炎症性肠病病人肠道菌丛结构特征的ERIC-PCR指纹图谱分析
|
摘要
炎症性肠病(Inflammatory Bowel Disease,IBD)是一组病因不明的慢性肠道炎症性疾病。微生物在IBD发病中的作用一直受到重视,但至今尚未找到某一特异病原微生物与IBD有恒定关系。由于目前能够分离培养的肠道细菌种类只占肠道细菌总数的少数,而以PCR技术为基础的DNA指纹图谱就可以用来分析肠道菌丛的组成。ERIC序列(Enterobacterial Repetitive Intergenic Consensus,肠杆菌基因间的重复共有序列)是主要存在于肠道细菌中的一段长为126 bp的反向重复序列,该序列高度保守,而且染色体定位具有种特异性,ERIC-PCR扩增的产物大小在50-3000bp之间,每种菌株均存在数目不等的各自独特的电泳带型,各特异性扩增的主带型能重复稳定出现,可以区别不同种类的细菌和同一种类细菌中不同的菌株。本研究应用ERIC-PCR技术建立10例IBD患者及10例正常对照者的肠道细菌DNA指纹图谱,分析IBD患者及正常对照者的肠道菌丛的整体差异。经分析发现IBD患者及正常对照者的肠道菌丛存在整体差异,正常对照组ERIC-PCR指纹图谱电泳DNA条带多,主带位置无统一趋势;IBD组ERIC-PCR指纹图谱电泳DNA条带较少,且所有样本主带分布非常一致,均出现在约1.1kb处。从而推断正常人肠道细菌种类多,IBD患者少。IBD组1.1kb处主带可能为某一种肠道细菌中特有的序列,或为不同序列或几个序列的混合物。
Inflammatory bowel disease (IBD) is a group of chronic enteric inflammatory diseases without definite etiopathogenisis. Microorganism plays an important role in pathogenesis of IBD. But no specific pathogenic microorganism has been found related to IBD so far. The number of enterobacteria species could be cultured in vitro only accounts for small percent of the total number of enterobacteria species. DNA fingerprinting based on PCR technology could be used to analyze the makeup of intestinal flora. Enterobacterial Repetitive Intergenic Consensus (ERIC) sequence is a kind of reverse and repeated sequence mostly located in the enterobacteria genome that is 126bp in size and highly conservative. ERIC-PCR generates multiple distinct amplification products of sizes ranging from approximately 50 to 3000 bp. Each kind of bacteria has special electrophoretic bands with different number and the main band could be appeared stably and repeatedly. The unique location of ERIC elements in bacterial genomes allows discrimination at genus, species and strain levels based on the electrophoretic pattern amplification products. In this study, we set up the DNA fingerprinting of IBD patients and healthy persons based on ERIC-PCR technology. We have identified the DNA fingerprintings of IBD patients and the healthy persons, and a significant difference was noticed between them.There are a lot of bands in the DNA fingerprinting of the healthy group but fewer in the DNA fingerprinting of the IBD patients. Strikingly, a same band of DNA fingerprinting was noticed in IBD patients. So the variaty of intestinal flora of healthy patients is more than it of IBD patients.The unique band might be the sequence of specific bacterium. Or it might be the sequence of different bacterium and the mixed sequence.
Inflammatory bowel disease (IBD) is a group of chronic enteric inflammatory diseases without definite etiopathogenisis. Microorganism plays an important role in pathogenesis of IBD. But no specific pathogenic microorganism has been found related to IBD so far. The number of enterobacteria species could be cultured in vitro only accounts for small percent of the total number of enterobacteria species. DNA fingerprinting based on PCR technology could be used to analyze the makeup of intestinal flora. Enterobacterial Repetitive Intergenic Consensus (ERIC) sequence is a kind of reverse and repeated sequence mostly located in the enterobacteria genome that is 126bp in size and highly conservative. ERIC-PCR generates multiple distinct amplification products of sizes ranging from approximately 50 to 3000 bp. Each kind of bacteria has special electrophoretic bands with different number and the main band could be appeared stably and repeatedly. The unique location of ERIC elements in bacterial genomes allows discrimination at genus, species and strain levels based on the electrophoretic pattern amplification products. In this study, we set up the DNA fingerprinting of IBD patients and healthy persons based on ERIC-PCR technology. We have identified the DNA fingerprintings of IBD patients and the healthy persons, and a significant difference was noticed between them.There are a lot of bands in the DNA fingerprinting of the healthy group but fewer in the DNA fingerprinting of the IBD patients. Strikingly, a same band of DNA fingerprinting was noticed in IBD patients. So the variaty of intestinal flora of healthy patients is more than it of IBD patients.The unique band might be the sequence of specific bacterium. Or it might be the sequence of different bacterium and the mixed sequence.
引文
1. Bhan AK, Mizoguchi E, Smith RN, Mizoguchi A. Colitis in transgenic and knockout animals as modles of human inflammatory bowel disease. Immunol Rev 1999;169: 195-207
2. Harper PH. Lee ECG, Kettlewell MGW, et al. Roles of the faecal stream in the maintenance of Crohn's colitis. Gut 1985;26: 279-84
3. Hulton CS, Higgins CF, Sharp PM. ERIC sequences: a novel family of repetitive elements in the genomes of Escherichia coli, Salmonella typhimurium and other enterobacteria. Mol Microbiol 1991;5: 825-834
4. Versalovic J, Koeuth T, Lupski JR. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res 1991;19: 6823-6831
5. Di Giovanni GD, Watrud LS, Seidler RJ, et al. Fingerprinting of mixed bacterial strains and BIOLOG gram-negative (GN) substrate communities by enterobacterial repetitive intergenic consensus sequence-PCR (ERIC-PCR). Curr Microbiol 1999;38: 217-223
6. Use of Repetitive (Repetitive Ectragenic Palindromic and Enterobacterial Repetitive Intergeneric Consensus)Sequences and the Polymerase Chain Reaction To Fingerprint the Genomes of Rhizobium meliloti Isolates and Other Soil Bacteria, Applied and Enviromental Microbiology, July 1992, 2180-2187
7. Short, Interspersed Repetitive DNA Sequences in Prokaryotic Genomes, Journal of Bacteriology, July 1992, 4525-4529
8. Seksik P, Rigottier-Gois L, Gramet G, et al. Alterations of the dominant faecal bacterial groups in patients with Crohn's disease of the colon. Gut 2003;52: 237-42
9.曹又方,赵立平.ER IC序列在不同细菌基因组中分布的分析.山西大学学报(自然科学)2002;25:354-357
10. Rahmati A, Gal M, Northey G, Brazier JS. Subtyping of Clostridium difficile polymerase chain reaction (pcr) ribotype 001 by repetitive extragenic palindromic PCR genomic fingerprinting. J Hosp Infect, 2005 May;60(1): 56-60
11. Jin LL,Wang QY, Hou X. ERIC-PCR on identification of Listeria species and strain. Yi Chuan. 2003 Mar;25(2): 195-7.
12. Song G, Wei G, Cao Y, et al. Use of ERIC-PCR as a community fingerprinting technology in elucidation of mode of action of a new probiotic formulation for prevention and treatment of bacterial diarrhea in piglets[J]. Book of abstracts of the Asia Pacific Conference On Plant Tissue Culture & Agrobiotechnology. 2000;1923: 166).
13. Ventura M, Meylan V, Zink R. Identification and tracing of Bifidobacterium species by use of enterobacterial repetitive intergenic consensus sequences. Appl Environ Microbiol 2003;69: 4296-4301.
14. McLellan SL, Daniels AD, Salmore AK. Genetic characterization of Escherichia coli populations from host sources of fecal pollution by using DNA fingerprinting. Appl Environ Microbiol 2003;69: 2587-2594
15. Femandez-Baca V, Ballesteros F, Hervas JA, et al. Molecular epidemiological typing of Enterobacter cloacae isolates from a neonatal intensive care unit: three-year prospective study. J Hosp Infect 2001;49: 173-182
16. Aarts HJ, Joosten RG, Henkens MH, et al. Rapid duplex PCR assay for the detection of pathogenic Yersinia enterocolitica strains. J Microbiol Methods 2001;47: 209-217,
17.鲁海峰等,ERIC-PCR分子杂交技术分析大熊猫肠道菌群结构,中国微生态学杂志,2005年4月第17卷第2期
18.潘莉等,腹泻儿童肠道菌群结构特征的ERIC-PCR指纹图分析,中国微生态学杂志,2003年6月第15卷第3期
19. Issemann Ⅰ, Green S1 Activation of a memberof the steroid hormone receptor superfamily byperoxisome proliferators [J]. Nature, 1990, 347: 645-6501
20. Asano T, Wakisaka M, Yoshinari M, et al, Peroxisome proliferator-activated receptorgammal (PPAR gammal) expresses in rat mesangial cells and PPARgamma agonists modulate its differentiation [J]. Biochim Biophys Acta, 2000. 1497 (1): 148-1541
