Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
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DOI：
10.3791/52620
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发表时间：
2015-03-01
影响因子：
1.2
通讯作者：
Richards-Kortum, Rebecca
中科院分区：
文献类型：
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作者：
Crannell, Zachary A.;Rohrman, Brittany;Richards-Kortum, Rebecca
最近证明，重组酶聚合酶扩增（RPA），一种用于病原体检测的等温扩增平台，可用于使用标准曲线定量DNA样品浓度。在这份手稿中，提供了一个详细的协议，用于开发和实施实时定量重组酶聚合酶扩增试验（qRPA试验）。以HIV-1 DNA定量为例，描述了实时RPA反应的组装、内部阳性对照（IPC）序列的设计以及IPC和目标靶标的共扩增。提供了使用来自多个实验的数据构建标准曲线的说明和数据处理脚本，其可用于预测未知样品的浓度或评估测定的性能。最后，描述了一种使用显微镜和载物台加热器收集实时荧光数据的替代方法，作为开发即时qRPA测定的一个步骤。所提供的方案和脚本可用于开发任何目标DNA靶标的qRPA测定。
It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.