多重实时定量PCR检测BCR—ABL mRNA方法的建立

Eatablishment of multiple real-time quantitative PCR for measuring BCR-ABL mRNA transcripts

目的建立一种快速、可靠的实时定量PCR（RQ-PCR）检测BCR-ABL mRNA的方法。方法应用LightCycler技术，设计在同一PCR反应管内可扩增b3a2、b2a2、ela2几种常见的BCR-ABL转录本和b3a3、b2a3等少见转录本，并对RQ-PCR主要要素进行优化，评价优化后RQ-PCR的敏感性、特异性和可靠性。结果建立的RQ-PCR方法可检出10^3健康人单个核细胞中的1个K562细胞，最低可检出100个拷贝BCR—ABL分子。BCR—ABL和ABL的循环域值（C1）值（拷贝数）批内变异系数分别为0．68％（12．93％）和0．59％（8．60％），批间变异系数分别为1．14％（18．89％）和1．01％（12．72％）。结论RQ-PCR可作为评价慢性粒细胞白血病（CML）疾病进展、预后判断和骨髓移植后微量残留白血病监测的灵敏、可靠的方法。

Objective To establish a rapid and reliable real-time quantitative polymerase chain reaction （RQ-PCR） for detection and quantification of BCR-ABL mRNA transcripts. Methods By applying LightCycler hybridization probes technolony, the primes were designed to amplify the common BCR-ABL transcripts including b3a2, b2a2, ela2 and some rare transcripts such as b3a3 and b2a3. After condition of RQ-PCR was optimized, sensitivity, specificity and reliability were evaluated. Results The RQ-PCR condition was optimized to amplify less than 100 target molecules/reaction and detect one K562 cell in 10% cells from healthy donors, The intrassay Ct value variation coefficient of BCR- ABL and ABL was 0, 68% （concentration of 12, 93%） and 0, 59% （concentration of 8.60%） respectively; the inter-assay Ct value variation coefficient was 1, 14% （concentration of 18, 89%） and 1.01 % （concentration of 12.72% ） respectively. Conclusion RQ-PCR assay may be used as a reliable and sensitive method for evaluating pathogenetic condition of chronic myeloid leukemia （CML）, assessing prognosis and monitoring minimal residual disease （MRD） after bone marrow transplantation.