| Background:Intaking the contaminated food or water causes foodborne disease. The usual nosogenetic factors are as follows:pathogens, natural toxins, parasites and poisonous chemicals. Although natural toxins and poisonous chemicals could cause foodborne disease, the pathogens are main factors.Foodborne infection has become a very important hygienic problem because of the rapid expansion of food trade and highly increased mobility of today's populations. Hundreds of outbreaks of foodborne infection cases occur every year around the world.The epidemic surveillance data show that the incidence of foodborne infective disease is increasing and the species of bacteria causing foodborne infection have become more diversified continuously. In addition, many kinds of bacteria with weak toxigenicity have also become foodborne pathogens except intestinal pathogenic bacteria in recent years. But many public health governments still can not wake up to the importance of the food safety. There is big challenge for prevention and control of the foodborne infection disease. The major property of many foodborne pathogens is the alteration in their biological characters, such as Gram staining, colony formation and antigenicity. These changes often make conventional detection methods inefficient. Another property of foodborne infection is that it often involve quite a large number of affected individual and can spread rapidly in schools, factories and other institutions if the foodborne infection disease could not be controlled in time.Rapid trace to the source, detection and identification of the pathogens are the key issues to control foodborne infections. With more study in pathogens, many conventional microbiological assay such as Gram staining, culturial, biochemical properities, biotype- or serotype- identification is often inefficient and time consuming. It is more important to identify the bacteria on type, subtype, strain and molecule. The advent of genetic-based technologies makes feasible to develop a sensitive and specific screening test for the detection of microbial pathogens and their drug-resistence. The development in molecular biological technologies provides an efficient tool for bacteria classification. It makes bacteria division to molecular level and provides more details for picking out pathogens and transmission routes. We cannot only rapidly identify pathogens but also study their heredity relations in subtype, serotype and gene level. Multilocus sequence typing (MLST) is based on determination of the DNA sequence of a series of selected housekeeping. MLST may provide an ideal balance of high discriminatory power and a powerful data analysis capability requiring minimal human input. This technique, made possible by the increased availability of robotic sequencers, is based on determinationof the nucleotide sequences of a series of predetermined housekeeping gene. Now the MLST data of several pathogenic bacteria, such as Neisseria meningitides, Streptococcus pneumoniae have been based in worldwide web. Pulsed-field gel electrophoresis (PFGE) is a new DNA finger technique in recent years. The technique is based on all genome of bacteria and can discriminate 20～500 kb DNA segment. The data of MLST and PFGE can be compared among different labs. Gene chip technology is a reverse solid hybridization technology. Firstly a large number of probes are fixed onto the supporting material, then are hybridized with marked sample. At last, targets of sample are detected by the intensity of hybridization signal. Because more probes are fixed on the supporting material at the same time, microarray technology is characterized by high throughput, parallel disposal, micro miniaturization and easy automatization. At present, microarray technology is largely applied in medicine and molecular biology. It can parallelly identify many sequences and rapidly detect gene expression. Microarray technology can be largely used in the pathogens.Recently a DNA and oligonucleotide microarray technique has been applied to screen multiple microbial organism isolates in diagnostic assays. But such gene chips require sophisticated instruments to prepare and the results can be read only by laser scanner, thus limiting its broad application in common laboratories. Therefore we tried to establish an oligonucleotide array method in order to eliminate the need for sophisticated equipments but retain the sensitivity and specificity of the technique.To select a common gene fragment for identification of multiple pathogens, such gene must contain conservative regions common to these pathogens, and on the other hand sufficient sequence diversity for species identification. In literature, the 16S rRNA,23S rRNA, and 16S-23S rRNA spacer region have been selected as identification targets of bacteria. The conservation of 16S rRNA makes it suitable for identification of different species (genera) of bacteria. The extent of mutation is great in the 16S-23S rRNA spacer region among bacterial species or genera, but this region is too short to identify certain species of bacteria. Heat shock protein is a highly conserved protein, whose encoding gene groEL constitutes to be the most conserved component in evolution. The groEL gene has been used as the target gene in the typing and identification of Salmonella, Campylobacter jejuni and Staphylococcus on account of its complete database. In this study a mutation region of groEL was selected to prepare oligonucleotide membrane array on a nylon membrane to detect 17common bacterial