title: Development of a quantitative real-time polymerase chain reaction targeted to the toxR for detection of Vibrio vulnificus.
abstract: The TaqMan assay, a quantitative real-time polymerase chain reaction (PCR), was developed to target the ToxR gene (toxR) of Vibrio vulnificus. The toxR of V. vulnificus was cloned and sequenced. Based on these results, we designed specific primers and a probe for use in the quantitative PCR assay. Twenty-nine strains of V. vulnificus that were obtained from various sources produced a single PCR product. The amount of final amplification product and threshold cycle number were the same among the strains. We used the method to detect V. vulnificus in seawater and oyster samples. We developed standard curves to quantitate V. vulnificus numbers using the PCR from seawater and oyster samples. The standard curves were not different from that of the pure culture of V. vulnificus. We found the assay was very sensitive detecting as few as 10 microbes per milliliter of seawater and oyster homogenate. Moreover, we evaluated the TaqMan assay to detect V. vulnificus in seawater samples. The numbers of V. vulnificus counted by the TaqMan assay were similar to those by a culture method in almost samples. The TaqMan assay was performed within 2 h compared to days using the culture method. The results indicate the TaqMan assay method used in this study was rapid, effective and quantitative for monitoring V. vulnificus contamination in seawater and seafoods such as oysters.
authors.affiliation: Division of Microbiology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
authors.forename: Hajime
authors.initials: H
authors.lastname: Takahashi
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Hara-Kudo
authors.forename: Jiro
authors.initials: J
authors.lastname: Miyasaka
authors.forename: Susumu
authors.initials: S
authors.lastname: Kumagai
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Konuma
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D005516:Food Microbiology; D008969:Molecular Sequence Data; D010125:Ostreidae / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012623:Seawater / Q000382:microbiology; D019914:Taq Polymerase; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics*; D041261:Vibrio vulnificus / Q000235:genetics / Q000302:isolation & purification*; D014871:Water Microbiology
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D014157:Transcription Factors; C066719:toxR protein, bacteria; D019914:Taq Polymerase