Rapid detection of Escherichia coli O157:H7 by using green fluorescent protein-labeled PP01 bacteriophage.
A previously isolated T-even-type PP01 bacteriophage was used to detect its host cell, Escherichia coli O157:H7. The phage small outer capsid (SOC) protein was used as a platform to present a marker protein, green fluorescent protein (GFP), on the phage capsid. The DNA fragment around soc was amplified by PCR and sequenced. The gene alignment of soc and its upstream region was g56-soc.2-soc.1-soc, which is the same as that for T2 phage. GFP was introduced into the C- and N-terminal regions of SOC to produce recombinant phages PP01-GFP/SOC and PP01-SOC/GFP, respectively. Fusion of GFP to SOC did not change the host range of PP01. On the contrary, the binding affinity of the recombinant phages to the host cell increased. However, the stability of the recombinant phages in alkaline solution decreased. Adsorption of the GFP-labeled PP01 phages to the E. coli cell surface enabled visualization of cells under a fluorescence microscope. GFP-labeled PP01 phage was not only adsorbed on culturable E. coli cells but also on viable but nonculturable or pasteurized cells. The coexistence of insensitive E. coli K-12 (W3110) cells did not influence the specificity and affinity of GFP-labeled PP01 adsorption on E. coli O157:H7. After a 10-min incubation with GFP-labeled PP01 phage at a multiplicity of infection of 1,000 at 4 degrees C, E. coli O157:H7 cells could be visualized by fluorescence microscopy. The GFP-labeled PP01 phage could be a rapid and sensitive tool for E. coli O157:H7 detection.
Oda
Masahito
M
Department of Bioengineering, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan.
Morita
Masatomo
M
Unno
Hajime
H
Tanji
Yasunori
Y
D000818:Animals; D001483:Base Sequence; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003090:Coliphages / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D019453:Escherichia coli O157 / Q000302:isolation & purification* / Q000821:virology; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013552:Swine / Q000821:virology; D013997:Time Factors
D036022:Capsid Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins