Droplet Cas12a Assay Enables DNA Quantification from Unamplified Samples at the Single-Molecule Level
Droplet Cas12a Assay Enables DNA Quantification from Unamplified Samples at the Single-Molecule Level
复制标题
液滴 Cas12a 检测可在单分子水平上对未扩增样品进行 DNA 定量
DOI：
10.1021/acs.nanolett.1c00715
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发表时间：
2021-05-26
期刊：
影响因子：
10.8
通讯作者：
Zhou, Xiaoming
中科院分区：
文献类型：
--
作者：
Yue, Huahua;Shu, Bowen;Zhou, Xiaoming
DNA定量对于生物医学研究是重要的，但是常规使用的技术依赖于核酸扩增，其具有固有的问题，如交叉污染风险和定量偏倚。在这里，我们报告了一种基于CRISPR-Cas 12 a的分子诊断技术，用于在单分子水平上对DNA进行无扩增和绝对定量。为了实现这一目标，我们首先筛选出了高效Cas 12 a检测的最佳反应参数，与报道的Cas 12 a检测相比，灵敏度提高了50倍以上。我们进一步利用微滴使能的限制效应来进行超定位液滴Cas 12 a测定，获得优异的特异性和单分子灵敏度。此外，我们通过直接计数来自具有广泛病毒滴度的临床血清样品的不同病毒的DNA（非洲猪瘟病毒、爱泼斯坦-巴尔病毒和肝炎B病毒）来证明其通用性和定量能力。鉴于crRNA的灵活可编程性，我们设想这种免扩增技术作为分子诊断的通用和定量平台。
DNA quantification is important for biomedical research, but the routinely used techniques rely on nucleic acid amplification which have inherent issues like cross-contamination risk and quantification bias. Here, we report a CRISPR-Cas12a-based molecular diagnostic technique for amplification-free and absolute quantification of DNA at the single-molecule level. To achieve this, we first screened out the optimal reaction parameters for high-efficient Cas12a assay, yielding over 50-fold improvement in sensitivity compared with the reported Cas12a assays. We further leveraged the microdroplet-enabled confinement effect to perform an ultralocalized droplet Cas12a assay, obtaining excellent specificity and single-molecule sensitivity. Moreover, we demonstrated its versatility and quantification capability by direct counting of diverse virus's DNAs (African swine fever virus, Epstein-Barr virus, and Hepatitis B virus) from clinical serum samples with a wide range of viral titers. Given the flexible programmability of crRNA, we envision this amplification-free technique as a versatile and quantitative platform for molecular diagnosis.