Multiplex PCR assays for the detection of Vibrio alginolyticus , Vibrio parahaemolyticus , Vibrio vulnificus , and Vibrio cholerae with an internal amplification control

• Published in: Diagnostic microbiology and infectious disease Vol. 79; no. 2; pp. 115 - 118
• Main Authors: Wei, Shuang, Zhao, Hui, Xian, Yuyin, Hussain, Malik A, Wu, Xiyang
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.06.2014
• Subjects: Bacterial Proteins - genetics; DNA Primers - genetics; Infectious Disease; Internal amplification control; Internal Medicine; Microbiological Techniques - methods; Microbiological Techniques - standards; Multiplex PCR; Multiplex Polymerase Chain Reaction - methods; Multiplex Polymerase Chain Reaction - standards; Reference Standards; Sensitivity and Specificity; Time Factors; Vibrio; Vibrio alginolyticus - classification; Vibrio alginolyticus - genetics; Vibrio alginolyticus - isolation & purification; Vibrio cholerae - classification; Vibrio cholerae - genetics; Vibrio cholerae - isolation & purification; Vibrio parahaemolyticus - classification; Vibrio parahaemolyticus - genetics; Vibrio parahaemolyticus - isolation & purification; Vibrio vulnificus - classification; Vibrio vulnificus - genetics; Vibrio vulnificus - isolation & purification; Multiplex PCR; Internal amplification control; Vibrio
• ISSN: 0732-8893; 1879-0070
• DOI: 10.1016/j.diagmicrobio.2014.03.012

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 105 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.