RESUMEN
A polymerase chain reaction (PCR) assay was developed for detection of pathogenic, virulent strains of Yersinia enterocolitica. By using both virulence loci virF and ail as markers for pathogenicity, detection of species with a virulence factor present was possible. DNA preparation in the presence of hexadecyl trimethy ammonium bromide (CTAB) was followed by two 44 cycle amplification reactions, one for each of the markers. As few as 10(2) Y. enterocolitica cells were detected in ground pork in the presence of 10(5)-10(6) bacteria of other species. The described PCR assay provides a sensitive robust assay for the detection of virulent Y. enterocolitica in food.
Asunto(s)
Microbiología de Alimentos , Reacción en Cadena de la Polimerasa , Yersinia enterocolitica/aislamiento & purificación , Sensibilidad y EspecificidadRESUMEN
The use of buoyant density centrifugation (BDC) to prepare samples for PCR analysis of food pathogens is described. Blue cheese and milk homogenates were inoculated with Shigella flexneri and layered on top of Percoll media. After BDC, the food homogenates remained in the upper part of the centrifuge tube, separated from the bacteria, which retained viability and were concentrated below the lighter Percoll layer. PCR inhibitors stayed in the homogenate and PCR analyses of treated samples consistently detected 10(4) cfu g-1 of blue cheese and 500 cfu ml-1 of milk, respectively. Differences in the density of live and killed Sh. flexneri and Yersinia enterocolitica were detected by BDC but were dependent on the mechanism of killing.