Detection of Salmonella spp. by TaqMan real-time PCR and comparison of nucleotide sequences of ompC gene among Salmonella

TaqMan 실시간 중합 효소 연쇄반응에 의한 살모넬라속의 검출 및 ompC 항원단백 유전자의 비교

  • Lee, Young-Sung (Bio-Safety Research institute, College of Veterinary Medicine, Chonbuk National University) ;
  • Choi, Kyoung-Seong (Bio-Safety Research institute, College of Veterinary Medicine, Chonbuk National University) ;
  • Kim, Myeong-Chul (Bio-Safety Research institute, College of Veterinary Medicine, Chonbuk National University) ;
  • Han, Jae-Cheol (Bio-Safety Research institute, College of Veterinary Medicine, Chonbuk National University) ;
  • Chae, Joon-Seok (Bio-Safety Research institute, College of Veterinary Medicine, Chonbuk National University)
  • 이영성 (전북대학교 수의과대학 생체안전성연구소) ;
  • 최경성 (전북대학교 수의과대학 생체안전성연구소) ;
  • 김명철 (전북대학교 수의과대학 생체안전성연구소) ;
  • 한재철 (전북대학교 수의과대학 생체안전성연구소) ;
  • 채준석 (전북대학교 수의과대학 생체안전성연구소)
  • Accepted : 2002.12.04
  • Published : 2002.12.30

Abstract

Antigenic ompC genes of S. gallinarum, S. pullorum and S. dublin were characterized among Salmonella spp. isolated from chickens and other animals to identify genetic variation. Salmonella ompC gene fragment (1,027 bp) was amplified by PCR and the amplicons were cloned for comparison of nucleotide sequences. The identity of the sequences between S. gallinarum and S. pullorum, S. gallinarum and S. dublin, S. pullorum and S. dublin was 99.8%, 97.6% and 97.8%, respectively. Also, we found that ompC has some diversity between S. gallinarum and S. pullorum, and other Salmonella spp. which may be useful to type the organisms. Similar to diagnosis in other organisms, the TaqMan PCR method can be applied to rapid and accurate diagnosis of salmonellosis in chickens and other animals. We designed PCR primers and TaqMan probe for flagellin gene (fliC) for detection of Salmonella spp. by TaqMan PCR. The TaqMan PCR method was 10,000 times more sensitive than conventional PCR.

Keywords

References

  1. Smith HW, Tucker JF. Ihe virulence of salmonella strains for chickens; their excetion by infected chicken. J Hyg, 84:479-488, 1989
  2. Cast RK, Beard CW. Age-related changes in the persistence and pathogenicity of Salmonella typhimurium in chickens. Poultry Sci, 68:1454-1460, 1989
  3. Li J, Smith NH, Nelson K, et al. Evolutionary origin and radiation of avian-adapted on-motile Salmonellae. J Med Microbiol, 38:129-139, 1993
  4. Olsen JE, Skov MN, Christensen JP, et al. Genomic lineage of Salmonella enterica serotype Gallinarum. J Med Microbiol, 45:413-418, 1996
  5. 김기석, 이희석, 모인필. 국내 닭에서의 가금티푸스 (Fowl typhoid) 발생. 농업과학논문집, 37:544-549, 1995
  6. 오강희, 최원필. 초생추 유래 Salmonella 속균의 생물학적 특성. 대학수의학회지, 34:501-510, 1994
  7. Barrow PA. Immunity to experimental fowl typhoid in chickens induced by a virulence plasmid-cured derivative of Salmonella gallinarum. Infect Immun, 58:2283-2288, 1990
  8. Barrow PA, Bumstead N. Resistance to Salmonella gallinarum, S. pullorum and S. enteritidis in inbred lines of chickens. Avian Dis, 37:189-193, 1993
  9. Ewing WH. Edward and Ewing's Identification of Enterobacterceae. Amsterdam Elsevier Biomedical press, 1986
  10. Gast RK, Detecting infections of chickens with recent Salmonella pullorum isolates using standard serological methods. Poultry Sci, 76:17-23, 1997
  11. Holt PS, Chaubal LH. Detection of motility and putative synthesis of flagellar proteins in Salmonella pullorum cultures. J Clin Microbiol, 35(4):1016-1020, 1997
  12. Gast RK, Holt PS. Application of flagellar-based immuonoassays for serologic of salmonella pullorum infection in chickens. Avian Dis, 42:807-811, 1998
  13. Tuchili LM, Kodama H, Izumoto Y, et al. Detection of Salmonella gallinarum and S. typhimurium DNA in experimetally infected chicks by polymerase chain reaction. J Vet Med Sci, 57:59-63, 1995
  14. Itoh Y, Hirose K, Miyake M, et al. Amplification of rfbE. and fliC genes by polymerase chain reaction for identification and detection of Salmonella serovar enteritidis, dublin and gallinarum-pullorum. Microbiol Immunol, 41:791-794, 1997
  15. Singh SP, Singh SR, Wlliams YU, et al. Antigenic determinants of the ompC porin from Salmonella typhimurium. Infect Immun, 63:4600-4605, 1995
  16. Puente JL, Juarez D, Bobadilla M, et al. The Salmonella ompC gene : structure and use as a carrier for heterologous sequences. Gene, 156:1-9, 1995
  17. Lobos SR, Mora GC. Alteration in the electrophoretic mobility of ompC due to variations in the ammonium persulfate concentration in sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Electrophoresis, 12:488-450, 1991
  18. Puente JL, Alvarez-Scherer V, Gosset G et al. Comparison analysis of the Salmonella typhi and Escherichia coli ompC genes, Gene, 83:197-206, 1989
  19. Puente JL, Flores V, Femandez M, et al. Isolation of an ompC-like outer membrane protein gene from Salmonella typhi. Gene, 156:1-9, 1995
  20. Park MK., Choi KS., Kim MC., et al. Differential diagnosis of Salmonella gallinarum and S. pullorum using PCR-RFLP. J Vet Sci, 2(3):213-219, 2001
  21. Sambrook J, Fritch EF, Molecular cloning. Although manual. 2nd ed, Cold Spring Harbor Laboratory Press. 1981;1.3-9.58.38. Bimboim HC, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res, 7:1513-1523, 1979
  22. Corpet F. Multiple sequence alignment with huerachical clustering. Nucl Acids Res, 16:10881-10890, 1988
  23. 김원용, 장영효, 박경윤 등. Polymerase chain reaction과 Southern hybridization을 이용한 Salmonella 속균의 신속한 검출. 대한수의학회지, 35:531-536, 1995
  24. 안종삼, 정석찬, 김종만, 등. Salmonella A 및 D group의 특이적 검색을 위한 rfbS 염기서열 분석에 기초한 PCR 기법의 적용. 대한위생학회지, 31:155-163, 1996
  25. 최경성, 박진호, 권오덕 등. 중합효소연쇄반응을 이용한 자돈에서 Salmonella typhimurium의 신속한 검출. 대한수의학회지, 38(4):763-770, 1998
  26. 박두희, 김원용, 김철중 등. Polymerase chain reaction에 의한 Salmonella 속균의 검출. 대한수의학회지, 34:115-125, 1994
  27. Cohen ND, Neibergs HL, Wallis DE, et al. Genusspecific detection of salmonellae in equine feces by use of the polymerase chain reaction. Am J Vet Res, 55:1049-1054, 1994
  28. Chen W, Martinez G, Mulchandani A. Malecular Beacons: A real-time polymerase chain reaction assay for detecting Salmonella. Anal Biochem, 280:166-172, 2000
  29. Bratin K, Saha. Baohong Tian, et al. Quantitation of HIV-1 by real-time PCR with a unique fluorogenic probe. J Virol Methods, 93:33-42, 2001
  30. Ma L, Bluyssen HA, De Raeymaeker M, et al. Rapid determination of adenoviral vector titers by quantitative real-time PCR. J Virol Methods, 93:181-188, 2001