생명과학회지 (Journal of Life Science)

  • 제22권2호
  • /
  • Pages.245-250
  • /
  • 2012
  • /
  • 1225-9918(pISSN)
  • /
  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
권호 표지
DOI QR코드

DOI QR Code

PCR-RFLP에 의한 Vibrio core group을 포함한 Vibrio 종의 구분

Differentiation of Vibrio spp. including Core Group Species by PCR-RFLP

  • 투고 : 2012.01.11
  • 심사 : 2012.02.23
  • 발행 : 2012.02.28
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

Vibrio속의 core 균주(Vibrio alginolyticus, Vibrio parahaemolyticus)를 포함하여 총 6 종의 Vibrio 균주(V. fluvialis, V. proteolyticus, V. vulnificus, V. mimicus)와 Grimontia (Vibrio) hollisae의 16S rDNA를 PCR 증폭하여 Alu I, Cfo I, Dde I, Hae III, Msp I, Rsa I의 6 종의 제한효소를 처리 후 RFLP 분석을 수행하였다. 2 종의 core 균주와 V. proteolyticus는 4 종의 제한효소(Cfo I, Dde I, Msp I, Rsa I)에서 동일한 제한효소 패턴을 나타내었다. 제한효소의 패턴의 조합에 의해 6 종의 Vibrio 종은 6 개의 RFLP type으로 구분되었다. 특히 Alu I의 경우, 실험된 6 종의 Vibrio속에 대하여 각기 다른 6 개의 종 특이적 RFLP type을 나타내었다. 제한효소 패턴에 근거하여 작성한 덴드로그램에서 Vibrio core group 균주인 V. alginolyticus 와 V. parahaemolyticus는 90% 이상의 매우 높은 유사도를 나타내었다. 반면 Grimontia hollisae는 실험된 모든 제한효소 패턴에서 Vibrio속 세균과는 분명히 구분되는 RFLP type을 나타내었다. 따라서 PCR-RFLP는 제한효소를 적절히 선택한다면 Vibrio 속 세균의 신속한 구분에 여전히 유용하다.

The 16S rDNA - RFLP types for six Vibrio species (V. fluvialis, V. proteolyticus, V. vulnificus, V. mimicus) including two core group members, V. alginolyticus and V. parahaemolyticu s, and Grimontia (Vibrio) hollisae were determined using PCR-RFLP analysis. Six tetrameric restriction enzymes (Alu I, Cfo I, Dde I, Hae III, Msp I, and Rsa I) were selected for RFLP analysis. V. alginolyticus, V. parahaemolyticus, and V. proteolyticus showed the same RFLP pattern following digestion with four of the six used restriction enzymes: CfoI, DdeI, MspI, and RsaI. Various restriction enzyme combinations generated digests recognizable as distinct RFLP types for each of the assayed Vibrio species. In particular, AluI single digestion produced species specific band patterns that enabled the differentiation between these Vibrio species. Dendrogram based on restriction patterns showed that two Vibrio core group members, V. alginolyticus and V. parahaemolyticus were closely related having a similarity over 90%. Although the observed RFLP pattern for Grimontia hollisae shared several common bands with other Vibrio spp., G. hollisae results were still clearly distinct from Vibrio spp. RFLP types for all restriction enzymes tested. If restriction enzymes are aptly selected, PCR-RFLP analysis is still a rapid and effective tool for differentiating Vibrio species.

