| Acute lymphoblastic leukemia is a malignant neoplastic disease caused by abnormal proliferation of lymphocytes in the bone marrow.The malignant tumor cells can not only damage the bone marrow,inhibit its normal hematopoietic function,but also damage other tissues outside the bone marrow(such as meninges,lymph nodes,liver,etc.)because of the easy spread of the tumor cells.According to reports,the incidence of acute lymphoblastic leukemia in China is 0.69 per 100,000,of which children are high-incidence groups and seriously threaten human life and health.Therefore,the development of detection methods for acute lymphoblastic leukemia with high specificity and sensitivity to achieve early diagnosis and timely and effective treatment is of great significance for reducing patient suffering and improving cure rate.The methods for early diagnosis of common acute lymphoblastic leukemia include immunoassay,gene detection,nanometer detection,and spectrometry.These methods limit the scope of application because of their low sensitivity,complicated operation process,and long experimental time.Among them,immunoassay methods for detecting tumor cells using specific recognition of antigens and antibodies are the most widely used,but effective techniques for specifically identifying tumor cell surface markers have yet to be improved.Aptamer,which refers to an artificially synthesized single-stranded DNA or RNA molecule that is capable of folding into a secondary or tertiary structure through intramolecular forces and thus has a strong specific binding to a certain target molecule.Performance.Because aptamers not only possess the main properties of antibodies,but also compensate for some of the defects of antibodies,but also has the advantages of easy synthesis,rich target,and simple modification,the researchers are known as "chemical antibodies." In addition,the application of nucleic acid isothermal amplification technology,which is often applied to high-sensitivity detection of nucleic acid strands,has been mature and improved,and it can rapidly amplify 106-109(in exponential form)target nucleus at a specific temperature.Aglycone chain.Based on the above analysis,this paper aims at the development of novel tumor cell detection methods,which can be used to detect nucleic acid strands with high sensitivity by combining the advantages of nucleic acid aptamers as novel tumor targeting recognition molecules and nucleic acid isothermal amplification techniques.A novel detection method for acute lymphoblastic leukemia cells(CCRF-CEM)has been constructed,and combined with real-time fluorescence quantification,highly sensitive and highly specific assays have been performed on the tumor cells.The specific research content and main conclusions are as follows:?.Aptamer-based loop-mediated isothermal amplification(LAMP)assay for the detection of acute lymphoid leukemia cells(CCRF-CEM)In conventional tumor cell assays,in order to reduce the influence of environmental factors on antibodies,nucleic acid aptamers are used as tumor cell target recognition molecules,combined with highly sensitive ring-mediated isothermal amplification technology to achieve high tumor cell Sensitive,highly specific detection.In this study protocol,CCRF-CEM cells were used as the test object,and their specific aptamer sgc8c was selected as the recognition molecule to capture the cell,and a double stem loop template for LAMP amplification reaction containing the sgc8c sequence was designed.After the double stem loop template is incubated with the CCRF-CEM cells for a period of time,the nucleic aptamer will specifically capture the tumor cells,and then the double stem loops not bound to the cells are removed by washing,and then the next LAMP amplification reaction is performed.Quantitative detection of double stem loops enables quantitative analysis of CCRF-CEM cells.This method can specifically detect the number of CCER-CEM cells is 1000,which provides a new idea for the detection of tumor cells with high specificity and high sensitivity.?.Based on the magnetic separation technology constant temperature exponential amplification(EXPAR)detection of tumor cellsBased on the above research ideas,CCRF-CEM cells were continuously used as the target cells for detection,and the detection of highly sensitive tumor cells using EXPAR amplification technology was explored.Compared with LAMP technology,EXPAR technology is simpler in design,requires shorter amplification time,and uses magnetic beads(MB)to connect nucleic acid aptamers more firmly.Magnetic separation technology is also easier to monitor and remove.The specific research scheme is as follows.Firstly,the modified magnetic ball is connected to the aptamer sgc8c,and an oligonucleotide chain is designed to complementarily hybridize to the nucleic acid aptamer.After incubation of the complex(magnetic sphere-nucleic acid aptamer-oligonucleotide chain)with CCRF-CEM cells for a period of time,the aptamer sgc8c captures CCRF-CEM cells,releasing oligonucleosides.Acid chain.This oligonucleotide chain was continued as a primer for the EXPAR amplification reaction and an EXPAR amplified bifunctional template strand was further designed to facilitate the next amplification reaction.The experimental results can realize the detection of tumor cells by real-time detection of oligonucleotide chains.This method combines the advantages of nucleic acid aptamers and isothermal exponential amplification to detect 10 target cells with high sensitivity.The linear range can span 3 orders of magnitude,creating a simple,efficient,and highly operable method.Tumor cell detection method.In addition,the capture of different tumor cells can be achieved by selecting different nucleic acid aptamers,thus enabling highly specific detection of different tumor cells,which has great potential for the development of tumor typing. |
PDF Full Text Request