title: Application of tetraplex PCR for detection of Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus in cockle.
abstract: A tetraplex PCR method was developed for simultaneous detection of Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus in cockle samples in comparison with conventional culture method. Specific primers targeting ompW of V. cholerae, tl of V. parahaemolyticus, hsp60 of V. vulnificus and sodB of V. mimicus were employed in the same PCR. Detection limit of the tetraplex PCR assay was 104 cfu/ml (400 cfu/PCR reaction) for pure cultures of all four species of Vibrio. In Vibrio spiked cockle samples, the limit of detection after 6 hours enrichment in alkaline peptone water was 1 cfu/10 g of cockle tissue for three Vibrio spp, except for V. mimicus that was 102 cfu/10 g of cockle tissue. When the tetraplex PCR and culture methods were applied to 100 cockle samples, V. parahaemolyticus, V. vulnificus, V. cholerae and V. mimicus were detected in 100, 98, 80 and 9% of the samples by tetraplex PCR and in 76, 42, 0 and 0% by the culture method, respectively. This developed tetraplex PCR method should be suitable for simultaneous and rapid detection of Vibrio species in food samples and for food safety assessment.
authors.affiliation: Department of Microbiology and Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Pachara
authors.initials: P
authors.lastname: Senachai
authors.forename: Chariya
authors.initials: C
authors.lastname: Chomvarin
authors.forename: Wises
authors.initials: W
authors.lastname: Namwat
authors.forename: Warawan
authors.initials: W
authors.lastname: Wongboot
authors.forename: Suwin
authors.initials: S
authors.lastname: Wongwajana
authors.forename: Waraluk
authors.initials: W
authors.lastname: Tangkanakul
mesh_terms: D000818:Animals; D049873:Cardiidae / Q000382:microbiology*; D005798:Genes, Bacterial; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014733:Vibrio / Q000235:genetics* / Q000302:isolation & purification; D014734:Vibrio cholerae / Q000302:isolation & purification; D044164:Vibrio mimicus / Q000302:isolation & purification; D014736:Vibrio parahaemolyticus / Q000302:isolation & purification; D041261:Vibrio vulnificus / Q000302:isolation & purification