Application Of Capture And Ligation-based Nucleic Acid Detection Methods In Molecular Diagnosi

Objective:With the development of social medical level,individualized precision medicine has gradually become the focus of public attention.It mainly refers to the screening,diagnosis,prognosis evaluation and medication guidance of diseases from the perspective of genetics.The increasingly mature molecular diagnosis technology occupies an important part in it.part.Through the molecular diagnostic technology based on PCR amplification technology for early screening of major infectious diseases,screening for tumor-specific markers,etc.,we can conduct large-scale epidemiological investigation and analysis and formulate personalized treatment plans,This has a far-reaching impact on improving the overall population health of our country.However,the current PCR-based nucleic acid amplification technology still has certain problems.Large-scale nucleic acid extraction after sample processing is time-consuming and labor-intensive,and the RNA target is prone to degradation or breakage during the extraction process,which limits the detection sensitivity to a certain extent;The fluorescence detection channel is restricted,and the PCR amplification multiple is limited.In summary,a sensitive and specific nucleic acid detection technology without nucleic acid extraction can solve some existing problems in molecular diagnosis.This study innovatively improved a high-specificity,high-sensitivity,nucleic acid detection method based on capture and ligation It was applied to the detection of fusion gene of childhood leukemia and human immunodeficiency virus,and successfully realized the high-throughput nucleic acid detection and quantitative research of these two diseases.Methods:In this study,a nucleic acid-free,sensitive and specific PCR nucleic acid detection method based on capture and ligation was applied to the qualitative and quantitative detection of fusion genes in childhood leukemia.Parallel detection of in vitro translation RNA(IVT-RNA)and patient blood samples was carried out,and the sensitivity and specificity of this method were evaluated by methodological comparison,and then 50 cases from Beijing Children’s Hospital affiliated to Capital Medical University Samples from children with acute lymphoblastic leukemia were tested and verified.On the basis of PCR,we used MALDI-TOF mass spectrometry to initially establish a multiple fusion gene mass spectrometry technology based on capture and ligation,and then used IVT-RNA to evaluate the feasibility of the method.In addition,this study applied a nucleic acid detection technology method based on capture and ligation to streptavidin-coated magnetic beads,combined with LAMP(loop-mediated isothermal amplife-ication)constant temperature amplification to establish a nucleic acid-free Extracted,sensitive and efficient loop-mediated isothermal amplification(capture and ligation probe-LAMP,CLIP-LAMP)method system based on magnetic bead capture and ligation,which is used for the qualitative and quantitative detection of infectious disease HIV IVT-RNA,while evaluating the sensitivity and specificity of the system.Results:In this study,the 96-well plate-based CLIP-PCR nucleic acid detection technology was successfully applied to the detection of fusion genes in childhood acute lymphoblastic leukemia,and qualitative,quantitative and dual detection of two common fusion genes,BCR-ABL1 and ETV6-RUNX1,was carried out.BCR-ABL1 can stably detect 192 copies,ETV6-RUNX1 can stably detect 24.24 copies,and the same detection limit can be obtained by methodological comparison with ordinary RT-PCR kits.We further combined CLIP-PCR and nucleic acid mass spectrometry to carry out preliminary feasibility analysis of multiplex detection of BCR-ABL1 and ETV6-RUNX1 fusion genes.The experimental results proved that this method was successfully applied to the detection of multiple fusion genes,and the detection results were good.In addition,the loopmediated isothermal amplification method based on magnetic bead capture and ligation constructed in this study has been successfully applied to the qualitative and quantitative detection of HIV,a major infectious disease,and can stably detect 32 CPs/μL.Viral load,the detection method has good specificity,and has the possibility of processing largevolume samples>1 mL,and the entire reaction can be controlled to complete within 2 hours.Conclusion:In this study,based on the nucleic acid detection technology based on capture and ligation,it was combined with the constant temperature amplification on magnetic beads and the PCR amplification method system in the capture plate,and successfully established the qualitative detection of RNA without nucleic acid extraction as the target.And quantitative detection technology,and applied to the detection of fusion genes and HIV in blood tumors.Through the analysis of the results,it was proved that the PCR amplification and nucleic acid mass spectrometry method system based on 96-well plate capture and ligation was applied to the qualitative and quantitative detection of fusion genes,and achieved high sensitivity and specificity;The mediated isothermal amplification method system can specifically capture RNA targets and perform highefficiency amplification,and has the potential to become a sensitive,efficient,simple and fast HIV screening technology in border areas.In conclusion,the nucleic acid detection method based on capture and ligation has been successfully applied in the fusion gene and human immunodeficiency virus in children’s hematological tumors,and provides new methods and ideas for the detection of other diseases.