Study On New Technologies Of Isothermal Nucleic Acid Amplification Based On Nicking Enzyme

Nucleic acid as an important genetic information vector, is widely used in the detection of food safety, biomedical science, environment and other fields. Currently,polymerase chain reaction(PCR) is the most widely used detection method of nucleic acid amplification. However, PCR technology requires specialized thermal cycler,which limits its application in point-of-care testing(POCT). Therefore, it is a great significance to develop a more sensitive and rapid detection method of isothermal nucleic acid amplification. In this thesis, we established several isothermal amplification methods that were simple, rapid and specific to detect DNA or RNA.In chapter II, we established a new isothermal amplification detection method of nucleic acids by a double-nicked beacon. The method cleverly designed a molecular beacon with double nicking enzyme sites, and the amplification reaction was initiated by polymerase and nicking endonuclease to achieve cascade signal amplification. To verify the method, the extracted Escherichia coli(E. Coli) 16 S r DNA of different concentrations were detected, and 10 amol target could be detected. The point of inflection(POI) was linearly related to the negative logarithmic(lg) value of the target amount in the range from 100 fmol to 10 amol. The method showed a strong specificity when the primer had mutated bases. In addition, it also showed a strong anti-jamming capability by detecting targets under complication reaction system.Additionally, it was a one-pot and isothermal strand displacement amplification method without the requirement of a stepwise procedure, which greatly simplified the experimental procedure and decreased the probability of contamination of samples.With its advantages, the method would be very useful to detect nucleic acids in POCT or field use.Among the conventional methods of RNA detection, most of them involve an extra step of reverse transcription, in which RNA reverse transcribed into the complementary DNA(c DNA) strand for next amplification reactions. In chapter III,we established three new methods of isothermal nucleic acid amplification, which could directly amplify and detect RNA no need of adding reverse transcriptase, due to Bst DNA enzyme not only possessing the DNA polymerase activity but also having intrinsic reverse transcriptase activity. Our method fully used the DNA polymerase with strand displacement activity and the nicking enzyme to achieve the multiple signal amplifications. The optimal method was used to detect E. coli 16 S r DNA, and the detection limit was 1 amol. Moreover, 16 S r RNA of E. coli could also be effectively detected at a certain concentration. Under isothermal and homogeneous conditions, these methods could directly detect RNA, which shorten the reaction time greatly, reduced experimental cost and decreased operation difficulty. The established new isothermal methods provide new methods and new ideas for other nucleic acid detection, and they also will have important significance for POCT.