키워드

참고문헌

  1. Aznar, R., W. Ludwig., R. I. Amann, and K. H. Schlefer. 1994. Sequence determination of rRNA genes of pathogenic Vibrio species and whole-cell identification of Vibrio vulnificus with rRNA-targeted oligonucleotide probes. Int. J. Syst. Bacteriol. 44, 330-337. https://doi.org/10.1099/00207713-44-2-330
  2. Dorsch, M., D. Lane, and E. Stackebrandt. 1992. Toward a phylogeny of the genus Vibrio based on 16S rRNA sequence. Int. J. Syst. Bacteriol. 42, 58-63. https://doi.org/10.1099/00207713-42-1-58
  3. Gomez-Gil, B., S. Soto-Rodriguez, A. Garcia-Gasc, A. Roque, T. Vazquez-Juarez, F. L. Thompson, and J. Swings. 2004. Molecular identification of Vibrio harveyi-related isolates associated with diseased aquatic organisms. Microbiol. 150, 1769-1777. https://doi.org/10.1099/mic.0.26797-0
  4. Gomez-Gil, B., F. L. Thompson, C. C. Thompson, A. Garcia-Gasca, A. Roque, and J. Swings. 2004. Vibrio hispanicus sp. nov., isolated from Artemia sp. and sea water in Spain. Inc. J. syst. Evol. Microbiol. 54, 261-265. https://doi.org/10.1099/ijs.0.02775-0
  5. Hernandez, G. and J. Olmos. 2003. Molecular identification of pathogenic and nonpathogenic strains of Vibrio harveyi using PCR and RAPD. Appl. Microbiol. Biotechnol. 63, 722-727. https://doi.org/10.1007/s00253-003-1451-z
  6. Ki, J. S. 2009. Heterogeneity analysis of the 16S rRNA gene sequence of the genus Vibrio. Korean J. Microbiol. 45, 430-434.
  7. Ki, J. S., R. Zhang, W. Zhang, Y. L. Huang, and P. Y. Qian. 2009. Analysis of RNA polymerase beta subunit (rpoB) gene sequences for the discriminative power of marine Vibrio species. Microb Ecol. 58, 679-691. https://doi.org/10.1007/s00248-009-9519-7
  8. Kim, D. G., S. H. Ahn, L. H. Kim, K. J. Park, Y. K. Hong, and I. S. Kong. 2008. Application of the rpoS gene for species-specific detection of Vibrio vulnificus by real-time PCR. J. Microbiol. Biotechnol. 18, 1841-1847.
  9. Kumar, N. R. and S. Nair. 2007. Vibrio rhizosphaerae sp. nov., a red-pigmented bacterium that antagonizes phytopathogenic bacteria. Inc. J. Syst. Evol. Microbiol. 57, 2241-2246. https://doi.org/10.1099/ijs.0.65017-0
  10. Lambert, M. A., F. W. Hickman-Brenner, J. J. FarmerIII, and C. W. Moss. 1983. Differentiation of Vibrionaceae species by their cellular fatty acid composition. Int. J. Syst. Bacteriol. 33, 777-792. https://doi.org/10.1099/00207713-33-4-777
  11. Lee, J. W., I. J. Jun, H. J. Kwun, K. L. Jang, and J. H. Cha. 2004. Direct identification of Vibrio vulnificus by PCR targeting elastase gene. J. Microbiol. Biotechnol. 14, 284-289.
  12. Lerat E, V. Daubin, and N. A. Moran. 2003. From gene trees to organismal phylogeny in prokaryotes: the case of the gammaproteobacteria. PLOS Biol. 1, E19.
  13. Pascual, J., M. C. Macian, D. R. Arahal, E. Garay, and M. J. Pujalte. 2010. Multilocus sequence analysis of the central clade of the genus Vibrio by using the 16S rRNA, recA, pyrH, rpoD, gyrB, rctB and toxR genes. Inc. J. Syst. Evol. Microbiol. 60, 154-165. https://doi.org/10.1099/ijs.0.010702-0
  14. Pedersen, K., L. Verdonck, B. Austin, D. A. Austin, A. R. Blanch, P. A. D. Grimont, J. Jofre, S. Koblavi, J. L. Larsen, T. Tiainen, M. Vigneulle, and J. Swings. 1998. Taxonomic evidence tha Vibrio carchariae grimes et al. 1985 is a junior synonym of Vibrio harveyi (Johnson and Shunk 1936) Baumann et al. 1981. Int. J. Syst. Bacteriol. 48, 749-758. https://doi.org/10.1099/00207713-48-3-749
  15. Rivas, R., P. Garcia-Fraile, P. F. Mateos, E. Martinez-Molina, and E. Velazquez. 2006. Photobacterium halotolerans sp. Nov., isolated from lake Martel in Spain. Inc. J. Syst. Evol. Microbiol. 56, 1067-1071. https://doi.org/10.1099/ijs.0.64099-0
  16. Thompson, F. L., B. Hoste, K. Vandemeulebroecke, and J. Swings. 2003. Reclassification of Vibrio hollisea as Grimontia hollisae gen. nov., comb. Nov. Inc. J. syst. Evol. Microbiol. 53, 1615-1617. https://doi.org/10.1099/ijs.0.02660-0
  17. Thompson, F. L., D. Gevers, C. C. Thompson, P. Dawyndt, S. Naser, B. Hoste, C. B. Munn, and J. Swings. 2005. Phylogeny and molecular identification of vibrios on the basis of multilocus sequence analysis. Appl. Microbiol. Biotechnol. 71, 5107-5115.
  18. Thompson, F. L., T. Iida, and J. Swings. 2004. Biodiversity of vibrios. Microbiol. Mol. Biol. Rev. 68, 403-431. https://doi.org/10.1128/MMBR.68.3.403-431.2004
  19. Urakawa, H., K. Kita-Tsukamoto, and K. Ohwada. 1997. 16S rDNA genotyping using PCR/RFLP (restriction fragment length polymorphism) analysis among the family Vibrionaceae. FEMS Microbiol. Lett. 152, 125-132. https://doi.org/10.1111/j.1574-6968.1997.tb10418.x
  20. Urakawa, H., K. Kita-Tsukamoto, and K. Ohwada. 1998. A new approach to separate the genus Photobacterium from Vibrio with RFLP patterns by HhaI digestion of PCR-amplified 16S rDNA. Curr. Microbiol. 36, 171-174. https://doi.org/10.1007/PL00006762
  21. Yeon, S. H., W. J. Jeong, and J. S. Park. 2005. The diversity of culturable organotrophic bacteria from local solar salterns. J. Microbiol. 43, 1-10.