Study On New Isothermal Amplification Methods For The Detection Of MiRNA And DsDNA

Nucleic acid, as the basic substance of life, widely exists in cells of plants, animals and bacteria. It plays the role of analytical marker in the areas of biological analysis, disease diagnosis and food safety. Thus, it becomes the main objective in the field of nucleic acid detection to develop new, simple, easy-to-use, sensitive and rapid detection methods of isothermal nucleic acid amplification. Here, we reported two new detection methods of isothermal nucleic acid amplification with simple design, which had been used to rapidly, sensitively and specifically detect miRNA and DNA, respectively.In chapter Ⅱ, we reported a double-nicked beacon mediated isothermal amplification (BAMP) method for miRNA detection. In the system, miRNA, as the primer, hybridized with double-nicked molecular beacon as the template, which initiated the amplification reaction mediated by polymerase and nicking endonuclease. After signal amplification of two cycles, BAMP reaction achieved cascade non-linear amplification results. A real-time fluorescence detection of the BAMP reaction originated from different concentrations of miR-Let-7a was performed.0.1 zmol target could be well identified from the blank. To evaluate the specificity of the proposed BAMP method, Let-7 miRNA family members were measured using BAMP system, respectively. To investigate the feasibility of BAMP to detect miR-Let-7a in a complex cell extract, the fluorescence signal of miR-Let-7a in the sample containing total RNA of human lung carcinoma cells (H1299) was evaluated. Therefore, BAMP would be very important to rapidly detect miRNA in clinical application and in the field of "POCT" owing to its simplicity, specificity, and anti-jamming.In chapter Ⅲ, we presented a single primer-triggered isothermal amplification (SAMP) method to detect dsDNA. The method successfully used nicking endonuclease and polymerase to linearly switch dsDNA target to ssDNA. The primer hybridized with ssDNA and extended. The extended primer could dynamically dissociate from the ssDNA to form "turn-back structure", which could further initiate another cyclic amplification reaction. The free ssDNA could bind to another primer and repeat the above-described reaction process. SAMP employed a single primer to initiate a non-linear cascade signal amplification reaction with polymerase and nicking enzyme for dsDNA detection by above-described three cycles amplification. Real-time fluorescence detection of SAMP was initiated by different amounts of pBlu2KSP target. The time corresponding to the maximum slope in the fluorescence curve as well as the point of inflection (POI) was linearly related to the negative logarithmic (lg) value of the target amount in the range from 10 amol to 0.01 zmol. Therefore, BAMP showed a good dynamic range of 6 orders of magnitude. BAMP had good specificity so that one-base mutation also could be well distinguished. Moreover, the method was anti-jamming because the plasmid could be detected in the presence of Escherichia coli genomic DNA. Finally, Cyprinus carpio and Gallus gallus were simultaneously detected in one pot by BAMP. In summary, BAMP provided a new way for the detection of dsDNA.