A broad-range PCR assay for the detection of bacteria belonging to the Enterobacteriaceae family was developed in this study. Primers targeting the bacterial 16S rRNA gene were newly designed and used in this PCR assay. To determine the specificity of the assay, 72 different bacterial species (of 49 genera), 2 fungi, 3 animals, and 4 plants were tested. Results were positive for every tested Vibrioaceae or Enterobacteriaceae strain except Proteus mirabilis. For all other bacterial strains and eukaryotes tested, results were negative. Bacterial DNA for PCR was prepared by a simple procedure with the use of Chelex 100 resin from culture after growth in brain heart infusion medium. To test this PCR assay for the monitoring of the Enterobacteriaceae family, either Escherichia coli or Salmonella Enteritidis was inoculated into various foods as an indicator. Prior to the PCR, the inoculation of 10 to 40 CFU of bacteria per g of food was followed by a 5-h enrichment culture step, and the PCR assay allowed the detection of bacterial cells. When actual examinations of the contamination of 15 noodle foods with Enterobacteriaceae by this PCR assay were conducted, 33% (5 of 15) of the samples tested positive. These results agreed with those of the Petrifilm Enterobacteriaceae Count Plate assay. Including the enrichment culture step, the entire PCR detection process can be completed within 7 h.

A broad-range PCR assay for the detection of bacteria belonging to Bacillus and Staphylococcus genera was developed. Primers targeting the bacterial 16S rRNA gene were newly designed and used in a PCR assay. To determine the specificity of the assay, 81 different bacterial strains (of 50 genera), 2 fungi, 3 animals, and 4 plants were tested. Results were positive for every tested Bacillus, Staphylococcus, or Aerococcus strain. In addition, the result for Listeria grayi was positive with lower PCR product. For all other bacterial strains and eukaryotes tested, results were negative. Bacterial DNA was prepared with the use of achromopeptidase and Chelex 100 resin from culture after growth in brain heart infusion medium. To test the sensitivity of this PCR assay for Bacillus or Staphylococcus genus, either Bacillus cereus or Staphylococcus aureus was inoculated into various foods with undetectable levels of endogenous microbial contamination as an indicator. Inoculation of bacteria at 10 to 30 CFU/g of food was followed by a 5-h enrichment culture step after which the PCR assay allowed the detection of bacterial cells. When the inoculation (B. cereus or S. aureus) of 10 to 90 CFU/g into noodle foods containing endogenous microflora (10(3) to 10(5) CFU/g) was followed by a 6-h enrichment culture step, the PCR assay detected the bacteria. Including the enrichment culture step, the entire PCR detection process can be completed within 8.5 h.