Rapid and sensitive detection method of a bacterium by using a GFP reporter phage.
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.
Funatsu
Takashi
T
Department of Physics, School of Science and Engineering, Waseda University, Tokyo, Japan. funatsu@waseda.jp
Taniyama
Tadayoshi
T
Tajima
Takashi
T
Tadakuma
Hisashi
H
Namiki
Hideo
H
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
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins