Comparing real-time and conventional PCR to culture-based methods for detecting and quantifying Escherichia coli O157 in cattle feces.
Detection of Escherichia coli O157 in cattle feces has traditionally used culture-based methods; PCR-based methods have been suggested as an alternative. We aimed to determine if multiplex real-time (mq) or conventional PCR methods could reliably detect cattle naturally shedding high (≥10(4) CFU/g of feces) and low (∼10(2) CFU/g of feces) concentrations of E. coli O157. Feces were collected from pens of feedlot cattle and evaluated for E. coli O157 by culture methods. Samples were categorized as (i) high shedders, (ii) immunomagnetic separation (IMS) positive after enrichment, or (iii) culture negative. DNA was extracted pre- and postenrichment from 100 fecal samples from each category (high shedder, IMS positive, culture negative) and subjected to mqPCR and conventional PCR assays based on detecting three genes, rfbE, stx1, and stx2. In feces from cattle determined to be E. coli O157 high shedders by culture, 37% were positive by mqPCR prior to enrichment; 85% of samples were positive after enrichment. In IMS-positive samples, 4% were positive by mqPCR prior to enrichment, while 43% were positive after enrichment. In culture-negative feces, 7% were positive by mqPCR prior to enrichment, and 40% were positive after enrichment. The proportion of high shedder-positive and culture-positive (high shedder and IMS) samples were significantly different from mqPCR-positive samples before and after enrichment (P < 0.01). Similar results were observed for conventional PCR. Our data suggest that mqPCR and conventional PCR are most useful in identifying high shedder animals and may not be an appropriate substitute to culture-based methods for detection of E. coli O157 in cattle feces.
Department of Population Health and Pathobiology, North Carolina State University, 1060 William Moore Drive, College of Veterinary Medicine, Raleigh, North Carolina 27607, USA. megan_jacob@ncsu.edu.
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Jacob
Department of Diagnostic Medicine/Pathobiology, Kansas State University, 1800 Denison Avenue, Manhattan, Kansas 66506, USA.
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Bai
Department of Diagnostic Medicine/Pathobiology, Kansas State University, 1800 Denison Avenue, Manhattan, Kansas 66506, USA.
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Renter
Department of Population Health and Pathobiology, North Carolina State University, 1060 William Moore Drive, College of Veterinary Medicine, Raleigh, North Carolina 27607, USA.
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Rogers
Department of Diagnostic Medicine/Pathobiology, Kansas State University, 1800 Denison Avenue, Manhattan, Kansas 66506, USA.
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Shi
Department of Diagnostic Medicine/Pathobiology, Kansas State University, 1800 Denison Avenue, Manhattan, Kansas 66506, USA.
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Nagaraja
D000818:Animals; D057138:Bacterial Shedding; D002417:Cattle; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000235:genetics; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification*; D029968:Escherichia coli Proteins / Q000235:genetics; D005243:Feces / Q000382:microbiology*; D018189:Immunomagnetic Separation / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012680:Sensitivity and Specificity; D022622:Shiga Toxin 1 / Q000235:genetics; D022641:Shiga Toxin 2 / Q000235:genetics
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2