%0 Journal Article %T 基于CRISPR对大肠埃希菌O157∶H7的检测<br>Detection of Escherichia coli O157∶H7 based on CRISPR locus %A 梁文娟 %A 张荣光 %A 段广才 %A 王颖芳 %A 洪丽娟 %A 张 %A 冰 %A 王鹏飞 %A 郗园林 %A 杨海燕 %J 西安交通大学学报(医学版) %D 2016 %R 10.7652/jdyxb201605027 %X 摘要：目的 基于成簇的规律间隔短回文重复序列(CRISPR)，建立对大肠埃希菌O157∶H7的新型检测方法。 方法 应用PCR扩增实验室保存的443株肠道细菌(310株非O157∶H7大肠埃希菌、35株大肠埃希菌157∶H7、89株志贺菌和9株沙门菌)的CRISPR1和CRISPR2，并将PCR产物测序；提取CRISPR database数据库中(标准法)100株肠道细菌(47株非O157∶H7大肠埃希菌、5株大肠埃希菌O157∶H7、9株志贺菌和39株沙门菌)的CRISPR1和CRISPR2序列。使用CRISPR Finder在线软件分析PCR产物测序序列和CRISPR database数据库的CRISPR序列。Clustal X软件进行间隔序列比对。比较标准法和PCR扩增CRISPR两种方法检测大肠埃希菌O157∶H7的一致性。结果 共分析543株肠道细菌，其中75.6%的非O157∶H7大肠埃希菌、75.5%志贺菌、91.7%沙门菌和95% O157∶H7大肠埃希菌含有CRISPR1，其间隔序列数目为3～26、2～9、2～32、3。57.1%的非O157∶H7大肠埃希菌、77.6%志贺菌、85.4%沙门菌和100% O157∶H7大肠埃希菌含有CRISPR2，其间隔序列数目为1～20、1～6、2～27、1或4个。95%的O157∶H7大肠埃希菌的CRISPR1和90% CRISPR2分别含有3条独特间隔序列(S1-1，S1-2，S1-3)和1条独特间隔序列(S2-1)。间隔序列比对结果显示，S1-1+S1-2+S1-3和S2-1检测O157∶H7大肠埃希菌的特异性分别是100%和99.6%。标准法检测和PCR扩增CRISPR1和CRISPR2检测大肠埃希菌O157∶H7的一致性分别达99.6%和99.3%。基于CRISPR检测大肠埃希菌O157∶H7在模拟样品中的应用，结果显示在原样品大肠埃希菌O157∶H7浓度约2.3CFU/mL时，经12h增菌后即能检测出来。大肠埃希菌O157∶H7聚类分析显示，40株O157∶H7大肠埃希菌可分为3类。结论 基于CRISPR的大肠埃希菌O157∶H7的检测方法，可以作为监测大肠埃希菌O157∶H7感染和高毒株大肠埃希菌O157∶H7有价值的流行病学工具。<br>ABSTRACT: Objective To establish a method for detection and identification of Escherichia coli O157∶H7 with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) locus as a target by PCR. Methods PCR amplification was used to detect the CRISPR locus of 443 strains (310 strains of non-O157∶H7 Escherichia coli, 35 strains of Escherichia coli O157∶H7, 89 Shigella strains, and 9 Salmonella strains). The CRISPR Finder was used to analyze the sequences including the 100 strains (47 strains of non-O157∶H7 Escherichia coli, 5 strains of Escherichia coli O157∶H7, 9 Shigella strains, and 39 Salmonella strains) in the CRISPR database. The spacers were aligned with Clustal X. Then the consistency was analyzed with the standard method and PCR amplification CRISPR1 detection of E.coli O157∶H7. Results We found that 75.6% of non-O157∶H7 Escherichia coli, 75.5% of Shigella, 91.7% of Salmonella and 95% of O157∶H7 Escherichia coli contained CRISPR1. The number of the spacers could be 3-26, 2-9, 2-32 and 3 among the 543 strains, respectively. 57.1% of non-O157∶H7 Escherichia coli, 77.6% of Shigella, 85.4% of Salmonella and 100% of O157∶H7 Escherichia coli contained CRISPR2; the number of the spacers was 1-20, 1-6, 2-27, and 1 or 4, respectively. Three unique spacers (S1-1, S1-2, S1-3) and one unique spacer (S2-1) could be detected in CRISPR1 and CRISPR2 of the Escherichia coli O157∶H7. The spacer alignment results showed that the specificity of the Escherichia coli O157∶H7 with the S1-1+S1-2+S1-3 and S2-1 was 100% and 99.6%, respectively. The consistency for CRISPR1 and CRISPR2 detecting O157∶H7 %K 大肠埃希菌O157∶H7 %K 成簇的规律间隔短回文重复序列(CRISPR) %K 聚合酶链反应 %K 检测< %K br> %K Escherichia coli O157∶H7 %K clustered regularly interspaced short palindromic repeat (CRISPR) %K PCR %K detection %U http://yxxb.xjtu.edu.cn//oa/darticle.aspx?type=view&id=201605027