反转录实时荧光定量PCR用于检测白血病相关融合基因分型的性能及应用评价

Performance and application evaluation of RT-q PCR assay for detecting leukemia related fusion gene

目的评价反转录实时荧光定量PCR(RT-qPCR)检测白血病相关融合基因的性能,以确定临床使用该方法的可行性。方法采用RT-qPCR检测临床骨髓标本中的白血病相关融合基因BCR-ABL、PML-RARα及AML1-ETO的RNA,验证准确性、特异性、最低检出限、精密度、抗干扰和融合基因亚型检出能力等性能指标,并对其临床应用进行初步评价。结果RT-qPCR检测白血病相关融合基因BCR-ABL、PML-RARα及AML1-ETO的阴阳性符合率为100%,特异性和准确性得到验证;该方法检测下限为100 copies/每反应,其批内和批间精密度CV值均<10%;精密度符合实验要求;胆红素、甘油三酯和血红蛋白对该方法无明显干扰作用;均能检出融合基因亚型;白血病患者治疗前融合基因表达为阳性,完全缓解、巩固、维持各期的相关融合基因表达均为阴性,此结果与疾病进展关系一致。结论采用RT-qPCR检测白血病相关融合基因试剂盒符合规定的性能指标,该检测方法具有较高的灵敏度和较好的准确性和重复性等优势,适合应用于临床。

Objective To evaluate the performance of RT-qPCR in the detection of leukemia related fusion genes and determine the feasibility of clinical application of this method.Methods RT-qPCR was used to detect RNA-associated fusion genes such as BCR-ABL,PML-RARαand AML1-ETO RNA in clinical bone marrow samples;and the performance indicators including accuracy,specificity,lowest detection limit,precision,anti-interference ability and the ability to detect fusion subtypes were verified;the clinical application was preliminarily evaluated.Results The negative/positive coincidence rates of RT-qPCR detection of leukemia associated fusion genes like BCR-ABL,PML-RAR and AML1-ETO were 100%,and their specificity and accuracy were verified.The lower limit of detection was 100 copies/test,and the precision CV value within and between batches was<10%.The sensitivity and precision meet the experimental requirements.Bilirubin,triglyceride and hemoglobin did not interfere with the method.All fusion subtypes can be detected.The expression of fusion gene was positive before treatment in patients with leukemia,and the expression of related fusion gene was negative in stages of complete remission,consolidation and maintenance,which was consistent with the disease progression.Conclusion The study shows that the RT-qPCR test kit for the detection of leukemia related fusion genes met the prescribed performance indexes,and the method has the advantages of higher sensitivity,better accuracy and repeatability.It was suitable for clinical application.