Multicolor Melting Curve Analysis For The Detection Of Leukemia Fusion Genes

Leukemia has attracted widespread attention for its high morbidity and mortality.Hematological malignancies are more susceptible to chromosomal aberrations than solid tumors,and chromosomal translocations and deletions lead to the production of fusion genes.Fusion genes are closely related to the occurrence,treatment and prognosis of leukemia.The Clinical screening of fusion genes for patients with newly diagnosed leukemia is crucial for their attending doctor to make decisions about diagnosis,treatment and prognosis.In this thesis,the multicolor mediator probe melting curve analysis technology was used to develop an efficient,accurate,low-cost and reliable fusion gene screening system which is suitable for clinical detection.In the first chapter,we briefly introduced leukemia and its pathogenesis,the production of fusion gene,and explained the molecular biology mechanism of fusion gene on the pathogenesis,treatment and prognosis of leukemia,and the significance of clinical detection.Several common fusion gene detection methods are compared at the same time,included their advantages and disadvantages.Then we introduced the principles of the mediator probe melting curve analysis technique used in this paper,and finally put forward the purpose,content and significance of this paper.In the materials and methods in Chapter 2,we first describe the selection principle of detection objects and the design strategy of the detection system.Subsequently,primers and probes were designed for each fusion gene that based on the breakpoints of the fusion genes.In combination with the design principles of the mediator probes,primers and mediator probes were designed and screened on the premise of ensuring specificity.On the basis of completing the establishment of the single-plex detection system of 46 fusion genes,we established a multiplex detection system for detecting 46 fusion genes of leukemia with two tubes and five colors.The system was optimized to examine the performance of the system.Finally,the system was examined using clinical samples.The third chapter is the result and analysis.The reaction A of the detectionsystem was a 32-plex PCR system and a total of 24 fusion genes were detected.The reaction B was a 24-plex PCR system that detected a total of 22 fusion genes.In the examination of the specificity of the system,the melting peaks of the two reactions were well-distributed,no cross-signal was generated between the positive samples,and the negative samples did not generate any non-specific signals,indicating that the specificity of the system was good.In the stability examination experiments,the Tm of melting peak in each reaction tube was stable(CV<1%).A gradient-diluted plasmid was used as a template for sensitivity analysis,the detection sensitivity of reaction A and reaction B was up to 1000 copies/reaction.We used the detection system to detect 340 clinical sample and a total of 90 positive samples were detected.The detect results were verified by single-plex real-time PCR and sequencing.The results were consistent,indicating that the detection results of the detection system were reliable and accurate.The multicolor melting curve analysis system of leukemia fusion genes established in this paper can carry out the simultaneous detection of 46 common fusion genes.It has many advantages such as sensitive,specific,reliable,cost-effective,comprehensive,and easy to perform.Clinically,it can be combined with chromosome karyotype analysis for fusion gene screening in patients newly diagnosed with leukemia.