【摘要】 在急性早幼粒细胞白血病 (APL)细胞中迅速、准确检出PML/RARα融合基因对于及时应用全反式维甲酸 (ATRA)或亚砷酸 (As2 O3 )提高诱导缓解率、减轻出血导致的早期死亡至关重要 ,但目前临床上采用的嵌套式RT PCR方法繁琐且费时 ,亟待改进以满足临床APL快速、准确诊断的需要。本研究采用TRIZOL 快速提取高质量的细胞总RNA ,随机引物反转录 ,热启动单轮PCR ,适当控制MgCl2 及引物浓度和Taq酶用量 ,4条引物 ,3个体系 ,相同循环参数 ,对 4 0例初诊APL患者骨髓标本同时扩增PML/RARα融合基因及内对照RARα。结果显示 ,4 0例APL患者标本均扩增出特异条带 ,非APL标本无特异扩增产物 ;阳性扩增片段清晰易辩、断裂点类型易于确定 ,检测可在 6小时内完成。结论 :此改进的RT PCR检测PML/RARα融合基因快速、特异、简便 ,完全满足临床对初诊APL诊断的需要更多还原

【Abstract】 Detection of the PML/RARα fusion gene by RT PCR in acute promyelocytic leukemia (APL) blasts is not only critical to commence promptly the specific therapy with all trans retinoic acid (ATRA) or arsenic trioxide (As 2O 3), but also essential for the definition of PML breakpoint type and subsequent monitoring of minimal residual disease (MRD). The current PML/RARα amplification techniques with conventional nested PCR are laborious and time consuming, which fails to meet the requirements for rapid diagnosis of APL in clinical practice. Therefore, an easily handled RT PCR methodology for the rapid and accurate amplification of PML/RARα fusion transcripts is needed. A modified one round RT PCR protocol was described with a few variations which includes rapid extraction of high quality cellular total RNA, cDNA synthesis with random hexamer and M MLV reverse transcriptase, optimal concentrations of MgCl 2 (1 mmol/L), PCR primers (0.4 μmol/L) and Taq polymerase (0.01 U/μl), hot start procedure, and concomitant amplification of PML/RARα fusion gene and RARα internal control under the identical thermocycle parameters. The results in 40 patients with newly diagnosed APL showed that the improved RT PCR protocol allowed the rapid detection of PML/RARα fusion gene and the accurate discrimination of its transcript types, and simultaneous amplification of RARα internal control under the identical program in less than 6 hours. There were no false positive or negative results found with the assay. In conclusion, the assay reported here is proved to be a simple, easily handled, and highly specific procedure for the diagnosis of APL cases, particularly those requiring such urgent therapeutic intervention as ATRA or As 2O 3 and meriting its further application in APL management.更多还原