Digital Droplet Loop-Mediated Isothermal Amplification Featuring a Molecular Beacon Assay, 3D Printed Droplet Generation, and Smartphone Imaging for Sequence-Specific DNA Detection
Digital Droplet Loop-Mediated Isothermal Amplification Featuring a Molecular Beacon Assay, 3D Printed Droplet Generation, and Smartphone Imaging for Sequence-Specific DNA Detection
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DOI：
10.1021/acs.analchem.2c02979
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发表时间：
2022-08-16
影响因子：
7.4
通讯作者：
Anderson，Jared L.
中科院分区：
文献类型：
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作者：
Hsieh，Shu-An;Shamsaei，Danial;Anderson，Jared L.
核酸检测广泛用于扩增和定量来自生物样品的核酸。虽然聚合酶链式反应（PCR）非常流行，但需要昂贵的热循环仪来精确控制温度。环介导等温扩增（LAMP）能够在恒定温度下进行高度灵敏、快速和低成本的核酸扩增。LAMP检测通常依赖于缺乏序列选择性的双链DNA结合染料或金属指示剂。分子信标（MB）是发夹形状的寡核苷酸探针，其在LAMP中的序列特异性提供区分单核苷酸多态性（SNP）的能力。数字液滴LAMP（ddLAMP）能够进行大量独立的LAMP反应，并提供靶DNA序列的定量。然而，ddLAMP的主要挑战是需要昂贵的液滴发生器来形成均匀的微滴。在这项研究中，我们首次证明了三维（3D）打印液滴生成平台可以与以MB为序列特异性探针的LAMP检测相结合。设计了低成本的3D打印液滴发生器系统，并在单分散ddLAMP油中测定微滴的形成中证明了其可定制性。此外，一个基于智能手机的成像系统被证明，以增加护理点应用的可访问性。显示MB-ddLAMP测定在不同扩增温度下区分两种SNP，以提供用于序列特异性、灵敏性和准确的DNA定量的有用平台。这项工作将MB的实用性扩展到ddLAMP用于SNP检测的定量研究，并利用3D打印技术的可定制性来优化水包油微滴的均匀性，大小和体积。
Nucleic acid detection is widely used in the amplification and quantitation of nucleic acids from biological samples. While polymerase chain reaction (PCR) enjoys great popularity, expensive thermal cyclers are required for precise temperature control. Loop-mediated isothermal amplification (LAMP) enables highly sensitive, rapid, and low-cost amplification of nucleic acids at constant temperatures. LAMP detection often relies on double-stranded DNA-binding dyes or metal indicators that lack sequence selectivity. Molecular beacons (MBs) are hairpin-shaped oligonucleotide probes whose sequence specificity in LAMP provides the capability of differentiating between single-nucleotide polymorphisms (SNPs). Digital droplet LAMP (ddLAMP) enables a large number of independent LAMP reactions to be performed and provides quantification of target DNA sequences. However, a major challenge with ddLAMP is the requirement of expensive droplet generators to form homogeneous microdroplets. In this study, we demonstrate for the first time that a three-dimensional (3D) printed droplet generation platform can be coupled to a LAMP assay featuring MBs as sequence-specific probes. The low-cost 3D printed droplet generator system was designed, and its customizability was demonstrated in the formation of monodisperse ddLAMP assay-in-oil microdroplets. Additionally, a smartphone-based imaging system is demonstrated to increase accessibility for point-of-care applications. The MB-ddLAMP assay is shown to discriminate between two SNPs at various amplification temperatures to afford a useful platform for sequence-specific, sensitive, and accurate DNA quantification. This work expands the utility of MBs to ddLAMP for quantitative studies in the detection of SNPs and exploits the customizability of 3D printing technologies to optimize the homogeneity, size, and volume of oil-in-water microdroplets.