pathogens causing foodborne infections.Our studies are as follows:(1) To study MLST and PFGE typing of foodborne infection pathogens for epidemiologic investigation. (2) To study the mutagenecity of pathogenic bacteria groEL gene and provide the materials to discriminative diagnosis of pathogen. (3) To construct a rapid and accurate detection method for common pathogenic bacteria in foodborne diseases with the technology of oligonucleotide membrane array. Materials and methods:Materials:1. Standard bacteria species:Seventeen common bacteria species causing foodborne infections were selected in our study. They were Escherichia coli, Escherichia coli, Campylobacter jejuni, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio alginolyticus, Staphylococcus aureus, Streptococcus hemolyticus, Yersinia enterocolitica,Proteus vulgaris, Bacillus cereus, Salmonella enterica, Salmonella typhimurium,Listeria monocytogenes, Shigella dysenteriae, Shigella flexneri,Clostridium perfringens,Clostridium botulinum Streptococcus pneumoniae, Klebsiella pneumoniae and Neisseria gonorrhoeaeserved as control species unrelated to foodborne infections. All strains above are stored in our lab.2. Foodborne infection mock samples:Pure cultures of two or three species of other bacteria were mixed and diluted as foodborne infection mock samples. Mock sample No.1 contains bacteria species of E. coli; mock sample No.2 contains bacteria species of Salmonella enterica; mock sample No.3 contains bacteria species of Listeria monocytogenes; mock sample No.4 contains bacteria species of Campylobacter jejuni; mock sample No.5contains bacteria species of Vibrio parahaemolyticus; mock sample No.6contains bacteria species of Proteus vulgaris3. Foodborne infection true samples:Ten foodborne infection true samples were collected from Guangzhou center for disease control and prevention between June 2000 and June 2010.4. Molecular biology software:Bioedit, DNA star, Cluster W, GeneDoc, Primer 5.0 and Primer express3.0Methods:1. To apply MLST based on housekeeping gene and PFGE based on all genome to type pathogens for epidemiologic investigation.2. With material of GenBank and biosoft and PCR amplification of groEL gene fragment of different bacteria, the groEL conservative regions, variable regions and their relation to the genetic development of bacteria strains were studied. 3. groEL gene was selected as target fragment for real-time PCR identification foodborne infection pathogens.4. Nylon membrane was used as array base. Oligonucleotide probes were synthesized and spotted onto nylon membrane as oligonucleotide array. The groEL gene amplifiaction products of different pathogenic bacteria from pure cultures, foodborne infection mock samples and foodborne samples were hybridized on oligonucleotide array.5. To apply digoxigenin developing color system for analysis the hybridization results.Results:1. To have acquired the data of Salmonella MLST and PFGE analysis.2. With gene amplfication and bioinformatic analysis,34 conservative regions were obtained. Conservative and mutant regions are distributed separately and most small mutant regions distributed like morsac among conservative regions. The phylogenetic trees among different bacteria drawn from the full length of groEL gene or partial fragment were the same from 16S rRNA.3. To have developed the real-time PCR based on groEL gene for identification of foodborne infection pathogens.4. With the same amplification and hybridization conditions, the groEL gene fragments of 17 species of pathogenic bacteria were amplified and hybridized onto the oligonucleotide array. Hybrizdization results manifested that 15 species {Escherichia coli, Campylobacter jejuni, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio alginolyticus, Staphylococcus aureus, Streptococcus hemolyticus, Yersinia enterocolitica, Proteus vulgaris, Bacillus cereus, Salmonella enterica, Salmonella typhimurium, Listeria monocytogenes, Clostridium perfringens and Clostridium botulinum.) showed high sensitivity and specificity. As to Shigella dysenteriae and Shigella flexneri, we found cross-reaction with the E. coli species- specific probe. Three species irrelated to foodborne infection disease showed no hybridization signal on the oligonucleotide array. The new method could also be applied in rapid identification of pathogen in foodborne infection mock samples and foodborne infection samples. Sensitivity in the assay mounts to 10 cfu/ml.5. To have developed digoxigenin developing color system for analysis the hybridization results.Conclusion:1. MLST based on housekeeping gene and PFGE based on all genome can type pathogens for epidemiologic investigation.2. The mutagenecity of groEL gene would provide suitable resolution for bacteria identification.3. Anticipated results could be obtained from foodbore infection mock samples with the oligonucleotide array. With foodbore infection samples, the new method could only identified to genus or some species level. Identification in subspecies or serotype level must be applied with traditional methods and antimicrobial resistance test. The method of oligonucleotide array has the priority of rapidity and accuracy in the diagnosis of foodborne diseases, and provides the efficiency way in the diagnosis, therapy and control of foodborne infection.4. Much study should be carried out on increasing hybridization probes and broadening the detection scope, and also on improving the specificity of the detection method. |
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