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Journal of microbiology and biotechnology v.15 no.2, 2005년, pp.310 - 320   피인용횟수: 3

A Membrane-Array Method to Detect Specific Human Intestinal Bacteria in Fecal Samples Using Reverse Transcriptase-PCR and Chemiluminescence

KIM PYOUNG IL    (Division of Microbiology, National Center for Toxicological Research, Food and Drug Administration   ); ERICKSON BRUCE D    (Division of Microbiology, National Center for Toxicological Research, Food and Drug Administration   ); CERNIGLIA CARL E.    (Division of Microbiology, National Center for Toxicological Research, Food and Drug Administration  );
  • 초록

    A membrane-based oligonucleotide array was used to detect predominant bacterial species in human fecal samples. Digoxygenin-labeled 16S rDNA probes were generated by PCR from DNA that had been extracted from fecal samples or slurries. These probes were hybridized to an array of 120 oligonucleotides with sequences specific for 40 different bacterial species commonly found in human feces, followed by color development using an alkaline phosphatase-conjugated antibody and NBT /BCIP. Twenty of the species were detected by this method, but E. coli, which was present at $\~$ 1 $\times 10 $^5$ CFU per gram feces, was not detected. To improve the sensitivity of this assay, reverse transcriptase-PCR was used to generate probes from RNA extracted from fecal cultures. Coupled with a chemiluminescence detection method, this approach lowered the detection limit for E. coli from $\~1$ $\times 10 $^6$ to ${\leq}$ 1 $\times 10 $^5$ These results indicate that the membrane-array method with reverse transcriptase-PCR and chemiluminescence detection can simultaneously identify bacterial species present in fecal samples at cell concentrations as low as ${\leq}$ 1 $\times 10 $^5$ CFU per gram.


  • 참고문헌 (38)

