The purpose of this study was to establish real-time based methods for detection of NPM1 gene mutauon m acute myeloid leukemia (AML). Primers/probes were designed according to the clustered region of NPM1 mutations on exon 12. Two real-time PCR assays, including high resolution melting curve (HRM) and allele-specific PCR (ASPCR), were developed and clinically evaluated with 89 AML samples, which were parallelly detected by capillary electrophoresis (CE) and sequencing. The results showed that a total of 17 mutation-positive samples were detected,including type A ( 15 cases), type B ( 1 case) and type Nm ( 1 case). HRM assay could detect all mutant types, and the analytical sensitivity was around 5％. In contrast, AS-PCR assay detected only 95％ mutant types, but its sensitivity was as high as 0. 01％. It is concluded that considering the characteristics of each method as well as the clinical evaluation results, HRM may be used for screening of NPM1 mutations at diagnosis, while the AS-PCR can be used for the MRD quantification during follow-up.%本研究旨在建立检测急性髓系细胞白血病(AML)患者NPM1基因突变的方法.针对NPM1基因突变集中区域设计引物/探针,建立高分辨熔解曲线方法(HRM)和等位基因特异PCR方法(AS-PCR),通过89份AML标本进行了临床评估,并来用毛细管电泳法(CE)和测序法作为对照进行了验证.结果表明,通过上述4种方法共检出阳性标本17例(19.1%),其中A型突变15例,B型和Nm型突变各1例.上述方法中HRM法检测全面,灵敏度为5%,而AS-PCR法有一定的漏检,但灵敏度高达0.01%.结论:考虑到操作的难易程度,同时也结合临床样品的检测情况,HRM在临床上可法用于NPM1突变筛查,而AS-PCR法可用于后续的微小残留病定量检测.
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