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As the important homogeneous detection technology, real-time PCR is a rapid, simple, sensitive, specific, and quantitative advantages, has been widely used in the field of molecular biology and medical research. However, subject to a limit on the number of real-time PCR instrument can detect fluorescence channel, real-time PCR to detect small capacity, difficult to detect multiple target sequences in a single reaction. To address this issue, the paper launched a multi-target detection technology for real-time PCR, research work mainly includes multicolor combination probe encoding technology research and probe melting curve analysis technology. The first part of a multi-color combination probe encoding technology, and examines the feasibility of the technology for multi-target identification and mutation detection. The part of the first chapter, for the detection of a variety of food-borne pathogens using modified molecular beacons as a detection probe, the color combination probe encoding technology is used to identify the eight kinds of food-borne pathogens established detection system can accurately identify any of eight food-borne pathogens, with a rapid, simple, high-throughput, specificity. Chapter II, a detailed examination of fluorescent double-stranded replacement probe by Taq DNA polymerase 5 '→ 3' exonuclease activity of the impact and the rule, for multicolor combination probe coding technique for a variety of mutations detected lay the foundation. Chapter III, a variety of mutations in the β-globin gene to detect objects by combining HAND system and a fluorescent double-stranded replacement probe technology, the the multicolor combination probe coding technique is used for the detection of multiple mutations in a single reaction tube in the detection of five kinds of Chinese common β-thalassemia mutations and 21 genotypes typing. The second part, the development of a new probe melting curve analysis technology. Multicolor fluorescence detection and T_m multiple detection is a powerful tool for multi-target detection to achieve real-time PCR. However, the present fluorescent probes having a difficult to simultaneously combination of multicolor fluorescence detection and T_m multiplex detection of defects. Therefore, in this part of the chapter, first systematically investigated the feasibility of commonly used fluorescent probes for melting curve analysis comparing the melting curve analysis of the advantages and disadvantages of each probe is used, and found that the modified molecular beacon probe particularly suitable for the melting curve analysis of a fluorescent probe. Chapter II, a variety of mutations in the β-globin gene to detect objects and effects of the modified molecular beacon probe melting curve method used to detect a variety of mutations, and on this basis, according to the common Chinese β-thalassemia mutation frequency of two multi-multi-color mutation detection system, to achieve a common 15 kinds of β-thalassemia mutation detection and genotyping.
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