艰难梭菌二元基因分型方法建立及方法学对比研究
发布时间：2018-07-20 20:22
【摘要】：目的:艰难梭菌(Clostridium difficile,CD)是一种能形成芽孢的革兰阳性厌氧粗大杆菌。上世纪70年代末被首次发现是引起抗生素相关性伪膜性肠炎的原因之一,近年来由艰难梭菌引起的感染(Clostridium difficile infection,CDI)已经成为抗生素相关性腹泻的主要原因。其致病原因可能是由于抗生素改变了肠道正常菌群,破坏了其保护作用,使得艰难梭菌可以在结肠定植繁殖,进而释放两种毒力因子-毒素A(Tcd A)和B(Tcd B)。临床症状表现为轻度的自限性腹泻到严重的伪膜性肠炎甚至是死亡。艰难梭菌分型技术对于追踪艰难梭杆菌相关性腹泻(CDAD)流行菌株的来源,调查其在全球范围内的传播途径,以及探索其致病机制等具有重要意义。随着致病性艰难梭菌的传播流行,已有核糖体分型(PCR ribotyping,PCR RT),脉冲场凝胶电泳(Pulsed Field Gel Electrophoresis,PFGE)和多位点序列分析(Multilocus Sequence Typing,MLST)等多种分子分型技术应用于艰难梭菌流行病学研究。但是,这些分型方法在分辨力、重复性、可操作性、费用和实验室间比对等方面存在不同的缺陷。二元基因分型(Binary Typing)是一种新型的基于不同菌株上某种基因存在与否的分型技术,即根据某一个基因在同一菌种上的存在与否都可以将这一菌种分为“有”和“无”两个群体,如果有多个类似的基因组合起来,就可以把一种细菌进行详细分型。具有这样特点的基因被称为“二元基因”,由这些基因分析得到的不同型别就是二元基因型。它已经成功应用于金黄色葡萄球菌、空肠弯曲杆菌等菌种的流行病学分析。并以其操作简单快速,费用低、高分辨力等特点受人们的关注。本研究的目的是建立一个全新的、快速、费用低、可操作性强的艰难梭菌二元基因分型方法,并将该方法与传统的MLST方法作比较研究。方法:应用Mauve软件对Genome数据库下载的4条艰难梭菌全基因组DNA序列进行比对分析,初步查找可能存在的大小介于500bp和1000bp之间的二元基因位点。以NCBI Genome数据库中已存在的50个艰难梭菌全基因组DNA序列作为测试样本,将每个可能的二元基因都进行BLAST(Basic Local Alignment Search Tool)检索,模拟分析筛选的基因位点在艰难梭菌上的存在情况,根据每个二元基因在50个全基因DNA序列上的存在与否,标记为(0,1)矩阵。应用最优组合查询软件(Optimal Combination Finder,OCF),以辛普森指数(Simpson's index,SI)作为评价分型方法分辨力的指标,计算分析不同二元基因位点组合的分辨力,初步筛选最优二元基因分型组合。以2010年2012年临床分离的99株艰难梭菌和1株参考菌株ATCC BAA-1870(RT 027)为测试样本,进一步优化分析二元基因分型位点组合。并以核糖体分型作为参考方法进行对比研究。确定最终的二元基因分型的位点组合。以挑选收集自2010至2014年间不同地域和不同人群的100株艰难梭菌为测试样本同时进行二元基因分析和多位点序列分型(Multilocus sequence typing,MLST),比较分析二元基因分析方法的优劣。结果:从Genome数据库下载得到4条艰难梭菌全基因组DNA序列,分别为:C.difficile R20291,QCD-63q42,QCD-76w55和C.difficile 630。用Mauve软件将4个全基因组DNA序列对比分析后,共发现50个可能存在的二元基因位点。以NCBI Genome数据库中已存在的50个艰难梭菌全基因组DNA序列作为测试样本进行模拟分析,对50个二元基因位点的不同组合进行评价分析后,发现其中28个二元基因位点组合具有最好的分辨力,SI值为0.908。将28个二元基因位点设计引物对99株艰难梭菌和1株参考菌株ATCC BAA-1870(RT 027型)进行PCR检测分型后,发现由其中10个基因位点组成最优分型方案可以达到28个基因位点的分型水平,SI值亦为0.908。以核糖体分型作为参考方法,对相同的100株测试菌株进行分型,并计算分析后发现其SI值仅为0.847。将二元基因分型与核糖体分型方法进行合并分析,其SI值为0.933。仅在二元基因分型的基础上提高了0.025。采用10位点二元基因分型和MLST分型方法对收集自2010至2014年间不同地域和不同人群的100株艰难梭菌进行比对分析后发现,两种分型方法的其SI值分为0.938和0.886。二元基因分型方法显示了很好的分辨力。结论:艰难梭菌二元基因分型方法是一种非常有用的分型方法,可以提供准确可信的分型信息。它不仅具有快速、实用、费用低廉等特点,而且具有较高的分辨力,可以在流行病学水平上提供有效的分型数据。此外,由于所选取的10个二元基因位点中的部分位点是涉及毒力和耐药相关的功能基因,二元基因的分型结果可以让临床医师直观地了解从患者样本中分离到菌株的功能基因,以便采取进一步的治疗措施。因此,相比较其他分型方法,二元基因分型具有很独特的临床意义。
[Abstract]:Objective: Clostridium difficile (CD) is a Gram-positive anaerobibacillus anaerobibacillus that forms spores. It was first discovered in the late 70s of last century as one of the causes of antibiotic related pseudolitis. The infection (Clostridium difficile infection, CDI) caused by Clostridium difficile (CDI) has become an antibiotic related in recent years. The main cause of sexual diarrhea may be because antibiotics have altered the normal flora of the intestines and destroyed its protective effect, making Clostridium difficile colonized and propagated in colon, and then releasing two virulence factors - toxin A (Tcd A) and B (Tcd B). Clinical symptoms are mild self limiting diarrhea to severe pseudoralenous enteritis and even It is fatal. The Clostridium difficile typing technique is of great significance for tracing the source of the strains of Clostridium difficile associated diarrhea (CDAD), investigating its transmission routes around the world, and exploring its pathogenic mechanism. With the spread of pathogenic Clostridium difficile, the PCR ribotyping, PCR RT, and pulse field gel have been found. Various molecular typing techniques, such as Pulsed Field Gel Electrophoresis (PFGE) and multipoint sequence analysis (Multilocus Sequence Typing, MLST), are applied to the epidemiological study of Clostridium difficile. However, these typing methods have different defects in resolution, repeatability, operability, cost and laboratory comparison. Two yuan basis Binary Typing