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。
Objective To establish a method for detection and identification of Escherichia coli O157∶H7with 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（47strains of non-O157∶H7 Escherichia coli,5strains of Escherichia coliO157∶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 Escherichia coli was 99.6% and 99.3% compared with the standard method.The Escherichia coli O157∶H7 was detected after 12 hours with a sensitivity of 2.3 CFU/mL milk sample.The cluster analysis showed that 40 strains of O157∶H7 E.coli could be divided into three groups.Conclusion The detection of E.coli O157∶H7 based on the CRISPR locus by PCR may become a valuable epidemiologic tool for the surveillance of E.coli O157∶H7 infections.
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2016年第5期748-753,共6页
Journal of Xi’an Jiaotong University（Medical Sciences）
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)~~
