RNA Based Isothermal Amplification Detection Method

Nucleic acid is the material basis of genetic variation and the supporter of genetic information.It plays an important role in protein synthesis.Genetic,variation,growing development,cells differentiation of organism are closely related to nucleic acid.Nucleic acid contains ribonucleic acid(RNA)and deoxyribonucleic acid(DNA).Especially,RNA is very important in the process of protein synthesis.Meanwhile,RNA is often used as a marker for analysis in the biological analysis,disease diagnosis and food safety.Therefore,RNA detection method that was simple,sensitive and rapid,which is the ultimate goal in the field of detection and main research contents of this paper.Here,we established a RNA detection method that simple,sensitive and rapid based on the isothermal nucleic acid amplification detection method in this paper.In the second chapter,we introduced the concept of strand exchange amplification(SEA)mediated by denaturation bubbles.Similar to traditional PCR,it only employed a DNA polymerase and a pair of common primers to realize a three-step cycle process,but the entire SEA reaction was performed at a single temperature.One-step detection of RNA can be achieved used the Bst 2.0 WarmstartTM DNA polymerase in the SEA reaction system based on the breakthrough that the Bst 2.0 WarmstartTM DNA polymerase possess the activity of reverse.We investigated the feasibility of SEA by detecting the specific sequence of Escherichia coli(E.coli)16S r DNA.The limit of detection can up to the 100 amol.We further performed RNA direct detection in 10% fetal bovine serum(FBS)without any pre-treatment to investigate the feasibility of SEA in biological samples.The fluorescence curves also showed good regularity corresponding to the amount of the RNA target,and 100 amol of target RNA was well distinguished from the negative control,showing that SEA functioned in 10% FBS too.A good capability of anti-jamming is excellent for its practical application.So,SEA is the simple RNA detection method.It can achieve one-step RNA detection.It had the important significance in the clinical application and point-of-care testing.However,the reaction efficiency was unsatisfactory owing to the melt of double-stranded DNA based on the structure of DNA bubble.To enhanced reaction efficiency the modified SEA had been established in the third chapter.In the SEA reaction system,primers with restriction sites were added.The restriction endonuclease made the long ds DNA to the short DNA segment that easy to open in the reaction temperature.However,due to the high concentration of primer with enzyme cutting site in the reaction system.It produces a high nonspecific.To speed up the reaction rate,it is the unavoidable to overcome the nonspecific.So,This paper discusses the nonspecific at last.