Thakku Sri Gowtham, Ackerman Cheri M, Myhrvold Cameron, Bhattacharyya Roby P, Livny Jonathan, Ma Peijun, Gomez Giselle Isabella, Sabeti Pardis C, Blainey Paul C, Hung Deborah T
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
PNAS Nexus. 2022 Apr 15;1(1):pgac021. doi: 10.1093/pnasnexus/pgac021. eCollection 2022 Mar.
Rapid and accurate diagnosis of infections is fundamental to individual patient care and public health management. Nucleic acid detection methods are critical to this effort, but are limited either in the breadth of pathogens targeted or by the expertise and infrastructure required. We present here a high-throughput system that enables rapid identification of bacterial pathogens, bCARMEN, which utilizes: (1) modular CRISPR-Cas13-based nucleic acid detection with enhanced sensitivity and specificity; and (2) a droplet microfluidic system that enables thousands of simultaneous, spatially multiplexed detection reactions at nanoliter volumes; and (3) a novel preamplification strategy that further enhances sensitivity and specificity. We demonstrate bCARMEN is capable of detecting and discriminating 52 clinically relevant bacterial species and several key antibiotic resistance genes. We further develop a simple proof of principle workflow using stabilized reagents and cell phone camera optical readout, opening up the possibility of a rapid point-of-care multiplexed bacterial pathogen identification and antibiotic susceptibility testing.
快速准确地诊断感染对于个体患者护理和公共卫生管理至关重要。核酸检测方法对这项工作至关重要，但在靶向病原体的广度或所需的专业知识和基础设施方面受到限制。我们在此展示了一种高通量系统，即细菌病原体快速鉴定微流控系统（bCARMEN），它利用：（1）基于模块化CRISPR-Cas13的核酸检测，具有更高的灵敏度和特异性；（2）一种液滴微流控系统，能够在纳升体积下同时进行数千次空间多重检测反应；（3）一种新型预扩增策略，进一步提高灵敏度和特异性。我们证明bCARMEN能够检测和区分52种临床相关细菌物种和几种关键抗生素耐药基因。我们进一步开发了一种使用稳定试剂和手机摄像头光学读数的简单原理验证工作流程，开启了快速床旁多重细菌病原体鉴定和抗生素敏感性测试的可能性。
