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

摘要:

【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CRISPR finder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。

Abstract:

[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, ipaBCD, virA 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 ipaH, ial, virA and ipaBCD is 100%, 100%, 98.2% and 87.7%, respectively. No statistical difference was found between the distribution of active-CRISPR/Cas system and the ipaBCD 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.