Plasmid Construction Of PML/RARa(L) Fusion Gene And Reference Gene ABL And Construction Of Duplex Real-time PCR Methods To Detect PML/RARa(L) Fusion Gene

Objective:1,to construct a recombinant plasmid containing PML/RARa(L) fusion gene andreference gene ABL.2,construction of duplex real-time quantitative PCR methods to detect PML/RARa(L) fusion gene and ABL gene.3,to develop a PML/RARa fusion gene detection reagent box to diagnosis acutepromyelocytis leukemia.Methods:1,Total RNA was extracted from the NB4cells using RT-PCR. To clone ABLgene into the plasmid PCMV4-PML/RARa (this plamid was obtained from Dr. ZhangXiaowei who is from Institute of Hematology, Shanghai Jiao Tong University). Thenthe recombinant plasmid was subjected to sequencing. After sequencing, name therecombinant plasmid PCMV4-PML/RARa-ABL. Assess the plasmid's stability andevaluate its' reaction performance.2,construct single real-time quantitative PCR methods to detect PML/RARa(L)fusion gene and ABL gene,On the basis of the establishment of single real-timequantitative PCR methods, construct stable duplex real-time quantitative PCRmethods and develop a PML/RARa fusion gene detection reagent box to diagnosisacute promyelocytis leukemia.Results：1,Successfully construct the standard plasmids containing PML/RARa (L)fusion gene and ABL gene for quantitative detection of leukemia patients with PML/RARa fusion gene.2,Successfully develop single and duplex real-time quantitative PCR reagentbox to detect leukemia patients with PML/RARa fusion gene.Conclusion：1,The recombinant standard plasmid with PML/RARa and ABL double gene is stable, and can be used for accurate quantification. for leukemia patients with PML/RARa fusion gene.2,Evaluate the reaction performance of single and duplex real-time quantitativePCR reaction system,and then successfully develop stable quantitative detectionreagent box.