Melt Array Technology For Detection Fusion Genes In Acute Lymphoblastic Leukemia

Leukemia is a hematological malignant tumor in which chromosome translocations and deletions are common mechanisms in fusion genes.ALL(Acute Lymphoblastic Leukemia)is a common subtype accounting for 15%of leukemia and 30%to 40%in AL(Acute Leukemia).Carrying out fusion gene screening is of key clinical significance for patients with early clinical diagnosis of ALL to determine the specific classification and select appropriate diagnosis and prognosis.In this paper,the MeltArray technology combined with HAND system(Homo-Tag Assisted Non-Dimer)were used to establish an ALL fusion gene screening system.In the first chapter,we briefly reviewed the pathogenesis,diagnosis,molecular genetic abnormalities and therapy of ALL.At the same time,several common fusion gene detection methods are listed and compared.Finally,the purpose,content and significance of this paper were summarized.The second chapter includes the principles of selecting the detection objects of the system,the principles of primers and probes design and the establishment of the PCR system.After a series of optimizations,the four-reaction,four-color PCR method was developed for screening of 49 detection targets involving more than 135 fusion forms.To further improve the sensitivity,a pre-amplification system was established on the basis of multiple systems,and a comprehensive performance inspection was conducted.In the Results and Analysis of chapter three,the the performance of the multiplex system was examined.The sensitivity of the screening system in four reaction tubes was 100 copies per reaction.In the stability examination experiments,the Tm vavle of melting peak of each fusion gene in each reaction tube was stable(CV<1%);In the specificity experiments,the melting peaks of the four reactions were well distinguished,and no cross-signal was generated between the positive samples and no non-specific signals were generated in the negative samples.We used 115 clinical blood samples to evaluate the system.A total of 8 positive samples were detected,involving 4 fusion genes.The test results were consistent after single PCR system and sequencing verification.In summary,this thesis established an ALL fusion genes screening system which proved to be a convenient,high-throughput and low-cost method with noticeable clinical significance.It is expected to be combined with chromosome karyotype analysis for the fusion gene screening of newly diagnosed patients of ALL which can assist physicians in diagnosis classification,prognosis judgment and selection of appropriate treatment.At the same time,the design method of this system is also applicable to the screening of other subtype fusion genes such as AML(Acute myeloid leukemia),which is expected to help patients achieve more systematic and comprehensive detection.