Sample preparation methods for PCR detection of Escherichia coli O157 : H7, Salmonella typhimurium, and Listeria monocytogenes on beef chuck shoulderusing a single enrichment medium

To improve the utility of the polymerase chain reaction (PCR) for food samp les, methods for preparing template DNA were developed to remove PCR inhibi tors. Beef chuck shoulder medallions, artificially contaminated, individual ly or in combination, with Escherichia coli serotype O157:H7 strain FSIS 45 753-35, Salmonella typhimurium DT104 strain 13HP, or Listeria monocytogenes strain Scott A at concentrations of 10, 1 and 0.5 cfu/cm(2) were swabbed w ith a sponge, and the sponges were enriched for 18 h at 37 degreesC in univ ersal pre-enrichment broth (UPB). Enriched broth cultures (EBC), cell pelle ts (CP), or phosphate-buffered saline-washed cell pellets (PBSCP) from enri ched sponge samples were compared for detection of E. coli O157:H7, S. typh imurium DT104, or L. monocytogenes by the PCR using the BAX(TM) system. Rec overy of the three organisms was effective for detection of each pathogen a t initial levels of 10, 1 and 0.5 cfu/cm(2) when inoculated separately, or in combination, onto the beef samples. Use of EBC, CP, or PBSCP of sponge-s wabbed samples eliminated problems associated with inhibition of the PCR by food components, time-consuming extraction of DNA, and inhibition due to l arge amounts of nontarget DNA derived from the food. The procedure involvin g enrichment of sponge-swabbed beef samples in UPB followed by PCR amplific ation using EBC with the BAX(TM) system is the most efficient and simple me thod for detection of E. coli O157:H7, S. typhimurium DT104, and L. monocyt ogenes. (C) 2001 Academic Press.