本文選題：CRISPR/Cas系統(tǒng)　切入點(diǎn)：插入序列　出處：《鄭州大學(xué)》2017年碩士論文

【摘要】：大腸桿菌是寄宿在人體腸道內(nèi)的正常菌群,但部分血清型的大腸桿菌對人體是致病的,致病性的大腸桿菌可對人體造成多種疾病,如輕度腹瀉、出血性結(jié)腸炎、溶血性尿毒癥、尿路致病性感染等疾病。CRISPR/Cas系統(tǒng)是原核生物中發(fā)現(xiàn)的一種適應(yīng)性免疫系統(tǒng),其具有高度的多態(tài)性,利用CRISPR位點(diǎn)的多態(tài)性可實(shí)現(xiàn)對大腸桿菌分型。目的1通過生物信息學(xué)方法揭示大腸桿菌的結(jié)構(gòu)特征,描述CRISPR/Cas系統(tǒng)與插入序列之間的位置關(guān)系,探討CRISPR1及CRISPR2.2位點(diǎn)之間的關(guān)系。2建立基于CRISPR的大腸桿菌分子分型方法,比較CCT、CLPST、MLST、Serotype四種分型方法的分型效果,分析四種分型方法之間的關(guān)系。3利用CRISPR分型方法預(yù)測大腸桿菌血清型,并評估其靈敏度及特異度。方法1 CRT軟件及CRISPR finder在線工具獲得CRISPR位點(diǎn)信息,并提取間隔序列及重復(fù)序列,RNA fold進(jìn)行重復(fù)序列二級結(jié)構(gòu)預(yù)測,CRISPR Target軟件進(jìn)行間隔序列外源同源性搜索,Mega 6.0繪制系統(tǒng)發(fā)育進(jìn)化樹。2 Serotype Finder在線工具獲得菌株血清學(xué)信息,Virulence Finder在線工具獲得菌株毒力基因信息,MLST Finder在線工具獲得菌株MLST信息。3 Excel表對間隔序列進(jìn)行編號并繪制間隔序列圖譜,R軟件獲得CRISPR分型型別。4辛普森指數(shù)被用于評估CCT、CLPST、MLST、Serotype四種分型方法的分型效果,Spearman相關(guān)性分析被用于獲得四種分型方法的相關(guān)系數(shù)。結(jié)果1大腸桿菌CRISPR位點(diǎn)的生物信息學(xué)分析:283株全基因組測序的大腸桿菌可識別7種CRISPR位點(diǎn),其中CRISPR1位點(diǎn)陽性率為81.62%,CRISPR2.1位點(diǎn)陽性率為88.69%,CRISPR2.2位點(diǎn)陽性率為88.70%,CRISPR2.3位點(diǎn)陽性率為7.42%,CRISPR3位點(diǎn)陽性率5.65%,CRISPR4位點(diǎn)陽性率為5.65%,CRISPR3-4位點(diǎn)陽性率為93.28%。IS可插入到CRISPR/Cas系統(tǒng)周圍,也可插入到Cas基因中,甚至于插入到重復(fù)序列之間。2基于CRISPR的大腸桿菌分子分型方法的建立:根據(jù)CCT分型方法可獲得430個(gè)型別,根據(jù)CLPST分型方法可獲得215個(gè)型別,CCT、CLPST、MLST及Serotype四種分型方法的辛普森指數(shù)分別為0.8864,0.8760,0.8581,0.8643。3間隔序列組成及排布方式與血清分型及stx噬菌體的關(guān)系:stx噬菌體可在各CRISPR亞型中出現(xiàn),并未發(fā)現(xiàn)菌株間隔序列排布方式與分離來源、分離時(shí)間及分離地區(qū)有關(guān)。4 CRISPR分型方法在預(yù)測大腸桿菌血清型中的應(yīng)用:CRISPR分型方法預(yù)測O157:H7或NM、O104:H4、O16:H48、O5:H9、O111:H8或NM、O121:H19、O103:H2及O45:H2血清型的靈敏度及特異度可達(dá)到95%以上,而對于預(yù)測O26:H11或NM血清型的靈敏度及特異度較低。結(jié)論1 CRISPR/Cas系統(tǒng)在大腸桿菌中廣泛存在。2大腸桿菌的CRISPR/Cas系統(tǒng)中存在著IS的插入現(xiàn)象。3 CRISPR分型方法區(qū)分大腸桿菌能取得較好的效果。4 CRISPR分型可用于預(yù)測部分大腸桿菌血清型。
[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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R378.21;Q78

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