Research On The Application Of Nucleic Acid Probe Technology In Identifying The Authenticity Of Tetrastigma Hemsleyanum Diels Et Gilg

Tetrastigma hemsleyanum Diels et Gilg is a member of the Magnoliophyta,Vitaceae,Albertisia Becc.It is a rare Chinese herbal medicine.It is mainly used to treat children with febrile convulsion,bronchitis,hepatitis,rheumatoid arthritis,Viral meningitis and other diseases.Is is also a commonly used anti-cancer antineoplastic drugs.Due to its high medicinal value,large demand,over exploitation and the difficulty of artificial cultivation,wild T.hemsleyanum plants are on the verge of extinction recently.There are increasing numbers of counterfeits on the market,and some people was poisoned when they used adulterants occasionally.Unfortunately there are few methods to identify the authenticity of T.hemsleyanum.DNA is the genetic material of most organisms and the difference in base sequence between species DNA is the main cause of the difference between species.Therefore,it is of decisive significance to carry out the DNA identification of T.hemsleyanum.In this thesis,by comparing the ITS2 sequence of T.hemsleyanum authenticity,we found that the specific sequence of T.hemsleyanum was different from other counterfeit products.The specific sequence was used as the target sequence,then three nucleic acid sensors of T.hemsleyanum are used to quickly identify the authenticity of T.hemsleyanum.This thesis is composed of four chapters,the specific content and research results are as follows:In chapter 1,this chapter is a literature review,which mainly discusses the research progress,present situation and identification technology of T.hemsleyanum,and puts forward the main research contents,research significance and purpose of this thesis.In chapter 2,this chapter constructs a DNA Peroxidase-mimicking DNAzyme sensor with a target sequence as a self-primers and RCA technology which is used for the identification of efficient visualization of the authenticity of T.hemsleyanum.The experimental results show that the reaction product can form G-quadruplex with the help of K+ after the target sequence initiates the RCA reaction.After adding hemin,it is assembled into a catalyzed peroxidic enzyme which catalyzes the reaction of ABTS2--H2O2 system,which can be used to identify the authenticity of the T.hemsleyanum.The limit of detection of the proposed sensor is 65 fM.In chapter 3,we improve the traditional RCA reaction step by using the properties of Exo ? and Exo ?.A RCA fluorescence sensor based on target sequence-induced seal probe structure transition was constructed by pre-synthesis seal probe,which is used to identify the authenticity of the T.hemsleyanum.The experimental results show that the RCA reaction can be triggered by the presence of the target sequence,and the Hairpin DNA products are obtained in series.The fluorescence intensity of the system is enhanced by the addition of the fluorescent dye SYBR Green I.So as to identify the authenticity of the T.hemsleyanum,The limit of detection of the proposed sensor is 0.98 pM.In chapter 4,this chapter combines the HRCA technique with the properties of dsDNA and SYBR Green I compounds to build a sensor with photocatalytic activity to construct a photocatalytic visualization for distinguishing the authenticity of T.hemsleyanum.The results indicated that the sensor can indirectly catalyze the oxidation of TMB under 465 nm LED irradiation,and the sensor has a very high sensitivity,the detection limit of the sensor is 1 pM.So as to identify the authenticity of the T.hemsleyanum.