临床分离志贺菌中CRISPR/Cas系统的分布及其与毒力基因的关系

Distribution of CRISPR/Cas system in Shigella clinical strains and its relationship with virulence genes

【目的】了解临床分离志贺菌中CRISPR/Cas系统的分布特征并分析其与毒力基因的关系。【方法】以聚合酶链式反应(PCR)方法,采用10对引物分别对57株临床分离志贺菌中CRISPR1、cas2-cas1、cas6e-cas5、cas7、cse2、cse1-cas3基因和毒力基因ipaH、ial、ipaBCD、virA进行检测。对CRISPR1的PCR结果进行测序,并用CRISPRfinder在线软件对CRISPR1基因座进行分析。通过卡方检验初步分析CRISPR/Cas系统与毒力基因的关系。【结果】测序结果显示,CRISPR1基因座中间隔序列数目较少且在不同菌株间一致性较高;57株志贺菌中,84.2%(48/57)的志贺菌中可检测到CRISPR/Cas系统,其中68.8%(33/48)的志贺菌中cas6e-cas5基因或(和)cse2基因中发现插入序列;毒力基因ipaH、ial、virA、ipaBCD的检出率依次为100%、100%、98.2%和87.7%;毒力基因ipaBCD的阳性率与活性CRISPR/Cas系统的分布无关(P>0.05)。【结论】CRISPR/Cas系统广泛存在于临床分离志贺菌中;部分cas基因中有插入序列;并未发现志贺菌中活性CRISPR/Cas系统与毒力基因的分布有关。

[Objective] To detect the distribution of CRISPR/Cas system in Shigella clinical strains and to analyze the relationship between CRISPR/Cas system and virulence genes.[Methods] We used 10 pairs of primers of PCR assay for detection of the CRISPR/Cas system including CRISPR1,cas2-cas1 genes,cas6e-cas5 genes,cas7 gene,cse2 gene,cse1-cas3 genes and virulence genes including ipaH,ial,ipa BCD,vir A in 57 Shigella strains.The CRISPR sequences were analyzed using CRISPR finder.The relativity between the presence of CRISPR/Cas system and virulence gene was analyzed by χ2-test.[Results] Among the 57 strains of Shigella,the number of spacers is little and its sequences have similarity among different strains.The positive rate of CRISPR/Cas system is 84.2%.However,68.8% of Shigella strains analyzed contained insertion sequencese in cse2 gene or cas6e-cas5 gene.The positive rates of ipa H,ial,vir A and ipa BCD is 100%,100%,98.2% and 87.7%,respectively.No statistical difference was found between the distribution of active-CRISPR/Cas system and the ipa BCD gene.[Conclusion] CRISPR/Cas system was widely distributed in Shigella clinical strains.Insertion sequences were presented in some strains.Our analysis of CRISPR/Cas system did not reveal a potential link between its presence and the virulence genes in Shigella clinical isolates.