Application of the rpoS gene for species-specific detection of Vibrio vulnificus by real-time PCR.
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.
Kim
Dong-Gyun
DG
Department of Biotechnology, Pukyong National University, Busan 608-737, Korea.
Ahn
Sun-Hee
SH
Kim
Lyoung-Hwa
LH
Park
Kee-Jai
KJ
Hong
Yong-Ki
YK
Kong
In-Soo
IS
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
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