bcr-abl P210和abl基因实时荧光定量PCR检测共用质粒标准品的制备及应用

 目的 建立一个荧光实时定量PCR（RQ-PCR）共用质粒标准品，用于同时检测bcr-abl P210白血病融合基因和abl内参基因。方法 分离K562细胞RNA，反转录为cDNA，以此为模板，设计引物进行PCR，PCR产物经琼脂糖电泳、胶回收，T-A载体克隆后，转化JM109工程菌，抽提质粒、测序、测定拷贝／μl，冻存备用。以RQ-PCR和定性巢式PCR进行验证，检测23例CML患者的骨髓标本，并同bcr-abl基因荧光原位杂交（FISH）结果比较。结果 获得了预期的模板质粒，以欧洲抗癌协会推荐的引物探针RQ-PCR检测bcr-abl P210和abl基因，浓度相同时，Ct值相同；反复检测日内差和日间差均<5 %。23例标本按FISH结果≥10%（n=8）、0.5 %～10 %（n=6）和阴性（n=9）分组，RQ-PCR结果分为0.492±0.263、0.023±0.033和（4.33±3.84）×10-4；FISH阴性组和其余两组间P值均＜0.001，三组间P值均＜0.05。结论 该研究建立了bcr-abl P210基因和abl基因共用的质粒标准品，定量准确，性质稳定，能简化操作，满足临床检测和科研的要求。

One plasmid standard for detecting both bcr-abl P210 and abl gene cDNA in real-time quantitative RT-PCR

Objective To establish a plasmid standard for detecting both bcr-abl P210 and abl gene cDNA in real-time quantitative RT-PCR (RQ-PCR). Methods 473 bp cDNA sequence was RT-PCR from K562 cell, which was subcloned into pMD18-T vector, after sequencing, the plasmid standard was tested by RQ-PCR and nested RT-PCR, 23 bone marrow samples of chronic myeloid leukemia (CML) patients were al－so examined. Results The plasmid quite well qualified for bcr-abl P210 and abl gene detection with Taqman primers and probes suggested by Europe Anti-Cancer group, coefficients of variation among different experi－ments are less than 5 %. 23 cases CML patients divide into 3 groups by FISH value≥10% (n=8), 0.5 %～10 % (n=6) and negative (n=9), the RQ-PCR ration of bcr-abl-abl are 0.492±0.263, 0.023±0.033 and (4.33±3.84)×10－4, P ＜0.05 between each group. Conclusion Being sensitive, specific, reliable and accurate, the plasmid is good for detecting both bcr-abl P210 and abl gene.