摘要
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。
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 pMDlg-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 ean be used to make the MSP results mor reliable.
出处
《临床检验杂志》
CAS
CSCD
北大核心
2006年第1期44-47,i0002,共5页
Chinese Journal of Clinical Laboratory Science
基金
江苏省政府"135"重点实验室科研基金(SK200205)。
关键词
质量控制
甲基化特异性PCR
APC基因
quality control
methylation specific PCR
adenomatous polyposis coli
