单核细胞增生李斯特菌规律成簇间隔短回文重复序列位点分子特征及其遗传进化分析  

Molecular characteristics and genetic evolution analysis of CRISPR loci in Listeriamonocytogenes

作　　者：杜波 吴莹[1] 蔡楠楠 任妍妍 修敏[1] 刘文新[1] DU Bo;WU Ying;CAI Nannan;REN Yanyan;XIU Min;LIU Wenxin(Fuxin Center for Disease Control and Prevention,Fuxin,Liaoning 123000,China)

机构地区：[1]阜新市疾病预防控制中心,辽宁阜新123000

出　　处：《中国热带医学》2025年第3期343-349,共7页China Tropical Medicine

摘　　要：目的 检测单核细胞增生李斯特菌(Listeria monocytogenes)中规律成簇间隔短回文重复序列(clustered regularly interspaced short palindromic repeats, CRISPR),对其位点的结构以及同源性进行分析。方法 对阜新市疾病预防控制中心2018—2023年分离出的34株单核细胞增生李斯特菌进行分离鉴定、PCR扩增和测序。利用生物信息学位软件搜索、鉴定和分析CRISPR位点中重复序列(repeat)结构和保守性,预测重复序列RNA二级结构以及间隔序列(spacer)同源性。结果 34株菌株共检测出7种CRISPR位点,CRISPR位点的重复序列前2个碱基和后2个碱基变异率较高,中间部分变异率较低。7种CRISPR位点组成8种CRISPR结构型,其中CRISPR1和CRISPR2的重复序列相对保守,CRISPR1和CRISPR5的重复序列能形成哑铃状二级结构。每个CRISPR位点含有的间隔序列个数为2~15个,平均为2.43个。检测出的136个间隔序列除了与李斯特菌质粒和噬菌体同源外,还与环境中未培养的病毒序列、葡萄球菌噬菌体和英诺克李斯特菌同源。相同CRISPR基因型未呈现大范围聚集,但在同一年份的一些菌株处在同一亲缘关系较近的进化簇。结论 本研究中的单增李斯特菌CRISPR结构具有高度特异性,此菌的CRISPR结构与噬菌体的同源性高,为应用噬菌体防控单增李斯特菌提供理论依据。Objective To detect clustered regularly interspaced short palindromic repeats(CRISPR)in Listeria monocytogenes,and analyze the structure and homology of CRISPR loci.Methods Totally 34 strains of Listeria monocytogenes isolated in our laboratory were identified,PCR amplified and sequenced.The repeat sequence structure and spacer sequence homology in CRISPR loci were analyzed by bioinformatics software.Results A total of 7 CRISPR loci were detected in 34 strains.The mutation rate of the first 2 and last 2 bases of the Repeat sequence of CRISPR loci was higher,while the mutation rate of the middle part was lower.Seven CRISPR sites form eight CRISPR structural types,among which the Repeat sequences of CRISPR1 and CRISPR2 are relatively conserved,while the Repeat sequences of CRISPR1 and CRISPR5 can form dumbbell shaped secondary structures.The number of Spacer sequences contained in each CRISPR site ranges from 2 to 15,with an average of 2.43.The 136 Spacer sequences detected were not only homologous to Listeria plasmids and bacteriophages,but also homologous to uncultured virus sequences,staphylococcal bacteriophages,and Listeria innocua.The same CRISPR genotype did not show large-scale clustering,but some strains in the same year were in the same evolutionary cluster with close genetic relationships.Conclusion The CRISPR structure of Listeria monocytogenes in this study exhibits high specificity,and its homology with bacteriophages provides a theoretical basis for the application of bacteriophages in the control and prevention of Listeria monocytogenes.

关 键 词：单核细胞增生李斯特菌 规律成簇间隔短回文重复序列 重复序列 间隔序列 噬菌体 

分 类 号：R378[医药卫生—病原生物学]