基于连接PCR高灵敏度定量检测融合基因转录本  

Quantitative detection with high sensitivity of gene fusion transcripts based on ligation-PCR

作　　者：洪伟祥 王辉 李正平 Weixiang Hong;Hui Wang;Zhengping Li(School of Chemistry and Biological Engineering,University of Science and Technology,Beijing 100083,China)

机构地区：[1]北京科技大学化学与生物工程学院,北京100083

出　　处：《中国科学：化学》2024年第10期1927-1934,共8页SCIENTIA SINICA Chimica

基　　金：国家重点研发计划(编号:2023YFC2413001);国家自然科学基金重点项目(编号:22234001)资助项目。

摘　　要：融合基因在间充质和血液系统恶性肿瘤中发挥着重要的作用.作为人类肿瘤疾病的重要生物标志物,融合基因转录本的分析检测在生物学研究、临床诊断、精准治疗和恶性肿瘤预后等方面具有广阔的应用前景.本文基于连接PCR建立了高灵敏度和高特异性检测融合基因mRNA的方法.在融合基因BCR-ABL mRNA的融合位点处设计两个特异性的寡核苷酸探针,只有当反应体系中存在相对应的靶标时,特异性的探针才会在融合位点相邻处与靶标互补配对并被连接形成新的DNA.通过对该DNA的PCR扩增可以检测低至0.1 fmolL^(-1)的mRNA,表现出了良好的灵敏度和特异性.此外,该方法还利用TaqMan探针实现在1个试管中同时检测融合基因BCR-ABL的不同亚型mRNA.Gene fusion plays an increasingly important role in mesenchymal and hematological malignancies.As an important biomarker of human tumor diseases,the analysis and detection of gene fusion transcripts have broad application prospects in biological research,clinical diagnosis,precision therapy,and prognosis of malignant tumors.In this paper,we establish a highly sensitive and specific method for the detection of gene fusion m RNA based on ligationPCR.Two specific oligonucleotide probes were designed at the fusion site of the BCR-ABL m RNA.Only when the corresponding target existed in the reaction system,the specific probes would complement with the target at the adjacent fusion site and be ligated to form new DNA.Our method can detect mRNA as low as 0.1 fmol L^(-1),showing good sensitivity and specificity.In addition,we used the Taq Man probe to simultaneously detect m RNA of different subtypes of BCR-ABL in a single test tube.

关 键 词：融合基因 连接PCR TAQMAN探针 

分 类 号：Q503[生物学—生物化学] R440[医药卫生—诊断学]