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目的建立免疫球蛋白重链(IgH)的实时定量(RQ)-PCR 检测体系,以用于儿童急性淋巴细胞白血病(ALL)微小残留病(MRD)检测。方法采用定性 PCR 筛选109例儿童 B 细胞 ALL(B-ALL)IgH 基因单克隆重排,根据连接区序列设计等位基因特异性引物,应用 RQ-PCR 技术,采用胚系 Taqman 探针与引物,在41例患儿中建立 RQ-PCR 定量检测方法,并分析其敏感度、特异性等。结果109例儿童 B-ALL 初诊标本中 IgH 单克隆重排有48例,经测序分析发现,V、D、J 片段频率最高的分别为 V3家族、D3家族和 J4家族。RQ-PCR 方法扩增48例 IgH 单克隆重排的初诊 DNA,其中41例可重复敏感度和最大敏感度均≤10~(-4),非特异性扩增的循环阈值(Ct 值)均>40,与特异性扩增 Ct值的差距均>3。标准曲线斜率均值为-3.31±0.19,截距均值为37.66±1.23,相关系数均为0.97以上。结论以 IgH 基因重排为靶分子,采用 RQ-PCR 方法和胚系探针策略检测儿童 B-ALL,敏感性≤10~(-4),并具有较高的特异性,适于 MRD 的定量研究。
Objective To establish a real-time quantitative (RQ) -PCR system for immunoglobulin heavy chain (IgH) detection for the detection of minimal residual disease (MRD) in children with acute lymphoblastic leukemia (ALL). Methods A total of 109 cases of childhood B cell ALL (B-ALL) IgH gene were screened by qualitative PCR. Allele-specific primers were designed according to the junctional region sequence. RQ- RQ-PCR quantitative assay was established in 41 children and its sensitivity, specificity and so on were analyzed. Results The IgH monoclonal rearrangements in 109 cases of children with B-ALL had 48 cases of rearrangement. According to the sequence analysis, the highest frequencies of V, D and J fragments were V3, D3 and J4, respectively. 48 cases of newly diagnosed IgH with monoclonal IgH rearrangement were amplified by RQ-PCR. The reproducibility and sensitivity of 41 cases were all ≤10 ~ (-4), and the non-specific amplification cycle threshold (Ct) was> 40, and the difference between the specific amplification Ct values were> 3. The mean slope of the standard curve was -3.31 ± 0.19, the intercept average was 37.66 ± 1.23, and the correlation coefficients were all above 0.97. Conclusion The IgH gene rearrangement as a target molecule, RQ-PCR method and germ-line probe strategy detection of children with B-ALL, the sensitivity ≤ 10 -4, and has high specificity, suitable for quantitative MRD the study.