Abstract
The polymerase chain reaction (PCR) technique was applied to detect L. monocytogenes in cheese samples. Two types of soft cheese were inoculated with various numbers of L. monocytogenes (serovar 1/2a and 4b). Then, 10g of each cheese sample was mixed 50ml of modified EB broth (MEB), buffered by the addition of MOPS buffer, and incubated at 30°C for 20 hours. An 0.5ml aliquot of each MEB culture was plated onto Oxford agar. After incubation at 35°C for 24 hours, the growth was removed and suspended in distilled water. This suspension was boiled, centrifuged and the resultant supernatant fluid was used as a DNA sample for PCR. Primers used in the study were designed to amplify a 702-bp region of the L. monocytogenes listeriolysin O (hly) gene, as previously reported by Border et al (1990). Fewer than 10 CFU of L. monocytogenes per g of cheese was detectable by this procedure, irrespective of cheese type and Listeria stain.
Seventy-six retailed cheese samples including naturally contaminated ones which had been stored in a freezer were assayed by the PCR procedure as described above. A conventional culture procedure (IDF standard) was also performed on the same cheese samples In 76 cheese samples, 12 were identified as being positive by both the PCR procedure and the conventional culture procedure. Four samples identified as being positive by the PCR procedure were negative by the conventional culture procedure. Among 60 samples that were negative in the PCR assay, 9 were shown by culture procedure to contain L. innocua.
These findings demonstrated that the PCR assay following a two-day broth and plate enrichment is useful for the rapid detection of L. monocytogenes in cheese samples.
