title: Automatic polymerase chain reaction product detection system for food safety monitoring using zinc finger protein fused to luciferase.
abstract: An automatic polymerase chain reaction (PCR) product detection system for food safety monitoring using zinc finger (ZF) protein fused to luciferase was developed. ZF protein fused to luciferase specifically binds to target double stranded DNA sequence and has luciferase enzymatic activity. Therefore, PCR products that comprise ZF protein recognition sequence can be detected by measuring the luciferase activity of the fusion protein. We previously reported that PCR products from Legionella pneumophila and Escherichia coli (E. coli) O157 genomic DNA were detected by Zif268, a natural ZF protein, fused to luciferase. In this study, Zif268-luciferase was applied to detect the presence of Salmonella and coliforms. Moreover, an artificial zinc finger protein (B2) fused to luciferase was constructed for a Norovirus detection system. In the luciferase activity detection assay, several bound/free separation process is required. Therefore, an analyzer that automatically performed the bound/free separation process was developed to detect PCR products using the ZF-luciferase fusion protein. By means of the automatic analyzer with ZF-luciferase fusion protein, target pathogenic genomes were specifically detected in the presence of other pathogenic genomes. Moreover, we succeeded in the detection of 10 copies of E. coli BL21 without extraction of genomic DNA by the automatic analyzer and E. coli was detected with a logarithmic dependency in the range of 1.0×10 to 1.0×10(6) copies.
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture & Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Yoshida
authors.forename: Aki
authors.initials: A
authors.lastname: Kezuka
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Murakami
authors.forename: Jinhee
authors.initials: J
authors.lastname: Lee
authors.forename: Koichi
authors.initials: K
authors.lastname: Abe
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Motoki
authors.forename: Takafumi
authors.initials: T
authors.lastname: Matsuo
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Shimura
authors.forename: Mamoru
authors.initials: M
authors.lastname: Noda
authors.forename: Shizunobu
authors.initials: S
authors.lastname: Igimi
authors.forename: Kazunori
authors.initials: K
authors.lastname: Ikebukuro
mesh_terms: D001331:Automation; D001426:Bacterial Proteins / Q000235:genetics*; D004279:DNA, Viral / Q000032:analysis / Q000302:isolation & purification; D019453:Escherichia coli O157 / Q000235:genetics; D059022:Food Safety / Q000379:methods*; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D029322:Norovirus / Q000235:genetics; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012475:Salmonella / Q000235:genetics; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D004279:DNA, Viral; D011993:Recombinant Fusion Proteins; D008156:Luciferases
keywords: Automatic analyzer; Luciferase; Pathogen detection; Polymerase chain reaction; Zinc finger protein