基于CRISPR对大肠埃希菌O157∶H7的检测

发布时间：2018-01-20 03:26

本文关键词： 大肠埃希菌O∶H 成簇的规律间隔短回文重复序列(CRISPR) 聚合酶链反应检测　出处：《西安交通大学学报(医学版)》2016年05期 　论文类型：期刊论文

【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O157∶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。
[Abstract]:Objective to study the regular interval short palindromes repeat sequence (CRISPRR) based on clustering. A new method for the detection of Escherichia coli O157: H7 was established. Methods 443 strains of enteric bacteria, 310 strains of non-O157: H7, were amplified by PCR. The CRISPR1 and CRISPR2 of 35 strains of Escherichia coli O157: H7F89 strains and 9 strains of Salmonella) were sequenced. CRISPR database database was used to extract 47 strains of non-O157: H7 Escherichia coli and 5 strains of Escherichia coli O157: H7 from CRISPR database database. 9 strains of Shigella and 39 strains of Salmonella). Analysis of PCR product sequence and CRISPR using CRISPR Finder online software. CRISPR sequence of database Database. Clustal. X software was used for interval sequence alignment. Two methods, standard method and PCR amplification CRISPR, were used to detect the consistency of Escherichia coli O157: H7. Results A total of 543 strains of enterobacteria were analyzed. Among them, 75.6% of non-O157: H7 Escherichia coli 75.5% Shigella spp. 91.7% Salmonella and 95 O157 7: H7 Escherichia coli contained CRISPR1. The number of the spacers was 30.26%, 32.57.1% and 77.6% of non-O157: H7 Escherichia coli. 85.4% Salmonella and 100 O157: H7 Escherichia coli contained CRISPR2, and the number of the spacers was 1 / 20 / 1 / 6 / 2 / 27. 1 or 4. 95% of O157: H7 Escherichia coli CRISPR1 and 90 CRISPR2 contained three unique spacer sequences, S1-1 and S1-2, respectively. S1-3) and a unique spacer sequence S2-1. S1-1 S1-2 S1-3 and S2-1 detect O157:. The specificity of H7 Escherichia coli was 100% and 99.6.The standard method, PCR amplified CRISPR1 and CRISPR2 were used to detect Escherichia coli O157. The consistency of H7 was 99.6% and 99.3 respectively. The application of CRISPR to the detection of Escherichia coli O157: H7 in simulated samples. The results showed that when the concentration of Escherichia coli O157: H7 was about 2.3 CFU / mL, the bacteria could be detected after 12 hours of inoculation. The cluster analysis of Escherichia coli O157: H7 showed that Escherichia coli O157: H7 was detected by cluster analysis. 40 strains of Escherichia coli O157: H7 can be classified into 3 groups. Conclusion the detection method of Escherichia coli O157: H7 based on CRISPR was studied. It can be used as a valuable epidemiological tool for monitoring Escherichia coli O157: H7 infection and high virulent Escherichia coli O157: H7 infection.
【作者单位】：郑州大学公共卫生学院流行病与卫生统计学系;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;河南科技大学预防医学教研室;
【基金】：国家科技重大专项(No.2013ZX100046070) 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(No.XTCX-2015-PY4)~~
【分类号】：R440
【正文快照】： 大肠埃希菌O157∶H7是大肠埃希菌中一个重要的致病血清型。O157∶H7菌株感染不仅可引起出血性结肠炎、阑尾炎、食管狭窄和结肠穿孔等严重胃肠道并发症[1],而且在儿童和老年人群中还可引起溶血性尿路综合征(hemolytic uremic syndrome,HUS)和血栓性血小板紫癜(thrombotic throm

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