智能手机成像的晶片上基于逆转录环介导等温扩增(RT-LAMP)技术的全血中HIV-1检测

Gregory L. Damhorst , Carlos Duarte-Guevara , Weili Chen , Tanmay Ghonge , Brian T. Cunningham , Rashid Bashir

工程(英文) ›› 2015, Vol. 1 ›› Issue (3) : 324 -335.

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工程(英文) ›› 2015, Vol. 1 ›› Issue (3) : 324 -335. DOI: 10.15302/J-ENG-2015072
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智能手机成像的晶片上基于逆转录环介导等温扩增(RT-LAMP)技术的全血中HIV-1检测

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Smartphone-Imaged HIV-1 Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) on a Chip from Whole Blood

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摘要

病毒载量测量对于人类免疫缺陷病毒 (HIV) 阳性患者长期临床护理来说是一个必不可少的工具。然而,考虑到病毒载量测量所需的仪器体积、成本和操作的复杂性,在医疗基础设施较差的偏远地区 (尤其是在被HIV感染人群比例较高的地区) 普及标准的病毒载量测量仪器是较为困难的。为提高该检测方法的普及性,人们已经开始开发可以进行即时检测的病毒载量检测平台,然而尚没有解决办法能够同时满足低成本、便携、易于操作等多种实际要求。本文通过运用微流体和微型硅晶片平台,对经过最低程度处理的含有HIV的全血样本进行了逆转录环介导等温扩增 (RT-LAMP),并利用智能手机进行了荧光检测。集成实验检测结果表明,一滴约60 nL的反应液滴中仅有的3个病毒依然可以通过RT-LAMP技术被检测到,这相当于每微升全血样品中只有670个病毒。该技术在数字化RT-LAMP方法上具有重要意义,扩展该技术能够实现对HIV阳性患者在临床护理中采集指血进行病毒载量检测。研究结果显示,病毒载量检测过程所需的各个步骤,从血滴的准备到RT-LAMP反应的成像,都可以集成为晶片实验并且可以和移动设备兼容。

Abstract

Viral load measurements are an essential tool for the long-term clinical care of human immunodeficiency virus (HIV)-positive individuals. The gold standards in viral load instrumentation, however, are still too limited by their size, cost, and sophisticated operation for these measurements to be ubiquitous in remote settings with poor healthcare infrastructure, including parts of the world that are disproportionately affected by HIV infection. The challenge of developing a point-of-care platform capable of making viral load more accessible has been frequently approached but no solution has yet emerged that meets the practical requirements of low cost, portability, and ease-of-use. In this paper, we perform reverse-transcription loop-mediated isothermal amplification (RT-LAMP) on minimally processed HIV-spiked whole blood samples with a microfluidic and silicon microchip platform, and perform fluorescence measurements with a consumer smartphone. Our integrated assay shows amplification from as few as three viruses in a ~ 60 nL RT-LAMP droplet, corresponding to a whole blood concentration of 670 viruses per μL of whole blood. The technology contains greater power in a digital RT-LAMP approach that could be scaled up for the determination of viral load from a finger prick of blood in the clinical care of HIV-positive individuals. We demonstrate that all aspects of this viral load approach, from a drop of blood to imaging the RT-LAMP reaction, are compatible with lab-on-a-chip components and mobile instrumentation.

关键词

人类免疫缺陷病毒(HIV) / 病毒载量 / 环介导等温扩增 / 智能手机 / 即时检测

Key words

human immunodeficiency virus (HIV) / viral load / loop-mediated isothermal amplification / smartphone / point-of-care

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Gregory L. Damhorst, Carlos Duarte-Guevara, Weili Chen, Tanmay Ghonge, Brian T. Cunningham, Rashid Bashir 智能手机成像的晶片上基于逆转录环介导等温扩增(RT-LAMP)技术的全血中HIV-1检测[J]. 工程(英文), 2015, 1(3): 324-335 DOI:10.15302/J-ENG-2015072

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基金资助

本研究受NIH的发现与发展经费(R21)(AI106024)资助。Gregory L. Damhorst受到来自Ruth L. Kirschstein全国研究服务奖对博士研究人员的支持(F30) (AI109825)。()

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