艱難梭菌二元基因分型方法建立及方法學(xué)對比研究
發(fā)布時(shí)間：2018-07-20 20:22
【摘要】：目的:艱難梭菌(Clostridium difficile,CD)是一種能形成芽孢的革蘭陽性厭氧粗大桿菌。上世紀(jì)70年代末被首次發(fā)現(xiàn)是引起抗生素相關(guān)性偽膜性腸炎的原因之一,近年來由艱難梭菌引起的感染(Clostridium difficile infection,CDI)已經(jīng)成為抗生素相關(guān)性腹瀉的主要原因。其致病原因可能是由于抗生素改變了腸道正常菌群,破壞了其保護(hù)作用,使得艱難梭菌可以在結(jié)腸定植繁殖,進(jìn)而釋放兩種毒力因子-毒素A(Tcd A)和B(Tcd B)。臨床癥狀表現(xiàn)為輕度的自限性腹瀉到嚴(yán)重的偽膜性腸炎甚至是死亡。艱難梭菌分型技術(shù)對于追蹤艱難梭桿菌相關(guān)性腹瀉(CDAD)流行菌株的來源,調(diào)查其在全球范圍內(nèi)的傳播途徑,以及探索其致病機(jī)制等具有重要意義。隨著致病性艱難梭菌的傳播流行,已有核糖體分型(PCR ribotyping,PCR RT),脈沖場凝膠電泳(Pulsed Field Gel Electrophoresis,PFGE)和多位點(diǎn)序列分析(Multilocus Sequence Typing,MLST)等多種分子分型技術(shù)應(yīng)用于艱難梭菌流行病學(xué)研究。但是,這些分型方法在分辨力、重復(fù)性、可操作性、費(fèi)用和實(shí)驗(yàn)室間比對等方面存在不同的缺陷。二元基因分型(Binary Typing)是一種新型的基于不同菌株上某種基因存在與否的分型技術(shù),即根據(jù)某一個(gè)基因在同一菌種上的存在與否都可以將這一菌種分為“有”和“無”兩個(gè)群體,如果有多個(gè)類似的基因組合起來,就可以把一種細(xì)菌進(jìn)行詳細(xì)分型。具有這樣特點(diǎn)的基因被稱為“二元基因”,由這些基因分析得到的不同型別就是二元基因型。它已經(jīng)成功應(yīng)用于金黃色葡萄球菌、空腸彎曲桿菌等菌種的流行病學(xué)分析。并以其操作簡單快速,費(fèi)用低、高分辨力等特點(diǎn)受人們的關(guān)注。本研究的目的是建立一個(gè)全新的、快速、費(fèi)用低、可操作性強(qiáng)的艱難梭菌二元基因分型方法,并將該方法與傳統(tǒng)的MLST方法作比較研究。方法:應(yīng)用Mauve軟件對Genome數(shù)據(jù)庫下載的4條艱難梭菌全基因組DNA序列進(jìn)行比對分析,初步查找可能存在的大小介于500bp和1000bp之間的二元基因位點(diǎn)。以NCBI Genome數(shù)據(jù)庫中已存在的50個(gè)艱難梭菌全基因組DNA序列作為測試樣本,將每個(gè)可能的二元基因都進(jìn)行BLAST(Basic Local Alignment Search Tool)檢索,模擬分析篩選的基因位點(diǎn)在艱難梭菌上的存在情況,根據(jù)每個(gè)二元基因在50個(gè)全基因DNA序列上的存在與否,標(biāo)記為(0,1)矩陣。應(yīng)用最優(yōu)組合查詢軟件(Optimal Combination Finder,OCF),以辛普森指數(shù)(Simpson's index,SI)作為評價(jià)分型方法分辨力的指標(biāo),計(jì)算分析不同二元基因位點(diǎn)組合的分辨力,初步篩選最優(yōu)二元基因分型組合。以2010年2012年臨床分離的99株艱難梭菌和1株參考菌株ATCC BAA-1870(RT 027)為測試樣本,進(jìn)一步優(yōu)化分析二元基因分型位點(diǎn)組合。并以核糖體分型作為參考方法進(jìn)行對比研究。確定最終的二元基因分型的位點(diǎn)組合。以挑選收集自2010至2014年間不同地域和不同人群的100株艱難梭菌為測試樣本同時(shí)進(jìn)行二元基因分析和多位點(diǎn)序列分型(Multilocus sequence typing,MLST),比較分析二元基因分析方法的優(yōu)劣。結(jié)果:從Genome數(shù)據(jù)庫下載得到4條艱難梭菌全基因組DNA序列,分別為:C.difficile R20291,QCD-63q42,QCD-76w55和C.difficile 630。用Mauve軟件將4個(gè)全基因組DNA序列對比分析后,共發(fā)現(xiàn)50個(gè)可能存在的二元基因位點(diǎn)。以NCBI Genome數(shù)據(jù)庫中已存在的50個(gè)艱難梭菌全基因組DNA序列作為測試樣本進(jìn)行模擬分析,對50個(gè)二元基因位點(diǎn)的不同組合進(jìn)行評價(jià)分析后,發(fā)現(xiàn)其中28個(gè)二元基因位點(diǎn)組合具有最好的分辨力,SI值為0.908。將28個(gè)二元基因位點(diǎn)設(shè)計(jì)引物對99株艱難梭菌和1株參考菌株ATCC BAA-1870(RT 027型)進(jìn)行PCR檢測分型后,發(fā)現(xiàn)由其中10個(gè)基因位點(diǎn)組成最優(yōu)分型方案可以達(dá)到28個(gè)基因位點(diǎn)的分型水平,SI值亦為0.908。以核糖體分型作為參考方法,對相同的100株測試菌株進(jìn)行分型,并計(jì)算分析后發(fā)現(xiàn)其SI值僅為0.847。將二元基因分型與核糖體分型方法進(jìn)行合并分析,其SI值為0.933。僅在二元基因分型的基礎(chǔ)上提高了0.025。采用10位點(diǎn)二元基因分型和MLST分型方法對收集自2010至2014年間不同地域和不同人群的100株艱難梭菌進(jìn)行比對分析后發(fā)現(xiàn),兩種分型方法的其SI值分為0.938和0.886。二元基因分型方法顯示了很好的分辨力。結(jié)論:艱難梭菌二元基因分型方法是一種非常有用的分型方法,可以提供準(zhǔn)確可信的分型信息。它不僅具有快速、實(shí)用、費(fèi)用低廉等特點(diǎn),而且具有較高的分辨力,可以在流行病學(xué)水平上提供有效的分型數(shù)據(jù)。此外,由于所選取的10個(gè)二元基因位點(diǎn)中的部分位點(diǎn)是涉及毒力和耐藥相關(guān)的功能基因,二元基因的分型結(jié)果可以讓臨床醫(yī)師直觀地了解從患者樣本中分離到菌株的功能基因,以便采取進(jìn)一步的治療措施。因此,相比較其他分型方法,二元基因分型具有很獨(dú)特的臨床意義。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R440
本文編號：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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R440
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 Perry Hookman;Jamie S Barkin;;Clostridium diffi cile associated infection,diarrhea and colitis[J];World Journal of Gastroenterology;2009年13期
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