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Evaluation of fecal dna purification methods for the detection of E. coli O157:H7 in feces of naturally infected feedlot cattle.

Several protocols have been used to purify DNA from animal feces for PCR; direct DNA extraction from feces, DNA extraction with previous enrichment in selective media, and the use of commercial kits that involve the use of spin columns. The objective of this study was to compare the accuracy of the QIAamp DNA Stool Mini Ktt (QIAamp Kit) in detecting E. coli O157:H7 in enrichment and non-enrichment cattle feces from naturally infected feedlot cattle.

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Using conventional culture methods (gold standard), 456 fecal samples were analyzed for detectable levels of E. coli O157:H7. QIAamp Kit was used to purify fecal DNA (from enrichment and non-enrichment feces) and E. coli O157:H7 genes (Stx1 and Stx 2) were targeted and amplified by PCR.

A total of 199/456 (43.6%) fecal samples were positive for E. coli O157:H7 by culture. Of the 456 enrichment fecal samples, E. coli O157:H7 Shiga toxin-like genes were detected from 159/456 (34.6%) enrichment fecal samples compared to 43/456 (9.4%) targeted from non-enrichment samples. There was a substantial agreement (Kappa > 0.61) between the gold standard and QIAamp Kit (enrichment) as opposed to a fair agreement (Kappa > 0.21) for non-enrichment samples. Positive agreement rates were greater for the enrichment samples (83.6%) compared to the non-enrichment samples (65%). Agreement rates were lower (86.4%) for QIAamp Kit for enrichment samples compared to non-enrichment samples (98.5%). These data indicate that the use of QIAamp DNA Stool Mini Kit as the select protocol for E. coli O157:H7 detection in cattle feces should take into account the respective accuracy of the test for enriched and non-enriched fecal samples in comparison to the conventional culture method.

Acknowledgments: We are grateful to USDA-APHIS (Agrosecurity: Disease Surveillance and Public Health) and USDA-CSREES (Food Safety Risk Assessment Grant) for providing funding for this project.

References:

1. Khaitsa, M. L., M. L. Bauer, P. S Gibbs, G. P Lardy, D. K. Doetkott, and R. B. Kegode. 2005. Comparison of two sampling methods for Escherichia coli O157:H7 detection in feedlot cattle. J. Food Prot. 68:1724-1728.

2. Gioffre, A., L. Meichtri, M. Zumarraga, R. Rodriguez, and A. Cataldi. 2004. Evaluation of QIAamp DNA stool purification kit for Shiga-toxigenic Escherichia coli detection in bovine fecal swabs by PCR. Revista Argentina de Microbiologia. 36:1-5.

Ebot S. Tabe *, James Oloya, Dawn K. Doetkott, Margaret L. Khaitsa.

* The Great Plains Institute of Food Safety, North Dakota State University, Fargo, N D 58105. Department

of Veterinary and Microbiological Sciences, North Dakota State University, 1523 Centennial Blvd,

Fargo, ND 58105
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Title Annotation:COMMUNICATIONS--GRADUATE
Author:Tabe, Ebot S.; Oloya, James; Doetkott, Dawn K.; Khaitsa, Margaret L.
Publication:Proceedings of the North Dakota Academy of Science
Article Type:Report
Geographic Code:1USA
Date:Apr 1, 2008
Words:433
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