Species-specific PCR detection of the food-borne pathogen Vibrio parahaemolyticus using the irgB gene identified by comparative genomic analysis.
Vibrio parahaemolyticus is an enteric pathogen, which can cause acute gastroenteritis in humans after consumption of raw or partially cooked seafood, and specific molecular markers are necessary for its accurate identification by PCR methods. In the present study, 23 protein-coding sequences were identified by the comparative genomics method as V. parahaemolyticus-specific candidate markers. We targeted the irgB gene (vp2603), coding for iron-regulated virulence regulatory protein IrgB, in order to develop a PCR method for the detection of V. parahaemolyticus. PCR specificity was identified by amplification of 293 V. parahaemolyticus templates and by the loss of a PCR product with 11 strains from other Vibrio species and 35 non-Vibrio bacterial strains. The PCR assay had the 369-bp fragment and the sensitivity of 0.17 pg purified genomic DNA from V. parahaemolyticus. Furthermore, a multiplex PCR assay for the detection of total and virulent strains of V. parahaemolyticus was developed by targeting irgB, tdh and trh genes. These data indicated that the irgB gene is a new and effective marker for the detection of V. parahaemolyticus. In addition, this study demonstrates that genome sequence comparison has a powerful application in identifying specific markers for the detection and identification of bacterial pathogens.
Yu
Shuijing
S
Department of Food Science and Technology, Joint Sino-US Food Safety Research Center & Bor Luh Food Safety Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China.
Chen
Wanyi
W
Wang
Dapeng
D
He
Xiaohua
X
Zhu
Xinna
X
Shi
Xianming
X
D001426:Bacterial Proteins / Q000235:genetics*; D001431:Bacteriological Techniques / Q000379:methods*; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005516:Food Microbiology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014736:Vibrio parahaemolyticus / Q000235:genetics / Q000302:isolation & purification*
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial