<OL> <LI> To develop procedures for food sample processing. <LI> To develop a sensitive and real-time RAM assay for detecting E. coli O157 :H7 and other Shiga toxin-producing E. coli (STEC). <LI> To evaluate the RAM system for detecting E. coli O157:H7 & other STEC in laboratory .
NON-TECHNICAL SUMMARY: Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) have emerged to be significant foodborne pathogenic microorganism. The microorganism is responsible for outbreaks of hemorrhagic diarrhea associated with consumption of undercooked ground beef and hemolytic uremic syndrome with a death rate as high as 1%. A number of methods have been developed for detecting microorganism in food including culture isolation, serological tests and polymerase chain reaction (PCR) assays. These methods lack sensitivity or specificity or require long period of time to detect the organism. We have recently developed a novel isothermal DNA amplification technology, ramification amplification (RAM) which amplifies minute amount of DNA target from a few pathogenic microorganisms to billion folds in a short time, therefore, increasing detection sensitivity. In this proposal we plan to develop an assay by combining rapid bacterial isolation from food without culture enrichment, magnetic bead-based target DNA capturing, isothermal DNA amplification and real-time fluorescence detection. One of the significant advantages of this technology is that the entire assay, including amplification, can be carried out at a single temperature in a closed test tube, unlike PCR that requires thermal cycling. Therefore, the assay can be performed in any environment at ambient temperature. The objective of the study is to develop a simple, rapid and sensitive assay to be used in field testing for detecting E. coli O157:H7 and other STEC in food without cultural enrichment in less than 3 hours.
APPROACH: Using ground beef as a model system, we developed a procedure that enabled a PCR and RAM to detect E. coli O157:H7 at 10 cells per gram of ground beef without enrichment We will modify the sample processing procedure for use in other food Systems to eliminate the requirement of culture enrichment. Genes encoding Shiga toxins 1 (stxl) and 2 (stx2), and O157-antigen biosynthesis (rfb) will be used as targets for the RAM assay to identify E. coli O157:H7 and other STEC. The molecular beacon will be designed based on the gene sequence and labeled with fluorescents and quenchers to develop a real time RAM assay. The assays will be evaluated in our laboratories by using a number of STEC serotypes to test their sensitivity and specificity.