Development the rapid and simple methods for Escherichia coli 0-157 detection in foods
1997
Sudsai Trevanich (Kyushu Univ., Fukuoka (Japan). Faculty of Agriculture. Dept. of Food Science)
Escherichia coli 0-157 (E. coli 0-157) was identified as a human pathogen and cause of hemorrhagic colitis and hemolytic uremic syndrome. Food products contaminated with E. coli 0-157 have commonly been identified as the source of infection. Therefore, the development of reagents and methods to detect E. coli 0-157 in food products and in clinical medicine specimens has recently been the focus of much research. While several methods have been developed to detect and isolate E. coli 0-157, there is currently no simple rapid, and accurate technique suitable for routine use by clinical medicine or food microbiology laboratories. A major limiting factor for detection has been the lack of high-quality immunodiagnostic reagents. The Recombinant Phage Antibody System (RPAS) is relatively usefulness system that offers a simple, quick, and economical production of recombinant antibody. This approach relies upon a phage-display system in which fragments of antibodies are expressed as fusion proteins displayed on the phage surface or soluble antibody fragments. Using the Polymerase Chain Reaction (PCR), the VH (variable heavy) and VL (variable light) genes are separately amplified from mouse spleen Blymphocyte mRNA, assembled into a single chain Fv fragment (ScFv) with a short linker DNA, (Gly)4(Ser)3. ScFv fragment digested with SfiI and NotI was cloned into a phagemid vector (pCANTAB 5E) and transformed into E. coli 0-157 TG1. The recombinant antibody is displayed at the tip of the helper phage (M13K07) as a fusion protein with the phage gene 3 protein (g3p). Antigen-positive phage are then selected based on their affinity for the antigen of interest. The antigen-positive phage are used to infect E. coli 0-157 HB 2151 cells for soluble antibody production. The resulting recombinant antibody can be used for many applications.
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