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Title: EVALUATION OF A REAL-TIME PCR KIT FOR DETECTING ESCHERICHIA COLI O157 IN BOVINE FECAL SAMPLES

Author
item Bono, James - Jim
item Keen, James
item Miller, Laura
item Fox, James
item Chitko-Mckown, Carol
item Heaton, Michael - Mike
item Laegreid, William

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/9/2003
Publication Date: 3/1/2004
Citation: BONO, J.L., KEEN, J.E., MILLER, L.C., FOX, J.M., CHITKO MCKOWN, C.G., HEATON, M.P., LAEGREID, W.W. EVALUATION OF A REAL-TIME PCR KIT FOR DETECTING ESCHERICHIA COLI O157 IN BOVINE FECAL SAMPLES. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. 2004. V. 70. P. 1855-1857.

Interpretive Summary: In the literature there are many reports of detection assays using real-time PCR for detection of organisms. This is a sensitive method and is capable of detecting less than 10 bacteria per reaction. We evaluated the Ruggedized Advanced Pathogen Identification Device (R.A.P.I.D.) System Escherichia coli O157 real-time PCR detection kit produced by Idaho Technology, Inc. for its ability to detect E. coli O157. The kit had a 99% sensitivity and specificity when using DNA from isolates in pure culture. Using bovine feces inoculated with E. coli O157, the kit can quantify the number of bacteria in the feces. The kit was also able to detect E. coli O157 from feces of naturally infected cattle. However, a drawback to the kit was its inability to distinguish between E. coli O157 that contain shiga toxins (STEC O157) and those that don't. STEC O157 bacteria, principally E. coli O157:H7, are potentially pathogenic in humans--while non-STEC O157 are not. Therefore, further testing of R.A.P.I.D. E. coli O157-positive samples would be needed to determine if they contained shiga toxins.

Technical Abstract: We evaluated the Ruggedized Advanced Pathogen Identification Device (R.A.P.I.D.) System Escherichia coli O157 detection kit (Idaho Technology, Inc., Salt Lake City, UT)for its ability to accurately detect E. coli O157 from pure culture and bovine fecal samples. One hundred seven E. coli O157 and 102 non-E. coli O157 isolates in pure culture were tested. Using a receiver operator characteristic derived cutoff threshold cycle (CT) value of 35 the sensitivity and specificity of the test were 99.1% and 99.0%, respectively. However, the E. coli O157 detection kit was unable to differentiate between shiga toxigenic E. coli (STEC) O157 and non-STEC O157 isolates. Based on artificial inoculation using two STEC O157 strains, the minimum detection limit was 500 bacteria g^-1 from non-enriched bovine feces and one bacteria g^-1 from bovine feces after selective broth enrichment. Finally, 75 bovine fecal samples from a herd naturally infected with STEC O157 were assayed for E. coli O157 by both an immunomagnetic bead-based (IMS) culture method and the R.A.P.I.D. System E. coli O157 detection kit. E. coli O157 was cultured from 13 fecal samples. The R.A.P.I.D. E. coli O157 detection kit detected eight of the 13 IMS positive samples before enrichment and all 13 IMS-positive samples after enrichment. These results demonstrate that the R.A.P.I.D. System E. coli O157 detection kit accurately identifies E. coli O157, does not discriminate between STEC and non-STEC O157, and can detect E. coli O157 in a complex sample matrix such as bovine feces.