在重组酶聚合酶扩增中使用扩增子产量进行核酸定量,Analytical Chemistry

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在重组酶聚合酶扩增中使用扩增子产量进行核酸定量
Analytical Chemistry ( IF 6.7 ) Pub Date : 2022-09-28 , DOI: 10.1021/acs.analchem.2c02810
Priyanka Valloly ₁ , Rahul Roy _{1,

2}

Affiliation

基于扩增的定量聚合酶链式反应 (qPCR) 提供准确和灵敏的核酸定量。然而，温度循环和实时监控的要求限制了它对许多设置的转换。定量等温扩增方法减少了对热循环仪的需求；然而，他们仍然需要对复杂阅读器上的核酸扩增进行持续监测。在这里，我们采用等温重组酶聚合酶扩增 (RPA) 反应来开发一种半定量方法，该方法依赖于最终扩增子产量来估计初始目标核酸拷贝数。为了实现这一点，我们开发了一个现象学模型来捕捉基本的 RPA 动力学。我们确定了限制与起始 DNA 模板浓度相对应的反应产量的反应条件。我们通过实验验证了这些预测，并表明 RPA 反应结束时的扩增子产量与起始 DNA 浓度密切相关，同时有力地减少了非特异性扩增。我们展示了这种称为定量端点 RPA (qeRPA) 的方法，用于检测超过 5 个对数级的 DNA，检测限为 100 个分子。我们使用归一化终点强度 (NEI) 标准曲线的线性回归模型，估计登革热病毒感染患者血清中的病毒载量，其性能与 qPCR 相当。与传统的等温定量方法不同，

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Nucleic Acid Quantification with Amplicon Yield in Recombinase Polymerase Amplification

Amplification-based quantitative polymerase chain reaction (qPCR) provides accurate and sensitive nucleic acid quantification. However, the requirement of temperature cycling and real-time monitoring limits its translation to many settings. Quantitative isothermal amplification methods alleviate the need for thermal cyclers; however, they still require continuous monitoring of the nucleic acid amplification on sophisticated readers. Here, we adapted an isothermal recombinase polymerase amplification (RPA) reaction to develop a semiquantitative method that relies on the final amplicon yield to estimate the initial target nucleic acid copy number. To achieve this, we developed a phenomenological model that captures the essential RPA dynamics. We identified reaction conditions that constrained the reaction yield corresponding to the starting DNA template concentration. We validated these predictions experimentally and showed that the amplicon yields at the end of the RPA reaction correlated well with the starting DNA concentration while reducing nonspecific amplification robustly. We demonstrate this approach, termed quantitative endpoint RPA (qeRPA), to detect DNA over five log orders with a detection limit of 100 molecules. Using a linear regression model of the normalized endpoint intensity (NEI) standard curve, we estimate the viral load from the serum of dengue virus-infected patients with comparable performance to qPCR. Unlike the conventional isothermal quantitative methods, qeRPA can be employed for robust and sensitive nucleic acid estimation at close to room temperature without real-time monitoring and can be beneficial for field deployment in resource-limited settings.

更新日期：2022-09-28

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