Abstract
目的 比较不同类型的逆转录荧光实时定量聚合酶链反应(RQ-PCR)检测急性早幼粒细胞白血病(APL)PML-RARα融合基因的方法.方法建立荧光染料SYBR Green Ⅰ和Eva GreenRQ-PCR技术,同时检测56例APL融合基因PML-RARα的表达水平,并且与传统巢式PCR方法进行敏感度比较.选择10例初诊患者随机分为两组,分别给予维甲酸(ATRA)加化疗或ATRA加三氧化二砷治疗.每3周抽取骨髓标本,检测治疗后PML-RARα表达的变化.结果两种RQ-PCR的灵敏度均可达到50拷贝,稍低于巢式PCR,但RQ-PCR定量准确,不易污染.分别取103拷贝和107拷贝的PML-RARα质粒以及患者cDNA同时做8次SYBR-Green Ⅰ平行扩增,批内变异系数分别为7.31%、8.26%和5.02%.不同APL患者PML-RARα表达水平有较大差异,初诊患者PML-RARα/GAPDH比值介于0.018～0.799,中位值为0.070;使用这两种染料建立的RQ-PCR方法进行定量检测,荧光染料Eva-Green荧光强度高于SYBR GreenⅠ 4倍.对接受ATRA加化疗或ATRA加三氧化二砷治疗两种不同治疗方案的APL患者动态观察发现,2～4周PML-RARα转录本均迅速减少,2个月后80%PML-RARα转阴,但两种治疗方式无明显差异.结论RQ-PCR可以准确检测微小残留病(MRD)融合基因表达水平;Eva Green和SYBR Green Ⅰ都可以用于RQ-PCR,但是Eva Green更敏感高效;监测融合基因表达可以有效观察不同治疗方法的治疗效果。
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