Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Application of the rpoS Gene for Species-Specific Detection of Vibrio vulnificus by Real-Time PCR

  • Kim, Dong-Gyun (Department of Biotechnology, Pukyong National University) ;
  • Ahn, Sun-Hee (Department of Biotechnology, Pukyong National University) ;
  • Kim, Lyoung-Hwa (Biotechnology Research Center, National Fisheries Research and Development Institute) ;
  • Park, Kee-Jai (Korea Food Research Institute) ;
  • Hong, Yong-Ki (Department of Biotechnology, Pukyong National University) ;
  • Kong, In-Soo (Department of Biotechnology, Pukyong National University)
  • Published : 2008.11.30
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Abstract

Vibrio vulnificus is a causative agent of serious diseases in humans, resulting from the contact of wound with seawater or consumption of raw seafood. Several studies aimed at detecting V. vulnificus have targeted vvh as a representative virulence toxin gene belonging to the bacterium. In this study, we targeted the rpoS gene, a general stress regulator, to detect V. vulnificus. PCR specificity was identified by amplification of 8 V. vulnificus templates and by the loss of a PCR product with 36 non-V. vulnificus strains. The PCR assay had the 273-bp fragment and the sensitivity of 10 pg DNA from V. vulnificus. SYBR Green I-based real-time PCR assay targeting the rpoS gene showed a melting temperature of approximately $84^{\circ}C$ for the V. vulnificus strains. The minimum level of detection by real-time PCR was 2 pg of purified genomic DNA, or $10^3$ V. vulnificus cells from pure cultured broth and $10^3$ cells in 1 g of oyster tissue homogenates. These data indicate that real-time PCR is a sensitive, species-specific, and rapid method for detecting this bacterium, using the rpoS gene in pure cultures and in infected oyster tissues.

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References

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