DOI QR코드

DOI QR Code

Detection of Gram-negative Bacteria in Broad-range PCR Amplifying 16S rRNA Gene with Semi-nested Primers and Its Application in Market Milk

16S rRNA 유전자의 Semi-nested Primer를 이용한 Broad-range PCR에 의한 그람음성세균의 검출과 시유에서의 응용

  • Choi, Suk-Ho (Dept of Biotechnology, Sangji University) ;
  • Choi, J.J. (Dept of Biotechnology, Sangji University) ;
  • Lee, S.B. (Dept of Biotechnology, Sangji University)
  • 최석호 (상지대학교 생명공학과) ;
  • 최정준 (상지대학교 생명공학과) ;
  • 이승배 (상지대학교 생명공학과)
  • Published : 2005.06.30

Abstract

A two-step broad-range PCR method detecting gram-negative bacteria at the level as low as 2 CFU was developed by using primers of GNFI and GNRI and then semi-nested primer of GNF2 and GNRI. The nucleotide sequences of the primers were determined based on l6S rRNA gene. The DNA fragments of 1173 bp and 169 bp were amplified in one-step PCRs with primer sets of GNFI-GNRI and GNF2-GNRl, respectively, using template DNA from seven strains of gram-negative bacteria including Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas spp., and Acinetobacter baumaii but not from Achromobacter lyticus, Alca/igens faecalis, and five strains of gram-positive bacteria. DNA fragments of 180 bp were amplified from LTLT-pasteurized milk and UHf-pasteurized milk in the two-step PCR. The DNA fragments were amplified from LTLT-pasteurized milk which was added with Pseudomonas j/uorescens and subsequently heated at 65 $^{\circ}C$, 80 $^{\circ}C$, and 100 $^{\circ}C$ for 30 min but they were not amplified from the milk autoclaved at 121$^{\circ}C$ for 15 min. It was suggested in PCR that Pseudomonas fluorescens heated at 65 $^{\circ}C$ for 30 min in milk was more sensitive to DNase treatment than viable bacteria.

Keywords

PCR;16S rRNA;Gram-negative bacteria;Market milk

References

  1. Bej, A. K., Mahbubani, M. H. and Atlas, R. M. 1991. Detection of viable Legionella pneumophila in water by polymerase chain reaction and gene probe methods. Appl. Environ. Microbiol. 57:597--600
  2. Cooray, K. J., Nishibori, T., Xiong, H., Matsuyama, T., Fujita, M and Mitsuyama, M. 1994. Detection of multiple virulence-associated genes of Listeria monocytogens by PCR in artificially contaminated milk samples. Appl. Environ. Microbiol. 60:3023-3026
  3. Duprey, E., Caprais, M. P., Derrien, A. and Fach, F. 1997. Salmonella DNA persistence in natural eawaters using PCR analysis. J. Appl. Microbiol. 82:507-510 https://doi.org/10.1046/j.1365-2672.1997.00143.x
  4. Greisen, K., Loeffelholz, M., Purohit, A and Leong, D. 1994. PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. J. Clin. Microbiol. 32:335-351
  5. Hashimoto, Y., Itho, Y., Fujinaga, Y., Khan, A. Q., Sultana, F., Miyake, M., Hirose, K., Yamamoto, H. and Ezaki, T. 1995. Development of nested PCR based on the ViaB sequence to detect Salmonella typhi. J. Clin. Microbiol., 33:775-777
  6. Herman, L. 1997. Detection of viable and dead Listeria monocytogenes by PCR Food Microbiol. 14:103-110 https://doi.org/10.1006/fmic.1996.0077
  7. Herman, L. M. F., De Block, J. H. G. E. and Moermans, R. J. B. 1995. Direct detection of Listeria monocytogenes in 25 milliliters of raw milk by a two-step PCR with nested primers. Appl. Environ. Microbiol. 61:817-819
  8. Holt, J. G. 1984. Bergey's manual of systematic bacteriology. Williams and Wilkins, Baltimore and London
  9. IDF. 1993. Catalogue of tests for the detection of post-pasteurization contamination of milk. Bulletin of the IOF 281, pp13-34
  10. Impraim, C. C., Saiki, R. K., Erlich, H. A. and Teplitz, R. L. 1987. Analysis of DNA extract from formalin-fixed, paraffin-embedded tissues by enzymatice amplification and hybridization with sequence-specific oligonucleotides. Biochem, Biophys. Res, Commun., 142:710-6 https://doi.org/10.1016/0006-291X(87)91472-0
  11. Masters, C. L., Shallcross, J. A. and Mackey, B. M. 1994. Effect of stress treatments on the detection of Listeria monocytogenes and enterotoxigenic Escherichia coli by the polymerase chain reaction J. Appl. Bacteriol. 77:73-9 https://doi.org/10.1111/j.1365-2672.1994.tb03047.x
  12. McKillip, J. L., Jaykus, L. -A. and Drake M. 1998. rRNA stability in heat-killed and UV-irradiated enterotoxigenic Staphylococcus aureus and Escherichia coli O157:H7. Appl. Environ. Microbiol. 64:4264-4268
  13. Paabo, S., Gifford, J. A. and Wilson, A. C. 1988. Mitochondrial DNA sequences from a 7000-year brain. Nucleic Acids Res., 16:0775-87 https://doi.org/10.1093/nar/16.2.775
  14. Rasmussen, H. N., Rasmussen, O. F., Christensen, H. and Olsen, J. E. 1995. Detection of Yersinia enterocolitica 0:3 in faecal samples and tonsil swabs from pigs using IMS and PCR. J. Appl. Bacteriol. 78:563-568 https://doi.org/10.1111/j.1365-2672.1995.tb03100.x
  15. Vaitilingom, M., Gendre, F. and Brignon, P. 1998. Direct detection of viable bacteria, molds, and yeasts by reverse transcriptase PCR in contaminated milk samples after heat treatment. Appl. Environ. Microbiol. 64:1157-1160
  16. Wiedmann, M., Czajka, J., Barany, F. and Batt, C. A. 1992. Discrimination of Listeria monocytogenes from other Listeria species by ligase chain reaction. Appl. Environ. Microbiol. 58:3443-3447
  17. Wiedmann, M., Barany, F. and Batt, C. A. 1993. Detection of Listeria monocytogenes with a nonisotopic polymerase chain reaction-coupled ligase chain reaction assay. Appl. Environ. Microbiol. 59: 2743-2745
  18. Woese, C. R. 1987. Bacterial evolution. Microbiol. Rev. 51:221-271
  19. Zimmermann, K., Volkei, D., Turceic, P., Schwarz, H. –P. and Rieger, M. 2001. Methods for the detection, quantification and and differentiation of infectious versus non-infectious pathogens in a sample. PCT, WO 01/46462 A2
  20. 최석호, 최정준, 이승배, 윤영호. 2004. 시유의 2차오염과 저장가능기간을 결정하기 위한 resazurin 환원시간검사. 한국동물자원과학회지, 46:999-1006
  21. Versalovic, J., Koeuth, T. and Lupski, J. R. 1991. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 19:6823-31 https://doi.org/10.1093/nar/19.24.6823
  22. Giovannoi, S. -J., Delong, E. F., Olsen, G. J. and Pace, N. R. 1988. Phylogenetic group-specific oligonucleotide probe for identification of single microbial cells. J. Bacteriol. 170:720-726