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Quantitative Analysis of Human- and Cow-Specific 16S rRNA Gene Markers for Assessment of Fecal Pollution in River Waters by Real-Time PCR

  • Jeong, Ju-Yong (Department of Agricultural Biotechnology, Seoul National University) ;
  • Park, Hee-Deung (School of Civil, Environmental and Architectural Engineering, Korea University) ;
  • Lee, Kyong-Hee (Department of Agricultural Biotechnology, Seoul National University) ;
  • Hwang, Jae-Hong (Department of Agricultural Biotechnology, Seoul National University) ;
  • Ka, Jong-Ok (Department of Agricultural Biotechnology, Seoul National University)
  • 발행 : 2010.02.28

초록

The base sequences representing human- and cow-specific 168 rRNA gene markers identified in a T-RFLP analysis were recovered from clone libraries. The human- and cow-specific primers were designed from these sequences and their specificities were analyzed with fecal DNAs from human, cow, and pig. The AllBac primer set showed positive results for all human, cow, and pig samples, whereas the human-specific primer set showed positive result only for the human sample but not for the cow or pig samples. Likewise, the cow-specific primer set showed positive results only for the cow sample but not for the human or pig samples. Real-time PCR assay with these primers was developed for the identification and quantification of fecal pollution in the river water. The human- and cow-specific markers were detected in the order of 9 $\log_{10}$ copies per gram wet feces, which were two orders of magnitude lower than those of total Bacteroidales. For the river water samples, the human-specific marker was detected in $1.7-6.2\;\log_{10}$ copies/100 ml water, which was 2.4-4.9 orders of magnitude lower than those of total Bacteroidales. There was no significant correlation between total Bacteroidales and conventional fecal indicators, but there was a high correlation between Bacteroidales and the human-specific marker. This assay could reliably identify and quantify the fecal pollution sources, enabling effective measures in the watersheds and facilitating water quality management.

키워드

참고문헌

  1. Ashelford, K. E., N. A. Chuzhanova, J. C. Fry, A. J. Jones, and A. J. Weightman. 2006. New screening software shows that most recent large 16S rRNA gene clone libraries contain chimeras. Appl. Environ. Microbiol. 72: 5734-5741. https://doi.org/10.1128/AEM.00556-06
  2. Bernhard, A. E. and K. G. Field. 2000. Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes. Appl. Environ. Microbiol. 66: 1587-1594. https://doi.org/10.1128/AEM.66.4.1587-1594.2000
  3. Bernhard, A. E. and K. G. Field. 2000. A PCR assay to discriminate human and ruminant feces on the basis of host differences in Bacteroides-Prevotella genes encoding 16S rRNA. Appl. Environ. Microbiol. 66: 4571-4574. https://doi.org/10.1128/AEM.66.10.4571-4574.2000
  4. Carson, C. A., J. M. Christiansen, H. Yampara-Iquise, V. W. Benson, C. Baffaut, J. V. Davis, et al. 2005. Specificity of a Bacteroides thetaiotaomicron marker for human feces. Appl. Environ. Microbiol. 71: 4945-4949. https://doi.org/10.1128/AEM.71.8.4945-4949.2005
  5. Jeong, J. Y., K. I. Gil, K. H. Lee, and J. O. Ka. 2008. Molecular identification of fecal pollution sources in water supplies by host-specific fecal DNA markers and terminal restriction fragment length polymorphism profiles of 16S rRNA gene. J. Microbiol. 46: 599-607. https://doi.org/10.1007/s12275-008-0174-3
  6. Kildare, B. J., C. M. Leutenegger, B. S. McSwain, D. G. Bambic, V. B. Rajal, and S. Wuertz. 2007. 16S rRNA-based assays for quantitative detection of universal, human-, cow-, and dog-specific fecal Bacteroidales: A Bayesian approach. Water Res. 41: 3701-3715. https://doi.org/10.1016/j.watres.2007.06.037
  7. Klappenbach, J. A., P. R. Saxman, J. R. Cole, and T. M. Schmidt. 2001. RRNDB: The Ribosomal RNA Operon Copy Number Database. Nucleic Acids Res. 29: 181-184. https://doi.org/10.1093/nar/29.1.181
  8. 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.
  9. Kreader, C. A. 1998. Persistence of PCR-detectable Bacteroides distasonis from human feces in river water. Appl. Environ. Microbiol. 64: 4103-4105.
  10. Layton, A., L. McKay, D. Williams, V. Garrett, R. Gentry, and G. Sayler. 2006. Development of Bacteroides 16S rRNA gene TaqMan-based real-time PCR assays for estimation of total, human, and bovine fecal pollution in water. Appl. Environ. Microbiol. 72: 4214-4224. https://doi.org/10.1128/AEM.01036-05
  11. 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. https://doi.org/10.1128/AEM.68.11.5445-5451.2002
  12. Mieszkin, S., J. P. Furet, G. Corthier, and M. Gourmelon. 2009. Estimation of pig fecal contamination in a river catchment by real-time PCR using two pig-specific Bacteroidales 16S rRNA genetic markers. Appl. Environ. Microbiol. 75: 3045-3054. https://doi.org/10.1128/AEM.02343-08
  13. Okabe, S., N. Okayama, O. Savichtcheva, and T. Ito. 2007. Quantification of host-specific Bacteroides-Prevotella 16S rRNA genetic markers for assessment of fecal pollution in freshwater. Appl. Microbiol. Biotechnol. 74: 890-901. https://doi.org/10.1007/s00253-006-0714-x
  14. Reischer, G. H., D. C. Kasper, R. Steinborn, A. H. Farnleitner, and R. L. Mach. 2007. A quantitative real-time PCR assay for the highly sensitive and specific detection of human fecal influence in spring water from a large alpine catchment area. Lett. Appl. Microbiol. 44: 351-356. https://doi.org/10.1111/j.1472-765X.2006.02094.x
  15. Reischer, G. H., D. C. Kasper, R. Steinborn, R. L. Mach, and A. H. Farnleitner. 2006. Quantitative PCR method for sensitive detection of ruminant fecal pollution in freshwater and evaluation of this method in alpine karstic regions. Appl. Environ. Microbiol. 72: 5610-5614. https://doi.org/10.1128/AEM.00364-06
  16. Schloss, P. D. and J. Handelsman. 2005. Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Appl. Environ. Microbiol. 71: 1501-1506. https://doi.org/10.1128/AEM.71.3.1501-1506.2005
  17. Seurinck, S., T. Defoirdt, W. Verstraete, and S. D. Siciliano. 2005. Detection and quantification of the human-specific HF183 Bacteroides 16S rRNA genetic marker with real-time PCR for assessment of human fecal pollution in freshwater. Environ. Microbiol. 7: 249-259. https://doi.org/10.1111/j.1462-2920.2004.00702.x
  18. Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24: 1596-1599. https://doi.org/10.1093/molbev/msm092
  19. Tillett, D., D. L. Parker, and B. A. Neilan. 2001. Detection of toxigenicity by a probe for the microcystin synthetase A gene (mcyA) of the cyanobacterial genus Microcystis: Comparison of toxicities with 16S rRNA and phycocyanin operon (phycocyanin intergenic spacer) phylogenies. Appl. Environ. Microbiol. 67: 2810-2818. https://doi.org/10.1128/AEM.67.6.2810-2818.2001
  20. Walters, S. P. and K. G. Field. 2009. Survival and persistence of human and ruminant-specific fecal Bacteroidales in freshwater microcosms. Environ. Microbiol. 11: 1410-1421. https://doi.org/10.1111/j.1462-2920.2009.01868.x

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