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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 new detection method for Escherichia coli O157: H7 based on clustered regularly spaced short palindrome repeats (CRISPR). Methods PCR-amplified CRISPR1 of 443 strains of intestinal bacteria (310 strains of non-O157: H7 Escherichia coli, 35 strains of Escherichia coli O157: H7, 89 strains of Shigella and 9 strains of Salmonella) CRISPR2, and PCR products were sequenced. 100 strains of enteric bacteria (47 strains of non-O157: H7 Escherichia coli, 5 strains of Escherichia coli O157: H7 and 9 strains of Shigella and 39 Salmonella) CRISPR1 and CRISPR2 sequences. The PCR product sequencing sequences and the CRISPR database of the CRISPR database were analyzed using the CRISPR Finder online software. Clustal X software for spacer sequence alignment. Comparison of standard methods and PCR amplification CRISPR two methods to detect Escherichia coli O157: H7 consistency. Results A total of 543 strains of enteric bacteria were analyzed. Among them, 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, 3 to 26,2 to 9,2 to 32,3.57.1% 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 spacer sequences is 1 to 20, 1 to 6, 2 to 27, 1, or 4. 95% of the O157: H7 Escherichia coli CRISPR1 and 90% CRISPR2 contain three unique spacer sequences (S1-1, S1-2, S1-3) and one unique spacer sequence (S2-1), respectively. The sequence alignment showed that the specificity of S1-1 + S1-2 + S1-3 and S2-1 for detecting O157: H7 Escherichia coli was 100% and 99.6%, respectively. The consistency of standard detection and PCR amplification of CRISPR1 and CRISPR2 detection Escherichia coli O157: H7 were 99.6% and 99.3% respectively. Based on CRISPR test Escherichia coli O157: H7 in simulated samples, the results showed that the original sample of Escherichia coli O157: H7 concentration of about 2.3CFU / mL, after 12h enrichment can be detected. Escherichia coli O157: H7 cluster analysis showed that 40 O157: H7 Escherichia coli can be divided into three categories. Conclusion The detection of Escherichia coli O157: H7 based on CRISPR can be used as a valuable epidemiological tool to monitor Escherichia coli O157: H7 infection and highly virulent Escherichia coli O157: H7.