ddRFC: A scalable multiplexed droplet digital nucleic acid amplification test platform.
ddRFC: A scalable multiplexed droplet digital nucleic acid amplification test platform.
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DOI：
10.1016/j.bios.2020.112499
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发表时间：
2020-11-01
影响因子：
12.6
通讯作者：
Wang TH
中科院分区：
文献类型：
--
作者：
Zhang Y;Zhang P;Chen L;Kaushik A;Hu K;Wang TH
数字核酸扩增测试（数字NAAT）因其高灵敏度和特异性而成为流行的核酸检测工具。然而，大多数当前的数字 NAAT 平台仅限于“单一颜色单一目标”方法，其中每个目标都用特定的荧光标记探针进行编码，用于单重荧光检测。由于任何其他荧光团之间的光谱重叠，这种方法很难进行多重分析，因此数字 NAAT 的多重分析能力受到限制。作为扩展多重性的一种手段，我们开发了一种多重数字 NAAT 平台，称为液滴数字比例荧光编码 (ddRFC)，通过基于挂锁探针的核酸检测分析，该分析用 2 个荧光团的独特组合对每个核酸靶标进行编码。我们通过在微流体液滴中进行数字放大来检测每个目标的这种编码的双色荧光特征。为了证明我们平台的实用性，我们合成了 6 个不同的挂锁探针，每个探针都向代表临床重要性传播感染 (STI) 的核酸靶标呈现独特的双色荧光特征。我们继续展示具有单分子分辨率的 STI 目标的广泛、二重、四重和六重检测。我们的设计提供了一种经济高效的方法，通过简单地调整挂锁探针上分子信标结合位点的数量来扩大多重性，而无需重新设计扩增引物或荧光分子信标。随着进一步的发展，我们的平台有潜力实现核酸靶标的高度多重检测，并具有潜在的不受限制的多重性，并在未来作为更多疾病的诊断工具。
Digital nucleic acid amplification tests (digital NAATs) have emerged as a popular tool for nucleic acid detection due to their high sensitivity and specificity. Most current digital NAAT platforms, however, are limited to a “one-color-one-target” approach wherein each target is encoded with a specific fluorescently-labeled probe for single-plex fluorometric detection. This approach is difficult to multiplex due to spectral overlap between any additional fluorophores, and multiplexability of digital NAATs has therefore been limited. As a means to scale multiplexability, we have developed a multiplexed digital NAAT platform, termed Droplet Digital Ratiometric Fluorescence Coding (ddRFC), via a padlock probe-based nucleic acid detection assay which encodes each nucleic acid target with a unique combination of 2 fluorophores. We detect this encoded two-color fluorescence signature of each target by performing digital amplification in microfluidic droplets. To demonstrate the utility of our platform, we have synthesized 6 distinct padlock probes, each rendering a unique two-color fluorescence signature to a nucleic acid target representing a clinically important sexually transmitted infection (STI). We proceed to demonstrate broad-based, two-plex, four-plex, and six-plex detection of the STI targets with single-molecule resolution. Our design offers a cost-effective approach to scale up multiplexability by simply tuning the number of molecular beacon binding sites on the padlock probe without redesigning amplification primers or fluorescent molecular beacons. With further development, our platform has the potential to enable highly multiplexed detection of nucleic acid targets, with potentially unrestricted multiplexability, and serve as a diagnostic tool for many more diseases in the future.
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影响因子：
6.1
作者：
Rane TD;Chen L;Zec HC;Wang TH
通讯作者：
Wang TH
影响因子：
4.6
作者：
Dobnik D;Štebih D;Blejec A;Morisset D;Žel J
通讯作者：
Žel J
影响因子：
48
作者：
Hindson, Christopher M.;Chevillet, John R.;Briggs, Hilary A.;Gallichotte, Emily N.;Ruf, Ingrid K.;Hindson, Benjamin J.;Vessella, Robert L.;Tewari, Muneesh
通讯作者：
Tewari, Muneesh
影响因子：
7.4
作者：
Hindson, Benjamin J.;Ness, Kevin D.;Masquelier, Donald A.;Belgrader, Phillip;Heredia, Nicholas J.;Makarewicz, Anthony J.;Bright, Isaac J.;Lucero, Michael Y.;Hiddessen, Amy L.;Legler, Tina C.;Kitano, Tyler K.;Hodel, Michael R.;Petersen, Jonathan F.;Wyatt, Paul W.;Steenblock, Erin R.;Shah, Pallavi H.;Bousse, Luc J.;Troup, Camille B.;Mellen, Jeffrey C.;Wittmann, Dean K.;Erndt, Nicholas G.;Cauley, Thomas H.;Koehler, Ryan T.;So, Austin P.;Dube, Simant;Rose, Klint A.;Montesclaros, Luz;Wang, Shenglong;Stumbo, David P.;Hodges, Shawn P.;Romine, Steven;Milanovich, Fred P.;White, Helen E.;Regan, John F.;Karlin-Neumann, George A.;Hindson, Christopher M.;Saxonov, Serge;Colston, Bill W.
通讯作者：
Colston, Bill W.
影响因子：
14.9
作者：
Göransson J;Wählby C;Isaksson M;Howell WM;Jarvius J;Nilsson M
通讯作者：
Nilsson M