Studies On The Enzymatic Basis Of “One-step” Detection Of Rnaand The Detection Of Thrombin By Isothermal Amplification

As the carrier of genetic information, nucleic acid detection has been widely used in food safety, biomedical testing, environmental testing, and many other fields. In recent years, the detection methods of isothermal nucleic acid amplification with the advantages of simplicity, rapidity and easy-to-operate have been rapidly developed,which is suitable for point-of-care testing(POCT) and they have become current research focuses. Thus, it becomes the main goal in the biochemical analysis field to develop new, simple, easy-to-use, sensitive and rapid detection methods of isothermal nucleic acid amplification.Here, we first reported the basic research of enzyme for RNA “one-step”detection. In recent years, RNA detection has become one of the most important parts in molecular biology, medical diagnostics and drug discovery fields. Conventional RNA detection methods involve an extra reverse transcription step, which is so complex that limits their further application for RNA rapid detection. In chapter II, we described the basic research on RNA rapid detection. It is a novel finding that Bst and Klenow DNA polymerases possess innate reverse transcriptase activities, which can make reverse transcription and amplification combined to “one-step”. In this chapter,we have demonstrated that Bst and Klenow DNA polymerases could be successfully used to reverse transcribe RNA within 65 nt length by real time RT-PCR and polyacrylamide gel electrophoresis(PAGE). Their reverse transcriptase activity could be comparable with the AMV reverse transcriptase within 65 nt, and the reverse transcription product could be used as an effective template for PCR amplification techniques. Our findings will spur the development of a myriad of simple and easy-to-use RNA detection technologies for isothermal RNA direct detection. This willjust meet the future needs of bioanalysis and clinical diagnosis to RNA rapid detection in POCT, inspection and quarantine.Nucleic acid aptamers can be synthesized and amplified in vitro and bined with a variety of target substances with high specificity and selectivity, so they are widely used in the detection of isothermal nucleic acid amplification technologies in recent years. Since appeared, aptamers have always been the research focus, and have a broad application prospects in laboratory diagnosis reagents, gene regulation and new drug research. In chapter III, thrombin as the star target in aptamers was selected to validate the selection process, which could lay the foundations for the later construction of aptamers selection. In this method, a stem-loop structure of nucleic acid containing aptamer sequences was used as the template for amplification. This hairpin acted as molecular switch, in the presence of the thrombin, the aptamer sequences would combine with its target to form a stable aptamer-target complex, and this combination forced the hairpin to undergo a structural change to release the free 3’-end at the same time. Then the strand displacement reactions proceeded with two primers, nicking enzyme and DNA polymerase. Therefore, the hairpin could be amplified in an exponential way. This method was simple and sensitive for thrombin detection, which could detect as low as 2.6×10-12 mol/L thrombin. To evaluate the anti-jamming capability and specificity, serum, BSA and bovine thrombin were measured,respectively. Thus, it had proved the feasibility of the technology. It will filter to other suitable ligand sequence of bio-molecular detection with new ideas and technical support. Therefore, this approach may offer a potentially useful tool in the fields of molecular selection and molecular evolution.