CRISPR1上游侧翼序列用于大肠埃希菌和志贺菌鉴定的效果评价

摘要

目的 探讨基于成簇规律间隔的短回文重复序列(CRISPR)4上游侧翼序列对大肠埃希菌和志贺菌鉴定和评价效果.方法 通过BLAST重复序列识别并获得全基因组测序大肠埃希菌和志贺菌的CRISPRs和CRISPR相关基因(CRISPR-associated,cas),并分析其种系;选取CRISPRs上、下游各500 bp侧翼序列,使用C1ustalX进行序列比对;采用PCR方法扩增CRISPR1上游侧翼序列,以确定其对大肠埃希菌和志贺菌鉴定和评价效果.结果 73.4％(149/203)的大肠埃希菌存在I-E型CRISPR/Cas系统,包含了A、B1、D种系;8.4％(17/203)的大肠埃希菌存在I-F型CRISPR/Cas、17.2％ (35/203)的大肠埃希菌不存在CRISPR/Cas,这2种大肠埃希菌均属于B2种系;9株志贺菌均存在I-E型CRISPR/Cas.在大肠埃希菌(B2种系外)、志贺菌CRISPR1上游和大肠埃希菌(B2种系)各存在61 bp侧翼序列,序列一致性为99％,且有种属特异性,PCR扩增此区域鉴定大肠埃希菌和志贺菌的灵敏度和特异度均＞91％.结论 基于CRISPR1上游序列可用来鉴定大肠埃希菌和志贺菌,且具有很好的效果.%Objective To analyze the effect of the identification and evaluation of Escherichia (E.) coli and Shigella,based on the upstream flanking sequences of CRISPR1.Methods Both CRISPR and cas sequences were obtained through the BLAST with repeating sequences against the publicly complete genome in GenBank that related to E.coli and Shigella.Clustal X was used to perform multi-sequences alignment of the flanking sequences.PCR method was used to amplify the upstream flanking sequences of CRISPR1 in order to appraise the effect of identification and evaluation of upstream flanking sequences on E.coli and Shigella,which were based on the upstream flanking sequences of CRISPR1.Results The results showed that 73.4％ of the strains containing the I-E CRISPR/Cas that belonged to the phylogroups A,B1,D while 8.4％ strains carried the I-F CRISPR/Cas.Another 17.2％ of the strains owned CRISPR3-4 (non-CRISPR/Cas) only belonged to the phylogroups B2.All the Shigella strains carried I-E CRISPR/Cas.More than 99％ of similarity the CRISPR1 upstream-flanking sequences was seen in E.coli (except B2) and Shigella and E.coli (B2).Both sensitivity and specificity were greater than 91％ after PCR amplification in the region to identify the E.coli and Shigella.Conclusion The upstream of CRISPR1 could achieve a preliminary identification effect on E.coli and Shigella.