Application of deoxyribonucleic acid (DNA) based techniques for the detection of Yersinia enterocolitica and Fusobacterium necrophorum.

摘要

Yersinia enterocolitica is a psychrotrophic pathogen. Culture methods for detecting virulent Y. enterocolitica require selective enrichment and a series of confirmatory tests that are time-consuming, expensive, and laborious. The development and evaluation of the fluorogenic 5′ nuclease assay to detect virulent Y. enterocolitica in food samples is of critical importance to the food industry.; The specificity of the chromosomal yst gene based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic serotypes of Y. enterocolitica; other species of Yersinia (Y. aldovae, Y. pseudotuberculosis, Y. mollaretti, Y. intermedia, Y. bercovieri, Y. ruckeri, Y. frederiksenii, and Y. kristensenii ); and other, enteric bacteria (Escherichia, Salmonella, Citrobacter, and Flavobacterium). The assay was found to be 100% specific in identifying the pathogenic strains of Y. enterocolitica. The sensitivity of the assay was found to be ≥10 2 CFU/ml in pure cultures and ≥103 CFU/g in spiked ground pork samples. The assay indicated a natural contamination rate of 35.5% (94/265) in various meats and tofu, whereas the CIN plating method indicated a contamination rate of 28.3% (75/265). Only 75% (60/80) of the Yersinia spp. Isolated on CIN were identified by autoagglutination and crystal binding methods as containing a virulence plasmid. This study demonstrates the potential of the 5′ nuclease assay for rapidly and specifically identifying virulent Y. enterocolitica in processed foods and can be completed within 5 hours after enrichment.; Fusobacterium necrophorum can cause infections in man and a variety of animals. In cattle, F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme are known as the primary pathogens of hepatic abscesses. Experiments dealing with the differences in the outer membrane profiles (OMP) of the two subspecies of F. necrophorum resulted in significant differences in their profiles. Major OMP bands between 39–78 KD were markedly different between the two subspecies. Serum samples from two test animals (both abscessed and unabscessed) recognized all the major OMP bands as observed on SDS-PAGE gels. Growth factors like pH, aerobic/anaerobic conditions, and culture media were found show differences in the OMP profiles of the two subspecies of F. necrophorum.