Construction Of Two Nucleic Acid Amplification Techniques Based On Circular "Polymerization-isomerization" Mechanism And Its Application In Nucleic Acid Detection

Nucleic acid detection plays an important role in disease diagnosis,identification of environmental microorganisms and pathogens.A variety of methods have been developed for the detection of nucleic acids.Among these methods,amplification-based nucleic acid amplification technology can achieve rapid and sensitive detection of low abundance nucleic acid biomolecules,which plays an important role in biomedical research and clinical diagnosis.However,how to further improve the application of existing nucleic acid amplification techniques in life science and medicine;How to develop new nucleic acid amplification techniques to solve the problems in the existing nucleic acid amplification techniques,and the new problems encountered in the process of life research are still a severe challenge for biomedical workers,but also provides new opportunities to strengthen the integration of analytical chemistry with life science and medicine.In this thesis,we aim at the above-mentioned scientific problem and build a thermostatic nucleic acid amplification(polymerization and isomerization cyclic amplification,PICA)based on the self-assembly technology of nucleic acid molecules.Based on the PICA technology,we established the PICA amplification system with reverse transcriptase for the ultrasensitive and specific detection of specific miRNAs in mouse tissues;Combined with the reverse transcription and polymerase characteristics of BST enzyme,we established the PICA amplification system with BST enzyme for the rapid and sensitive detection of miRNAs related to acute myocardial infarction and Boka virus single-stranded nucleic acids;Combined with CRISPR technology,a CRISPR-PICA amplification system was established for sensitive and specific detection of double-stranded nucleic acid of Mycobacterium tuberculosis virus double-stranded nucleic acids.Further,we designed linear hairpin variable primers and constructed an RT-qPCR system based on circular "polymerizationisomerization" mechanism to achieve ultra-sensitive detection of miRNAs by circular reverse transcription(RT)and PCR.The details of the study are as follows.Part Ⅰ: Thermostatic nucleic acid amplification based on cyclic polymerization-isomerization mechanism and its application in nucleic acid detection.1.Construction of cyclic "polymerization-isomerization" thermostatic nucleic acid amplification(PICA)methodThe existing nucleic acid amplification technology,including PCR variation and thermostatic amplification technology,mainly consists of two stages:(1)Target signal conversion process.The target signal is converted to the template signal by various primers(such as PCR primers,LAMP primers,RCA-linked fragments,etc.);(2)Amplification process.The template is amplified exponentially or non-exponentially,mediated by primers,to achieve the specific sequence amplification purpose.These amplification techniques achieve the ability to amplify target molecules millions of times for sensitive detection.However,there is a risk of false positives during nucleic acid amplification.This comes from two main sources:(1)The amplification reaction is between a template molecule and a primer molecule.More primer molecules than the template are often added during amplification,and the excess free primer molecules may react to form dimers and give false positives.(2)The template used for each amplification is different,and it is an exponential or linear replication with the constantly newly generated product as the template.During the amplification process,especially at the early stage of amplification,a mismatch may cascade amplification and a false positive may occur.To this end,we have developed a novel thermostatic nucleic acid amplification method,namely the cyclic "polymerization-isomerization" thermostatic amplification(PICA)method.This method embeds a template and primers in a DNA hairpin and uses the strand-swapping activity of Bst polymerase to generate long single-stranded DNA(ss DNA)with tandem repeats in a programmable and autonomous manner by cyclic alternating polymerization and isomerization.Self-extension amplification of nucleic acid molecules without the involvement of an applied primer is achieved.The correctness of PICA amplification was verified by various means such as polyacrylamide gel electrophoresis(PAGE),atomic force microscopy(AFM),enzymatic digestion,and sequencing.2.Reverse transcriptase-PICA amplification system for ultra-sensitive and specific detection of specific miRNAs in mouse tissuesPICA is a nucleic acid amplification system,and a complete detection platform can be built by designing a recognition system that can identify and convert the target into PICA.The detection of the target can be achieved by designing a recognition system that can identify and convert the target into PICA and construct a complete detection platform.In this chapter,a specific structured linear hairpin variable primer is designed for miRNA detection.Under the action of reverse transcriptase,the primer is recognized with the target and then reverse transcribed to generate asymmetric hairpin DNA,which is amplified by PICA to achieve target miRNA detection.The whole detection is divided into two processes: the reverse transcription