Research On Detection Technology Of Isothermal Amplification DNA Based On Selective Position Probe Mediated Cleavage

The 21st century is the century of life science.As the basic genetic material in life,nucleic acid plays a decisive role in life activities.Therefore,innovative research on nucleic acid detection technology is very necessary.The cleavage and amplification technology,which is the core and difficult point of nucleic acid detection technology,has become a global research hotspot.The main technologies for nucleic acid-specific cleavage are zinc finger nucleases(ZFNs),transcription activator-like effector nucleases(TALEN),Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9(CRISPR/Cas 9)and restriction endonucleases(REase).ZFNs and TALEN are based on Fokl enzyme protein-mediated DNA-specific scission technology,which requires the design of the corresponding protein structure for each cleavage sequence,the experimental process is complicated;although CRISPR/Cas 9 simplifies the experimental process,its guide RNA sequence is long and may form a secondary structure during the operation,which has an effect on the experiment.REase is widely used in nucleic acid cleavage because of its good shear specificity,ease of availability,and low experimental cost.Following Polymerase Chain Reaction(PCR),a large number of isothermal amplification techniques have been developed.Among them,the exponential isothermal amplification reaction(EXPAR)is favored because of its simple template design and no need for primers,but it will produce a strong non-specificity for nucleic acids with long target sequences.In summary,based on a large number of literature studies,this paper has carried out a selective position probe-mediated isothermal amplification assay(SPPMC-EXPAR),single base mismatch detection based on SPPMC-EXPAR,new detection methods based on aptamer-magnetic beads-mediated SPPMC-EXPAR,the main innovative research results are as follows:1.A selective position probe(SPP)-mediated cleavage(SPPMC)technique has been developed.The core technology is to design a novel asymmetric hairpin structure selective shear probe.The probe structure is more stable,the shearing efficiency is higher,and the C-base selective binding site can be realized.Programmable arbitrary site cleavage of the target sequence without the recognition site can be achieved,and with good versatility2.A SPPMC-EXPAR amplification detection technique was developed.SPPMC cleaves the target sequence,and the resulting short sequence for the EXPAR reaction can inhibit non-specific amplification to some extent.Experiments prove that SPPMC-EXPAR can realize indirect detection of target sequences,and it is expected to achieve high sensitivity and standardization of detection3.A method based on SPPMC for C base mismatch detection has been developed.The method is divided into SPPMC and EXPAR combined detection method,the minimum detection concentration can reach 10-12 M;and SPPMC combined with gel electrophoresis screening method can detect target sequence with concentration greater than 10-7 M,the method is fast Simple,with good application prospects.4.A new aptamer-magnetic bead-mediated SPPMC-EXPAR assay was developed.The method has the advantages of high specific detection target recognition without sample pretreatment,rapid separation of target objects in complex systems,detection signal amplification,and high sensitivity indirect detection.In theory,this method can achieve highly sensitive and even single molecule detection.The detection of methamphetamine drugs proved the effectiveness of the method.