title: Multiplex real-time PCR assay for detection of pathogenic Vibrio parahaemolyticus strains.
abstract: Foodborne disease caused by pathogenic Vibrio parahaemolyticus has become a serious public health problem in many countries. Rapid diagnosis and the identification of pathogenic V. parahaemolyticus are very important in the context of public health. In this study, an EvaGreen-based multiplex real-time PCR assay was established for the detection of pathogenic V. parahaemolyticus. This assay targeted three genetic markers of V. parahaemolyticus (species-specific gene toxR and virulence genes tdh and trh). The assay could unambiguously identify pathogenic V. parahaemolyticus with a minimum detection limit of 1.4 pg genomic DNA per reaction (concentration giving a positive multiplex real-time PCR result in 95% of samples). The specificity of the assay was evaluated using 72 strains of V. parahaemolyticus and other bacteria. A validation of the assay with clinical samples confirmed its sensitivity and specificity. Our data suggest the newly established multiplex real-time PCR assay is practical, cost-effective, specific, sensitive and capable of high-throughput detection of pathogenic V. parahaemolyticus.
authors.affiliation: Jiaxing Center for Disease Control and Prevention, No. 486, Wen Qiao Road, Jiaxing, Zhejiang 314050, China.
authors.forename: Peiyan
authors.initials: P
authors.lastname: He
authors.affiliation: Jiaxing Center for Disease Control and Prevention, No. 486, Wen Qiao Road, Jiaxing, Zhejiang 314050, China. Electronic address: czw@jxcdc.gov.cn.
authors.forename: Zhongwen
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Jiaxing Center for Disease Control and Prevention, No. 486, Wen Qiao Road, Jiaxing, Zhejiang 314050, China.
authors.forename: Jianyong
authors.initials: J
authors.lastname: Luo
authors.affiliation: Jiaxing Center for Disease Control and Prevention, No. 486, Wen Qiao Road, Jiaxing, Zhejiang 314050, China.
authors.forename: Henghui
authors.initials: H
authors.lastname: Wang
authors.affiliation: Jiaxing Center for Disease Control and Prevention, No. 486, Wen Qiao Road, Jiaxing, Zhejiang 314050, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Yan
authors.affiliation: Jiaxing Center for Disease Control and Prevention, No. 486, Wen Qiao Road, Jiaxing, Zhejiang 314050, China.
authors.forename: Lixia
authors.initials: L
authors.lastname: Chen
authors.affiliation: Jiaxing Center for Disease Control and Prevention, No. 486, Wen Qiao Road, Jiaxing, Zhejiang 314050, China.
authors.forename: Wenjie
authors.initials: W
authors.lastname: Gao
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D017931:DNA Primers / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014157:Transcription Factors / Q000235:genetics; D014735:Vibrio Infections / Q000175:diagnosis* / Q000821:virology; D014736:Vibrio parahaemolyticus / Q000145:classification / Q000235:genetics* / Q000472:pathogenicity; D014774:Virulence / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D014157:Transcription Factors; C066719:toxR protein, bacteria
keywords: Multiplex real-time PCR; Vibrio parahaemolyticus; tdh; toxR; trh