Integrated Comprehensive Droplet Digital Detection (IC 3D) system for rapid detection of bacteria andantimicrobial resistance

 描述（由申请人提供）：抗菌素耐药性是美国日益严重的健康问题，每年有超过200万人感染，超过23，000人死亡。耐药细菌介导的血流感染（BSI）的一个主要挑战是诊断：当医生确定细菌种类以改进治疗时，可能B已晚。事实上，目前细菌检测的金标准，血液培养，需要几天才能得到结果，而其他分子诊断方法，如PCR，往往不够灵敏，无法直接检测血液中低浓度（1-100 CFU/mL）的细菌。我们开发了一种平台技术，称为集成综合液滴数字检测（IC 3D）。该系统能够以单细胞灵敏度在一步中快速（1-3小时）和选择性地检测毫升（mL）未处理全血中的细菌，并培养- 和无扩增反应。拟议的研究旨在开发和验证用于快速检测碳青霉烯类耐药肠杆菌科（CRE）和超广谱β-内酰胺酶（ESBL）的新型IC 3D检测，因为它们作为革兰氏阴性病原体至关重要，很少有抗菌剂对其具有活性。我们还将与包括ISS Inc、Dolomite Microfluidics和BioVenture Services（BVS），LLC在内的行业合作伙伴合作，开发和验证集成、自动化、便携式IC 3D系统，并为未来的临床批准开展产品开发活动。具体而言，我们将1）分离和优化特异性检测ESBL和克雷斯的DNAzyme传感器（目标1），2）整合和验证IC 3D，用于血液中的快速、单一CRE和ESBL检测（目标2），3）将IC 3D用于临床标本中的CRE和ESBL检测（目标3）。到本研究结束时，我们将开发并验证1）用于快速检测CRE和ESBL的新型IC 3D诊断检测试剂盒，以及2）第一代仪器，准备提交FDA并商业化。

 DESCRIPTION (provided by applicant): Antimicrobial resistance is a growing health problem in the United States with more than two million people are infected annually and >23,000 deaths. A major challenge in antibiotic-resistant bacterium-mediated bloodstream infections (BSI) is diagnosis: by the time doctors identify the bacteria species to refine treatment, it can b too late. Indeed, the current gold standard for bacteria detection, blood culture, takes days to get a result while other molecular diagnosis methods such as PCR are often not sensitive enough to directly detect bacteria that occur at low concentrations in blood (1-100 CFU/mL). We have developed a platform technology called Integrated Comprehensive Droplet Digital Detection (IC 3D). This system is able to rapidly (1-3 h) and selectively detect bacteria from milliliters (mLs) of unprocessed whole blood at single-cell sensitivity in a one-step, and culture- and amplification-free reaction. The proposed study aims to develop and validate new IC 3D tests for rapid detection of carbapenem-resistant Enterobacteriaceae (CRE) and extended spectrum beta lactamase (ESBL) because of their critical importance as Gram-negative pathogens for which few antimicrobials are active. We will also work with our industrial partners including ISS Inc, Dolomite Microfluidics and BioVenture Services (BVS), LLC to develop and validate an integrated, automated, portable IC 3D system and to perform product development activities towards future clinical approval. Specifically, we will 1) isolate and optimize DNAzyme sensors that specifically detect ESBLs and CREs (Aim 1), 2) Integrate and validate the IC 3D for rapid, single CRE and ESBL detection in blood (Aim 2), and 3) Validate the IC 3D for CRE and ESBL detection from clinical specimens (Aim 3). By the conclusion of this study, we will have developed and validated 1) a novel IC 3D diagnostic assay for rapid CRE and ESBL detection, and 2) a first generation instrument, which are ready for FDA submission and commercialization.