Detection of bcr-abl transcript in chronic myelogenous leukemia patients by reverse-transcription-polymerase chain reaction and capillary electrophoresis.
Detection of bcr-abl transcript in chronic myelogenous leukemia patients by reverse-transcription-polymerase chain reaction and capillary electrophoresis.
复制标题
通过逆转录聚合酶链反应和毛细管电泳检测慢性粒细胞白血病患者的 bcr-abl 转录本。
作者：
G. Martinelli;N. Testoni;V. Montefusco;M. Amabile;G. Saglio;E. Ottaviani;C. Terragna;F. Bonifazzi;A. de Vivo;F. Pane;G. Rosti;S. Tura
背景和目标
毛细管电泳法(CE)已经成为平板凝胶分析的一种有吸引力的替代方法，可以直接和准确地检测扩增产物，而几个循环的聚合酶链式反应(PCRS)就足以进行定量和定性分析。我们试图评估：1)CE是否是一种实用的非同位素方法，可以直接检测逆转录-聚合酶链式反应(RT-PCR)扩增的bcr-abl的存在(定性分析)；2)是否有可能用竞争性RT-PCR测量CE图谱的峰面积来定量PCR产物(定量分析)。
设计与方法
从34例确诊的慢性粒细胞白血病(CML)患者(中位年龄47.5岁，范围18~65岁；中位Sokal‘s评分0.9分；范围0.53~2.78)的骨髓总RNA中提取1微克总RNA，用RT-PCR(定性分析)扩增出两种类型的bcr-abl相关转录产物。扩增产物用2%琼脂糖凝胶电泳法(SGE)和毛细管电泳法(CE)分析(128次，中位数3.3次)。此外，我们用竞争性RT-PCR方法和CE(bcr-abl转录本以每微克总RNA的转录本表示)来评估PCR产物的数量(定量分析)。
结果
定性RT-PCR扩增产物的CE分离显示b2-a2型(343个碱基对，10例患者)和b3-a2型(418个碱基对，24例患者)分别在9.33min和10.03min(SD=0.25)出现基线分辨。通过定量分析，我们发现BCR-ABL在诊断时的表达存在很大的患者间差异：BCR-ABL转录本数量的中位数为78,000 BCR-ABL转录本/微克总RNA，范围在17,300到750,000之间。诊断时bcr-abl转录量与原始细胞数(0%原始细胞和1%原始细胞分别为128,859和331,722，p=0.004)和Sokal评分(分别为156,865和408,800，P=0.003)有关。
解读和结论
我们的结果证实，CE分析在检测和定量bcr-abl聚合酶链式反应产物方面提供了更高的分辨率和更高的灵敏度。用毛细管电泳法对BCR-ABL产物进行定性分析为亚纳克级DNA片段的快速分析提供了一种快速技术(不到20分钟)。CE运行时间短，毛细管可重复使用，通过计算机控制的步骤进行数据采集，可能实现全自动化。用毛细管电泳法对bcr-abl进行竞争性/定量分析，反应更少，定量更准确。
BACKGROUND AND OBJECTIVE
Capillary electrophoresis (CE) has become an attractive alternative to SLAB gel analysis for direct and accurate detection of amplified product, and a few cycles of polymerase chain reactions (PCRs) could be sufficient for both quantitative and qualitative analysis. We try to assess: 1) whether CE could be a practical, non-isotopic method for direct detection of the presence of amplified bcr-abl obtained by a reverse transcription (RT)-PCR (qualitative analysis) and 2) whether it is possible to quantify PCR products using a competitive RT-PCR measuring peak areas of CE electropherograms (quantitative analysis).
DESIGN AND METHODS
The two types of bcr-abl chronic myelogenous leukemia (CML) associated transcript products were generated by RT-PCR (qualitative analysis) from 1 microgram of total RNA extracted from bone marrow samples of 34 CML patients at diagnosis (median age 47.5; range 18-65; median Sokal's score 0.9; range 0.53-2.78). The PCR products were analyzed by SLAB-gel electrophoresis (SGE) on 2% agarose gels and by CE (128 runs; median 3.3 times for each sample). Furthermore, we assessed the amount of PCR product (quantitative analysis) by a competitive RT-PCR approach and by CE (bcr-abl transcripts were expressed as transcript per microgram of total RNA examined).
RESULTS
CE separation of PCR products obtained by qualitative RT-PCR showed baseline resolution for the two peaks corresponding to the two types of bcr-abl junctions: the b2-a2 type (343 base pairs, 10 patients) was revealed at 9.33 min [standard deviation (SD) = 0.1] and the b3-a2 type (418 base pair, 24 patients) at 10.03 min (SD = 0.25). By quantitative analysis we found that there is great interpatient variability in bcr-abl expression at diagnosis: the median value of the amount of bcr-abl transcript was 78,000 bcr-abl transcript/microgram total RNA ranging from 17,300 to 750,000. The amount of bcr-abl transcript at diagnosis was related to the number of blast cells (mean value 128,859 vs. 331,722 in patients with 0% blast cells and > 1% blast cells, respectively; p = 0.004) and Sokal's score (mean value 156,865 vs. 408,800 in patients with Sokal's score < 0.8 and > 1.2, respectively; p = 0.003).
INTERPRETATION AND CONCLUSIONS
Our results confirm that CE analysis offers greater resolution and enhanced sensitivity for detection and quantification of bcr-abl PCR product in the study of this leukemia. Qualitative analysis by CE of bcr-abl product provides a rapid technique (less than 20 min) for the analysis of subnanogram amounts of DNA fragments. CE run times are short, the capillary can be re-used and full automation may be feasible with data acquisition by a computer-controlled step. Competitive/quantitative analysis of bcr-abl as analyzed by CE allowed fewer reactions and more precise quantification.
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