title: A pentaplex PCR assay for detection and characterization of Vibrio vulnificus and Vibrio parahaemolyticus isolates.
abstract: Vibrio parahaemolyticus and Vibrio vulnificus are the leading causes of seafood-related illnesses and also can cause wound infections. These bacteria often co-exist in marine and estuarine environments. However, there have been no reported protocols that can detect and characterize (i.e. pathogenic or nonpathogenic) them in a single PCR. In this study, we developed a pPCR assay with a combination of two species-specific and three pathogenic-specific PCR primers to simultaneously detect virulent (viuB in V. vulnificus and tdh/trh in V. parahaemolyticus) and nonvirulent (vvhA in V. vulnificus and tlh in V. parahaemolyticus) markers of the two species in bacterial isolates. The assay was validated by three methods. First, the pPCR was used to characterize 300 bacterial isolates consisting of seven reference strains and 293 environmental strains isolated from the Gulf of Mexico water. Results were compared with characterizations based on single-gene PCR amplifications and previously published multiplex PCR protocols. Second, 51 isolates characterized with the pPCR were analysed by 16S rRNA sequencing to confirm any false-negative/positive reaction. Finally, the effectiveness of the assay for heterogeneous bacterial samples was validated. The pPCR correctly characterized isolates from the Gulf with an efficiency of 96·6-98·7%.
authors.affiliation: Department of Environmental Sciences, School of the Coast and Environment, Louisiana State University, Baton Rouge, LA 70803, USA.
authors.forename: N
authors.initials: N
authors.lastname: Bhattacharyya
authors.forename: A
authors.initials: A
authors.lastname: Hou
mesh_terms: D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004784:Environmental Monitoring / Q000379:methods*; D005819:Genetic Markers; D060851:Gulf of America; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014736:Vibrio parahaemolyticus / Q000235:genetics / Q000302:isolation & purification*; D041261:Vibrio vulnificus / Q000235:genetics / Q000302:isolation & purification*; D014871:Water Microbiology
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D005819:Genetic Markers; D012336:RNA, Ribosomal, 16S
keywords: bacterial isolates; pathogenic vibrios; pentaplex PCR; seafood; simultaneous detection