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Quantitative isothermal amplification on paper membranes using amplification nucleation site analysis
Lab on a Chip ( IF 6.1 ) Pub Date : 2022-04-27 , DOI: 10.1039/d2lc00007e
Benjamin P Sullivan 1 , Yu-Shan Chou 2 , Andrew T Bender 1 , Coleman D Martin 2 , Zoe G Kaputa 3 , Hugh March 3 , Minyung Song 1 , Jonathan D Posner 1, 2, 4
Affiliation  

Quantitative nucleic acid amplification tests (qNAATs) are critical in treating infectious diseases, such as in HIV viral load monitoring or SARS-CoV-2 testing, in which viral load indicates viral suppression or infectivity. Quantitative PCR is the gold standard tool for qNAATs; however, there is a need to develop point-of-care (POC) qNAATs to manage infectious diseases in outpatient clinics, low- and middle-income countries, and the home. Isothermal amplification methods are an emerging tool for POC NAATs as an alternative to traditional PCR-based workflows. Previous works have focused on relating isothermal amplification bulk fluorescence signals to input copies of target nucleic acids for sample quantification with limited success. In this work, we show that recombinase polymerase amplification (RPA) reactions on paper membranes exhibit discrete fluorescent amplification nucleation sites. We demonstrate that the number of nucleation sites can be used to quantify HIV-1 DNA and viral RNA in less than 20 minutes. An image-analysis algorithm quantifies nucleation sites and determines the input nucleic acid copies in the range of 67–3000 copies per reaction. We demonstrate a mobile phone-based system for image capture and onboard processing, illustrating that this method may be used at the point-of-care for qNAATs with minimal instrumentation.

中文翻译:

使用扩增成核位点分析在纸膜上进行定量等温扩增

定量核酸扩增测试 (qNAAT) 对于治疗传染病至关重要,例如 HIV 病毒载量监测或 SARS-CoV-2 测试,其中病毒载量表明病毒抑制或感染性。定量 PCR 是 qNAAT 的黄金标准工具;然而,需要开发即时护理 (POC) qNAAT 来管理门诊、低收入和中等收入国家以及家庭的传染病。等温扩增方法是 POC NAAT 的一种新兴工具,可替代传统的基于 PCR 的工作流程。之前的工作主要集中在将等温扩增大量荧光信号与目标核酸的输入拷贝相关联以进行样品定量,但取得的成功有限。在这项工作中,我们表明纸膜上的重组酶聚合酶扩增(RPA)反应表现出离散的荧光扩增成核位点。我们证明,成核位点的数量可用于在 20 分钟内量化 HIV-1 DNA 和病毒 RNA。图像分析算法可量化成核位点并确定每个反应 67-3000 个拷贝范围内的输入核酸拷贝。我们演示了一种基于移动电话的图像捕获和机载处理系统,表明该方法可以在 qNAAT 护理点以最少的仪器使用。
更新日期:2022-04-27
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