建立实时定量PCR检测急性早幼粒细胞白血病PML／RARα融合基因转录本  

Quantification of PML/RARα fusion gene transcripts in patients with acute promyelocytic leukemia by using realtime quantitative PCR

作　　者：林江[1] 韩兰秀[2] 钱军[2] 王雅丽[2] 钱震[2] 杨小飞[2] 盛晓静[2] 

机构地区：[1]江苏大学附属人民医院实验血液学重点实验室,江苏省镇江市212002 [2]江苏大学附属人民医院血液科,江苏省镇江市212002

出　　处：《中华医学遗传学杂志》2008年第3期319-321,共3页Chinese Journal of Medical Genetics

基　　金：镇江市科委社会发展基金（SH2005026）

摘　　要：目的建立实时定量PCR（real—time quantitative PCR，RQ-PCR）法检测急性早幼粒细胞白血病（acute promyelocytic leukemia，APL）PML／RARa融合基因转录本，评价其在APL患者诊断和微小残留病（minimal residual disease，MRD）监测中的意义。方法设计TaqMan探针和引物，建立RQ-PCR法对长型（L-form）、短型（S-form）、变异型（V—form）不同类型PML／RARα阳性模板进行扩增，并检测6例APL患者PML／RARα融合基因转录本含量。结果RQ-PCR法最低可检测到10个拷贝／μE的阳性模板，但重复性较差，而10^8～10^2拷贝／αL阳模的重复性良好，正常对照无扩增信号。6例初诊APL患者不同类型PML／RARa融合基因转录本绝对含量为4．27×10^3～3．36×10^5拷贝／50ng（中位4．33×10^4拷贝／50ng），ABL纠正后的相对含量为29．38％～600．53％（中位48．12％）。1例患者诱导缓解后PML／RARa融合基因转录本下降，复发时又明显升高。结论RQ-PCR方法敏感、可靠，可用于APL患者的诊断和MRD监测。Objective To establish and evaluate a real time quantitative PCR （RQ-PCR） method for detection and quantification the PML/RARα fusion gene transcripts in patients with acute promyelocytic leukemia （APL）. Methods Three pairs of primers and TaqMan probe were designed for detecting the most frequent PML/RARα transcripts （L- form, S-form and V-form） and normal ABL was used as an internal control. A real time PCR condition was established to detect PML/RARα and ABL positive templates with a series of dilutions. To evaluate this assay, bone marrow samples from 6 APL patients were detected. Results In repeated tests, maximal sensitivities of 10 copies/μL were obtained, while reproducible maximal sensitivity achieved 100 copies/μL. In 10 normal controls, no amplified fluorescent signals were detected. 3he median absolute and normalized amount of PML/RAα fusion gene transcripts were 4.27 × 10^3-3.36 × 10^5 copies/50 ng （median 4.33×10^4 copies/50ng） and 29.38%-600.53% （median 48.12%） respectively. One case showed significant decrease of PML/RARα fusion gene transcripts after induction therapy compared to that at the time of diagnosis, while the fusion transcripts significandy increased after relapsed. Conclusion RQ-PCR is a sensitive, reliable quantitative assay and can be used in the diagnosis of APL and measurement of MRD.

关 键 词：实时定量PCR 急性早幼粒细胞白血病 融合基因 

分 类 号：R686[医药卫生—骨科学]