多重RT-PCR联合毛细管电泳法检测BCR-ABL融合基因

摘要

目的：建立多重逆转录-聚合酶链反应（RT-PCR）联合毛细管电泳法检测BCR-ABL融合基因，以期应用于慢性粒细胞白血病（CML）辅助诊断。方法采用重叠延伸法构建BCR-ABL融合基因阳性模板，设计并优化检测BCR-ABL融合基因的多重RT-PCR联合毛细管电泳体系，对检测体系进行初步的性能评估。结果多重RT-PCR技术检测BCR-ABL融合基因（e1a2、e13a2和e14a2）的最低检测限在102～103拷贝/μL；50例临床确诊CML的患者应用该法检测BCR-ABL融合基因的阳性率为86．0％，其中e13a2型（20．0％），e14a2型（66．0％）；本方法与骨髓细胞培养染色体核型分析相比，阳性符合率为97．6％，阴性符合率为75．0％，总符合率为94．0％，2种方法具有较高的一致性（Kappa＝0．765，P＞0．05）。结论成功建立检测BCR-ABL融合基因的多重RT-PCR联合毛细管电泳方法。该方法操作简单快捷、特异性好、灵敏度高，适合于临床上CML的辅助诊断和分子分型。%Objective To establish a method for detecting BCR-ABL fusion gene by multiplex reverse transcription polymerase chain reaction (RT-PCR),and to provide a useful tool for chronic myeloid leukemia (CML)auxiliary diagnosis.Methods The construction of BCR-ABL fusion gene as positive template was made by overlap extension method.The method of multiplex RT-PCR combining with capillary electrophoresis was designed and optimized.Its primary performance was evaluated.Results The lower detection limit for BCR-ABL fusion gene (ela2,e13a2 and e14a2)by multiplex RT-PCR was 102-103 copies/μL.The positive rate of this method to detect 50 CML patients was 86.0%(e13a2:20.0% and e14a2:66.0%).Compared with the results of chromosome karyotype analysis,the positive coincidence rate was 97.6%,and the negative coincidence rate was 75.0%.The 2 methods have high consistency (Kappa=0.765,P>0.05).Conclusions The method of multiplex RT-PCR combining with capillary electrophoresis to detect BCR-ABL fusion gene is simple and rapid with good specificity and sensitivity and is very suitable for the clinical detection of CML.