    1. Harmsen, H. M., G. C. Raangs, T. He, J. E. Degener, and G. W. Welling. 2002. Extensive set of 16S rRNA-based probes for detection of bacteria in human feces. Appl. Environ. Microbiol. 68: 2982- 2990 
    2. Malinen, E., A. Kassinen, T. Rinttila, and A. Palva. 2003. Comparison of real-time PCR with SYBR Green I or 5'nuclease assays and dot-blot hybridization with rDNAtargeted oligonucleotide probes in quantification of selected faecal bacteria. Microbiology 149: 269- 277 
    3. Manz, W., U. Szewzyk, P. Ericsson, R. Amann, K. H. Schleifer, and T. A. Stenstrom. 1993. In situ identification of bacteria in drinking water and adjoining biofilms by hybridization with 16S and 23S rRNA-directed fluorescent oligonucleotide probes. Appl. Environ. Microbiol. 59: 2293-2298 
    4. Sghir, A., G. Gramet, A. Suau, V. Rochet, P. Pochart, and J. Dore. 2000. Quantification of bacterial groups within human fecal flora by oligonucleotide probe hybridization. Appl. Environ. Microbiol. 66: 2263- 2266 
    5. Wang, R. F., W. W. Cao, and C. E. Cerniglia. 1997. PCR detection of Ruminococcus spp. in human and animal fecal samples. Mol. Cell. Probes 11: 259- 265 
    6. Wang. R. F., M. L. Beggs, L. H. Robertson, and C. E. Cerniglia. 2002. Design and evaluation of oligonucleotidearray method for the detection of human intestinal bacteria in fecal samples. FEMS Microbiol. Lett. 213: 175- 182 
    7. Wang, R. F., M. L. Beggs, B. D. Erickson, and C. E. Cerniglia. 2004. DNA microarray analysis of predominant human intestinal bacteria in fecal samples. Mol. Cell. Probes 18: 223- 234 
    8. Chadwick, R. W., S. E. George, and L. P. Claxton. 1992. Role of gastrointestinal mucosa and microflora in the bioactivation of dietary and environmental mutagens or carcinogens. Drug Metab. Rev. 24: 425- 492 
    9. Kok, R. G., A. de Waal, F. Schut, G. W. Welling, G. Weenk, and K. J. Hellingwerf. 1996. Specific detection and analysis of a probiotic Bifidobacterium strain in infant feces. Appl. Environ. Microbiol. 62: 3668- 3672 
    10. Alfreider, A., J. Pernthaler, R. Amann, B. Sattler, F. O. Glockner, A. Wille, and R. Psenner. 1996. Community analysis of the bacterial assemblages in the winter cover and pelagic layers of a high mountain lake by in situ hybridization. Appl. Environ. Microbiol. 62: 2138- 2144 
    11. Juretschko, S., G. Timmermann, M. Schmid, K. H. Schleifer, A. Pommerenning-Roser, H. P. Koops, and M. Wagner. 1998. Combination molecular and conventional analyses of nitrifying bacterium diversity in activated sludge: Nitrosococcus mobilis and Nitrospira-likc bacteria as dominant populations. Appl. Environ. Microbiol. 64: 3042- 3051 
    12. Schwiertz, A., G. L. Blay, and M. Blaut. 2000. Quantification of different Eubacterium spp. in human fecal samples with species-specific 16S rRNA-targeted oligonucleotide probes. Appl. Environ. Microbiol. 66: 375- 382 
    13. Lee, K.-H., G. S. Moon, J.-Y An, H. J. Lee, H. C. Chang. D. K. Chung, J. H. Lee, and J. K. Kim. 2002. Isolation of nisin-producing Lactobacillus lactis strain from Kimchi and characterization of its nisZ gene. J. Microbiol. Biotechnol. 12: 389- 397 
    14. Gibson, G. R., J. H. Cummings, and G. T. Macfarlane. 1988. Use of three-stage continuous culture system to study the effect of mucin on dissimilatory sulfate reduction and methanogenesis by mixed populations of human gut bacteria. Appl. Environ. Microbiol. 54: 2750- 2755 
    15. Cerniglia, C. E., J. P. Freeman, W. Franklin, and L. D. Pack. 1982. Metabolism of azo dyes derived from benzidine, 3,3'dimethylbenzidine and 3,3'-dimethoxybenzidine to potentially carcinogenic aromatic amines by intestinal bacteria. Carcinogenesis 3: 1255- 1260 
    16. Simon, G. L. and S. L. Gorbach. 1984. Intestinal flora in health and disease. Gastroenterology 86: 174- 193 
    17. Becker, S., P. Boger, R. Oehlmann, and A. Ernst. 2000. PCR bias in ecological analysis: A case study for quantitative taq nuclease assays in analyses of Microbiol communities. Appl. Environ. Microbiol. 66: 4945- 4953 
    18. Drasar, B. S. and A. K. Roberts. 1990. Control of the large bowel microflora. pp. 95- 100. In Hill, M. J. and Marsh, B. S. (eds.), Human Microbiol Ecology. Boca Raton, CRC Press Inc 
