Journal of Microbiology

The Microbiological Society of Korea (한국미생물학회)
권호 표지

Rapid Identification of Vibrio vulnificus in Seawater by Real-Time Quantitative TaqMan PCR

  • Wang, Hye-Young (Mol-BioNet Research Institute, Scientec Lab Center Co., Ltd. College of Veterinary Medicine, Chonbuk National University) ;
  • Lee, Geon-Hyoung (Dept. of Biology, College of Natural Sciences, Kunsan National University)
  • Published : 2003.01.01
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Abstract

In order to identify Vibrio vulnificus in the Yellow Sea near Gunsan, Korea during the early and late summers, the efficiency of the real-time quantitative TaqMan PCR was compared to the efficiency of the conventional PCR and Biolog identification system^TM. Primers and a probe were designed from the hemolysin/cytolysin gene sequence of V. vulnificus strains. The number of positive detections by real-time quantitative TaqMan PCR, conventional PCR, and the Biolog identification system from seawater were 53 (36.8%), 36 (25%), and 10 strains (6.9%), respectively, among 144 samples collected from Yellow Sea near Gunsan, Korea. Thus, the detection method of the real-time quantitative TaqMan PCR assay was more effective in terms of accuracy than that of the conventional PCR and Biolog system. Therefore, our results showed that the real-time TaqMan probe and the primer set developed in this study can be applied successfully as a rapid screening tool for the detection of V. vulnificus.

Keywords

References

  1. Athena, C., D. Webster, and M.L. Christine. 2000. Cloning and characterization of vuuA, gene encoding the Vibrio vulnificus ferric vulnibactin receptor. Infect. Immun. 68, 526-534.
  2. Bassler, H.A., S.J. Flood, K.J. Livak, J. Marmaro, R. Knorr, and C.A. Batt. 1995. Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogens. Appl. Environ. Microbiol. 61, 3724-3728.
  3. Blake, P.A., M.H. Merson, R.E. Weaver, D.G. Hollis, and P.C. Heublein. 1979. Disease cause by a marine Vibrio: clinical characteristics and epidemiology. N. Eng. J. Med. 300, 1-5.
  4. Chen, S., A. Yee, M. Griffiths, C. Larkin, T. Yamashiro, R. Behari, C. Paszko-Kolva, Chuang, Y.C., T.M. Chang, and M.C. Chang. 1997. Cloning and characterization of the gene(empV) encoding extracellular metalloprotease from Vibrio vulnificus. Gene 189, 163-168.
  5. Covadonga, R.A., G. Esperanza, and A. Rosa. 1995. Nested PCR Method for Rapid and sensitive detection of Vibrio vulnificus in fish, sediments, and water. Appl. Environ. Microbiol. 61, 3476-3478.
  6. Heid, C.A., J. Stevens, K.J. Livak, and P.M. Williams. 1996. Real time quantitative PCR. Genome Res. 6, 986-994.
  7. Kalinina, O., I. Lebedeva, J. Brown, and J. Silver. 1997. Nanoliter scale PCR with TaqMan detection. Nucleic Acids Res. 25, 1999-2004.
  8. Keya, S. 2000. Rapid identification of Yersinia enterocolitica in blood by the 5' nuclease PCR assay. J. Clin. Microbiol. 38, 1953-1958.
  9. Kim, M.S. and H.D. Jeong. 2001. Development of 16S rRNA targeted PCR methods for the detection and differentiation of Vibrio vulnificus in marine environments. Aquaculture 193, 199-211.
  10. Lee, S.E., S.Y. Kim, S.J. Kim, H.Y. Kim, J.H. Shin, S.H. Choi, S.S. Chung, and J.H. Rhee. 1998. Direct identification of Vibrio vulnificus in clinical specimens by nested PCR. J. Clin. Microbiol. 36, 2887-2892.
  11. Lee, S.E., S.H. Shin, S.Y. Kim, Y.R. Kim, D.H. Shin, S.S. Chung, Z.H. Lee, J.Y. Lee, K.C. Jeong, S.H. Choi, and J.H. Rhee. 2000. Vibrio vulnificus has the transmembrane transcription activator ToxRS stimulating the expression of the hemolysin gene vvhA. J. Bacteriol. 182, 3405-3415.
  12. Livak, K.J., S.J. Flood, J. Marmaro, W. Giusti, and K. Deetz. 1995. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl. 4, 357-362.
  13. Lyon, W.J. 2001. TaqMan PCR for Detection of Vibrio cholerae O1, O139, non-O1, and non-O139 in pure cultures, raw oysters, and synthetic seawater. Appl. Environ. Microbiol. 67, 4685-4693.
  14. McLaughlin, J.C. 1995. Vibrio. p. 465-476. In Murrary, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C., and Yolken, R.H. (eds), Manual of clinical microbiology, 6th ed. American Society for Microbiology, Washington, D.C.
  15. Nogva, H.K., K. Rudi, K. Naterstad, A. Holck, and D. Lillehaug. 2000. Application of 5'-nuclease PCR assay for quantitative detection of Listeria monocytogenes in pure cultures, water, skim milk, and unpasteurized whole milk. Appl. Environ. Microbiol. 66, 4266-4271.
  16. Norton, D.M. and C.A. Batt. 1999. Detection of viable Listeria monocytogens with a 5'-nuclease PCR assay. Appl. Environ. Microbiol. 65, 2122-2127.
  17. Paik, K.W., B. Moon, C.W. Park, K.T. Kim, M.S. Ji, S.K. Choi, J.S. Rew, and C.M. Yoon. 1995. Clinical characteristics of ninetytwo cases of Vibrio vulnificus infections. Kor. J. Infect. Dis. 27, 355-365.
  18. Park, S.D., H.S. Shon, and N.J. Joh. 1991. Vibrio vulnificus septicemia in Korea: clinical and epidemiologic findings in seventy patients. J. Am. Acad. Dermatol. 24, 397-403.
  19. Rahn, K. and S.A. De Grandis. 1997. The evaluation of fluorogenic polymerase chain reaction assay for the detection of Salmonella species in food commodities. Int. Food Microbiol. 35, 239-250.
  20. Shirley, S.C., D.M. Melanson, E.G. Biosca, and J.D. Oliver. 1996. Detection of Vibrio vulnificus biotypes 1 and 2 in eels and oysters by PCR amplification. Appl. Environ. Microbiol. 62, 1378-1382.
  21. Tison, D.L. and M.T. Kelly. 1984. Vibrio species of medically importance. Diag. Microbiol. Infect. Dis. 2, 263-276.
  22. Vishnubhatla, A.D., Y.C. Fung, R.D. Oberst, M.P. Hays, T.G. Nagaraja, and S.J.A. Flood. 2000. Rapid 5' nuclease(TaqMan) assay for detection of virulent strains of Yersinia enterocolotica. Appl. Environ. Microbiol. 66, 4131-4135.