is a new type of typing based on the presence or absence of certain genes on different strains, that is, according to the presence or absence of one gene in the same strain, this strain can be divided into two groups: "yes" and "no". If there are many similar genes, a bacterium can be carried out. The gene, which has this characteristic, is called the "two gene", and the different types of these genes are two yuan genotypes. It has been successfully applied to the epidemiological analysis of Staphylococcus aureus, Campylobacter jejuni and other strains, and is characterized by its simple and rapid operation, low cost, high discrimination and so on. The purpose of this study is to establish a new, fast, low cost and maneuverable two meta genotyping method of Clostridium difficile, and compare this method with the traditional MLST method. Method: Mauve software is used to compare and analyze the whole genome DNA sequence of 4 Clostridium difficile downloaded by Genome database. The two gene loci can exist between 500bp and 1000bp. The whole genome DNA sequence of 50 Clostridium difficile existing in the NCBI Genome database is used as the test sample, and each possible two gene is retrieved by BLAST (Basic Local Alignment Search Tool). The simulated analysis of the gene loci is on Clostridium difficile. The existence of each two gene in the 50 full gene DNA sequence is marked as (0,1) matrix. The optimal combination query software (Optimal Combination Finder, OCF) is used as the Simpson index (Simpson's index, SI) as an indicator of the resolution of the classification method, and the analysis of the composition of different two gene loci combinations is calculated and analyzed. The optimal two genotyping combination was screened. 99 strains of Clostridium difficile and 1 reference strains ATCC BAA-1870 (RT 027), which were clinically isolated in 2012 2010, were used as the test samples to further optimize the analysis of the two genotyping site combinations. The ribosome typing was used as a reference method to determine the final two genotyping. A combination of 100 strains of Clostridium difficile collected from different regions and different populations from 2010 to 2014 were selected to perform two yuan gene analysis and multiple point sequence typing (Multilocus sequence typing, MLST). The results were compared and analyzed by two meta gene analysis methods. Results: 4 hard shuttles were downloaded from the Genome database. The whole genome DNA sequence of the bacteria: C.difficile R20291, QCD-63q42, QCD-76w55 and C.difficile 630. were compared and analyzed by Mauve software for the 4 whole genome DNA sequences, and 50 possible two gene loci were found. The whole genome DNA sequence of 50 Clostridium difficile in NCBI Genome database was used as the test sample. After the simulation analysis, after evaluating the different combinations of 50 two gene loci, it was found that 28 of the two gene loci combinations had the best resolution. The SI value was 0.908., and 28 two yuan loci were designed for PCR detection of 99 Clostridium difficile and 1 reference strain ATCC BAA-1870 (RT 027), and 10 was found. The optimal genotyping scheme can reach 28 genotyping levels, and the SI value is also 0.908. with ribosome typing as the reference method. The same 100 strains of test strains are typed, and the SI value is only 0.847. to merge the two element genotyping and ribosome typing method, and its SI value The comparison and analysis of 100 strains of Clostridium difficile collected from different regions and different populations from 2010 to 2014 from 2010 to 2014 were compared and analyzed on the basis of 0.933. genotyping only on the basis of the two element genotyping. The