荧光定量逆转录-聚合酶链反应检测白血病bcr/abl mRNA

目的　建立荧光定量RT PCR法检测白血病融合基因bcr ablmRNA的方法 ,为白血病临床诊断及微量残留病监测提供有用工具。方法　RT PCR扩增培养的K5 6 2细胞bcr abl融合基因 ,A T载体克隆法构建定量标准模板 ,用PE770 0型检测仪 ,建立荧光定量RT PCR方法 ,对方法的灵敏性、稳定性、重复性进行测定 ;定量检测 14例慢性髓细胞白血病 (CML)患者和 4例急性淋巴细胞白血病(ALL)患者外周血样本。结果　建立的荧光定量RT PCR方法 ,可检测出约 10 - 5μgK5 6 2总RNA中的bcr abl融合基因和 10个bcr abl重组质粒拷贝 ,重复性的CT值 (Cyclethreshold)管间、批间变异系数 (CV)分别为 :2 0 % ,3.7%。 14例CML患者bcr abl融合基因表达量中位数为 5 .15× 10 4 拷贝 μgRNA ,产物经电泳分析 ,其中 11例为b2a2 ,3例为b3a2 ,1例 12× 10 4 拷贝 μgRNA的CML患者 ,骨髓移植后 1个月变为 0 .2 3× 10 4 拷贝 μgRNA。 4例ALL患者中 1例有bcr abl融合基因的表达 ,为b2a2 ,表达量为 8.2×10 5拷贝 μgRNA。 结论　建立的荧光定量RT PCR方法灵敏、特异、重复性好 ,结果用拷贝数表示 ,准确可靠 ,利于统一标准。该方法能检测出b2a2、b3a2两种类型的融合基因 ,可广泛用于CML的诊断和微量残留病的检测

Quantification of bcr/abl mRNA expression level in leukemia cells by FQ-RT-PCR

OBJECTIVE: To establish a fluorogenic probe quantitative RT-PCR (FQ-RT-PCR) method for detection of bcr/abl mRNA fusion gene expression level in leukemia cells, and provide a useful tool for leukaemia diagnosis and minimal residual disease inspectation. METHOD: The conventional RT-PCR was used to amplify bcr/abl gene from cultured K562 cells, the quantitative standard template was constructed with A-T clone method. The fluo-rogenic quantitative RT-PCR method by using Applied Biosystems 7700 Sequence Detector for detecting the expression of bcr/abl fusion gene was successfully. The sensitivity, stability and repetitiveness of this method was determined. The peripheral blood samples from 14 CML patients, one of whom before and one month after bone marrow transplantation (BMT) and 4 cases of ALL in the early stages were detected. RESULTS: The sensitivity of FQ-RT-PCR for detecting bcr/abl fusion gene was about 10(-5) micro g RNA from K562 cell and 10 copies recombined plasmid. The repetition CT value (cycle threshold) and the coefficient variation (CV) among tubes and batches were 2.0% and 3.7%, respectively. The median bcr/abl fusion gene expression level of 14 CML patients was 5.15 x 10(4) copies/ micro g RNA. The products analyzed by electrophoresis showed that 11 cases were b2a2 and 3 cases b3a2. 1.2 x 10(5) copies/ micro g RNA in one CML patient before BMT was changed to 2.3 x 10(3) copies/ micro g RNA one month after BMT. B2a2 was observed in one of the four (25.0%) patients with ALL, and its level of expression was 8.2 x 10(5) copies/ micro g RNA. CONCLUSION: The established FQ-RT-PCR method is sensitive, specific, reliable, accurate and good at repetitiveness. The results expressed in copies were easy for evaluation and comparation. Two different bcr/abl fusion gene form - b2a2, b3a2 can be detected by the method. It can be widely applied to diagnosis and detection of minimal residual disease for CML and some ALL patients.