1,721,194 research outputs found
USE OF A CULTURE-INDEPENDENT MOLECULAR METHOD FOR THE DIRECT DETECTION ANIDENTIFICATION OF YERSINIA SPP. IN FOOD
No full textA culture-independent method for the direct detection in food of Yersinia spp. was developed in this study. It is based on the
amplification of a 359 bp PCR product from the RNA polymerase beta-subunit gene (rpoB) and subsequent analysis by
denaturing gradient gel electrophoresis (DGGE). Direct detection of Yersinia spp. by PCR–DGGE was carried out in ready-toeat
vegetables and the results compared with the results of the traditional, culture-dependent method. The DGGE profiles were
determined to be species-specific. As a matter of fact, Yersinia enterocolitica, Yersinia intermedia, Yersinia frederiskenii and
Yersinia kristensenii showed differential migrations in the gels. Moreover, Y. enterocolitica serotypes O:3, O:5 and O:9 were
distinguishable, as well. Only for a limited number of traditionally isolated strains, the biochemical and molecular identification
agree. In particular, an overestimation of Y. enterocolitica, as determined biochemically, was observed. Finally when the
protocol was applied to 27 food samples, a good correlation was obtained when the results of traditional and direct methods
were analyzed. The molecular method was able to identify Y. enterocolitica, not detected by plating analysis. However, for 4
samples, that, by plating analysis, were determined to contain Yersinia spp., no PCR product could be obtained after
enrichment, probably due to low numbers of target cells, thereby not allowing the possibility to perform DGGE analysis.
The protocol described here represents a reliable tool for the detection of Yersinia spp. in food, which can be used to obtain the
needed results faster than with traditional culturing methods
Direct identification in Food of Listeria spp. by polimerase chain reaction (PCR) and denaturing Gradient Gel Electrophoresis (DGGE) without the need of traditional isolation
No full text
The late blowing in cheese: a new molecular approach based on PCR and DGGE to study the microbial ecology of the alteration process
No full textA molecular biology method based on polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) was developed to detect Clostridium spp. in cheese samples suspected of late blowing. Strains of Clostridium spp. and different Lactic Acid Bacteria species, obtained from international collections, were used to determine the experimental conditions for the PCR amplification and DGGE differentiation. DNA extracted directly from cheeses with late blowing symptoms was subjected to PCR and DGGE analysis and traditional agar plating was performed for samples pasteurized and enriched overnight. Moreover, volatile fatty acids were determined for comparison purposes. The PCR-DGGE results were in agreement with the plating performed, and only samples presenting DGGE bands migrating at the same position as Clostridium spp. bands, showed the presence of Clostridium colonies on Reinforced Clostridial Medium plates. Butyric acid contents were high (>100 mg/kg) in the cases of positive DGGE results, underlining the suitability of the protocol for the study of cheese spoilage. The sensitivity of the method is estimated to be 104 CFU/g. © 2003 Elsevier B.V. All rights reserved
A PCR-Microplate Capture Hybridization Method to Detect Listeria monocytogenes in Blood
No full textn order to improve the diagnosis of Listeria monocytogenes infection, we have developed a polymerase chain reaction (PCR)-based assay combined with microplate capture hybridization technique. The system is based on selective amplification of L. monocytogenes with two specific primers based on the iap gene. The amplicon produced, with digoxigenin 11-dUTP incorporated during PCR, is hybridized in streptavidin-coated microtitre plates prepared with biotinylated specific DNA probe. The method involved requires approximately 6-8 h, and its high sensitivity, rapidity and simplicity should make it valuable for diagnosis and for epidemiological studies of listeriosis
- …
