| Molecular biology-based pathogen identification assays have the potential of decreasing the time necessary for and increasing the specificity of pathogen determinations, making them efficacious alternatives to traditional biochemical and microbiological culture techniques. At present, genotype identification has been widely recognized as an effective tool in identification and characterization of infectious organisms. The gene chip technology is a method of genetype identification based on nucleic acid detection, which is fast, sensitive, effective, and with high flux. At present, some problems of techniques limit the application of gene chip in clinic diagnostics. At the probe design, the pathogen specific gene fragments are used as the probe in general diagnostic chips, their structure are complicated, the Tm, GC%, and the length are different greatly, which make it difficult for all probes to fully hybridize in the same condition and reduce the reliability of the detection. At the target labeling, the fluorescein is commonly used as reporter, but its sensibility is low, the detection condition is high, and the devices of signal read and analyze are very expensive. So no gene chip technology in pathogen identification is broadly used for clinical practice. In order to avoid these shortcomings, the oligonucleotide probe and nanoparticle gold are used in our research, to produce a gene chip for clinic pathogen detection, which is simple, fast, accurate and reliable, many pathogens can be detected simultaneously. This study was designed as two parts. First, according to the literature and the clinical investigation, some common pathogens and some rare but important in clinic are designed as targets, recognized-conservative gene fragments of pathogens are chosed as the characteristic molecular marker for pathogens detection and identification. Then the specific oligonucleotide probes were designed and their reliability was verified by reverse membrane hybridization. Second, the gene chip is made by fixing aminated oligonucleotide probe to aldehyde modified glass. The optimal spotting concentration was determined through comparing the effect of different capture probe concentration on its covalent fixation and on the hybridization efficiency. Nanogold- nucleic acid labeling method andsilver enhancement technology were applicated. Then, this gene chip was applied to identify clinical specimens; the practicability and reliability were studied.The main results were as follows:1. In Accordance with the literature report and the clinical investigation, characteristic nucleic acid marker of some bacteria, virus, Fungus and drug-resistant gene were determined and the amplification primer was designed.2. The amplification methods of 12 pathogens and a drug resistant gene were established. They are Staphylococcus Aureus, Escherichia.coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Enterococcus, Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria meningitides, Salmonella typhi, Candida albicans, Hepatitis B virus, Hepatitis C virus, and mecA. The all amplification reactions were optimized in the same condition. 3. The specific oligonucleotide probes of target pathogens were designed in the same condition and screened by bioinformatical technology using computer. Specificity and reliability of the probes were verified by hybridization with 32P labeled target. The results indicate that there is only 0.4℃ difference of Tm between probes. The parameters of probes such as GC%, Hairpin dG, Dimer dG and Repeat length fit the demand of design.4. The membrane chip fixed detection probe, positive control and negative control was used to identify clinical samples. The results showed that the positive signal in corresponding detection probe and positive control could be seen while the negative control, blank control and other detection probe without any signal, and the background is clear. It is proved that this method is accurate and reliable, can be used for pathogens detection.5. To determine...
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