SI values of the two typing methods were divided into 0.938 and 0.886. two genotyping methods. Good resolution. Conclusion: the two element genotyping method of Clostridium difficile is a very useful typing method, which can provide accurate and reliable typing information. It not only has the characteristics of rapid, practical, low cost and so on, but also has high resolution and can provide effective classification data at the epidemiological level. In addition, due to the selection of the classification data. The partial loci of the 10 two gene loci are functional genes related to virulence and resistance, and the typing results of the two gene can allow clinicians to intuitively understand the functional genes isolated from the patient's sample in order to take further treatment. Therefore, a comparison of other typing methods and two genotyping is compared. It has a unique clinical significance.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R440
本文编号：2134667
[Abstract]:Objective: Clostridium difficile (CD) is a Gram-positive anaerobibacillus anaerobibacillus that forms spores. It was first discovered in the late 70s of last century as one of the causes of antibiotic related pseudolitis. The infection (Clostridium difficile infection, CDI) caused by Clostridium difficile (CDI) has become an antibiotic related in recent years. The main cause of sexual diarrhea may be because antibiotics have altered the normal flora of the intestines and destroyed its protective effect, making Clostridium difficile colonized and propagated in colon, and then releasing two virulence factors - toxin A (Tcd A) and B (Tcd B). Clinical symptoms are mild self limiting diarrhea to severe pseudoralenous enteritis and even It is fatal. The Clostridium difficile typing technique is of great significance for tracing the source of the strains of Clostridium difficile associated diarrhea (CDAD), investigating its transmission routes around the world, and exploring its pathogenic mechanism. With the spread of pathogenic Clostridium difficile, the PCR ribotyping, PCR RT, and pulse field gel have been found. Various molecular typing techniques, such as Pulsed Field Gel Electrophoresis (PFGE) and multipoint sequence analysis (Multilocus Sequence Typing, MLST), are applied to the epidemiological study of Clostridium difficile. However, these typing methods have different defects in resolution, repeatability, operability, cost and laboratory comparison. Two yuan basis Binary Typing is a new type of typing based on the presence or absence of certain genes on different strains, that is, according to the presence or absence of one gene in the same strain, this strain can be divided into two groups: "yes" and "no". If there are many similar genes, a bacterium can be carried out. The gene, which has this characteristic, is called the "two gene", and the different types of these genes are two yuan genotypes. It has been successfully applied to the epidemiological analysis of Staphylococcus aureus, Campylobacter jejuni and other strains, and is characterized by its simple and rapid operation, low cost, high discrimination and so on. The purpose of this study is to establish a new, fast, low cost and maneuverable two meta genotyping method of Clostridium difficile, and compare this method with the traditional MLST method. Method: Mauve software is used to compare and analyze the whole genome DNA sequence of 4 Clostridium difficile downloaded by Genome database. The two gene loci can exist between 500bp and 1000bp. The whole genome DNA sequence of 50 Clostridium difficile existing in the NCBI Genome database is used as the test sample, and each possible two gene is retrieved by BLAST (Basic Local Alignment Search Tool). The simulated analysis of the gene loci is on Clostridium difficile. The existence of each two gene in the 50 full gene DNA sequence is marked as (0,1) matrix. The optimal combination query software (Optimal Combination Finder, OCF) is used as the Simpson index (Simpson's index, SI) as an indicator of the resolution of the classification method, and the analysis of the composition of different two gene loci combinations is calculated and analyzed. The optimal two genotyping combination was screened. 