Detection Of Escherichia.coli O157 By Multiplex PCR And Preparation Of Monoclonal Antibodies Against Vero Toxin

E.coli O157 is a one of the newly emerged foodborne human pathogens of animal origin and a leading cause of haemorrhagic uremic syndrome in humans. A sensitive test that can accurately and rapidly detect the organism in the food anima production enviroment is critically needed to monitor the emergence, transmission, and colonization of this pathogen in the animal reservoir. Vero toxin, produced by some E.coli, is the major factor of E.coli O157 associated with hemolytic uremic syndrome .In this study, a multiplex PCR system for rapid and sensitive identification of E.coli O157 was developed and two strains of monoclonal antibosies against VT2-B subunit were prepared, which allowd simultaneous identification of virulence factors and its production.A multiplex PCR was developed by using 5 sets of primers that identifies the gene sequences of E coli 16s rRNA, O157 antigen (rfbE), Vero toxin 1 (vt1), Vero toxin 2(vt2) and intimin(eaeA). Sensitivity of the assay was 10⁴CFU/ml of bacteria samples and 3-4 CFU/g of chicken meat that underwent enrichment Culturing for 8 hours. The method was then used for the examination of 64 E.coli isolates recovered from cattle, chikens, pigs and rabbits between 2000 and 2004. Among them, 10(15.6%) were rfbE positive. 8 E.coli isolates of the positive strains possessed the five genes, while the other two positive strains possessed four genes except vt1 gene. Moreover, 54 E.coli isolates only possessed the sequence of 16s rRNA. This multiplex PCR assay successfully identifed the Vero toxin-producing E. coli (VTEC) and their main virulence maker genes, and may be applied to rapid and sensitive detection of E.coli O157 in food when combined with an enrichement step.The segment of VT2-B subunit was amplified by PCR, then purified and digested with the endonucleases EcoR I and Xho I .The digested segment was inserted into the double-cleavaged expression vector pGEX_4T-2., and the recombinant expression vector named as pGEX_4T-2-VT2-B. The expression vector was transformed into E.coli BL21...