基于CRISPR1上游侧翼序列鉴定沙门菌的效果评价

Evaluation on methods of the effectiveness with which Salmonella enterica is identified based on flanking sequences upstream of CRISPR1

目的评价基于成簇规律间隔的短回文重复序列(Clustered regularly interspaced short palindromic repeats,CRISPR)1上游侧翼序列鉴定沙门菌的效果。方法在NCBI里通过已知的重复序列BLAST识别并获得全基因组测序沙门菌的CRISPRs和CRISPR相关基因(CRISPR-associated,cas)信息;选择并提取确定CRISPRs的上、下游各500bp的侧翼序列,使用Clustal X软件比对侧翼序列;采用PCR方法扩增沙门菌的CRISPRs序列和CRISPR1上游特异侧翼序列,与金标准API方法鉴定的菌株比较,评价CRISPR1上游特异侧翼序列检测对鉴定沙门菌的效果。结果329株全基因测序和38株实验室保存的沙门菌均存在I-E CRISPR/Cas,除猪霍乱沙门菌C500外,其余所有菌株均存在CRISPR1和2两个位点。在沙门菌的CRISPR1上游侧翼序列存在61bp的特异序列,一致性为99%,且具有种属特异性,通过PCR扩增此序列鉴定沙门菌的灵敏度为97.4%,特异度为99.1%。结论基于CRISPR1上游侧翼序列鉴定沙门菌的方法灵敏特异,效果良好。

Objective To evaluate the effectiveness with which Salmonella enterica can be identified based on flanking sequences upstream of clustered regularly interspaced short palindromic repeat 1(CRISPR1). Methods Sequences of both CRISPR and cas were obtained via a search of GenBank for repeat sequences using BLAST;the search was limited to the S.enterica genome.Five-hundred-bp flanking sequences upstream and downstream of CRISPR were identified and confirmed,and then Clustal X was used to perform multi-sequence alignment of the flanking sequences.Strains were identified using API(the gold standard).PCR was used to amplify CRISPRs and the flanking sequences upstream of CRISPR1.The latter were used to assess the effectiveness with which S.enterica was identified.Results Results indicated that all of the strains,including 329 strains with complete genome sequences and 38 strains kept in the laboratory,contained I-E CRISPR/Cas;CRISPR1 and 2 sites were present in all of the strains except S.enterica C500.The flanking sequences upstream of CRISPR1 contained a particular sequence 61 bp in length,with a consistency of 99%;the sequence was only found in S.enterica.When amplified with PCR,this region identified S.enterica with a sensitivity of 97.4%and specificity of 99.1%. Conclusion A method of identifying S.enterica based on the sequences upstream of CRISPR1 is sensitive,specific,and effective.