Sample preparation methods for PCR detection of Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes on beef chuck shoulder using a single enrichment medium.
To improve the utility of the polymerase chain reaction (PCR) for food samples, methods for preparing template DNA were developed to remove PCR inhibitors. Beef chuck shoulder medallions, artificially contaminated, individually or in combination, with Escherichia coli serotype O157:H7 strain FSIS 45753-35, Salmonella typhimurium DT104 strain 13HP, or Listeria monocytogenes strain Scott A at concentrations of 10, 1 and 0.5 cfu/cm(2)were swabbed with a sponge, and the sponges were enriched for 18 h at 37 degrees C in universal pre-enrichment broth (UPB). Enriched broth cultures (EBC), cell pellets (CP), or phosphate-buffered saline-washed cell pellets (PBSCP) from enriched sponge samples were compared for detection of E. coli O157:H7, S. typhimurium DT104, or L. monocytogenes by the PCR using the BAX(TM)system. Recovery of the three organisms was effective for detection of each pathogen at 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-swabbed samples eliminated problems associated with inhibition of the PCR by food components, time-consuming extraction of DNA, and inhibition due to large amounts of non-target DNA derived from the food. The procedure involving enrichment of sponge-swabbed beef samples in UPB followed by PCR amplification using EBC with the BAX system is the most efficient and simple method for detection of E. coli O157:H7, S. typhimurium DT104, and L. monocytogenes.
Bhaduri
S
S
Microbial Food Safety Research Unit, Eastern Regional Research Center, USDA, 600 E. Mermaid Lane, Wyndmoor, PA 19038, USA. sbhaduri@arserrc.gov
Cottrell
B
B
D000818:Animals; D002417:Cattle; D003470:Culture Media / Q000737:chemistry; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification*; D005516:Food Microbiology; D006801:Humans; D008089:Listeria monocytogenes / Q000235:genetics / Q000302:isolation & purification*; D008460:Meat / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012486:Salmonella typhimurium / Q000235:genetics / Q000302:isolation & purification*
D003470:Culture Media