基于CRISPR的大肠杆菌分子分型方法的建立

发布时间：2018-03-23 15:27

本文选题：CRISPR/Cas系统　切入点：插入序列　出处：《郑州大学》2017年硕士论文

【摘要】：大肠杆菌是寄宿在人体肠道内的正常菌群,但部分血清型的大肠杆菌对人体是致病的,致病性的大肠杆菌可对人体造成多种疾病,如轻度腹泻、出血性结肠炎、溶血性尿毒症、尿路致病性感染等疾病。CRISPR/Cas系统是原核生物中发现的一种适应性免疫系统,其具有高度的多态性,利用CRISPR位点的多态性可实现对大肠杆菌分型。目的1通过生物信息学方法揭示大肠杆菌的结构特征,描述CRISPR/Cas系统与插入序列之间的位置关系,探讨CRISPR1及CRISPR2.2位点之间的关系。2建立基于CRISPR的大肠杆菌分子分型方法,比较CCT、CLPST、MLST、Serotype四种分型方法的分型效果,分析四种分型方法之间的关系。3利用CRISPR分型方法预测大肠杆菌血清型,并评估其灵敏度及特异度。方法1 CRT软件及CRISPR finder在线工具获得CRISPR位点信息,并提取间隔序列及重复序列,RNA fold进行重复序列二级结构预测,CRISPR Target软件进行间隔序列外源同源性搜索,Mega 6.0绘制系统发育进化树。2 Serotype Finder在线工具获得菌株血清学信息,Virulence Finder在线工具获得菌株毒力基因信息,MLST Finder在线工具获得菌株MLST信息。3 Excel表对间隔序列进行编号并绘制间隔序列图谱,R软件获得CRISPR分型型别。4辛普森指数被用于评估CCT、CLPST、MLST、Serotype四种分型方法的分型效果,Spearman相关性分析被用于获得四种分型方法的相关系数。结果1大肠杆菌CRISPR位点的生物信息学分析:283株全基因组测序的大肠杆菌可识别7种CRISPR位点,其中CRISPR1位点阳性率为81.62%,CRISPR2.1位点阳性率为88.69%,CRISPR2.2位点阳性率为88.70%,CRISPR2.3位点阳性率为7.42%,CRISPR3位点阳性率5.65%,CRISPR4位点阳性率为5.65%,CRISPR3-4位点阳性率为93.28%。IS可插入到CRISPR/Cas系统周围,也可插入到Cas基因中,甚至于插入到重复序列之间。2基于CRISPR的大肠杆菌分子分型方法的建立:根据CCT分型方法可获得430个型别,根据CLPST分型方法可获得215个型别,CCT、CLPST、MLST及Serotype四种分型方法的辛普森指数分别为0.8864,0.8760,0.8581,0.8643。3间隔序列组成及排布方式与血清分型及stx噬菌体的关系:stx噬菌体可在各CRISPR亚型中出现,并未发现菌株间隔序列排布方式与分离来源、分离时间及分离地区有关。4 CRISPR分型方法在预测大肠杆菌血清型中的应用:CRISPR分型方法预测O157:H7或NM、O104:H4、O16:H48、O5:H9、O111:H8或NM、O121:H19、O103:H2及O45:H2血清型的灵敏度及特异度可达到95%以上,而对于预测O26:H11或NM血清型的灵敏度及特异度较低。结论1 CRISPR/Cas系统在大肠杆菌中广泛存在。2大肠杆菌的CRISPR/Cas系统中存在着IS的插入现象。3 CRISPR分型方法区分大肠杆菌能取得较好的效果。4 CRISPR分型可用于预测部分大肠杆菌血清型。
[Abstract]:Escherichia coli is a normal group of bacteria living in the human gut, but some of the serotypes of E. coli are pathogenic to the human body. The pathogenic Escherichia coli can cause a variety of diseases, such as mild diarrhea, hemorrhagic colitis, hemolytic uremia, etc. CRISPRP cas system is a kind of adaptive immune system found in prokaryotes. Objective 1 to reveal the structural characteristics of Escherichia coli by bioinformatics, and to describe the position relationship between CRISPR/Cas system and insertion sequence. To explore the relationship between CRISPR1 and CRISPR2.2 loci. To establish a molecular typing method of Escherichia coli based on CRISPR, to compare the typing effect of four typing methods, to analyze the relationship between the four typing methods and to predict the serotype of Escherichia coli by CRISPR typing method. Methods 1 CRT software and CRISPR finder online tools were used to obtain CRISPR locus information. The secondary structure prediction of repeat sequence was carried out by extracting spacer sequence and repeat sequence fold. CRISPR Target software was used to search the exogenous homology of interval sequence and draw phylogenetic tree .2 Serotype Finder online tool to obtain serological information of the strain. Virus Finder online tool to obtain virulence Gene Information MLST Finder online tool to obtain strain MLST Information. 3 Excel Table numbers the interval sequence and draws the spacer sequence Map / R software to obtain CRISPR typing type .4 Simpson index is used for evaluation. Results 1 bioinformatics analysis of CRISPR loci of Escherichia coli can identify 7 CRISPR loci in E. coli genome sequencing. The positive rate of CRISPR2. 2 was 81.62and the positive rate of CRISPR2.1 was 88.699.The positive rate of CRISPR2.2 was 88.70. The positive rate of CRISPR2.3 was 7.42%. The positive rate of CRISPR3 was 5.65%. The positive rate of CRISPR4 was 5.65%. The 93.28%.IS could be inserted around the CRISPR/Cas system and inserted into the Cas gene. Even the establishment of a molecular typing method for Escherichia coli based on CRISPR inserted between repeats: 430 types can be obtained according to the CCT typing method, According to the CLPST typing method, the Simpson index of 215 CCTV-CLPST-MLST and Serotype genotyping methods were 0.8864 4, 0.8760,0.8581n 0.8643.3, respectively, and the relationship between the sequence composition, serotyping and stx phage typing and stx bacteriophage could be found in each CRISPR subtype. The distribution of the spacer sequences and the source of the isolates were not found. Application of 4 CRISPR typing method in predicting serotype of Escherichia coli the sensitivity and specificity of O157:H7 or NMH104: H4: O16: H48: O5: H9: O111: H8 or NMMO121H19O103ClH2 and O45:H2 serotype can be over 95%. The sensitivity and specificity of predicting the serotypes of O26:H11 or NM were low. Conclusion 1 the insertion phenomenon of is in the CRISPR/Cas system in which the CRISPR/Cas system is widely present in Escherichia coli. 3. 3 CRISPR typing method can distinguish E. coli from Escherichia coli. 4. 4 CRISPR typing can be used to predict the serotypes of some Escherichia coli.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378.21;Q78

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