21.陈迎春等,大肠杆菌MG1655菌株ERIC-PCR图谱主带序列组成分析,微生物学通报,2002年29(6)
2. Harper PH. Lee ECG, Kettlewell MGW, et al. Roles of the faecal stream in the maintenance of Crohn's colitis. Gut 1985;26: 279-84
3. Hulton CS, Higgins CF, Sharp PM. ERIC sequences: a novel family of repetitive elements in the genomes of Escherichia coli, Salmonella typhimurium and other enterobacteria. Mol Microbiol 1991;5: 825-834
4. Versalovic J, Koeuth T, Lupski JR. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res 1991;19: 6823-6831
5. Di Giovanni GD, Watrud LS, Seidler RJ, et al. Fingerprinting of mixed bacterial strains and BIOLOG gram-negative (GN) substrate communities by enterobacterial repetitive intergenic consensus sequence-PCR (ERIC-PCR). Curr Microbiol 1999;38: 217-223
6. Use of Repetitive (Repetitive Ectragenic Palindromic and Enterobacterial Repetitive Intergeneric Consensus)Sequences and the Polymerase Chain Reaction To Fingerprint the Genomes of Rhizobium meliloti Isolates and Other Soil Bacteria, Applied and Enviromental Microbiology, July 1992, 2180-2187
7. Short, Interspersed Repetitive DNA Sequences in Prokaryotic Genomes, Journal of Bacteriology, July 1992, 4525-4529
8. Seksik P, Rigottier-Gois L, Gramet G, et al. Alterations of the dominant faecal bacterial groups in patients with Crohn's disease of the colon. Gut 2003;52: 237-42
9.曹又方,赵立平.ER IC序列在不同细菌基因组中分布的分析.山西大学学报(自然科学)2002;25:354-357
10. Rahmati A, Gal M, Northey G, Brazier JS. Subtyping of Clostridium difficile polymerase chain reaction (pcr) ribotype 001 by repetitive extragenic palindromic PCR genomic fingerprinting. J Hosp Infect, 2005 May;60(1): 56-60
11. Jin LL,Wang QY, Hou X. ERIC-PCR on identification of Listeria species and strain. Yi Chuan. 2003 Mar;25(2): 195-7.
12. Song G, Wei G, Cao Y, et al. Use of ERIC-PCR as a community fingerprinting technology in elucidation of mode of action of a new probiotic formulation for prevention and treatment of bacterial diarrhea in piglets[J]. Book of abstracts of the Asia Pacific Conference On Plant Tissue Culture & Agrobiotechnology. 2000;1923: 166).
13. Ventura M, Meylan V, Zink R. Identification and tracing of Bifidobacterium species by use of enterobacterial repetitive intergenic consensus sequences. Appl Environ Microbiol 2003;69: 4296-4301.
14. McLellan SL, Daniels AD, Salmore AK. Genetic characterization of Escherichia coli populations from host sources of fecal pollution by using DNA fingerprinting. Appl Environ Microbiol 2003;69: 2587-2594
15. Femandez-Baca V, Ballesteros F, Hervas JA, et al. Molecular epidemiological typing of Enterobacter cloacae isolates from a neonatal intensive care unit: three-year prospective study. J Hosp Infect 2001;49: 173-182
16. Aarts HJ, Joosten RG, Henkens MH, et al. Rapid duplex PCR assay for the detection of pathogenic Yersinia enterocolitica strains. J Microbiol Methods 2001;47: 209-217,
17.鲁海峰等,ERIC-PCR分子杂交技术分析大熊猫肠道菌群结构,中国微生态学杂志,2005年4月第17卷第2期
18.潘莉等,腹泻儿童肠道菌群结构特征的ERIC-PCR指纹图分析,中国微生态学杂志,2003年6月第15卷第3期
19. Issemann Ⅰ, Green S1 Activation of a memberof the steroid hormone receptor superfamily byperoxisome proliferators [J]. Nature, 1990, 347: 645-6501
20. Asano T, Wakisaka M, Yoshinari M, et al, Peroxisome proliferator-activated receptorgammal (PPAR gammal) expresses in rat mesangial cells and PPARgamma agonists modulate its differentiation [J]. Biochim Biophys Acta, 2000. 1497 (1): 148-1541
21.陈迎春等,大肠杆菌MG1655菌株ERIC-PCR图谱主带序列组成分析,微生物学通报,2002年29(6)
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |