- Volume 41 Issue 4
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
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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Heid, C.A., J. Stevens, K.J. Livak, and P.M. Williams. 1996. Real time quantitative PCR. Genome Res. 6, 986-994.
- Kalinina, O., I. Lebedeva, J. Brown, and J. Silver. 1997. Nanoliter scale PCR with TaqMan detection. Nucleic Acids Res. 25, 1999-2004.
- Keya, S. 2000. Rapid identification of Yersinia enterocolitica in blood by the 5' nuclease PCR assay. J. Clin. Microbiol. 38, 1953-1958.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Tison, D.L. and M.T. Kelly. 1984. Vibrio species of medically importance. Diag. Microbiol. Infect. Dis. 2, 263-276.
- 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.