PCR assay for detection of the E. coli O157:H7 eae-gene and effect of the sample preparation method on PCR detection of heat-killed E. coli O157:H7 in ground beef.
A PCR assay targeting the 3' end of the eae-gene of E. coli O157:H7 has been developed. It was shown to be specific for the E. coli O157:H7 eae-gene. Sensitivity of the PCR assay was 1 pg DNA or 10(3) cfu per PCR reaction. Subsequently, a study was conducted to examine the effect of the food matrix and the sample preparation method on PCR detection of non-viable cells using heat-killed E. coli O157:H7 in ground beef as a model system. Inoculated ground beef samples were subjected to either selective enrichment or immediately prepared for PCR analysis. Four sample preparation methods were applied: centrifugation, buoyant density centrifugation (BDC), immunomagnetic separation (IMS), and chelex extraction. Production of false-positive results due to amplification of the DNA of dead cells occurred if the number of heat-killed cells exceeded 10(8) cfu/g. For inocula <10(8) cfu/g, PCR results for heat- killed E. coli O157:H7 cells depended upon the sample preparation method. It was shown that the inclusion of two washings steps of the bacterial pellet before DNA extraction was the critical factor for elimination of false-positive results due to heat-killed cells in the centrifugation and BDC procedure. IMS did not produce false-positive results due to heat-killed cells (two washing steps were included in the IMS procedure). With the chelex-extraction method, false-positive results due to heat-killed cells were obtained. An effect of the ground beef food matrix on the presence of amplifiable DNA was noted.
Uyttendaele
M
M
Department of Food Technology and Nutrition, University of Gent, Belgium. mieke.uyttendaele@rug.ac.be
van Boxstael
S
S
Debevere
J
J
D018829:Adhesins, Bacterial; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D002352:Carrier Proteins; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002614:Chelating Agents / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D004927:Escherichia coli Infections / Q000517:prevention & control*; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification*; D029968:Escherichia coli Proteins; D005517:Foodborne Diseases / Q000517:prevention & control*; D006358:Hot Temperature; D018189:Immunomagnetic Separation; D008460:Meat / Q000382:microbiology*; D008863:Microspheres; D016133:Polymerase Chain Reaction / Q000379:methods; D011137:Polystyrenes / Q000737:chemistry; D011145:Polyvinyls / Q000737:chemistry; D012680:Sensitivity and Specificity
D018829:Adhesins, Bacterial; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D002614:Chelating Agents; D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011137:Polystyrenes; D011145:Polyvinyls; C073819:eaeA protein, E coli; C006960:chelex