engleski

Direktno odredjivanje Yersina spp. u hrani pomocu molekularnih metoda

Yersinia is well established as a foodborne pathogen of human concern and is most frequently associated with raw and cooked meat products. The genus Yersinia comprises 11 species, of which Y. pestis, Y. pseudotubercolosis and Y. enterocolitica posses the potential to be pathogenic in humans and animals. The pathogenicity of these three strains is controlled by the common 64- to 75-Kb virulence plasmid. Strains of Y. enterocolitica non- plasmid-bearing are not virulent. Those isolates formerly called Y. enterocolitica-like isolates were reclassified and assigned to eight different species (Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. mollareti, Y. bercoveri, Y. aldovae, Y. rhodei and Y. ruckeri). Traditional methods employ the use of prolonged enrichments at refrigeration temperatures to take advantage of phychrotrophic nature of Yersinia and to suppress the growth of background flora. Due to the extended time period needed for this type of method, efforts have been made to devise selective enrichment techniques employing shorter incubation times and higher temperatures, thus making more practical for routine use. With the development of new molecular techniques more powerful protocols have became available to improve the quality of the food microbiological analysis. The recent developed techniques for amplifying specific DNA sequences in vitro allow the detection of infinitesimally small amounts of target DNA in various environment and food specimens. In this study the Yersinia spp., more frequent isolated from food, were considered to optimize a PCR-Denaturing Gradient Gel Electrophoresis (DGGE) to allow their direct detection in food samples. A couple of primers targeting the rpoB gene were used to produce a 350 bp PCR amplicon that was subsequently subjected to DGGE analysis. The protocol was optimized by using strains from international collections, then applied for the identification of Yersinia spp. isolated traditionally from food samples and at last for the direct identification of yersiniae. The application of the PCR-DGGE protocol allowed to obtain species-specific patterns for the species used in the study, highlighting the possibility to apply the method for a direct detection and identification of Yersinia spp. in food samples.

Yersinia spp.; food; detection

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