The Research In New Methods For Drug And Drug Target Detection Based On The Nucleic Acid Amplification Technologies

Nucleic acid amplification test(NAAT)is a highly sensitive molecular diagnostic technique,based on the basic principle of polymerase chain reaction(PCR),which was developed in 1985,a series of new nucleic acid amplification techniques and detection methods have been developed and gradually become basic research methods of molecular biology,genomics,disease diagnosis.To meet the current development of the clinical and point-of-care testing(POCT),it is of great practical significance to develop a kind of simple,rapid,sensitive and specific nucleic acid amplification technologies and it has a promising application prospect.In this study,a novel nucleic acid amplification detection method with practical application prospect was constructed.We proposed a new method for the detection of platinum drugs and miRNA based on the helper qPCR technology,and established a quantitative detection of T4 polynucleotide kinase(T4PNK)based on the terminal hairpin formation and self-priming extension technology.The whole thesis is divided into three parts:1.IntroductionThe introduction part mainly elaborates the research purpose,significance,main content and technical route of the paper.2.A new method for detection of Pt ions and miRNA based on the helper qPCR systemThe traditional platinum ion detection technique,mainly utilizing optical detection,which requires complicated derivatization processes and were hard to avoid interference of biological matrix.The LC-MS method is the most widely used in the detection of platinum drugs,but it needs expensive technical instruments,the process of separation and enrichment before sample detection is time-consuming and laborious,therefore,we integrated the detection of platinum ions into the polymerase chain reaction(PCR)technology,and developed a new technology of platinum ion detection which is simple,fast,highly sensitive and selective.What's more,the system can be used to quantitatively detect miRNA.The method could detect the minimum concentration of platinum ions was as low as 1ng/mL and the detection limit for miRNA was up to 1pmol.3.A new quantitative method for detection of T4PNK based on the phosphorothioated self-priming extension strategyIn nucleic acid amplification reactions,there are various ways to initiate reactions,most of which rely on primers to initiate amplifications,but in the DNA amplification mechanism,a new variant,namely the 3'-termini hairpin sequence of single-stranded nucleotides,can automatically self-folding and initiating the self-extension reaction.This chapter describes a quantitative detection of T4PNK based on the terminal hairpin formation and self-priming extension technology.We optimized the experimental conditions of the method to improve the self-folding and expansion efficiency,the detection limit is 1mU/mL by the optimized method.