摘要
目的利用自身淬灭探针技术建立一种能检测慢性粒细胞白血病（CML）bcr／abl融合基因mRNA的实时荧光定量PCR方法，为CML的诊断、疗效观察以及微量残留白血病（MRD）的监测提供有效手段。方法用逆转录PCR方法（RT—PCR）扩增K562细胞的bcr／abl融合基因，A-T克隆法构建定量标准模板；设计自身淬灭荧光引物（探针），建立自身淬灭荧光定量逆转录PCR方法（FQ—RT—PCR），并对该方法的线性检测范围、灵敏度、重复性、稳定性进行检测；然后检测临床白血病患者骨髓标本bcr／abl mRNA。结果建立的FQ—RT—PCR方法可检测10copies／μl的bcr／abl重组质粒，并能从10^5个正常细胞中检出1个白血病细胞，该方法的批内、批间变异系数（CV）分别为2．1％、6．1％，线性检测范围为10^2-10^9copies／μl。25例CML患者bcr／abl融合基因mRNA表达量中位数为4．50×10^4[（0．45—89．00）×10^4]copies／μg RNA，其中11例慢性期初诊患者bcr／abl mRNA表达量中位数为5．45×10^4[（2．95—19．30）×10^4]copies／μg RNA，6例急变期患者bcr／abl mRNA表达量中位数为13．00×10^4[（4．10～89．00）×10^4]copies／μg RNA，8例慢性期治疗后复查患者bcr／abl mRNA表达量中位数为2．35×10^4[（0．45—5．12）×10^4]copies／μg RNA，CML急变期患者bcr／abl融合基因表达水平与慢性期患者之间差异有统计学意义（q=3．41，P〈0．05）。PCR产物经电泳分析，其中21例CML患者为b3a2型，4例CML患者为b2a2型；3例急性淋巴细胞白血病（ALL）患者中，1例有bcr／abl mRNA表达，为b2a2型。结论所建立的基于自身淬灭探针技术的实时荧光定量PCR检测方法灵敏、特异、重复性好，结果用拷贝数表示，准确可靠，利于标准统一。可广泛用于CML的诊断、疗效观察以及MRD的监测。
Objective To establish a quantitative RT-PCR method with self-quenched fluorogenic probe for detection of ber/abl mRNA in patients with chronic myeloid leukemia for providing a useful tool for diagnosis of CML, evaluation of therapeutic effect and monitoring of minimal residual disease （MRD）. Methods bcr/abl gene from cultured K562 cells was amplified by conventional RT-PCR. The standard quantitative plasmid was constructed by A-T clone method. The self-quenched fluorogenic quantitative RT- PCR method（FQ-RT-PCR） for determination of bcr/abl mRNA was established successfully using the ABI PRISM 7000 PCR Detector. The linear range, sensitivity, stability, and repetitiveness of the method were determined. The marrow samples from 25 CML patients and 3 ALL patients were assessed. Results The sensitivity of the FQ-RT-PCR was 10 copies/μl recombined plasmid, and ber/abl mRNA can be detected from 1 K562 cell in 10^5 normal ceils. The linear range was 10^2 - 10^9copies/μl recombined plasmid. The coefficient variation （CV） value was 2. 1% in intra-assay and 6. 1% in inter-assay. The median bcr/abl mRNA expression level was 4. 50 × 10^4 copies/μg RNA [ （ 0.45 - 89.00 ） × 10^4 ], 5.45×10^4 copies/μg RNA [（2.95-19.30）×10^4],13.00×10^4 copies/μg RNA[（4.10-89.00）×10^4] and 2.35×10^4 copies/μg RNA [ （0.45 -5.12）×10^4] in 25 CML patients, 11 patients in the incipient chronic phase, 6 patients in blastic crisis, 8 patients in chronic period after treatment, respectively. The bcr/abl mRNA level in blastic crisis was significantly higher than that in chronic phase （ q = 3.41, P 〈 0.05 ）. Gel electrophoresis showed that 21 cases were b3a2 and 4 cases b2a2. B2a2 was observed in one of three patients of acute lymphoblastic leukemia （ALL）. Condusions The established FQ-RT-PCR method is specific, sensitive, accurate, and reliable. It can be widely used in diagnosis of CML, evaluation of therapeutic effect and monitoring of MRD.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2007年第11期1227-1231,共5页
Chinese Journal of Laboratory Medicine
基金
国家自然科学基金(30470744)
