The Establishment And Application Of The Compound Pre-enrichment Technology And The Gene Chip Method To Detect The Main Pathogenic Bacteria Of The Food Of Animal Origin

Presently, the security of food of animal origin is already a significant strategic question assisted with the people's health and the construction of harmonious society. The harmfulness and affections of pathogenic bacteria occupy the most important status. The key question is that the detection system of food of animal origin pathogen in our country have not adapted to the development of economy and the improving of people living standard.In order to developing a high-through, fast and parallel detection system for the main pathogenic bacteria in food of animal origin, a kind of bacterial compound pre-enrichment technology was studied, which enhanced the accuracy and the sensitivity of the detection system. By analyzing and comparing the ingredient of twenty-two mediums which were commonly used to culture the main pathogenic bacteria such as salmonella, EHEC O157: H7, Staphylococcus aureus , Sh, Yersinia enterocolitica and so on, and two mediums namely NB and BPW were selected to studied the effects of bacterial compound pre-enrichment. In order to compare the effects of single and mixture bacterial enrichment of seven pathogenic bacteria in NB and BPW medium, 12 bacteria were cultured and diluted into 1 - 10cfu/ml,and experimental samples were prepared by using 5 food samples manual contaminated with the bacterial liquid. The results suggested that there were seven pathogenic bacteria cultured in N.B medium and only five in BPW were enriched with the mean standard bacteria count increased to 10~3cfu/ml within 24 hours. Therefore, the NB medium is more suitable for the combination growth of seven pathogenic bacteria than BPW medium. The accuracy of the detection by PCR amplification was increased with the increase of the number of target bacteria in the experiment samples.More than ten 16SrRNA and 23SrRNA complete sequences of the close origin genus of the pathogenic bacteria were selected, and analyzed with a multiple sequence alignment and cluster analysis, and three pairs consensus primers were designed according to the conservative regions of 16SrRNA and 23SrRNA genes and specific probes were designed a according to the variable region between the difference bacteria. Considering the utility of the multiple PCR amplification of seven pathogenic bacteria, the primers and the probes were designed with the same Tm value with the annealing temperature of 56℃.In detection of the seven pathogenic bacteria, the sensibility of the multiple PCR can reach 100 cfu/ml.The research designs sample application map of 14×4 chip point lattice. After amination modifications in 5'terminatio of the probes, the probes were doted on the chip of aldehyde group brood, and then non-specificity hybridization with salmon sperm DNA fragment were carried out to detect the probe whether having the problem of quality, such as nulli-point and the size of point. The multiple PCR amplification products were labeled with the Two Colour Fluorescence tumor marker Cy3 and Cy5, and hybridized with the gene chip. By optimizing hybridization condition of gene chip, we determine the hybridization temperature was 55℃of the tritus gene chip and the hybridization solution was NH. After scanning the hybridization, we adopt the method of S/N ratio to analyze the scanning results, and by the statistics analysis for scanning result, we obtain the signal standard judgment of valid hybridization, specificity hybridization, non-specificity hybridization and reduplicative experiment. The scanning results suggested that positive hybridization signal of the seven pathogenic bacteria hybridization were obtained correctly, the positive signals were above 10 times of the background and the ratios of positive focus and quality control focus was above 0.4.The probe sequences of 16SrRNA and 23SrRNA were studied with massive BLAST alignment and the specificity of the probe for seven pathogenic bacteria was appraised. Under the condition of non-target bacteria and target bacteria alone or mixing interference, the S/N ratio of target pathogenic bacteria by the detection of gene chip hybridization scanning were above 10, and the average ratios of positive focus and quality control focus were more than 0.4. The gene chip hybridization detection sensitivity of seven pathogenic bacteria was about 670cfu/ml.The experiment have proved, the number of bacterial template will influence the accuracy of hybridization of the chip. Increasing proper template number would avoid false-negative result. Three chips were random selected from each batch of the three batches were used to study the reproducibility and the stability of the chips, and the results indicated that the reproducibility and stability of the chips from the three batches were better, and the same results were obtained by testing the reproducibility and the stability of the chip from the same patch every two months in a year. Comparing the gene chip method with traditional standard method, PCR method and the verification method of tachy-instrumentation, the results proved that only gene chip method could detect several pathogenic bacteria in one time with the characteristics of fast, accuracy, high sensibility and specificity.By combinative pre-enrichment of the bacteria, multiple PCR amplification, purification and label of the DNA products, hybridization, scanning and result assessment, the gene chip could detect the seven pathogenic bacteria very soon. A kit for gene chip detection of main pathogenic bacteria in food of animal origin was developed based on the experiment, and thus a perfect detection system for the generalization of the detection method was set up. The gene chip detection method of main pathogenic bacteria in food of animal origin and the kit have been applied in the inspection and medical inspection of food of animal origin of import and export in Liaoning province during the year of 2005 and 2006, we make use of gene chip method to detect the food of animal origin which easily contaminated with medical inspection pathogenic bacteria, such as frozen pig ear, frozen port, frozen chicken claw and so on. For13 patches samples in total of more than two hundred patches in import (about 0.8% of total amount) were detected containing Salmonella typhimurium , EHEC O157: H7 and Staphylococcus aureus, and 21 batches of the two hundred batches in export (about 0.01% of total amount) containing Salmonella typhimurium. All the result were checked with the national standard methods ,and there were well consistencies between the two methods. The reasonable and effective measures were conducted in directing fast detection, prompt pre-warning, products segregation and pathogenic bacteria isolation, and the convincing and reasonable results were obtained, the unqualified products were destroyed which have saved the economic loss of nearly a hundred million Yuan for the country. The gene chip has played a very significant role in the monitoring import and export.