). Conclusion MLVA genotyping was significant for analyzing the sources, epidemics and outbreaks of brucellosis. Analy-zing the panel 2B loci was a simple and efficient way for genotyping the Brucella melitensis strains isolated in Shanxi province. The 41 strains belonged to two genotypes on the basis of rpoB typing, including 39 strains of rpoB-2 genotype and 2 strains of a new genotype rpoB-2 variant. There might be a correlation between ML-VA and rpoB genotyping.%目的：采用多位点可变数目串联重复序列分析( MLVA-2B)方法及rpoB基因分型方法对山西省分离的布鲁氏菌进行基因型分析。方法2014—2015年在山西省布鲁菌病(简称布病)监测点采集已确诊的布病患者血液进行血培养,对培养出的布鲁氏菌进行传统的生物分型鉴定。选取MLVA panel 2的8个位点进行分型,并对分型结果进行聚类分析。同时采用PCR方法对分离菌株rpoB基因4个相关突变位点进行扩增,PCR 产物测序结果与标准菌16M 进行比对。结果采用MLVA panel 2B分型方法对41株羊3型布氏菌进行聚类分析,所有菌株可分为5个群,31个基因型,基因型相似度在64.78%~100%。 rpoB基因检测发现39株为 rpoB-2型(629-GTG/985-GTC/1309-CTA,95.12%),2株为rpoB-2 variant型(629-GTG/1309-CTA,4.88%)。结论 MLVA基因分型对追溯传染源,分析布病的流行和暴发有重要意义。 MLVA panel 2B位点变异度更高,提示用panel 2B的5个位点对分析研究同一生物型菌株的变异情况更简便和高效。应用rpoB基因多态性分析发现,我省的菌株有39株为rpoB-2型,还有2株为新发现的rpoB-2突变型。同时发现了rpoB基因分型与MLVA分型方法可能存在相关性。