Integrated centrifugal reverse transcriptase loop-mediated isothermal amplification microdevice for influenza A virus detection.
Integrated centrifugal reverse transcriptase loop-mediated isothermal amplification microdevice for influenza A virus detection.
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DOI：
10.1016/j.bios.2014.12.043
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发表时间：
2015-06-15
影响因子：
12.6
通讯作者：
Seo TS
中科院分区：
文献类型：
--
作者：
Jung JH;Park BH;Oh SJ;Choi G;Seo TS
介绍了一种集成化的逆转录酶环介导等温扩增（RT-LAMP）微型装置，该装置由微珠辅助RNA纯化和RT-LAMP组成，并通过微型光学检测器进行实时监测。集成的RT-LAMP微型装置包括用于病毒RNA样品（纯化的甲型流感病毒RNA或裂解物）、洗涤溶液（70%乙醇）、洗脱溶液（无RNA酶的水）和RT-LAMP混合物的四个储器，以及两个室（废物室和RT-LAMP反应室）。用于洗涤溶液、洗脱溶液和RT-LAMP混合物的单独的储器设计有用于稳定储存的毛细管阀。以A/H1N1、A/H3 N2和A/H5 N1 3株流感病毒为RNA模板，设计RT-LAMP引物，检测HA和M基因。通过优化毛细管阀和虹吸通道，通过离心力实现样品顺序流向微珠用于RNA纯化。此外，通过改变旋转方向成功地从废液中分离纯化的RNA溶液，并在RT-LAMP反应室中与RT-LAMP混合物结合用于靶基因扩增。从进样到结果的整个过程在47 min内完成。即使使用10个病毒RNA拷贝，在集成的RT-LAMP微型装置上也确认了甲型H1N1流感病毒，其显示出比常规RT-PCR高10倍的灵敏度。还进行了甲型H1N1流感病毒裂解物的亚型和特异性测试，并成功地对临床样本进行了基因分型，以在我们提出的集成微设备上确认甲型流感病毒。本文介绍了一种集RNA纯化、RT-LAMP和实时监测于一体的微型装置。通过离心力和优化的微通道自动控制连续的样品运输和纯化的RNA分离。流感病毒等温扩增，并通过使用小型化荧光检测器鉴定产物。甲型H1N1流感病毒的检测限为10个拷贝。在微型设备上成功分析了临床样本。
An integrated reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) microdevice which consists of microbead-assisted RNA purification and RT-LAMP with real-time monitoring by a miniaturized optical detector was demonstrated. The integrated RT-LAMP microdevice includes four reservoirs for a viral RNA sample (purified influenza A viral RNA or lysates), a washing solution (70% ethanol), an elution solution (RNase-free water), and an RT-LAMP cocktail, and two chambers (a waste chamber and an RT-LAMP reaction chamber). The separate reservoirs for a washing solution, an elution solution, and an RT-LAMP cocktail were designed with capillary valves for stable storage. Three influenza A virus strains (A/H1N1, A/H3N2, and A/H5N1) were used for RNA templates, and RT-LAMP primer sets were designed to detect hemagglutinin (HA) and conserved M gene. Sequential sample flow to the microbeads for RNA purification was achieved by centrifugal force with optimization of capillary valves and a siphon channel. Furthermore, the purified RNA solution was successfully isolated from the waste solution by changing the rotational direction, and combined with the RT-LAMP cocktail in the RT-LAMP reaction chamber for target gene amplification. Total process from the sample injection to the result was completed in 47 min. Influenza A H1N1 virus was confirmed on the integrated RT-LAMP microdevice even with 10 copies of viral RNAs, which revealed 10-fold higher sensitivity than that of a conventional RT-PCR. Subtyping and specificity test of influenza A H1N1 viral lysates were also performed and clinical samples were successfully genotyped to confirm influenza A virus on our proposed integrated microdevice. An integrated microdevice cosistng of RNA purification and RT-LAMP with real-time monitoring was demonstrated. Sequential sample transportation and purified RNA separation were automatically controlled by centrifugal force and optimized microchannels. Influenza viruses were isothermally amplified and the products were identified by using a miniaturized fluorescence detector. Limit of detection for influenza A H1N1 was 10 copies. Clinical samples were successfully analyzed on the microdevice.
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影响因子：
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作者：
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通讯作者：
Garcia-Sastre, Adolfo
影响因子：
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作者：
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von Stetten, Felix
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