甲基化特异性基因扩增检测过程中的质量控制

目的构建adenomatous polyposis coli(APC)基因启动子1A的甲基化阳性质粒标准品,对甲基化特异性基因扩增(meth-ylation specific PCR,MSP)的检测灵敏度进行质量控制.方法对脐血淋巴细胞DNA转甲基并化学修饰,制备甲基化阳性和阴性模板,MSP后获得目的片段,克隆到pMD18-T质粒载体,测序筛选阳性和阴性质粒标准品,紫外分光法对标准品定量,对10倍梯度稀释的质粒进行SYBR Green Ⅰ荧光定量PCR来获得弱阳性质控品,对36份组织样本进行MSP检测.结果将MSP后电泳能显示的质粒(105拷贝/ml)作为弱阳性质控品.在加入弱阳性质控品后,2例阴性的样本最终定为阳性;11例未定结果的样本最终7例定为阳性,4例定为阴性.36例样本无质控品前的甲基化阳性率为25%,有质控品后的阳性率为50%,两次结果差异显著(P=0.001).结论构建了携带甲基化阳性的APC基因启动子的质粒标准品并制备了低拷贝的弱阳性质控品,可以对MSP检测灵敏度进行质量控制,降低了检测的假阴性率.

Quality control of the procedure of methylation-specific PCR

Objective To develop a methylated plasmid control of promoter 1A of adenomatous polyposis coli (APC),and control the sensitivity of methylation specific PCR (MSP) of clinical samples.Methods Methylated and unmethylated PCR products were cloned into plasmid pMD18-T.Liner plasmid for lowest sensitive control (cut-off control) of MSP was quantified with the method of UV absorption and quantitative MSP was carried out using SYBR Green I.Thirty-six samples were detected with the improved MSP.Results The methylated and unmethylated plasmid controls of promoter 1A of APC were made.The sensitivity of FQ-MSP was 104 copy /ml and the cut-off control of MSP was 105 copy /ml.With the weak positive cut-off control,two negative samples detected formerly were confirmed as positive;uncertain 11 samples were confirmed as 7 positives and 4 negatives.The methylation rate of 36 samples was 50%.This result showed very significant difference compared to the methylation rate of 25% without the cut-off control (P=0.001).Conclusion The methylated plasmid cut-off control of promoter 1A of APC was developed,which can be used to make the MSP results more reliable.