Characteristics Analysis And Identification Immunization Proteomic Of Two Pathogens In Food

In the current study,total 900 samples of Chinese dishes including frozen dumpling,flavored raw meat,roast meat,braised meat,and cold vegetable dish in sauce were collected from large supermarket,retail food market and delicatessen in three cities in northern China(City A,City B,and cities C).The presence of the two pathogens was detected in the food samples and the results showed that E.coli 0157:H7 bacteria was not detected and 21 samples were positive for L.Monocytogenes in total 900 samples.The samples from City A exhibited the highest pathogenic bacteria detection rate(3%),followed by samples from cities C(3.33%).Chinese braised meat showed the highest pathogenic bacteria detection rate(4.44%),which is higher than cold vegetable dish in sauce and flavored raw meat(3.33%).The positive rate of Chinese braised meat and Chinese cold vegetable dish in sauce from delicatessen reached 8.33%,which is higher than samples from retail food market and supermarket.This survey provides a basis for monitoring food poisoning and tracking pollution sources..Multilocus sequence typing was performed to analyze the isolated L.Monocytogenes and the results showed the strains were divided into 12 kinds of subtype,which include ST5(7 strains,31.58%),ST121(2 strains,10.53%),ST9(3 strains,10.53%)and 9 other ST types(each has one isolate).This result indicated that ST5 type is the most predominant type of L.monocytogenes.Antimicrobial resistance of the 21 L.monocytogenes isolates was also examined and the result showed that all the strains were completely insensitive to trimethoprim-sulfamethoxazole and multi polymyxin B;85.71%of the strains exhibited resistance to to cefoxitin,cefotaxime and cefuroxim;All of the strains showd resistance to ampicillin.The results of virulence factor test showed that InlA,InlB,ActA,HlyA,PlcA,PlcB were all positive except InlJ.In this study,E.coli 0157:H7 ATCC3515 and L.monocytogenes ATCC 19111 were selected for immunoproteomic analysis.The whole cells of the two strains were used to immunize rabit to prepare antibodies.Whole-cell proteins of two strains were extracted and applied to Two-dimensional electrophoresis(2-DE)analysis.The prepared polyclonal antibodies were used to carry out western blot analysis.PDquest software was used to compare the results of SDS-PAGE and western blot,and to select the protein points that exhibited obvious immunogenicity.The candidate points were picked out from the SDS-PAGE gels and identified with MALDI-TOF MS/MS.The results showed a total of 17 immunoreactive proteins were identified,including 1 outer membrane proteins and 16 cytoplasmic proteins in E.coli 0157:H7 ATCC3515.In L.monocytogenes ATCC 19111,18 immunoreactive proteins were identified,including 2 membrane proteins and 14 cytoplasmic proteins.The current study lays a foundation for monitoring,charactering and preventing of E.coli and L.monocytogenes.This study also provides a reference for surface protein identification and immune detection method development of the two pathogens.