|
|
- 2016
基于CRISPR对大肠埃希菌O157∶H7的检测
|
Abstract:
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。
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
| [1] | MORI Y, WADA H, TAMAKI S, et al. Outcome of thrombotic thrombocytopenic purpura and hemolytic uremic syndrome in Japan[J]. Clin Appl Thromb Hemost,1999, 5(2):110-112. |
| [2] | FRANZ E, KLERKS MM, DE VOS OJ, et al. Prevalence of Shiga toxin-producing Escherichia coli stx1, stx2, eaeA, and rfbE genes and survival of E.coli O157∶H7 in manure from organic and low-input conventional dairy farms[J]. Appl Environ Microbiol, 2007, 73(7):2180-2190. |
| [3] | JANSEN R, EMBDEN JD, GAASTRA W, et al. Identification of genes that are associated with DNA repeats in prokaryotes[J]. Mol Microbiol, 2002, 43(6):1565-1575. |
| [4] | DELANNOY S, BEUTIN L, FACH P. Use of clustered regularly interspaced short palindromic repeat sequence polymorphisms for specific detection of enterohemorrhagic Escherichia coli strains of serotypes O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157∶H7 by real-time PCR[J]. J Clin Microbiol, 2012, 50(12):4035-4040. |
| [5] | PAI CH, GORDON R, SIMS HV, et al. Sporadic cases of hemorrhagic colitis associated with Escherichia coli O157∶H7. Clinical, epidemiologic, and bacteriologic features[J]. Ann Intern Med, 1984, 101(6):738-742. |
| [6] | TOUCHON M, CHARPENTIER S, CLERMONT O, et al. CRISPR distribution within the Escherichia coli species is not suggestive of immunity-associated diversifying selection[J]. J Bacteriol, 2011, 193(10):2460-2467. |
| [7] | FABRE L, ZHANG J, GUIGON G, et al. CRISPR typing and subtyping for improved laboratory surveillance of Salmonella infections[J]. PLoS One, 2012, 7(5):e36995. |
| [8] | ISHINO Y, SHINAGAWA H, MAKINO K, et al. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product[J]. J Bacteriol, 1987, 169(12):5429-5433. |
| [9] | 郭向娇，王颖芳，段广才，等. 临床分离志贺菌中CRISPR/Cas系统的分布及其与毒力基因的关系[J]. 微生物学通报, 2015, 55(3):543-549. |
| [10] | 王琳琳，王颖芳，段广才，等. 志贺菌CRISPR的检测及其与耐药的关系[J].微生物学报, 2015, 55(4):476-483. |
| [11] | LI H, JING H, PANG B, et al. Study on diarrhea disease caused by enterohemorrhagic Escherichia coli O157∶H7 in Xuzhou city, Jiangsu province in 2000[J]. Chin J Epidemiol, 2002, 23(2):119-122. |
| [12] | TOUCHON M, ROCHA EP. The small, slow and specialized CRISPR and anti-CRISPR of Escherichia and Salmonella[J]. PLoS One, 2010, 5(6):e11126. |
| [13] | NOZAWA T, FURUKAWA N, AIKAWA C, et al. CRISPR inhibition of prophage acquisition in Streptococcus pyogenes[J]. PLoS One, 2011, 6(5):e19543. |
| [14] | MARRAFFINI LA, SONTHEIMER EJ. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA[J]. Science, 2008, 322(5909):1843-1845. |
| [15] | MOJICA FJ, GARC？PA-MART？PNEZ J, SORIA E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements[J]. J Mol Evol, 2005, 60(2):174-182. |
| [16] | MAKAROVA KS, HAFT DH, BARRANGOU R, et al. Evolution and classification of the CRISPR-Cas systems[J]. Nat Rev Microbiol, 2011, 9(6):467-477. |
| [17] | GUO X, WANG Y, DUAN G, et al. Detection and analysis of CRISPRs of Shigella[J]. Cur Microbiol, 2015, 70(1):85-90. |
