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目的应用脉冲场电泳(PFGE)分型技术和16S rRNA基因测序分析技术分别对贵州省3株动物宿主钩端螺旋体分离菌株进行分子分型和基因种鉴定,了解贵州省钩端螺旋体的分子流行病学特征。方法应用DNA限制性内切酶NotI对钩端螺旋体染色体DNA酶切后,用PFGE将DNA片段分离,采用BionumericsV4.0将3株钩体菌株PFGE图谱与中国15群15型参考菌株进行聚类分析;同时,应用PCR扩增几乎全长的钩体16S rRNA基因片段,并将扩增产物进行双向序列测定,并与GenBank数据库已注册的核酸序列进行同源性比对、确定基因种、分析亲缘进化关系。结果来自贵州省的3株动物宿主分离钩体菌株PFGE带型命名为LepNot I 002和LepNot I 003,经聚类分析,3株菌株与黄疸出血群黄疸出血型赖株的相似性大于95%。16S rRNA基因测序和分析表明,3株贵州分离钩体菌株之间的同源性为100%,与致病性钩体问号钩端螺旋体种(L.interrogans)不同血清型参考菌株的同源性达100%。结论 3株贵州动物分离钩体菌株经PFGE分型鉴定与黄疸出血群赖型赖株的相似性大于95%,经16S rRNA基因测序分析鉴定为L.interrogans种,上述两种方法对贵州省钩体分离菌株的鉴定结果一致,有助于贵州省钩端螺旋体病的主动监测、暴发调查和传染源追踪。
Objective To investigate the molecular typing and genotyping of three strains of Leptospira interrogans in Guizhou province by using pulsed-field electrophoresis (PFGE) and 16S rRNA gene sequencing analysis, and to understand the molecular epidemiology of Leptospira Learn characteristics. Methods DNA fragment was digested with DNA restriction endonuclease NotI and PFGE was used to isolate the DNA fragment. The PFGE patterns of three strains of Leptospira were analyzed by cluster analysis with 15 Chinese reference strains of 15 strains using Bionumerics V4.0. ; At the same time, PCR amplification of nearly full-length hooks 16S rRNA gene fragment, and the amplified products were two-way sequence analysis, and the GenBank database has been registered nucleic acid sequence homology comparison to determine the genotype, analysis of kinship Evolutionary relations. Results The PFGE bands of three isolates from Guizhou Province were named as LepNot I 002 and LepNot I 003, and the cluster analysis showed that the similarity of the three isolates to the jaundice hemorrhagic jaundice hemorrhagic strain was greater than 95%. 16S rRNA gene sequencing and analysis showed that the homology between the three isolates of Guizhou isolates was 100%, which was homologous with the homologous serotype reference strains of L. interrogans Up to 100%. Conclusion The similarity of PFGE genotypes and strains of Leptospira interrogans in Guizhou isolates from three Guizhou isolates was more than 95%, which was identified as L.interrogans by 16S rRNA gene sequencing analysis. The results of the identification of the isolates of the isolates are consistent, which is helpful for the active surveillance, outbreak investigation and source tracking of leptospirosis in Guizhou Province.