title: Comparison of a membrane surface adhesion recovery method with an IMS method for use in a polymerase chain reaction method to detect Escherichia coli O157:H7 in minced beef.
abstract: In this study, enrichment procedures and two recovery methods, a membrane surface adhesion technique and an immunomagnetic separation (IMS), were compared for use in conjunction with a multiplex polymerase chain reaction (PCR) method with a view to describing a fast (24 h) and economical test for detection of Escherichia coli O157:H7 in meat samples. The study showed no significant difference between three different enrichment media (BHI, E. coli (E.C.) broth+novobiocin, modified tryptone soya broth (mTSB)+novobiocin) or two incubation temperatures (37 or 41.5 degrees C) for growth of E. coli O157:H7 in minced beef. Minced beef samples inoculated with E. coli O157:H7 at 40 cfu g(-1) were incubated at 37 degrees C for 16 h in E.C. broth+novobiocin reaching numbers of (log(10)7.82-8.70). E. coli O157:H7 were recovered by attachment to polycarbonate membranes immersed in the enriched cultures for 15 min or by immunomagnetic separation. Subsequent treatment of recovered membranes or IMS beads with lysis buffer and phenol/chloroform/isoamyl alcohol was used to extract the DNA from the extracted E. coli O157:H7 cells. The results show when E. coli O157:H7 was present at high levels in the enriched meat sample (log(10)9.6-7.5 cfu ml(-1); >16-h enrichment), the membrane and IMS techniques recovered similar levels of the pathogen and the microorganism was detectable by PCR using both methods. At lower levels of E. coli O157:H7 (log(10)6.4), only the IMS method could recover the pathogen but at levels below this neither method could recover sufficient numbers of the pathogens to allow detection. The conclusion of the study is that with sufficient enrichment time (16 h) the membrane surface adhesion membrane extraction method used in combination with multiplex PCR has the potential for a rapid and economical detection method.
authors.affiliation: The National Diagnostics Centre, BioResearch Ireland, National University of Ireland, Galway, Ireland.
authors.forename: J
authors.initials: J
authors.lastname: Fitzmaurice
authors.forename: G
authors.initials: G
authors.lastname: Duffy
authors.forename: B
authors.initials: B
authors.lastname: Kilbride
authors.forename: J J
authors.initials: JJ
authors.lastname: Sheridan
authors.forename: C
authors.initials: C
authors.lastname: Carroll
authors.forename: M
authors.initials: M
authors.lastname: Maher
mesh_terms: D000818:Animals; D001422:Bacterial Adhesion / Q000502:physiology*; D002417:Cattle; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification*; D005516:Food Microbiology; D018189:Immunomagnetic Separation / Q000379:methods*; D008460:Meat / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D011108:Polymers
chemical_list: D004269:DNA, Bacterial; D011108:Polymers; C017902:lexan