贵州省黑线姬鼠钩端螺旋体分离株MLVA分型研究

MLVA genotyping of Leptospira strains isolated from A podemus agrarius in Guizhou Province

目的采用多位点可变数目串联重复序列分析技术(MLVA)分析贵州省黑线姬鼠钩端螺旋体(简称钩体)分离株的分子流行病学特征,为贵州省钩体病的防控提供依据。方法采用硅胶膜型TM细菌基因组DNA提取试剂盒提取56株致病性钩体DNA,选择7个可变数目串联重复序列VNTR位点进行PCR扩增,扩增产物经2%琼脂糖凝胶电泳后用凝胶成像系统成像并计算各VNTR位点的扩增长度,分析菌株各VNTR位点的重复数目;采用BioNumerics软件分析,用UPGMA系数分析分离株与参考菌株间的聚类关系。结果 MLVA分析结果显示,56株钩端螺旋体有VNTR4、VNTR36、VNTR50位点出现重复数目差别,被分为6个MLVA型(A1、B1、B2、B3、B4、B5型)。聚类分析显示56株钩端螺旋体与参考菌株黄疸出血群56601株聚类关系较近,与其他参考株聚类关系较远。结论贵州省近年黑线姬鼠钩端螺旋体分离株均为黄疸出血群,MLVA型别具有多样性。

Objectives To molecularly type Leptospira strains isolated from Apodemus agrarius using multiple loci VNTR analysis（MLVA）and to analyze their epidemiological characteristics in order to provide strategies for the prevention and control of leptospirosis in Guizhou Province. Methods DNA from 56 strains of Leptospira was extracted by alkaline lysis.Seven loci of tandem repeats（variable number tandem repeats or VNTRs）were selected and were amplified with PCR primers.PCR products were electrophoresed on 2% agarose gel and then photographed using agel imaging instrument.The length of the amplified fragment and the loci of repeats were determined and clustering among isolates and reference strains was analyzed.UPGMA cluster analysis was performed with the software BioNumerics. Results MLVA analysis indicated differences in VNTR4,VNTR36,and VNTR50 loci among 56 strains,and these strains were divided into six MLVA types（A1,B1,B2,B3,B4,and B5）.Cluster analysis indicated that these 56 strains were divided into two clusters and were closest to the strain 56601. Conclusion The Leptospirastrain isolated fromA.agrarius in Guizhou Province in recent years was L.icterohaemorrhagiae.Results indicated that there are obvious MLVA polymorphisms in Leptospiraspp.in Guizhou Province.