99 strains of Clostridium difficile and 1 reference strains ATCC BAA-1870 (RT 027), which were clinically isolated in 2012 2010, were used as the test samples to further optimize the analysis of the two genotyping site combinations. The ribosome typing was used as a reference method to determine the final two genotyping. A combination of 100 strains of Clostridium difficile collected from different regions and different populations from 2010 to 2014 were selected to perform two yuan gene analysis and multiple point sequence typing (Multilocus sequence typing, MLST). The results were compared and analyzed by two meta gene analysis methods. Results: 4 hard shuttles were downloaded from the Genome database. The whole genome DNA sequence of the bacteria: C.difficile R20291, QCD-63q42, QCD-76w55 and C.difficile 630. were compared and analyzed by Mauve software for the 4 whole genome DNA sequences, and 50 possible two gene loci were found. The whole genome DNA sequence of 50 Clostridium difficile in NCBI Genome database was used as the test sample. After the simulation analysis, after evaluating the different combinations of 50 two gene loci, it was found that 28 of the two gene loci combinations had the best resolution. The SI value was 0.908., and 28 two yuan loci were designed for PCR detection of 99 Clostridium difficile and 1 reference strain ATCC BAA-1870 (RT 027), and 10 was found. The optimal genotyping scheme can reach 28 genotyping levels, and the SI value is also 0.908. with ribosome typing as the reference method. The same 100 strains of test strains are typed, and the SI value is only 0.847. to merge the two element genotyping and ribosome typing method, and its SI value The comparison and analysis of 100 strains of Clostridium difficile collected from different regions and different populations from 2010 to 2014 from 2010 to 2014 were compared and analyzed on the basis of 0.933. genotyping only on the basis of the two element genotyping. The SI values of the two typing methods were divided into 0.938 and 0.886. two genotyping methods. Good resolution. Conclusion: the two element genotyping method of Clostridium difficile is a very useful typing method, which can provide accurate and reliable typing information. It not only has the characteristics of rapid, practical, low cost and so on, but also has high resolution and can provide effective classification data at the epidemiological level. In addition, due to the selection of the classification data. The partial loci of the 10 two gene loci are functional genes related to virulence and resistance, and the typing results of the two gene can allow clinicians to intuitively understand the functional genes isolated from the patient's sample in order to take further treatment. Therefore, a comparison of other typing methods and two genotyping is compared. It has a unique clinical significance.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R440
【参考文献】
相关期刊论文 前1条
1 Perry Hookman;Jamie S Barkin;;Clostridium diffi cile associated infection,diarrhea and colitis[J];World Journal of Gastroenterology;2009年13期
，本文编号：2134667
本文链接：https://www.wllwen.com/kejilunwen/jiyingongcheng/2134667.html
最近更新
教材专著
