Vyvoj metody rychle detekce salmonel ve vzorcich potravin

Abstract

Pathogenic bacteria occurring in foods cause various human illnisses. Standard methods for the detection of pathogenic microorganisms are time consuming (e. g. the determination of Salmonella in a food sample takes more than 5 days). In the food industry and health service there is a demand for a rapid pathogen detection method. The aim of this work was to develop a rapid method for the detection of Salmonella in food samples. The first step of the process optimisation was the connection of non-selective pre-enrichment of bacteria and filtration. Various materials were tested for the construction of the filtration bag, which would allow to passage the bacteria into the cultivation medium and at the same time avoid releasing of food particles. Usage of a polyamide material with pores of defined size for the construction of filtration bags was not successful. Better results were obtained with materials of paper with polyethylene (used for production of tea bags), or filtration membranes of commercially produced homogenisation bags (A. E. S. Laboratoire). The cultivation time required for filtration pre-enrichment was estimated to seven hours. Optimisation of shortened cultivation procedure for Salmonella enrichment in food samples was evaluated. Both the addition of iron compounds to buffered peptone water and the growth in various selective media did not enhance the growth rate of Salmonella cells significantly. Commercial kits for the shortened enrichment of Salmonella cells did not fulfil the demand for efficacy of selectivity. Good results were obtained using a combination of shortened enrichment in buffered peptone water followed by selective cultivation in Rappaport-Vassiliadis medium. The polymerase chain reaction (PCR) was chosen for the final specific detection of Salmonella. Primers were prepared according to sequences of the chromosomal gene invA and plasmid gene spvC. PCR is often inhibited by compounds from food matrices. To prevent this inhibition it is necessary to separate target bacteria or DNA from the sample. After the enrichment of bacteria from food sample the following separation methods were applied: immunomagnetic separation using anti-Salmonella Dynabeads (Dynal), two-step centrifugation, magnetic separation of DNA using Dynabeads DNA Direct (Dynal) and centrifugation on sucrose layer. Results differed according to the composition of sample food matrices. Immunomagnetic separation was not successful when applied to samples with high contents of fat and competitive microflora. Centrifugation methods provided good results for meat and egg samples. The optimised protocol including shortened non-selective enrichment in buffered peptone water, selective enrichment in Rappaport-Vassiliadis medium, two-step centrifugation and final detection by PCR was applied on real food samples. The reached sensitivity of the method were units of Salmonella cells added to 25g of food sample. Positive determination of Salmonella occurrence in entrails of infected animals was achieved by this method. Real-time fluorescence PCR was used for determination of Salmonella in samples of minced meat and chicken carcass rinse. The efficacy of four separation methods for sample pre-treatment before PCR was compared: gradient centrifugation using BactxtractorTM solution (Quintessence Research AB), magnetic separation of DNA using Dynabeads DNA Direct (Dynal), DNA isolation by DNeasyTM Tissue kit (Qiagen) and the combination of BactxtractorTM with DNeasyTM Tissue kit. BactxtractorTM and DNeasyTM Tissue kit provided reproducible results with the required sensitivity. Results obtained during this work can be exploited for the automation of food pathogen detection by PCR. This could be used in the future for routine determinations allowing detection time to be shortened to just one dayAvailable from STL Prague, CZ / NTK - National Technical LibrarySIGLECZCzech Republi