Study On New Detection Methods Of Isothermal Nucleic Acid Amplification Based On Enzyme Signal Amplification

With the development of molecular biology, nucleic acid amplification detection technology has been rapidly developed. The PCR technology is one of the current widely used detection techniques for nucleic acid amplification. However this technique relies on the temperature conditions, and it needs temperature-variable sophisticated equipment, and the experimental environment for it is harsh, all of this cannot make it to satisfy the modern society’s demand of the application of analytical techniques which are simple, fast and easy-operated. The isothermal nucleic acid amplification technology which is established with the efficient tool enzyme is able to make up for these shortcomings, and it demonstrates its advantage more and more obviously in the site analysis and emergency accident.In this paper, two enzyme-based isothermal nucleic acid detection new methods were constructed. In the second chapter, a method which can response quickly in a simple system was built for detecting the single-stranded RNA combined with molecular beacons, and the method was further verified by detecting microRNA Let-7a as the target. In this method, molecular beacons designed with nicking site, followed the principle of strand displacement and achieved a double cyclic amplification of the detection signal through the utilization of the two tool enzymes. This method had good linearity in the detection of Let-7a target with the range from 10 zmol to 1 fmol, and the lowest detection limit was 8.5×10-15 M. And it had showed a great practicability in the detection of human brain total RNA assay.In view of the present nucleic acid in most organisms existing in the form of double-stranded DNA, in the third chapter, the single primer isothermal nucleic acid amplification method was chosen from the three designed programs. In this method, the nicking enzyme recognized sequence-specific and nicked the double-stranded DNA, then the polymerase with strand displacement activity amplified the double-stranded DNA into single-stranded target which was as the template to design single primer amplification method for triple real-time fluorescence signal reaction. In this method, the experimental conditions was optimized by PBS nucleic acid target, and it reached the 1×10-13 M sensitive detection of targets ultimately. This method only need to design one primer, and it had simple system, rapid response, and could be applied usefully in the site detection.In a nutshell, the two tool enzyme-based isothermal nucleic acid amplification methods could provide for the analysis of nucleic acid samples of various emergencies and field testing as tool with simplicity, sensitivity and specificity.