Electrochemical Branched-DNA Assay for Polymerase Chain Reaction-Free Detection and Quantification of Oncogenes in Messenger RNA
Electrochemical Branched-DNA Assay for Polymerase Chain Reaction-Free Detection and Quantification of Oncogenes in Messenger RNA
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DOI：
10.1021/ac801263r
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发表时间：
2008-12-15
影响因子：
7.4
通讯作者：
Lin, Yuehe
中科院分区：
文献类型：
--
作者：
Lee, Ai-Cheng;Dai, Ziyu;Lin, Yuehe
我们描述了一种新的电化学支链dna （bDNA）检测方法，用于从细胞系中提取的信使核糖核酸（mRNA）群体中p185 BCR-ABL白血病融合转录物的无聚合酶链反应（PCR）检测和定量。利用携带高负荷碱性磷酸酶（AILP）示踪剂的bDNA放大器放大目标信号。在标记bDNA之前，通过协同夹层杂交在微孔板孔表面捕获目标。捕获的ALP的活性是通过方波伏安（SWV）分析电活性酶产物在1-磷酸萘酯存在下的监测。对底物和酶产物的伏安特性以及SWV分析参数进行了系统优化。电化学bDNA检测的检出限为1 fM (1 × 10(-19) mol靶转录本/ 100 μ L)，靶浓度动态范围为3个数量级。这一极限与p185 BCR-ABL融合转录本的17 fg相似。该方法的特异性和敏感性可直接检测到4.6 ng mRNA群体的目标转录物，而无需PCR扩增。结合使用定量良好的标准，电化学bDNA分析能够直接用于mRNA群体中目标转录物的无pcr定量分析。平均转录本拷贝数为62,900/ng，比实时定量PCR （qPCR）的转录本拷贝数高出至少50倍。这一发现与之前报道的qPCR对靶标的低估一致。此外，基于bDNA技术的独特设计增加了检测的特异性，因为只有p185 BCR-ABL融合转录本对检测有反应。因此，该方法为早期疾病诊断提供了一种简单、灵敏、准确的定量工具，可替代qPCR。
We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcripts in the population of messenger ribonucleic acid (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (AILP) tracers was used to amplify the target signal. The targets were captured on microplate well surfaces through cooperative sandwich hybridization prior to the labeling of bDNA. The activity of captured ALP was monitored by square-wave voltammetric (SWV) analysis of the electro-active enzymatic product in the presence of 1-napthyl phosphate. The voltammetric characteristics of substrate and enzymatic product as well as the parameters of SWV analysis were systematically optimized. A detection limit of 1 fM (1 x 10(-19) mol of target transcripts in 100 mu L) and a 3-order-wide dynamic range of target concentration were achieved by the electrochemical bDNA assay. Such limit corresponded to similar to 17 fg of the p185 BCR-ABL fusion transcripts. The specificity and sensitivity of assay enabled direct detection of target transcripts in as little as 4.6 ng of mRNA population without PCR amplification. In combination with the use of a well-quantified standard, the electrochemical bDNA assay was capable of direct use for a PCR-free quantitative analysis of target transcripts in mRNA population. A mean transcript copy number of 62,900/ng of mRNA was determined, which was at least 50-fold higher than that of real-time quantitative PCR (qPCR). The finding was consistent with the underestimation of targets by qPCR reported earlier. In addition, the unique design based on bDNA technology increases the assay specificity as only the p185 BCR-ABL fusion transcripts will respond to the detection. The approach thus provides a simple, sensitive, accurate, and quantitative tool alternative to the qPCR for early disease diagnosis.