美国麻省理工学院和哈佛大学Paul C. Blainey和Cameron Myhrvold研究团队近日取得一项新成果。他们研发了使用Cas13进行大规模多重核酸检测的技术。这一研究成果在线发表在2020年4月29日的《自然》上。
研究人员开发了核酸多元评估的组合阵列反应（CARMEN），这是一个可扩展的、多种病原体检测的平台。在CARMEN平台中，含有基于CRISPR核酸检测试剂的纳升液滴会自组装为微孔矩阵以与扩增样品的液滴配对，从而针对每个样品重复测试其CRISPR RNA（crRNA）。
CARMEN和Cas13检测（CARMEN-Cas13）的组合可在单个阵列上对超过4,500个crRNA-靶点对进行有效的测试。使用CARMEN-Cas13，研究人员研发了一种多重分析方法，该方法可同时区分所有169种具有≥10个已公开基因组序列的人类相关病毒，并快速整合其他crRNA以检测COVID-19的病原体。
CARMEN-Cas13进一步实现了对A型流感病毒株的全面亚型化，并能多重鉴定数十种HIV耐药性突变。CARMEN固有的多路复用和通量功能使其可扩展规模；小型化的检测方法可为每次测试的试剂成本降低300倍以上。基于CRISPR的可扩展、高度复用的核酸检测将诊断和监测工作从针对高优先级样品的测试转移到对大型样品集的全面检测，这极大地有利于患者和公共健康。
研究人员表示，绝大多数能在全球传播的病原体未被发现，这损害了对患者的护理并阻碍了对疾病爆发的准备和响应。为了能够进行常规监视和全面的诊断应用，需要新的检测技术，该技术可以同时测试许多样品，以及检测许多病原体。
附：英文原文
Title: Massively multiplexed nucleic acid detection using Cas13
Author: Cheri M. Ackerman, Cameron Myhrvold, Sri Gowtham Thakku, Catherine A. Freije, Hayden C. Metsky, David K. Yang, Simon H. Ye, Chloe K. Boehm, Tinna-Slveig F. Kosoko-Thoroddsen, Jared Kehe, Tien G. Nguyen, Amber Carter, Anthony Kulesa, John R. Barnes, Vivien G. Dugan, Deborah T. Hung, Paul C. Blainey, Pardis C. Sabeti
Issue&Volume: 2020-04-29
Abstract: The overwhelming majority of globally circulating pathogens go undetected, undermining patient care and hindering outbreak preparedness and response. To enable routine surveillance and comprehensive diagnostic applications, there is a need for detection technologies that can scale to test many samples1–3 while simultaneously testing for many pathogens4–6. Here, we develop Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection. In the CARMEN platform, nanoliter droplets containing CRISPR-based nucleic acid detection reagents7 self-organize in a microwell array8 to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN-Cas13) enables robust testing of >4,500 crRNA-target pairs on a single array. Using CARMEN-Cas13, we developed a multiplexed assay that simultaneously differentiates all 169 human-associated viruses with ≥10 published genome sequences and rapidly incorporated an additional crRNA to detect the causative agent of the 2020 COVID-19 pandemic. CARMEN-Cas13 further enables comprehensive subtyping of influenza A strains and multiplexed identification of dozens of HIV drug-resistance mutations. CARMEN’s intrinsic multiplexing and throughput capabilities make it practical to scale, as miniaturization decreases reagent cost per test >300-fold. Scalable, highly-multiplexed CRISPR-based nucleic acid detection shifts diagnostic and surveillance efforts from targeted testing of high-priority samples to comprehensive testing of large sample sets, greatly benefiting patients and public health9–11.
DOI: 10.1038/s41586-020-2279-8
Source: https://www.nature.com/articles/s41586-020-2279-8
Nature：《自然》，创刊于1869年。隶属于施普林格·自然出版集团，最新IF：69.504
官方网址：http://www.nature.com/
投稿链接：http://www.nature.com/authors/submit_manuscript.html
本期文章：《自然》：Online/在线发表
