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The efficacy of confirmatory tests recommended by a PrPN EN ISO 11290-1:1999 standard for identification of Listeria spp. and L. monocytogenes strains in food products was examined in our studies. Confirmatory assays consisted of catalase and motility (a characteristic 'umbrella- -shape' outgrowth) tests for Listeria spp. as well as hemolytic activity and sugar fermentation (D-xylose and L-rhamnose) tests for L. monocytogenes. They were compared with results of multiplex PCR (polymerase chain reaction) designed for confirmation of genus and species. Seventy strains were tested. In the great majority (52 strains, 74.3%) they were of beef and pork carcasses origin. Eighteen strains (25.7%) were collected by swabbing in meat-processing environment. All presumptive strains were motile in ambient temperature, catalase-positive, presented hemolytic activity and characteristic morphological features, which enabled to classify them as L. monocytogenes. The comparison of pathogen identification results carried out with the standard tests and genetic analysis revealed that 84.3% of results was conformable (45 strains recognized as L. monocytogenes and 14 strains identified as Listeria spp. using both procedures). Variations referred to over 15% of results. Nine strains (12.9%) were identified as L. monocytogenes based on the sugar fermentation pattern whereas not confirmed by multiplex PCR. The affiliation of two strains (2.8%) identified as L. monocytogenes by multiplex PCR was not confirmed in the sugar fermentation test. The number of inaccurately classified strains in tests recommended by ISO standard highlights its limited efficacy for identification of L. monocytogenes strains.
A multiplex PCR assay designed by Bansal [1996] was applied in a routine screening test of a meat-processing environment aimed at detection of Listeria monocytogenes and species of the genus Listeria. Listeria spp. strains yielded a single 938-bp product indicating presence of 16S rRNA conservative sequence typical of the genus, whereas L. monocytogenes strains yielded not only the 938-bp product but also a 750-bp product - a result of amplification within region of the listeriolysin (hly A) gene. The assay was used to verify identification of 50 colonies performed using classical tests, including catalase and hemolytic activity, motility and API®LISTERIA (bioMérieux) biochemical tests. Among isolates, 4 strains (8%) were found characterised by contradictory results of biochemical and genetic tests. The possibility of simultaneous identification and differentiation of potentially pathogenic L. monocytogenes strains from other Listeria spp. during one reaction performed was proved.
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