β-Lactamase (VPA0477) 유전자를 표적으로 Polymerase chain reaction에 의한 장염비브리오(Vibrio parahaemolyticus)의 검출

Application of the β-Lactamase (VPA0477) Gene for the Detection of Vibrio parahaemolyticus by Polymerase Chain Reaction

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  • ABSTRACT

    In this study, the β-lactamase (VPA0477) gene was used as a new target for the PCR-based detection of Vibrio parahaemolyticus. Primers specific for the β-lactamase (VPA0477) gene of V. parahaemolyticus, were designed and incorporated into a PCR-based assay. The assay was able to specifically detect all of the 191 V. parahaemolyticus strains tested, but did not result in amplification of 39 other Vibrio spp. and non-Vibrio spp. strains tested. The detection limit of the assay was 10 CFU of V. parahaemolyticus RIMD2210633 from pure culture broth. The β-lactamase (VPA0477) gene-based assay developed in this study was sensitive and specific, and has great potential for the accurate detection and identification of V. parahaemolyticus in seawater or seafood samples.


  • KEYWORD

    Vibrio parahaemolyticus , β-lactamase , Polymerase chain reaction , Detection

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  • [Table 1.] Bacteria strains used in this study
    Bacteria strains used in this study
  • [Fig. 1.] Nucleotide sequence alignment of the VPA0477 gene of V. parahaemolyticus, Vibrio sp. EX25, Vibrio sp. EJY3, V. harveyi, V. harveyi ATCC BBA-1116, V. campbellii ATCC BAA-1116, and V. alginolyticus NBRC15630. Nucleotides identical to those of V. parahaemolyticus are indicated with dots. Primer sets (VPA0477-F and VPA0477-R) are indicated by the arrows.
    Nucleotide sequence alignment of the VPA0477 gene of V. parahaemolyticus, Vibrio sp. EX25, Vibrio sp. EJY3, V. harveyi, V. harveyi ATCC BBA-1116, V. campbellii ATCC BAA-1116, and V. alginolyticus NBRC15630. Nucleotides identical to those of V. parahaemolyticus are indicated with dots. Primer sets (VPA0477-F and VPA0477-R) are indicated by the arrows.
  • [Fig. 2.] Agarose gel electrophoresis of V. parahaemolyticus-specific DNA products amplified in PCR using primer set VPA0477-F and VPA0477-R.
    Agarose gel electrophoresis of V. parahaemolyticus-specific DNA products amplified in PCR using primer set VPA0477-F and VPA0477-R.
  • [Fig. 3.] Agarose gel electrophoresis of Vibrio spp. and non-Vibrio spp. DNA products amplified in PCR using primer set VPA0477-F and VPA0477-R. M, 100 bp DNA ladder; lane 1, V. aerogene; lane 2, V. aestuarianus; lane 3, V. alginolyticus; lane 4, V. anguillarum; lane 5, V. campbellii; lane 6, V. carchariae; lane 7, V. cholerae; lane 8, V. diazotrophicus; lane 9, V. fischeri; lane 10, V. fluvialis; lane 11, V. furnissii; lane 12, V. gazogenes; lane 13, V. halioticoli; lane 14, V. harveyi; lane 15, V. hollisae; lane 16, V. mimicus; lane 17, V. natriegens; lane 18, V. nigripulchritudo; lane 19, V. ordalii; lane 20, V. orientalis; lane 21, V. parahaemolyticus; lane 22, V. pectenicida; lane 23, V. pelagius; lane 24, V. proteolyticus; lane 25, V. rumoiensis; lane 26, V. splendidus; lane 27, V. tubiashii; lane 28, V. vulnificus; lane 29, Aeromonas hydrophila; lane 30, Bacillus cereus; lane 31, Citrobacter freundii; lane 32, Enterobacter aerogenes; lane 33, Escherichia coli; lane 34, Listeria monocytogenes; lane 35, Photobacterium damselae; lane 36, Salmonella choleraesuis; lane 37, Salmonella typhimurium; lane 38, Shigella sonnei; lane 39, Staphylococcus aureus; lane 40, Yersinia enterocolitica.
    Agarose gel electrophoresis of Vibrio spp. and non-Vibrio spp. DNA products amplified in PCR using primer set VPA0477-F and VPA0477-R. M, 100 bp DNA ladder; lane 1, V. aerogene; lane 2, V. aestuarianus; lane 3, V. alginolyticus; lane 4, V. anguillarum; lane 5, V. campbellii; lane 6, V. carchariae; lane 7, V. cholerae; lane 8, V. diazotrophicus; lane 9, V. fischeri; lane 10, V. fluvialis; lane 11, V. furnissii; lane 12, V. gazogenes; lane 13, V. halioticoli; lane 14, V. harveyi; lane 15, V. hollisae; lane 16, V. mimicus; lane 17, V. natriegens; lane 18, V. nigripulchritudo; lane 19, V. ordalii; lane 20, V. orientalis; lane 21, V. parahaemolyticus; lane 22, V. pectenicida; lane 23, V. pelagius; lane 24, V. proteolyticus; lane 25, V. rumoiensis; lane 26, V. splendidus; lane 27, V. tubiashii; lane 28, V. vulnificus; lane 29, Aeromonas hydrophila; lane 30, Bacillus cereus; lane 31, Citrobacter freundii; lane 32, Enterobacter aerogenes; lane 33, Escherichia coli; lane 34, Listeria monocytogenes; lane 35, Photobacterium damselae; lane 36, Salmonella choleraesuis; lane 37, Salmonella typhimurium; lane 38, Shigella sonnei; lane 39, Staphylococcus aureus; lane 40, Yersinia enterocolitica.
  • [Fig. 4.] Agarose gel electrophoresis of DNA products amplified from V. parahaemolyticus RIMD2210633 in PCR assay using VPA0477 primers. M, 100 bp ladder marker; lane 1, 1.0×103 CFU; lane 2, 1.0×102 CFU; lane 3, 1.0×101 CFU; lane 4, 1.0×100 CFU.
    Agarose gel electrophoresis of DNA products amplified from V. parahaemolyticus RIMD2210633 in PCR assay using VPA0477 primers. M, 100 bp ladder marker; lane 1, 1.0×103 CFU; lane 2, 1.0×102 CFU; lane 3, 1.0×101 CFU; lane 4, 1.0×100 CFU.