Detection of Listeria monocytogenes using genetic and immunological tools
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A PCR assay targeting the genes encoding internalinAB (inlAB) was developed for detecting L. monocytogenes in foods. One primer set, targeting a 902-bp region of the inlAB, was most specific among those tested. The specific PCR product was detected in 51 L. monocytogenes strains belonging to 4 different serogroups. In contrast, the PCR product was not detected in other Listeria species and gram positive, indicating that the primer set was highly specific. The detection limit of the PCR assay was 105 CFU per ml of pure culture. However, the assay could detect as few as 10 CFU of L. monocytogenes in 25 g of frankfurters within 6 h after samples were enriched in modified Listeria enrichment broth (LEB) at 37?C. The total assay time, including enrichment, was approximately 24 h. |The developed PCR method was evaluated for detecting L. monocytogenes at levels ranging from ca. 1 to 100 cfu/g in with two enrichment procedures. Depending on the food type, i. e. ground beef or Brie cheese, PCR detection was achieved with different enrichment procedures. Ground beef samples enriched in tryptic soy broth (TSB) at 37ºC for 6 h, then transferred to LEB with further incubating for 18 h at 30ºC was efficient for detecting L. monocytogenes cells while we could detect as low as <1 cfu per gram in Brie cheese with enriched in LEB medium for 24 h at 37 ºC after PCR detection. Among 8 tested foods, the PCR method could detect as low as ca. <1 cells/g of food sample after enrichment, which had complete agreement with results obtained by using the conventional cultural method. These data suggest that the PCR method is specific and sensitive for detecting L. monocytogenes in various foods. |A rapid detection method for L. monocytogenes using immuno magenetic separation with flow cytometry was investigated. The efficiency of immuno capturing using magnetic beads was also determined. None of antibodies tested in our protocol differentiated between negative and positive sample. Attempts to optimize the concentration of magnetic beads or blocking step did not improve the signal difference. Plating results confirmed that cells were captured even with low recovery rate ranging from 7 to 21%. However, when the same antibody (KPL) was used as a fluorescent labeling antibody, the differentiation between negative and positive sample was not obtained.