贵州省3株钩端螺旋体分离株PFGE分型与基因种鉴定

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Not I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 。

Leptospira interrogans isolates by pulsed-field gel electrophoresis typing and genospecies identification in Guizhou Province

The aim of the study was to perform a molecular epidemiological investigation on the Leptospira isolates from animal hosts in Guizhou province by using pulsed-field gel electrophoresis（PFGE） molecular typing and 16S rRNA gene sequencing based species-specific identification.The extracted chromosomal DNA from leptospiral isolates were digested with restriction endonuclease Not I and the DNA segments were separated by using PFGE.By BioNumerics V4.0 software and 75% similarity as the standard,the obtained PFGE images from leptospiral isolates were managed and then the PFGE maps of leptospiral isolates were compared with those of reference strains belonging to l5 Serovar l5 serogroups of L.interrogans for cluster analysis.Besides,species-specific identification of those three Leptospira isolates were performed by 16S rRNA gene sequencing and analysis.PFGE analysis indicated that 3 strains of L.interrogans from Guizhou province were classified in to two PFGE types,and both of the two PFGE types belonged to the Serovar-groups Icterohaemorrhagiae Serovar Lai strain Lai.16S rRNA gene sequencing and analysis demonstrated the sequences of 16S rRNA gene of the 3 strains were identical,with homogeny as 100%.And the homogeny of 16S rRNA gene between the three isolated strains and Leptospira reference strains of different serovar types contained in L.interrogans species was 100%.The results revealed that 3 strains of L.interrogans isolated from Guizhou province belonged to Serovar-groups Icterohaemorrhagiae Serovar Lai,and belonged to L.interrogans genospecies.The identification results of PFGE typing are consistent with that of 16S rRNA analysis.It would contribute to the active surveillance,investigation of outbreak and source tracking for leptospirosis in Guizhou province.