Lee Ai-Cheng, Dai Ziyu, Chen Baowei, Wu Hong, Wang Jun, Zhang Aiguo, Zhang Lurong, Lim Tit-Meng, Lin Yuehe
Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Anal Chem. 2008 Dec 15;80(24):9402-10. doi: 10.1021/ac801263r.
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 (ALP) 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 electroactive enzymatic product in the presence of 1-naphthyl 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 microL) and a 3-order-wide dynamic range of target concentration were achieved by the electrochemical bDNA assay. Such limit corresponded to approximately 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.
我们描述了一种新型电化学分支DNA（bDNA）检测方法，用于在从细胞系提取的信使核糖核酸（mRNA）群体中对p185 BCR-ABL白血病融合转录本进行无聚合酶链反应（PCR）检测和定量。携带高负载碱性磷酸酶（ALP）示踪剂的bDNA放大器用于放大目标信号。在标记bDNA之前，通过协同夹心杂交将目标捕获在微孔板孔表面。在1-萘基磷酸存在下，通过对电活性酶产物进行方波伏安法（SWV）分析来监测捕获的ALP的活性。系统地优化了底物和酶产物的伏安特性以及SWV分析参数。电化学bDNA检测方法实现了1 fM的检测限（100 μL中1×10⁻¹⁹摩尔目标转录本）和3个数量级宽的目标浓度动态范围。该检测限对应于约17 fg的p185 BCR-ABL融合转录本。该检测方法的特异性和灵敏度使得无需PCR扩增就能直接在低至4.6 ng mRNA群体中检测目标转录本。结合使用定量良好的标准品，电化学bDNA检测方法能够直接用于对mRNA群体中的目标转录本进行无PCR定量分析。测定的平均转录本拷贝数为62,900/ng mRNA，这比实时定量PCR（qPCR）至少高50倍。这一发现与先前报道的qPCR对目标的低估一致。此外，基于bDNA技术的独特设计提高了检测特异性，因为只有p185 BCR-ABL融合转录本会对检测产生反应。因此，该方法为早期疾病诊断提供了一种简单、灵敏、准确且定量的工具，可替代qPCR。
