| 梁文娟,张荣光,段广才,洪丽娟,张冰,郗园林,杨海燕,陈帅印,娄婷叶,赵永新.基于CRISPR/Cas的大肠埃希菌分子标志物的监测研究[J].Chinese journal of Epidemiology,2016,37(8):1080-1086 |
| 基于CRISPR/Cas的大肠埃希菌分子标志物的监测研究 |
| A surveillance study on CRISPR/Cas molecular biomarker in Escherichia coli |
| Received:March 21, 2016 |
| DOI：10.3760/cma.j.issn.0254-6450.2016.08.005 |
| KeyWord: 大肠埃希菌 成簇的规律间隔短回文重复序列 分子标志物 |
| English Key Word: Escherichia coli Clustered regularly interspaced short palindromic repeats/cas Molecular biomarker |
| FundProject:国家科技重大专项（2013ZX10004607）；新乡医学院分子诊断与医学检验技术河南省协同创新中心（XTCX-2015-PY4） |
| Author Name | Affiliation | E-mail | | Liang Wenjuan | Department of Epidemiology, College of Public Health, Zhengzhou University, Zhengzhou 450001, China
Henan Innovation Center of Molecular Diagnosis and Laboratory Medicine, Xinxiang Medical University, Xinxiang 453003 | | | Zhang Rongguang | Department of Epidemiology, College of Public Health, Zhengzhou University, Zhengzhou 450001, China | | | Duan Guangcai | Department of Epidemiology, College of Public Health, Zhengzhou University, Zhengzhou 450001, China
Henan Innovation Center of Molecular Diagnosis and Laboratory Medicine, Xinxiang Medical University, Xinxiang 453003 | gcduan@zzu.edu.cn | | Hong Lijuan | Department of Epidemiology, College of Public Health, Zhengzhou University, Zhengzhou 450001, China | | | Zhang Bing | Department of Epidemiology, College of Public Health, Zhengzhou University, Zhengzhou 450001, China | | | Xi Yuanlin | Department of Epidemiology, College of Public Health, Zhengzhou University, Zhengzhou 450001, China | | | Yang Haiyan | Department of Epidemiology, College of Public Health, Zhengzhou University, Zhengzhou 450001, China | | | Chen Shuaiyin | Department of Epidemiology, College of Public Health, Zhengzhou University, Zhengzhou 450001, China | | | Lou Tingye | Laboratory Department, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang 453100, China | | | Zhao Yongxin | Laboratory Department, The Third Affiliated Hospital of Xinxiang Medical University, Xinxiang 453003, China | |
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| Abstract: |
| 目的 探讨基于CRISPR/Cas的大肠埃希菌分子标志物的监测研究。方法 通过BLAST收集GenBank数据库中135株全基因组测序大肠埃希菌、203株鸟枪法测序大肠埃希菌的CRISPR/Cas和PCR扩增、测序获得本实验室保存361株大肠埃希菌（包括38株大肠埃希菌O157：H7）的CRISPR序列，应用CRISPR Finder在线软件分析CRISPR特征、DNAMAN软件进行间隔序列的比对，使用Clustal Ⅹ进行cas多序列比对和Mega 5.1软件构建系统进化树。结果 本研究以全新的视角对大肠埃希菌的CRISPR/Cas位置进行描述；结果显示，135株全基因组测序、203株鸟枪法测序和361株本实验室测序的大肠埃希菌中分别有77.04%、100.00%和75.62%的大肠埃希菌具有CRISPR1，分别有74.81%、100.00%和92.24%的大肠埃希菌具有CRISPR2，分别有11.85%、0和1.39%的大肠埃希菌具有CRISPR3和CRISPR4；GenBank数据库下载的全基因组测序的1株和本实验室测序的2株大肠埃希菌存在4个CRISPR位点；缺少cas的CRISPR1下游有插入序列存在。在699株大肠埃希菌中，8株O55：H7、180株O157：H7、8株O157：HNM、40株O104：H4、4株O145：H28有独特的CRISPR；间隔序列的缺失可发生在CRISPR中间；依据I-E和I-F的cas构建系统发育树，均可分为两类。结论 大肠埃希菌的CRISPR/Cas可能作为鉴定强毒株大肠埃希菌或者新型菌株的分子标志物。间隔序列的缺失或获得可能与噬菌体有关。 |
| English Abstract: |
| Objective A new method related to molecular biomarker with CRISPR/Cas (clustered regularly interspaced short palindromic repeats-cas) in Escherichia (E.) coli was developed and used for surveillance programs. Methods CRISPR/Cas sequence that containing 135 strains with complete sequence and 203 strains with whole genome shotgun sequence of E. coli in GenBank by BLAST and 361 strains of E. coli (including 38 strains of E. coli O157:H7) in laboratory were identified by PCR and analyzed with the CRISPR Finder. Spacers were compared with DANMAN and the phylogenetic trees of cas gene were constructed under Clustal Ⅹ and Mega 5.1. Results With new perspective, a descriptive method was developed targeting on the position of CRISPR/cas in E. coli. The CRISPR1 was detected in 77.04%, 100.00% and 75.62% and the CRISPR2 was detected in 74.81%, 100.00% and 92.24% and the CRISPR3 and CRISPR4 were detected in 11.85%, 0 and 1.39% for 135 strains with complete sequence, 203 strains with whole genome shotgun sequence and 361 strains in the laboratory, respectively. One strain downloaded in GenBank with whole genome sequencing and 2 strains in the our laboratory were identified that containing four CRISPR locus. The other E. coli strain was with insertion sequence in downstream of the non-cas CRISPR1. The unique CRISPR was found in 8 strains of O55:H7, in 180 strains of O157:H7, in 8 strains of O157:HNM, in 40 strains of O104:H4, in 4 strains of O145:H28, in all the 699 E. coli strains. The phylogenetic tree could be divided into two groups-cas with type I-E or type I-F. Conclusions CRISPR/Cas might be used as a valuable molecular biomarker in epidemiological surveillance studies to identify the high virulent strains or new strains of E. coli. Phage night be related to the missing or obtaining of spacers. |
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