title: Rapid and sensitive detection method of a bacterium by using a GFP reporter phage.
abstract: A rapid, sensitive, and convenient method for detecting a specific bacterium was developed by using a GFP phage. Here we describe a model system that utilizes the temperate Escherichia coli-restricted bacteriophage lambda, which was genetically modified to express a reporter gene for GFP to identify the colon bacillus E. coli in the specimen. E. coli infected with GFP phage was detected by GFP fluorescence after 4-6 hr of incubation. The results show that a few bacteria in a specimen can be detected under fluorescence microscopy equipped with a sensitive cooled CCD camera. When E. coli and Mycobacterium smegmatis were mixed in a solution containing GFP phage, only E. coli was infected, indicating the specificity of this method. The method has the following advantages: 1) Bacteria from biological samples need not be purified unless they contain fluorescent impurities; 2) The infection of GFP phage to bacteria is specific; 3) The fluorescence of GFP within infected bacteria enables highly sensitive detection; 4) Exogenous substrates and cofactors are not required for fluorescence. Therefore this method is suitable for any phage-bacterium system when bacteria-specific phages are available.
authors.affiliation: Department of Physics, School of Science and Engineering, Waseda University, Tokyo, Japan. funatsu@waseda.jp
authors.forename: Takashi
authors.initials: T
authors.lastname: Funatsu
authors.forename: Tadayoshi
authors.initials: T
authors.lastname: Taniyama
authors.forename: Takashi
authors.initials: T
authors.lastname: Tajima
authors.forename: Hisashi
authors.initials: H
authors.lastname: Tadakuma
authors.forename: Hideo
authors.initials: H
authors.lastname: Namiki
mesh_terms: D010582:Bacteriophage lambda / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000302:isolation & purification*; D004927:Escherichia coli Infections / Q000382:microbiology*; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics*; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D012680:Sensitivity and Specificity
chemical_list: D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins