title: PCR-based DNA amplification and presumptive detection of Escherichia coli O157:H7 with an internal fluorogenic probe and the 5' nuclease (TaqMan) assay.
abstract: Presumptive identification of Escherichia coli O157:H7 is possible in an individual, nonmultiplexed PCR if the reaction targets the enterohemorrhagic E. coli (EHEC) eaeA gene. In this report, we describe the development and evaluation of the sensitivity and specificity of a PCR-based 5' nuclease assay for presumptively detecting E. coli O157:H7 DNA. The specificity of the eaeA-based 5' nuclease assay system was sufficient to correctly identify all E. coli O157:H7 strains evaluated, mirroring the previously described specificity of the PCR primers. The SZ-primed, eaeA-targeted 5' nuclease detection assay was capable of rapid, semiautomated, presumptive detection of E. coli O157:H7 when >/=10(3) CFU/ml was present in modified tryptic soy broth (mTSB) or modified E. coli broth and when >/=10(4) CFU/ml was present in ground beef-mTSB mixtures. Incorporating an immunomagnetic separation (IMS) step, followed by a secondary enrichment culturing step and DNA recovery with a QIAamp tissue kit (Qiagen), improved the detection threshold to >/=10(2) CFU/ml. Surprisingly, immediately after IMS, the sensitivity of culturing on sorbitol MacConkey agar containing cefeximine and tellurite (CT-SMAC) was such that identifiable colonies were demonstrated only when >/=10(4) CFU/ml was present in the sample. Several factors that might be involved in creating these false-negative CT-SMAC culture results are discussed. The SZ-primed, eaeA-targeted 5' nuclease detection system demonstrated that it can be integrated readily into standard culturing procedures and that the assay can be useful as a rapid, automatable process for the presumptive identification of E. coli O157:H7 in ground beef and potentially in other food and environmental samples.
authors.affiliation: Food Animal Health & Management Center, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas 66506, USA. oberst@vet.ksu
authors.forename: R D
authors.initials: RD
authors.lastname: Oberst
authors.forename: M P
authors.initials: MP
authors.lastname: Hays
authors.forename: L K
authors.initials: LK
authors.lastname: Bohra
authors.forename: R K
authors.initials: RK
authors.lastname: Phebus
authors.forename: C T
authors.initials: CT
authors.lastname: Yamashiro
authors.forename: C
authors.initials: C
authors.lastname: Paszko-Kolva
authors.forename: S J
authors.initials: SJ
authors.lastname: Flood
authors.forename: J M
authors.initials: JM
authors.lastname: Sargeant
authors.forename: J R
authors.initials: JR
authors.lastname: Gillespie
mesh_terms: D018829:Adhesins, Bacterial; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D002352:Carrier Proteins; D002417:Cattle; D015169:Colony Count, Microbial; D003470:Culture Media; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis; D004927:Escherichia coli Infections / Q000382:microbiology; D019453:Escherichia coli O157 / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D029968:Escherichia coli Proteins; D005069:Evaluation Studies as Topic; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D018189:Immunomagnetic Separation; D008460:Meat / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000378:metabolism
chemical_list: D018829:Adhesins, Bacterial; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D003470:Culture Media; D015342:DNA Probes; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; C073819:eaeA protein, E coli; D019914:Taq Polymerase; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V