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Research of ’Real-time’ Quantitative Reverse-transcriptase Polymerase Chain Reaction (RQ-PCR) to Detect bcr/abl Fusion Gene in Chronic Myeloid Leukemia

Author: ZhangYinHong
Tutor: ZhuBaoSheng
School: Kunming Medical College
Course: Biochemistry and Molecular Biology
Keywords: CML bcr/abl fusion gene RQ-PCR minimal residual disease(MRD)
CLC: Q78
Type: Master's thesis
Year: 2007
Downloads: 71
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Abstract


Objective To establish a real-time quantitative RT-PCR (RQ-PCR)method for detecting the bcr/abl fusion gene expression level of chronicmyeloid leukemia (CML), and provide a significant reference for CMLdiagnosis and minimal residual disease (MRD) inspectation.Methods The conventional RT-PCR was used to amplify housekeepinggene GUS and bcr/abl fusion gene of cultured K562 cells. Step-diluted PCRproducts were used to establish the quantitative standard curves. TheRQ-PCR method for detecting the expression of bcr/abl fusion gene wasconstructed successfully, then the detection limit, sensitivity,stability and repetition of this method were tested. The peripheral bloodor bone marrow from 19 CML patients were detected by the RQ-PCR method.Polyacrylamide gel electrophoresis was performed to identify thegenotypes of the PCR products.Results Both of the correlations of GUS and bcr/abl standard curveswere up to 0.999; The detection limit of RQ-PCR for detecting bcr/ablfusion gene was about 10-5μg RNA from K562 cells, and the coefficientvariation(CV) of repetition and stability were 2.59% and 3.25%,respectively. Bcr/abl fusion gene was not detected in the normal controls ,the false positivity was 0%; 94.7%(16/19) of 19 patients with CML weretested with positive results. The median of the bcr/abl fusion geneexpression level in the 18 patients was l.58×10-2 ( range 1.20×10-4 - 3.90×10-1 ) . The normalized copy number of bcr/abl fusion gene in chronicphase, accelerated phase and blast crisis phase of CML patients were (1.22±1.50)×10-2 , (l.72±0.86)×10-1 and (2.87±0.90)×10-1 , respectively.The expression level of bcr/abl mRNA in the blast crisis and acceleratedphases of CML patients increased markedly compared to the chronic phase.There were significant difference between the three phases (P<0.05) . ThePCR products analyzed by polyacrylamide gel electrophoresis showed that66.7% (12/18) of bcr/abl fusion genes have b3a2 junction, 27.8%(5/18) haveb2a2(+) and 5.5%(1/18) have both b3a2 and b2a2 types.Conclusions 1.Both Ph chromosome and bcr/abl fusion gene wereimportant diagnosis markers for CML; 2.The RQ-PCR method has goodsensitivity and repetition for detecting bcr/abl fusion gene in CMLpatients, its detection rate is higher than cytogenetic analysis method;3.The bcr/abl fusion gene expression level was highly correlated to thedevelopment of the disease, it is useful in evaluation leukemia cell load,assessing response to treatment and predicating the prognosis of thedisease; 4.Two bcr/abl fusion gene transcripts b3a2 and b2a2 can bedetected by the method. The RQ-PCR method can be used for diagnosis ofCML and for MRD detection during follow-up to evaluate treatmenteffectiveness.

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