Separation And Rapid Detection Of Shiga Toxin-producing Escherichia Coli In Food

The objective of this study was to use Immunomagnetic beads( IMBs) based technique multiplex PCR combined with denaturing high-performance liquid phase chromatography(DHPLC) and matrix assisted laser desorption ionization time of flight mass spectrometry(MALDI TOF MS) for the enrichment and separation and rapidly detection of Shiga Toxin-producing Escherichia coli in food.The standard strains of Shiga Toxin-producing Escherichia coli O111 and O157 were used as representative object in this method. Mainly from four aspects for the project research, First of all, separation of the target strains, immunomagnetic beads were used to enrich the target strains, specificity and sensitivity of magnetic beads would be verified. Experiments of the actual samples, drugs and chromogenic medium would be combined with the magnetic beads for the quick and accurate separation of the target strains. Secondly, genes of the rfbE and wzx were used as the target genes to establish the conditions and system in the research of the multiple PCR-DHPLC method, specificity and the sensitivity of the method were verified, the results of the sensitivity would be compared with the RT-PCR, 270 food samples were selected randomly to detect by this method. Thirdly, the method of the MALDI-TOF MS was established, the different pretreatment methods and culture media and culture times would be analysed on the impact of the protein map, the best method should be used to achieve the protein fingerprint maps of the target strains and add the database. Different culture conditions were set up to verify the accuracy of the method. Finally, in order to solve the problems of the interference on microbial activity from different substrates and degradation factors, the uniformity and stability would were analysed, proficiency testing of O111 and O157 should be carried out in the whole country.In the experiment of the separation and enrichment for the target strains, research results showed that immunomagnetic beads with better specificity and sensitivity could be applied to separate pathogenic microorganisms with a higher efficiency. The Multiple PCR-DHPLC that had been established showed a good specificity and sensitivity in the same of the gradient with RT-PCR, which O111 and O157 could be detected at the same time and reached a detection limit of 25 CFU/mL. In the experiment of the food samples, 1 case of O111 and 3 cases of O157 were detected in 129 beeves samples, 1 case of O111 and 1 case of O157 were positive from 74 chickens samples, while O111 and O157 were not be found in 67 vegetables samples. In the method of MALDI-TOF MS, the protein fingerprints of O111 and O157 that were culturing 24-36 h in the liquid medium of TSB were achieved, the two target strains were identified as Escherichia coli. However, for the protein fingerprints of the two target strains were similar, therefore, the experiments did not completely exactly identify the two target strains each other, molecular biology and serological techniques should be used to identify accurately. In the research of proficiency testing, a good uniformity and stability were obtained by using mung bean flour as the matrix. The correct rate from 138 laboratories who participated in the project research was 98%.