Sunday, July 26, 2015
Exhibit Hall (Oregon Convention Center)
Introduction: There is an increasing call by authorities for regulation of pathogenic E. coli O157:H7 and non-O157 “Big 6” E. coli in foods, particularly beef products. Real-time PCR represents a powerful tool for specific and rapid detection of foodborne pathogens with time to results of less than 24 hours, particularly desirable for products with short time-to-market requirements.
Purpose: The purpose of the current study was to develop a fully automated method for the purification and real-time PCR detection 1) of E. coli virulence factors O157, stx1/stx2, and eae as a rapid screening assay for the virulence factors and 2) E. coli O157:H7 and non-O157 O-serotypes in a further screen for pathogenic E. coli O-serotypes.
Methods: E. coli DNA was purified from enrichment cultures of raw beef trim, ground beef and spinach. Cultures were performed for 6 - 18 hours in mTSB at 41°C. A one-for-all fully automated purification method developed for Salmonella spp. and Listeria spp. was extended to E. coli using mericon E. coli O157 Screen Plus and mericon E. coli STEC O-type pathogen detection assays.
Results: Time course studies were performed for each matrix type to determine the enrichment time required. E. coli was detected for all 3 matrices at low level within 8 h. Limit of detection studies demonstrated that these new assays are equivalent to the standard reference methods in their ability to detect E. coli virulence factors and STEC O-types. The inclusivity and exclusivity of the two assays are as expected. A scheme for E. coli virulence factor screening with subsequent STEC O-type screen using these assays is presented.
Significance: The fully automated QIAsymphony Rotor-Gene Q provides a powerful, rapid, reproducible, and sensitive method for the detection of E. coli virulence factors and STEC O-type in beef products and spinach.
Purpose: The purpose of the current study was to develop a fully automated method for the purification and real-time PCR detection 1) of E. coli virulence factors O157, stx1/stx2, and eae as a rapid screening assay for the virulence factors and 2) E. coli O157:H7 and non-O157 O-serotypes in a further screen for pathogenic E. coli O-serotypes.
Methods: E. coli DNA was purified from enrichment cultures of raw beef trim, ground beef and spinach. Cultures were performed for 6 - 18 hours in mTSB at 41°C. A one-for-all fully automated purification method developed for Salmonella spp. and Listeria spp. was extended to E. coli using mericon E. coli O157 Screen Plus and mericon E. coli STEC O-type pathogen detection assays.
Results: Time course studies were performed for each matrix type to determine the enrichment time required. E. coli was detected for all 3 matrices at low level within 8 h. Limit of detection studies demonstrated that these new assays are equivalent to the standard reference methods in their ability to detect E. coli virulence factors and STEC O-types. The inclusivity and exclusivity of the two assays are as expected. A scheme for E. coli virulence factor screening with subsequent STEC O-type screen using these assays is presented.
Significance: The fully automated QIAsymphony Rotor-Gene Q provides a powerful, rapid, reproducible, and sensitive method for the detection of E. coli virulence factors and STEC O-type in beef products and spinach.