PCR assay for detection of the E-coli O157 : H7 eae-gene and effect of thesample preparation method on PCR detection of heat-killed E-coli O157 : H7in ground beef

A PCR assay targeting the 3' end of the eae-gene of E. coli O157:H7 has bee n 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. Su bsequently, a study was conducted to examine the effect of the food matrix and the sample preparation method on PCR detection of non-viable cells usin g heat-killed E. coli O157:H7 in ground beef as a model system. Inoculated ground beef samples were subjected to either selective enrichment or immedi ately prepared for PCR analysis. Four sample preparation methods were appli ed: centrifugation, buoyant density centrifugation (BDC), immunomagnetic se paration (IMS), and chelex extraction. Production of false-positive results due to amplification of the DNA of dead cells occurred if the number of he at-killed cells exceeded 10(8) cfu/g. For inocula < 10(8) cfu/g, PCR result s for heat-killed E. coli O157:H7 cells depended upon the sample preparatio n method. It was shown that the inclusion of two washings steps of the bact erial pellet before DNA extraction was the critical factor for elimination of false-positive results due to heat-killed cells in the centrifugation an d BDC procedure. IMS did not produce false-positive results due to heat-kil led cells (two washing steps were included in the LMS procedure). With the chelex-extraction method, false-positive results due to heat-killed cells w ere obtained. An effect of the ground beef food matrix on the presence of a mplifiable DNA was noted, (C) 1999 Elsevier Science B.V. All rights reserve d.