    19. Falk, P. G., L. V. Hooper, T. Midtvedt, and J. I. Gordon. 1998. Creating and maintaining the gastrointestinal ecosystem: What we know and need to know from gnotobiology. Microbiol, Mol. Biol. Rev. 62: 1157- 1170 
    20. Wang, R. E., W. W. Cao, and C. E. Cerniglia. 1996. PCR detection and quantitation of predominant anaerobic bacteria in human and animal fecal samples. Appl. Environ. Microbiol. 62: 1242- 1247 
    21. Hopkins, M. J., R. Sharp, and G. T. Macfarlane. 2001. Age and disease related changes in intestinal bacterial populations assessed by cell culture, 16S rRNA abundance, and community cellular fatty acid profiles. Gut 48: 198- 205 
    22. Dore, J., A. Sghir, G. Gramet, G. Corthier, and P. Pochart. 1998. Design and evaluation of a 16S rRNA-targeted oligonucleotide probe for specific detection and quantitation of human faecal Bacteroides populations. Syst. Appl. Microbiol. 21: 65-71 
    23. Matsuki, T., K. Watanabe, R. Tanaka, M. Fukuda, and H. Oyaizu. 1999. Distribution of bifidobacterial species in human intestinal microflora examined with 16S rRNA-genetargeted species-specific primers. Appl. Environ. Microbiol. 65: 4506-4512 
    24. Tannock, G. W. 1995. Normal Microflora: An Introduction to Microbes Inhabiting the Human Body. Chapman & Hall, London, United Kingdom 
    25. Cerniglia, C. E., P. C. Howard, P. P. Fu, and W. Franklin. 1984. Metabolism of nitropolycyclic aromatic hydrocarbons by human intestinal microllora. Biochem. Biophys. Res. Commun. 123: 262- 270 
    26. Moore, W. E. C. and L. H. Moore. 1995. Intestinal floras of populations that have a high risk of colon cancer. Appl. Environ. Microbiol. 61: 3202- 3207 
    27. Fuller, R. 1989. Probiotics in man and animals. J. Appl. Bacteriol. 66: 365- 378 
    28. Matsuki, T., K. Watanabe, J. Fujimoto, Y. Miyamoto, T. Takada, K. Matsumoto, H. Oyaizu, and R. Tanaka. 2002. Development of 16S rRNA-gene targeted group-specific primers for the detection and identification of predominant bacteria in human feces. Appl. Environ. Microbiol. 68: 5445-5451 
    29. Hwang, Y.-H. and H.-S. Lee. 2002. Antibacterial activity of Pinus densiflora leaf-derived components toward human intestinal bacteria. J. Microbiol. Biotechnol . 12: 610- 616 
    30. Kreader, C. A. 1995. Design and evaluation of Bacteroides DNA probes for the specific detection of human fecal pollution. Appl. Environ. Microbiol. 61: 1171- 1179 
    31. Marteau, P., P. Pochart, J. Dore, C. Maillet, A. Bernalier, and G. Corthier. 2001. Comparative study of bacterial groups within the human cecal and fecal microbiota. Appl. Environ. Microbiol. 67: 4939- 494 
    32. Wang, R. F., S.-J. Kim, L. H. Robertson, and C. E. Cerniglia. 2002. Development of a membrane-array method for the detection of human intestinal bacteria in fecal samples. Mol. Cell. Probes 16: 341- 350 
    33. Kenzaka, T., N. Yamaguchi, K. Tani, and M. Nasu, 1998. rRNA-targeted fluorescent in situ hybridization analysis of bacterial community structure in river water. Microbiology 144: 2085- 2093 
    34. Kim, K. Y., P. M. Davidson, and H. J. Chung. 2000. AntiMicrobiol effectiveness of pine extract on foodborne illness bacteria. J. Microbiol. Biotechnol. 10: 227- 232 
    35. Langendijk, P. S., F. Schut, G. J. Jansen, G. C. Raangs, G. R. Kamphuis, M. F. Wilkinson, and G. W. Welling. 1995. Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16S rRNA-targeted probes and its application in fecal samples. Appl. Environ. Microbiol. 61: 3069- 3075 
    36. Bae, E.-A., N.-Y. Kim, M. J. Han, M.-K. Choo, and D.-H. Kim. 2003. Transformation of ginsenosides to compound K (IH-901) by lactic acid bacteria of human intestine. J. Microbiol. Biotechnol. 13: 9- 14 
    37. Song, Y.-L., L. Kato, C. X. Liu, Y. Matsumya, H. Kato, and K. Watanabe. 2000. Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA. FEMS Microbiol. Lett. 187: 167- 173 
    38. Drasar, B. S. and B. I. Duerden. 1991. Anaerobes in the normal flora of man, pp. 162- 179. In Duerden, B. I. and Drasar, B. S. (eds.), Anaerobes in Human Disease. WileyLiss, New York, NY 
  • 이 논문을 인용한 문헌 (3)

    1. 2007. "" Journal of microbiology and biotechnology, 17(1): 5~14     
    2. 2007. "" Journal of microbiology and biotechnology, 17(11): 1885~1889     
    3. 2007. "" Journal of microbiology and biotechnology, 17(10): 1629~1637     

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