诱导耐药沙门菌CRISPR检测及cas基因表达分析

[目的]通过对诱导耐药沙门菌成簇间隔的短回文重复序列（CRISPR）比对分析，以及cas（CRISPR associated）mRNA表达水平的变化，探讨CRISPR/Cas与沙门菌耐药性的关系。[方法]对鼠伤寒沙门菌ATCC13311进行耐药诱导分别获得3株体外诱导耐药（环丙沙星、庆大霉素、氨苄西林）菌和1株体内诱导耐药（环丙沙星）菌，设计引物对CRISPR，进行PCR扩增并检测，应用CRISPR Finder分析CRISPR序列，对5株沙门菌的CRISPR序列进行比对；利用RT-qPCR检测耐药前后沙门菌cas1、cas6、casA mRNA表达水平的变化。[结果]成功扩增出2段CRISPR序列（CRISPR1、CRISPR2），耐药菌碱基的突变均发生在前导区和序列末端；CRISPR1序列的突变发生在前导区的1~4 bp和序列末端的1 136~1 144 bp区域内；CRISPR2序列的突变发生在前导区的1~14 bp和序列末端的780~796 bp区域内；重复间隔序列保守且发现有重复序列的退化现象（degeneration repeats，DRs），同时耐药菌cas1、cas6、casA mRNA的表达水平下降。[结论]提示：沙门菌CRISPR序列突变和cas mRNA表达水平下降与其耐药性有关。
[Objectives]By comparing and analyzing the clustered regularly interspaced short palindromic repeats sequence(CRISPR),and detecting the expression of mRNA in drug resistant Salmonella,the relationship between CRISPR/Cas and drug resistance was explored. [Methods]Salmonella typhimurium ATCC13311 strains were induced by in vitro methods to obtain ciprofloxacin-resistant strain,gentamicin-resistant strain,and ampicillin-resistant strain,and ciprofloxacin-resistant strain was induced in vivo. The CRISPR were obtained by PCR amplification method,and the CRISPR sequences were analyzed by CRISPR Finder. The CRISPR sequences of five Salmonella strains were compared and the mRNA expression of cas1,cas6 and casA of them were detected before and after drug resistance by RT-qPCR. [Results]Two CRISPR sequences(CRISPR1 and CRISPR2) were amplified successfully and the base mutation mainly happened at the leader and sequence ends. The mutation of the CRISPR1 sequence occurred in the area of 1-4 bp and 1 136-1 144 bp. The mutation of the CRISPR2 sequence occurred in the area of 1-14 bp and 780-796 bp. Repeat and spacer sequences were relatively conservative,the degeneration of repeats was found. Simultaneously,the expression level of cas1,cas6 and casA mRNA decreased. [Conclusions]The results suggested that the mutation of CRISPR sequences and the decrease of cas mRNA expression are related to drug resistance in Salmonella