title: Multiplex PCR assays for the detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae with an internal amplification control.
abstract: A multiplex polymerase chain reaction (PCR) assay that can simultaneously detect 4 major Vibrio spp., Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae, in the presence of an internal amplification control (IAC) was developed. Species-specific PCR primers were designed based on the gyrB gene for V. alginolyticus, the collagenase gene for V. parahaemolyticus, the vvhA gene for V. vulnificus, and the ompW gene for V. cholerae. Additionally, an IAC primer pair was designed in conserved regions of the bacterial 16S rRNA gene that is used to indicate false-negative results. A multiplex PCR method was developed after optimization of the reaction conditions. The specificity of the PCR was validated by using 83 Vibrio strains and 10 other non-Vibrio bacterial species. The detection limit of the PCR was 10 CFU per tube for V. alginolyticus, V. parahaemolyticus, V. vulnificus, and 10(5) CFU per tube for V. cholerae in mixed conditions. This method was used to identify 69 suspicious Vibrio isolates, and the results were consistent with physiological and biochemical tests. This multiplex PCR method proved to be rapid, sensitive, and specific. The existence of IAC could successfully eliminate false-negative results for the detection of V. alginolyticus, V. parahaemolyticus, V. vulnificus, and V. cholerae.
authors.affiliation: Department of Food Science and Engineering, Jinan University, Guangzhou, China.
authors.forename: Shuang
authors.initials: S
authors.lastname: Wei
authors.affiliation: Hubei Entry-Exit Inspection and Quarantine Bureau, Inspection and Quarantine Technology Center, Wuhan, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Department of Food Science and Engineering, Jinan University, Guangzhou, China.
authors.forename: Yuyin
authors.initials: Y
authors.lastname: Xian
authors.affiliation: Department of Wine, Food and Molecular Biosciences, Lincoln University, Christchurch, New Zealand.
authors.forename: Malik A
authors.initials: MA
authors.lastname: Hussain
authors.affiliation: Department of Food Science and Engineering, Jinan University, Guangzhou, China; Food Safety and Health Emergency Technical Research Center of Guangdong Province, Guangzhou, China. Electronic address: tkentwu@jnu.edu.cn.
authors.forename: Xiyang
authors.initials: X
authors.lastname: Wu
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D008828:Microbiological Techniques / Q000379:methods* / Q000592:standards; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D012680:Sensitivity and Specificity; D013997:Time Factors; D044149:Vibrio alginolyticus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D014734:Vibrio cholerae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D014736:Vibrio parahaemolyticus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D041261:Vibrio vulnificus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers
keywords: Internal amplification control; Multiplex PCR; Vibrio