Study On The Point Mutation And Hogwash Oil Detection Based On Signal Amplification By Tool Enzyme

In biochemical analysis, there are many molecules that can not be detected for alow concentration by the present methods. So, it’s very important to develop moresensitive signal amplification technology for these molecules. In this study, based onthe enzyme signal amplification technology in combination with chemiluminescentdetection and fluorescence quantitative PCR （FQ-PCR）, the novel and sensitivedetection methods for point mutation and hogwash oil have been presented,respectively. The major contents of the thesis were as follows:1. Highly sensitive chemiluminescent point mutation detection by circularstrand-displacement amplifcation reactionIn this chapter, we have developed a highly sensitive and selectivitychemiluminescence biosensor based on circular strand-displacement amplifcationand the separation by magnetic beads reducing the background signal forpoint mutation detection at room temperature. Here, we made the singlepoint mutation as the connection site of T4DNA ligase. When the target DNA wasthe mutant DNA, T4DNA ligase ligated the hairpin DNA and report DNA, then thesignal could be amplified by Klenow fragment （exo^-） polymerase. Finally, thechemiluminescence signal was detected. In the presence of wild type target DNA,5′-PO4of hairpin probe and3′-OH of reporter probe were unable to be ligatedbecause of the specifcity of T4DNA ligase. The target was displaced from hairpin probe followed by the reporter probe separated from magnetic beads after the primertriggered a polymerization reaction. So the CL signal did not occur. This methodtook advantage of both the T4DNA ligase recognizing single-base mismatch withhigh selectivity and the strand-displacement reaction of polymerase to performsignal amplifcation, and could achieve1.3×10^–16mol/L（3σ） detection limit.2. The study on hogwash oil detection based on exogenous characteristic mitochondriaDNA and FQ-PCRIn this study, we presented a sensitive detection method of hogwash oil basedon characteristic mitochondria DNA sequence and FQ-PCR signal amplification. Inthe chapter, characteristic DNA from exogenous animal mitochondrial was used asthe detection target for hogwash oil detection, and the primers that coulddiscriminate the characteristic mitochondrial DNA sequences in fish and ruminantwere designed. Mitochondrial DNA was extracted from the oil sample and amplifiedby Real-time quantitative PCR for quantitatively detecting oil sample by the Ct value.This method combined the multi-copy number, small molecular weight andstructural stability of mitochondrial DNA with high sensitivity and selectivity ofFQ-PCR. As a result, characteristic DNA from exogenous animal mitochondrial wasused as the detection target to provide a fast, sensitive and specific detection methodof hogwash oil.