| With the features of high-speed, high-efficiency and high-throughput, gene chip technology has a promising prospect in the rapid diagnosis of clinical disease. Currently a variety of gene chip technologies are commonly applied in medical field, however, they also have demerits in low sensitivity, too complicated technique in detection, or requisition for special conditions and equipments. On the basis of our previous work, the new gene chip detection system with patent technology of nano-enlargment was optimized. The optimized system can organically combine gene technology with nanotechnology to fully take the advantage of 106 fold of signal amplification by the interaction between nano-gold and silver to supersede the currently used signal reporter materials such as fluorescence, isotope, and enzyme-conjugation, which results in the following advantages: simple equipment, easily-grasped technique, high sensitivity and wide application and so on. With these advantages, the new detection system of nanogold-based gene chip could be easily used in common laboratories or even complicated situation—war field, for example. Additionally, in order to improve the preparation technique of samples,two methods were established, one was one-time PCR with common primer based on ribotyping and the other was non PCR with restriction enzyme digestion based on restriction differential display. Then by combining the methods and nanogold-based gene chip, the researcher successfully developed two kinds of practical gene chips which aimed to detect bacterial pathogens and non bacterial pathogens, such as mycoplasma, chlamydia, fungus and virus, respectively. It is hoped to establish a quick, sensitive, accurate method for detecting pathogens in clinical.Main research methods and findings: Section 1: Optimization of nanogold-based gene chip detection system1. In order to get an ideal PCR amplification result, thiol-modified primer must be deoxidized in advance. And the experiments also showed that the deoxidized primer had no negative influence on amplification efficiency and specificity, nor did it change the conditions for PCR amplification. 2. Optimization experiments showed that the reaction time for marking could be decreased by properly raising the ratio of nanogold and nucleic acid. Though agitation had minor effect on marking efficiency (p>0.05), it still significantly fastened the speed of labeling reaction and shortened the time to reach the maximum marking efficiency (about 4 hours).3. According to the optimization of hybridization kinetic, it was clear that the best hybridization results could be obtained when the samples were hybridized for 8 hours at 45℃ with 500nM's probe concentration, 0.8M's ionic strength, and then strictly rinsed. Furthermore, slight circumgyratetion could increase the hybridization efficiency remarkably, which included significantly shortening the hybridization time and increasing the hybridization intensity as well. 4. An increasing temperature could further improve the speed of chromatic reaction. The experiment showed that when it was 37℃, the reaction way in 3min×3 resulted in a better effect. 5. The fragments' number and length of the plant gene's plasmid digested by Nco I /EcoR I, Nco I/Hind III and Nco I, respectively, all coincided with the results of experiment design, so did the PCR amplification fragment of positive control. These results confirmed that the quality assurance gene and the positive control fragment were reliable.6. The experiments showed that the sensitivity (96.7%), specificity (100%) and reliability (95%) could be achieved after the chip system was optimized. Its detection sensibitity reached 50fM, which was 100 times higher than fluorescence labeling signal's, also 2 times than pre-optimization system' s. 7. Compared with tradition detection, the chip detection of clinical samples is faster and simpler and its results could be easily recognized by naked eyes. Though their detection results were almost consistent, the further corroboration...
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