Methodological Establishment And Preliminary Application Of Loop-mediated Isothermal Amplification In The Rapid Detection Of Leukemia PML/RAR?

Background:The CR in clinical AML mainly refers to the recovery of bone marrow function and peripheral blood cell count after chemotherapy.Patients with CR,a so-called cytomorphology,may carry as many as 10¹⁰ leukemia cells in their bone marrow.There are many technical methods for MRD detection of AML,and there is no single standard technique for this sensitive detection.For overexpression of genes or characteristic chromosomal translocations,fluorescence in situ hybridization?FISH?and multiparameter flow cytometry,and real-time quantitative PCR?qPCR?are all viable detection strategies.Regardless of the MRD assay used,it is now generally accepted that MRD positive?MRD+?in CR of cellular morphology predicts a higher risk of recurrence.Similarly,patients with MRD before HSCT have a higher risk of recurrence after transplantation.In recent years,with the development of immunological and molecular biology experimental techniques,the sensitivity and specificity of MRD assay have greatly improved,and its clinical application value has gradually been affirmed.Specific starter mutations in stem cells drive enhanced cloning predominance and leukemia potential,whereas subsequent joint mutations drive progression and transformation and form the basis of the genetic framework.Genetic characteristics have been incorporated into the classic model of AML prognosis,and molecular genetic evaluation has also become a routine part of clinical detection of leukemia.Specifically,the detection of genetic factors of AML can guide clinical remission treatment strategies.In particular,many studies have shown that MRD testing can assess the risk of leukemia prognosis.Current risk assessment related factors include cytogenetics,epigenetics,and gene mutation detection.Cytogenetic abnormality classification is an important basis for judging the risk of leukemia.Acute promyelocytic leukemia?APL?is the most dangerous type of non-lymphocytic leukemia in clinical practice.The complete remission rate of APL has reached more than 80%due to the application of allotropic retinoic acid and arsenic trioxide.However,leukemia relapse has been a major obstacle to post-resolve consolidation,maintenance therapy and affecting patients'overall survival.The root cause of recurrence is mainly from residual leukemic cells in the body,namely acute minimal leukemia?MRD?^[1,2].The translocation of chromosome t?15;17??q22;q21?and the formation of PML-RAR?fusion gene are specific markers of APL,with a positive rate of approximately 98%^[3,4].The detection of fusion gene products and the understanding of the depletion of leukemic cells during therapy are important for monitoring MRD.At present,the detection of minimal residual disease fusion gene mainly through PCR or real-time fluorescence quantitative PCR^[5,6].PCR takes a long time,has low sensitivity and specificity,and requires high instrumentation.Although the specificity and sensitivity are improved,the real-time fluorescence quantitative PCR method requires the generation of fluorescent primers and probes,as well as expensive detection equipment,and requires 2-3 hours.In order to solve the problem of false positives,high detection costs,complicated operation,and long detection time in the detection of leukemia minimal residual disease,we established a loop-mediated isothermal amplification LAMP method to detect PML-RAR?gene.Achieves one-step completion of gene amplification and result determination,simple operation,accurate and intuitive results,high specificity and sensitivity,safety to humans,and non-pollution of the environment.It is suitable for the rapid diagnosis of minimal leukemia of acute leukemia at all levels of hospitals,which exclude primary hospitals that difficult to carry out such inspections.Objective:To establish a loop-mediated isothermal amplification of LAMP to detect the PML-RAR?fusion gene for monitoring minimal residual disease?MRD?.Methods:Log NCBI access to the conserved gene sequence of the PML-RAR?fusion gene,sequence alignment by software,design multiple sets of LAMP primers online and design PCR primers required for the validation phase.A total of 4 LAMP primers,including a pair of specific primers and a pair of specific primers,and according to the reaction time and specificity of different LAMP primer sets of the reaction process and results were detected and screened.PCR primers were designed based on the same nucleic acid sequence to detect gene expression.At the same time,the outer primers F3 and B3 were used as PCR primers for the comparison of the LAMP method and the PCR method.The expression of PML-RAR?gene was detected by LAMP assay and the specificity or sensitivity of the method was analyzed.The naked eye was observed according to the color change of the reaction solution,and the amplification result was judged.Results:LAMP method was used to detect the fusion gene of PML-RAR?,and the time needed was shortened,and the results of the amplification could be observed directly with the naked eye,and the lower detection limit of LAMP method was lower than that of PCR method;The clinical samples were selected for LAMP and nested PCR detection,and the detection results of the two methods were not significantly different,with a higher agreement.Conclusion:1.LAMP method is simple and convenient.Just mix the sample?containing the target gene?with the prepared reagent and incubate at 65°C for 1 hour to detect the amplification product or determine whether the amplification reaction occurs or not.2.LAMP method does not need to change the temperature,only the reagents are added to the reaction tube,including the entire process of denaturation step can be completed in a short time under isothermal conditions,can detect the amplification product or determine the occurrence of amplification reaction.3.LAMP detection PML-RAR?fusion gene method,convenient,simple,one-step rapid detection of LAMP method is a simple,rapid gene amplification method.And LAMP method recognizes six segments by four kinds of primers,and only amplifies the target gene,which is an accurate gene amplification method.4.As the LAMP method does not require special reagents,it becomes a gene detection method that can effectively control the cost.The LAMP method is a simple genetic assay that allows real-time detection.5.After the use of loop primers,the amplification time can be shortened to 1/2 to1/3 of the original.6.LAMP method for detecting PML-RAR?fusion gene with higher Specificity,sensitivity,and clinical fit.