title: Application of the rpoS gene for species-specific detection of Vibrio vulnificus by real-time PCR.
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 degrees for the V. vulnificus strains. The minimum level of detection by real-time PCR was 2 pg of purified genomic DNA, or 103 V. vulnificus cells from pure cultured broth and 103 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.
authors.affiliation: Department of Biotechnology, Pukyong National University, Busan 608-737, Korea.
authors.forename: Dong-Gyun
authors.initials: DG
authors.lastname: Kim
authors.forename: Sun-Hee
authors.initials: SH
authors.lastname: Ahn
authors.forename: Lyoung-Hwa
authors.initials: LH
authors.lastname: Kim
authors.forename: Kee-Jai
authors.initials: KJ
authors.lastname: Park
authors.forename: Yong-Ki
authors.initials: YK
authors.lastname: Hong
authors.forename: In-Soo
authors.initials: IS
authors.lastname: Kong
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D052160:Benzothiazoles; D003470:Culture Media; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D003959:Diamines; D006801:Humans; D008969:Molecular Sequence Data; D009930:Organic Chemicals; D010125:Ostreidae / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012680:Sensitivity and Specificity; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics*; D013045:Species Specificity; D041261:Vibrio vulnificus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D052160:Benzothiazoles; D003470:Culture Media; D017931:DNA Primers; D004269:DNA, Bacterial; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012808:Sigma Factor; C069567:sigma factor KatF protein, Bacteria; C098022:SYBR Green I