Catalan Institute of Nanoscience and Nanotechnology (ICN2) , CSIC and Barcelona Institute of Science and Technology , Campus UAB , 08193 Barcelona , Spain.
On leave from Agricultural Research Center (ARC) , Ministry of Agriculture and Land Reclamation , Giza , Egypt.
Anal Chem. 2019 Apr 2;91(7):4790-4796. doi: 10.1021/acs.analchem.9b00340. Epub 2019 Mar 14.
Solid-phase isothermal recombinase polymerase amplification (RPA) offers many benefits over the standard RPA in homogeneous phase in terms of sensitivity, portability, and versatility. However, RPA devices reported to date are limited by the need for heating sources to reach sensitive detection. With the aim of overcoming such limitation, we propose here a label-free highly integrated in situ RPA amplification/detection approach at room temperature that takes advantage of the high sensitivity offered by gold nanoparticle (AuNP)-modified sensing substrates and electrochemical impedance spectroscopic (EIS) detection. Plant disease ( Citrus tristeza virus (CTV)) diagnostics was selected as a relevant target for demonstration of the proof-of-concept. RPA assay for amplification of the P20 gene (387-bp) characteristic of CTV was first designed/optimized and tested by standard gel electrophoresis analysis. The optimized RPA conditions were then transferred to the AuNP-modified electrode surface, previously modified with a thiolated forward primer. The in situ-amplified CTV target was investigated by EIS in a Fe(CN)/Fe(CN) red-ox system, being able to quantitatively detect 1000 fg μL of nucleic acid. High selectivity against nonspecific gene sequences characteristic of potential interfering species such as Citrus psorosis virus (CPsV) and Citrus caxicia viroid (CCaV) was demonstrated. Good reproducibility (RSD of 8%) and long-term stability (up to 3 weeks) of the system were also obtained. Overall, with regard to sensitivity, cost, and portability, our approach exhibits better performance than RPA in homogeneous phase, also without the need of heating sources required in other solid-phase approaches.
固相等温重组聚合酶扩增（RPA）在均相中的灵敏度、便携性和多功能性方面优于标准 RPA。然而，迄今为止报道的 RPA 设备受到需要加热源以达到灵敏检测的限制。为了克服这种限制，我们提出了一种无标记的高度集成的原位 RPA 扩增/检测方法，该方法利用了金纳米粒子（AuNP）修饰的传感基底提供的高灵敏度和电化学阻抗谱（EIS）检测。选择植物病害（柑橘衰退病毒（CTV））诊断作为证明概念的相关目标。首先设计/优化了用于扩增 CTV 特征性 P20 基因（387-bp）的 RPA 检测，并通过标准凝胶电泳分析进行了测试。然后将优化的 RPA 条件转移到先前用巯基化正向引物修饰的 AuNP 修饰电极表面。通过在 Fe(CN)/Fe(CN)氧化还原体系中进行 EIS 研究了原位扩增的 CTV 靶标，能够定量检测 1000 fg μL 的核酸。对潜在干扰物种（如柑橘溃疡病毒（CPsV）和柑橘裂皮病类病毒（CCaV））特征性非特异性基因序列表现出高选择性。还获得了该系统的良好重现性（8%的 RSD）和长期稳定性（长达 3 周）。总体而言，就灵敏度、成本和便携性而言，我们的方法比均相中的 RPA 表现出更好的性能，而且不需要其他固相方法所需的加热源。
