创伤弧菌16S-23S rDNA间区变形梯度凝胶电泳多态性分析

目的 建立创伤弧菌基因分型的新方法,了解创伤弧菌的分子特征.方法 应用PCR-变形梯度凝胶电泳(PCR-DGGE)技术对创伤弧菌的16S-23S rDNA间区(16S-23S rDNA intergenic spacer regions,ISR)多态性进行分析比较,结合药敏试验,以进一步验证分离菌株之间的亲缘关系.结果 ISR-PCR将创伤弧菌菌株扩增出4条约900、750、650、550 bp大小不同的条带；同时,创伤弧菌的ISR-DGGE序列表现出明显的株间差异,18株共产生了16种不同的指纹图谱；聚类分析将所有的细菌分为两大类,相似性分别为0.65和0.71;亲缘关系较近的菌株具有不同于关系较远的菌株的耐药谱,与ISR-DGGE指纹图谱分析相一致.结论 这种分子操作技术完全能够用于创伤弧菌株型的调查与鉴定,为创伤弧菌的基因分型提供了一种新的方法.

Analysis of the polymorphism of 16S-23S rDNA intergenic spacer regions from Vibrio vulnificus strains by denaturing gradient gel electrophoresis

To establish a new method for geneotyping of Vibrio vulnificus.Methods PCR-denaturing gradient gel electrophoresis(PCR-DGGE)was used to elucidate the molecular characteristics of the polymorphism of 16S-23S rDNA intergenic spacer regions(ISR)from 18Vibrio vulnificus strains. Meanwhile, detection of virulence factors and antimicrobial susceptibility test of isolates were conducted to verify the relationship of the strains.Results The Vibrio vulnificus strains could be amplified into 4different bands by ISR-PCR, including 900,0, 650and 550bp. At the same time, Vibrio vulnificus ISR-DGGE sequences showed significant differences between the strains, and all 18strains were typed into 16types by DGGE. Clustering analysis divided them into two categories with similarity of 0.65and 0.71. The results of antimicrobial susceptibility test implied that drug resistant types of sj6, sj11and sj12were different from the other 15strains, which was consistant to the ISR-DGGE.ConclusionThis molecular technique could be applied to investigation and identification of Vibrio vulnificus, and provide a new method for geneotyping.