Quantification of HIV-1 DNA Using Real-Time Recombinase Polymerase Amplification
Quantification of HIV-1 DNA Using Real-Time Recombinase Polymerase Amplification
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DOI：
10.1021/ac5011298
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发表时间：
2014-06-17
影响因子：
7.4
通讯作者：
Richards-Kortum, Rebecca
中科院分区：
文献类型：
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作者：
Crannell, Zachary Austin;Rohrman, Brittany;Richards-Kortum, Rebecca
尽管重组酶聚合酶扩增(RPA)在医疗现场应用中检测致病核酸具有许多优点，但RPA尚未被用于使用标准曲线来量化样本浓度。在这里，我们描述了一种带有内部阳性对照的实时RPA检测和一种分析实时荧光数据以量化HIV-1DNA的算法。我们发现DNA浓度与可检测到的扩增开始时间呈指数标准曲线相关。在一系列实验中，使用标准曲线和算法来分析和量化额外的DNA样本，该算法预测所有测试的HIV-1 DNA浓度的平均浓度都在正确浓度的1个数量级以内。这些结果表明，定量RPA(QRPA)可作为量化核酸的有力工具，并可用于单样本护理点诊断系统。
Although recombinase polymerase amplification (RPA) has many advantages for the detection of pathogenic nucleic acids in point-of-care applications, RPA has not yet been implemented to quantify sample concentration using a standard curve. Here, we describe a real-time RPA assay with an internal positive control and an algorithm that analyzes real-time fluorescence data to quantify HIV-1 DNA. We show that DNA concentration and the onset of detectable amplification are correlated by an exponential standard curve. In a set of experiments in which the standard curve and algorithm were used to analyze and quantify additional DNA samples, the algorithm predicted an average concentration within 1 order of magnitude of the correct concentration for all HIV-1 DNA concentrations tested. These results suggest that quantitative RPA (qRPA) may serve as a powerful tool for quantifying nucleic acids and may be adapted for use in single-sample point-of-care diagnostic systems.