EvaGreen染料法实时定量PCR检测急性髓系白血病PRAME基因转录本

Quantification of PRAME transcripts in patients with acute myeloid leukemia using real time PCR with EvaGreen dye

目的：采用实时定量聚合酶链反应（RQ-PCR）法检测急性髓系白血病（AML）中黑色素瘤优先表达抗原（PRAME）基因转录本,初步评价其在AML中的临床意义。方法：设计PRAME基因引物,建立扩增PRAME基因转录本的EvaGreen染料法RQ-PCR法,对PRAME转录本克隆质粒进行扩增,评价其特异性、重复性和灵敏性。并对10例AML患者的骨髓单个核细胞标本进行初步检测。结果：所建立的EvaGreen RQ-PCR法具有良好的特异性,最低可检测到10个拷贝/μl的阳性模板,但其重复性差,而10^8～10^2拷贝/μl阳模的重复性良好。10例初诊AML患者PRAME转录本绝对含量为4.91×10^2～8.07×10^5拷贝/50ng（中位7.26×10^3拷贝/50ng）,ABL标化后的相对含量为4.42%～13 170.01%（中位67.98%）。而对照组8例缺铁性贫血患者中PRAME转录本相对含量为0.00%～0.01%（中位0）。1例AML患者诱导缓解后PRAME转录本下降,复发时又明显升高。结论：EvaGreen染料法RQ-PCR检测PRAME转录本方法敏感可靠,可用于AML患者临床监测。

Objective： To evaluate the method of real-time quantitative polymerase chain reaction（RQ-PCR） with EvaGreen dye for detecting the preferentially expressed antigen of melanoma（PRAME） transcripts in patients with acute myeloid leukemia（AML）.To investigate preliminarily the characteristics and clinical implication of PRAME transcripts in AML.Methods： The primers of PRAME transcript were designed.The condition of RQ-PCR with EvaGreen was established to detect PRAME and ABL positive templates.To evaluate the utility of this assay, the samples of bone marrow mononuclear cells from 10 patientswith AML were detected. Results： There was a good specificity in the EvaGreen RQ-PCR. In repeated tests, maximal sensitivities of 10 copies/μl were obtained, while reproducible maximal sensitivities achieved 100 copies/μl. The median absolute and normalized amount of PRAME transcripts were 7.26 × 10^3 （4.91 ×10^2 ～ 8.07 ×10^5 ） copies/50ng and 67.98% （4.42% ～ 13 170.01% ） respectively. In one AML case, the PRAME transcript level significantly decreased after induction therapy compared to the moment of diagnosis, but significantly increased after relapse. In the control group, consisting of eight samples from patients with iron deficient anemia, the normalized PRAME transcript level was 0.00% ～ 0.01% （ median 0）. Conclusion： EvaGreen RQ-PCR for PRAME transcript was a sensitive, reliable quantitative assay and can be used in the diagnosis of AML and the disease follow-up.