Detection Of Mutations In The Tyrosine Kinase Domain Of Chimerical BCR-ABL1 By Multicolor Melting Curve Analysis

The BCR-ABL1 fusion gene is a common genetic abnormality marker in leukemia patients,accounting for 95%of patients with chronic myeloid leukemia(CML),20%-25%of adult patients with acute lymphoblastic leukemia(ALL),5%of children patients with ALL,1%of patients with acute myeliod meukemia(AML).Leukemia patients with BCR-ABL1 fusion gene have poor prognosis,The emergence of molecular targeted drugs has improved the survival rate of patients.However,due to the large-scale use of molecular targeted drugs,drug resistance has been observed in some patients.Studies have shown that patients carrying mutations in the tyrosine kinase domain(TKD)of chimerical BCR-ABL1 are clinically resistant or intolerant to molecular targeted drugs.Therefore,detection of resistant mutations in the TKD of chimerical BCR-ABL1 is of great significance.Sanger sequencing is the gold standard for detecting the drug-resistant mutations in the TKD of chimerical BCR-ABL1.However,the selectivity of Sanger sequencing can only reach 15%-25%,which is often causes missed detection.Therefore,exploring a rapid,accurate,and highly selective mutation detection technique is of great significance for the patients under individualized treatment.The first chapter mainly introduces the characteristics,abnormalities,diagnostic methods and treatments of CML,and the clinical significance of mutation detection.We also introduce the current common mutation detection methods and their advantages and disadvantages.The principle of the multicolor melting curve analysis(MMCA)adopted in this study,and the significance of this study are finally presented.The second chapter is the part of materials and methods,which introduces the source of the plasmid and clinical specimen used in the research,and expounds the design scheme of this thesis.After designing the primers and probes,the target sequence of the molecular beacon probe was examined.The single and multiple assay were established,the components and conditions of the PCR reaction were optimized,the sensitivity and selectivity of the MMCA assay were analyzed,and the applicability of the assay was evaluated by testing the clinical specimens.The third chapter is the part of results and analysis.The MMCA assay is divided into two tubes,A and B.The tube A detects 14 mutant types,and the tube B detects 11 mutant types.Without pre-amplification,the sensitivity of MMCA assay is in the range of 10-100 copies/reaction,the selectivity is between 5%-20%.The clinical samples of 59 leukemia patients carrying BCR-ABL1 fusion gene were detected,three positive samples were detected by MMCA assay.The results of the MMCA assay were consistent with the Sanger sequencing results,indicating that the assay has good detection accuracy and specificity.The fourth chapter is the part of discussion.The MMCA assay based on multiplex real-time PCR technology is one of the effective methods for clinical detection of the drug-resistant mutations in the TKD of chimerical BCR-ABL1,and providing patients with more accurate detection.In summary,we explored the application of molecular beacon probes and mediator probes to detect the drug-resistant mutations in the TKD of chimerical BCR-ABL1,and we established the MMCA assay that can simultaneously detect 23 common resistant mutations.The MMCA assay has the advantages of simple operation,high sensitivity and selectivity and low cost.It is suitable for the detection of the drug-resistant mutations in the TKD of chimerical BCR-ABL1,also it can guide the treatment of the patients,and achieve the purpose of precision treatment.