Detection of viable Shiga toxin-producing Escherichia coli by quantitative competitive polymerase chain reaction.
With the use of Escherichia coli O157:H7 as a model, a procedure for the quantitative detection of viable Shiga toxin-producing E. coli (STEC) in broth and cooked ground beef enrichments with multiple-time point quantitative competitive polymerase chain reaction (QC-PCR) was developed. The A subunit (a 401-bp fragment) of the stx2 gene was chosen as a target sequence. Immunomagnetic separation (IMS) was used to isolate and concentrate cells from ground beef enrichments. Cell viability was confirmed on the basis of the quantitative increase in the signal of target bands from QC-PCR across multiple time points. The application of IMS increased detection limits relative to those for QC-PCR without IMS. E. coli O157:H7 inoculated at 0.20 CFU/g of cooked ground beef (25 g of ground beef plus 225 ml of Bacto modified EC medium plus novobiocin) was detected and confirmed to be viable in <15 h. A DNA-based molecular approach can be used to determine cell viability.
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Department of Food Science, North Carolina State University, Box 7624, Raleigh, North Carolina 27695-7624, USA.
Drake
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MA
D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000235:genetics; D019453:Escherichia coli O157 / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification* / Q000378:metabolism; D005516:Food Microbiology; D018189:Immunomagnetic Separation; D008461:Meat Products / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D022621:Shiga Toxin / Q000096:biosynthesis; D022641:Shiga Toxin 2 / Q000235:genetics
D004269:DNA, Bacterial; D022641:Shiga Toxin 2; D022621:Shiga Toxin