Electric field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2.
Electric field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2.
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DOI：
10.1073/pnas.2010254117
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发表时间：
2020-11-24
影响因子：
11.1
通讯作者：
Santiago JG
中科院分区：
文献类型：
--
作者：
Ramachandran A;Huyke DA;Sharma E;Sahoo MK;Huang C;Banaei N;Pinsky BA;Santiago JG
在大流行期间，快速、早期筛查对于早期识别受感染患者和控制疾病传播至关重要。CRISPR生物学为快速准确的病原体检测提供了新方法。尽管它们具有多功能性和特异性，但现有的CRISPR诊断方法受到前期核酸提取、大试剂体积和几个手动步骤的要求的影响，这些因素延长了过程并阻碍了在低资源环境中的使用。我们在这里将联合收割机微流体、片上电场控制和CRISPR结合起来，直接解决当前CRISPR诊断方法的局限性。我们将该方法应用于临床样本中SARS-CoV-2 RNA的快速检测。我们的方法从原始样本到结果大约需要35分钟，这比现有的基于核酸的COVID-19诊断方法有了显著的改进。COVID-19在全球的迅速蔓延暴露了我们应对新的致命病原体的能力存在重大差距。快速、准确和易于配置的分子诊断测试对于防止新疾病的全球传播至关重要。基于CRISPR的诊断方法被证明是可现场部署的解决方案。在该测定的一种基本形式中，CRISPR-Cas 12酶与合成的指导RNA（gRNA）复合。该复合物只有在与靶DNA特异性结合并切割靶DNA时才被激活。激活的复合物随后非特异性地切割用荧光团-猝灭剂对标记的单链DNA报告探针。我们发现，电场梯度可用于控制和加速这种CRISPR检测，方法是在微流控芯片内共聚焦Cas 12-gRNA、报告基因和靶标。我们实现了一个适当的电场梯度使用选择性离子聚焦技术称为等速电泳（ITP）上实现的微流控芯片。与之前的CRISPR诊断检测不同，我们还使用ITP从原始鼻咽拭子样本中自动纯化靶RNA。我们在这里将这种ITP纯化与环介导的等温扩增和ITP增强的CRISPR检测相结合，以实现在约35分钟内检测人工和临床鼻咽拭子样本的严重急性呼吸综合征冠状病毒2（SARS-CoV-2）RNA（从原始样本到结果）。这种电场控制为一套基于微流体CRISPR的诊断测定提供了替代模式。
Rapid, early-stage screening is crucial during pandemics for early identification of infected patients and control of disease spread. CRISPR biology offers new methods for rapid and accurate pathogen detection. Despite their versatility and specificity, existing CRISPR diagnostic methods suffer from the requirements of up-front nucleic acid extraction, large reagent volumes, and several manual steps—factors which prolong the process and impede use in low-resource settings. We here combine microfluidics, on-chip electric field control, and CRISPR to directly address limitations of current CRISPR diagnostic methods. We apply our method to the rapid detection of SARS-CoV-2 RNA in clinical samples. Our method takes about 35 min from raw sample to result, a significant improvement over existing nucleic acid-based diagnostic methods for COVID-19. The rapid spread of COVID-19 across the world has revealed major gaps in our ability to respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based diagnostic approaches are proving to be useful as field-deployable solutions. In one basic form of this assay, the CRISPR–Cas12 enzyme complexes with a synthetic guide RNA (gRNA). This complex becomes activated only when it specifically binds to target DNA and cleaves it. The activated complex thereafter nonspecifically cleaves single-stranded DNA reporter probes labeled with a fluorophore−quencher pair. We discovered that electric field gradients can be used to control and accelerate this CRISPR assay by cofocusing Cas12–gRNA, reporters, and target within a microfluidic chip. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. Unlike previous CRISPR diagnostic assays, we also use ITP for automated purification of target RNA from raw nasopharyngeal swab samples. We here combine this ITP purification with loop-mediated isothermal amplification and the ITP-enhanced CRISPR assay to achieve detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA (from raw sample to result) in about 35 min for both contrived and clinical nasopharyngeal swab samples. This electric field control enables an alternate modality for a suite of microfluidic CRISPR-based diagnostic assays.
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DOI：
10.1073/pnas.1205004109
发表时间：
2012-07-10
影响因子：
11.1
作者：
Bercovici, Moran;Han, Crystal M.;Santiago, Juan G.
通讯作者：
Santiago, Juan G.
影响因子：
64.8
作者：
Ackerman, Cheri M.;Myhrvold, Cameron;Sabeti, Pardis C.
通讯作者：
Sabeti, Pardis C.
影响因子：
6.1
作者：
Borysiak, Mark D.;Kimura, Kevin W.;Posner, Jonathan D.
通讯作者：
Posner, Jonathan D.
影响因子：
--
作者：
Kaneko, Hisatoshi;Kawana, Takashi;Suzutani, Tatsuo
通讯作者：
Suzutani, Tatsuo
影响因子：
6.1
作者：
Kaigala, G. V.;Bercovici, M.;Backhouse, C. J.
通讯作者：
Backhouse, C. J.