BCR-ABL融合基因检测试剂盒质控品的建立  

Establishment of control materials for human BCR-ABL fusion gene

作　　者：曲守方[1] 于婷[1] 张娟丽 胡小许[3] 郭李平[3] 赵金银 高尚先[1] 黄杰[1] 

机构地区：[1]中国食品药品检定研究院,北京100050 [2]河南省医疗器械检验所,郑州450003 [3]中国科学院北京基因组研究所,北京100101 [4]大连晶泰医学检验所,大连116635

出　　处：《药物分析杂志》2016年第9期1629-1633,共5页Chinese Journal of Pharmaceutical Analysis

摘　　要：目的:建立BCR-ABL融合基因质控品,评价人类BCR-ABL融合基因突变检测试剂盒的性能。方法:根据人类BCR-ABL e1a2和e14a2型融合基因m RNA序列,设计覆盖断裂点序列,合成目的融合基因序列。用限制性内切酶BglⅡ和HindⅢ对含有目的融合基因的质粒和表达载体进行酶切,连接酶切产物。将连接产物转化DH5α感受态细胞,筛选单个阳性菌落进行验证。将正确的单个菌落进行超声诱导,制备假病毒溶液。用荧光逆转录-聚合酶链反应(RT-PCR)法对假病毒溶液提取的RNA进行检测。结果:PCR结果和测序结果表明成功构建人类BCR-ABL融合基因e1a2和e14a2型假病毒质粒;荧光RT-PCR结果表明构建的e1a2和e14a2型假病毒质粒能够表达目的 RNA,并且能被市售试剂盒正确检出,可以作为RNA质控品。结论:本研究建立人类BCR-ABL融合基因RNA质控品,用于评价BCR-ABL融合基因突变检测试剂盒的性能,为BCR-ABL融合基因相关白血病的临床诊断及靶向治疗提供指导。Objective：To establish the control materials for human BCR-ABL fusion gene,and further evaluate the performance of detection kits.Methods：According to the m RNA sequences for BCR-ABL fusion gene e1a2 and e14a2,the sequences covering break point cluster region were designed and synthesized.The plasmids with target gene and expression vectors were respectively digested by the restriction enzyme of Bgl Ⅱ and Hind Ⅲ.Then,the restricted DNA products were ligated and transformed into DH5α.The positive clone was selected and verified.The right clone was induced by ultrasound.The pseudotyped virus solutions were collected.The RNA obtained from pseudotyped virus solutions was detected by fluorescence reverse transcription-polymerase chain reaction（RT-PCR）.Results：The results by PCR and sequencing showed that the pseudotyped viral plasmids with e1a2 and e14a2 were successfully established.The results by fluorescence RT-PCR showed that they could express target RNA,which were correctly detected by the commercial kit as the control materials.Conclusion：The control materials for human BCR-ABL fusion gene could evaluate the performance of detection kits and provide the guide for clinical diagnosis and targeted therapy of leukemia associated with BCR-ABL fusion gene.

关 键 词：靶向药物 分子靶向治疗 慢性粒细胞白血病标志物 埃布尔森小鼠白血病病毒癌基因同源物1 BCR-ABL 融合基因 RNA检测 荧光逆转录-聚合酶链反应 基因检测试剂 标准物质研究 

分 类 号：R917[医药卫生—药物分析学]