Nanogold-Based Gene Chip For Rapid Pathogen Detection

Rapid determination and discrimination of pathogen has the vital significance for the diagnosis and treatment of the infectious disease. 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. The gene chip technology is a method of pathogen identification based on nucleic acid detection, which is fast, sensitive, effective, and with high flux. Nanoparticle gold refer to the gold particle or compound whose diameter lie in 1-100nm. For their stable chemical property, Nanoparticle gold can have been used to label the protein or nucleotide. Meanwhile, it could be observed through eyes or photography because silver deposition on gold nanoparticle can produce the bigger black particle than the single gold nanoparticle. So gold nanoparticle labeling and subsequent silver enhancement have received much attention and have been commonly used to visualize protein-, antibody-conjugated gold nanoparticles in histochemical electron microscopy studies. Inspired by such novel use of gold nanoparticles, we aim at developing an analogous detection assay of pathogen. In the present study the gene chip is prepared by fixing aminated oligonucleotide probe to aldehyde modified glass. Then DNA probe with thiol group in 5'terminus was covalently linked to gold nanoparticle by self-assemble technique and was hybridized with the many sample bacterium gene amplified by universal primer PCR. Main research methods and findings:1. In Accordance with the literature report and the clinical investigation, characteristic nucleic acid marker of some bacteria gene were determined, and the amplification primer was designed .which aimed at consensus sequence. of 16S rDNA and 23S rDNA .2. The amplification methods of 16 pathogens gene were established. They are Staphylococcus Aureus, Staphylococcus haemolyticus, Staphylococcus saprophyticus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Bacillus subtilis, Pseudomonas aeruginosa, Bordetella pertussis, Acinetobacter calcoaceticus, Neisseria gonorrhoeae, Pseudomonas cepacia , K.peumoniae, A.lwoffi , Proteus mirabilis. 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 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. Comparative study did between two coloration methods in inverse direction dot-blot ,including DAB and chemiluminescence.。The former is rapid and convenient, the latter is time-consuming and elaborate, but the former was improved much better than latter in sensitivity.6. Based on prophase experiment, the optimal spotting concentration was determined, while the spotting concentration changed from 5nM to 3000nM, coloration intensity increasing rapidly, and then be gently, until reached 1000nM, it descended instead. So the conclusion is the optimal spotting concentration was determined in 500 nM.7. Study did on the relationship of temperature and the speed of chromatic reaction. The experiment shows that when it is 25℃, the reaction way in 5min×5 results in a better effect.8. This gene chip was applied to identify clinical specimen. The corresponding results were found for detection probe and positive control, while negative control, blank control and other detection probe remainded clear. The results are consistent with that of traditional biochemical and microbiological culture techniques. It is suggested that this method is accurate and reliable, can be used for the pathogens detection in clinic.9. Comparative study did between two nano-gold mark method, one was marked on thiol-modified PCR amplification product, the other was marked on antibiotin and then combined with biotin-modified PCR amplification product. The result shows that the latter was more efficient in combination and coloration.The above experimental results show that this chip detection technology has high sensibility and specificity and can be easily operated without special device. It can meet the flux demand of clinical detection and has good prospect of clinical application.