step and the PICA reaction step.The linear hairpin variable primers are designed to extend the initial hairpin DNA to form PICA in the presence of target miRNA and reverse transcriptase;then,the target miRNA is detected through the PICA amplification process.The results showed that the method has a linear range of up to 6 orders of magnitude and a detection limit of 1.2 a M,which can effectively detect single base mutations and has good multiplex target detection ability.In addition,the reliability of the method was verified by comparison with Taqman RT-qPCR.3.BST-PICA amplification system enables rapid and accurate detection of miRNAs related to acute myocardial infarctionAcute myocardial infarction(AMI)is a critical clinical type of coronary heart disease,which progresses rapidly and has serious consequences.If not diagnosed and treated promptly within hours,the myocardium can become ischemic and hypoxic resulting in irreversible necrosis and death.Recent studies have identified specific circulating miRNAs that are significantly altered in the blood of patients with early AMI and are potential early detection markers for AMI.The above-mentioned high sensitivity and specificity of miRNA detection was achieved by reverse transcriptase-PICA amplification system,however,because reverse transcription and amplification are performed in steps,it takes a long time and is not conducive to the rapid diagnosis of AMI.Based on this,the BST-PICA amplification system was constructed using the feature that Bst 3.0 DNA has both polymerase activity and reverse transcriptase activity,and the reverse transcription and PICA amplification processes were carried out simultaneously to achieve rapid and sensitive detection of AMIrelated miRNAs.The detection time was controlled within 60 min,the detection limit was 1.5 a M,and the homologous miRNAs and miRNA precursors with single base mismatches could be effectively distinguished.The detection of three miRNAs(mi R-133 a,mi R-208 b,and mi R-499a)associated with AMI in clinical serum samples enabled the effective discrimination between healthy individuals and AMI patients.4.BST-PICA amplification system enables sensitive and specific detection of Boka virus single-stranded DNA nucleic acidBoca virus(HBo V)is a single-stranded envelope-free DNA virus,which is closely related to human respiratory infections and is one of the important respiratory pathogens for respiratory infections.By detecting HBo V,we further investigated the ability of the PICA amplification system to detect singlestranded DNA.We introduced a cyclic replication(CR)step,through which linear hairpin variable primers hybridize to the target single-stranded sequence and extend to form asymmetric hairpin DNA,which passes through the PICA amplification process for single-stranded DNA nucleic acid detection.The results showed that the method has a linear range of 6 orders of magnitude and a detection limit of 4 a M for detecting single-stranded DNA nucleic acid,which can effectively distinguish the single-stranded nucleic acid of different viruses with good detection specificity.5.CRISPR-PICA amplification system for accurate detection of Mycobacterium tuberculosis double-stranded DNA nucleic acidThe sensitive detection of Mycobacterium tuberculosis is important for the early diagnosis and treatment of tuberculosis.The ability of PICA amplification system to detect double-stranded DNA was further investigated by detecting Mycobacterium tuberculosis with a double-stranded nucleic acid structure.A CRISPR-based PICA method for ds DNA detection was constructed by transforming ds DNA into the initial DNA hairpin structure of PICA using the specific recognition and cleavage ability of CRISPR/Cas9 protein for doublestranded DNA.The results showed that by combining CRISPR/Cas9 and PICA,the dual specificity of ds DNA detection was achieved with a detection limit as low as 540 z M,and the detection of nucleic acid extracts from sputum could effectively differentiate between healthy individuals and patients with tuberculosis.Part Ⅱ: PCR amplification based on circular "polymerizationisomerization" mechanism and its application in nucleic acid detection6.Linear hairpin variable primer RT-qPCR amplification system for specific and sensitive miRNA detectionStudies have shown that some specific miRNAs are early detection markers for diseases and play an important role in early detection,diagnosis and treatment of diseases.However,miRNAs have problems of small size and high similarity.Traditional RT-qPCR has designed two specific types of primers for solving this problem.However,for linear primers,the specificity is insufficient;for hairpin primers,the background signal is relatively high.To this end,we designed linear hairpin variable primers and constructed a variable primer RTqPCR amplification system based on cyclic "polymerization-isomerization" to effectively reverse transcribe miRNAs by cyclic reverse transcription RT.The method was used to achieve ultra-sensitive detection of miRNAs.The results showed that the method is sensitive down to 4 target miRNA molecules,with a linear range of 8 orders of magnitude and high selectivity for miRNA sequences with single-base differences.The ability of the method to detect multiplexed miRNAs was demonstrated by the simultaneous detection of seven miRNA targets in seven mouse tissues using multiplexed RT.