Multiplexed, Non-Amplified, Nucleic Acid-Based Identification of Multidrug Resistant Pathogens Using an Integrated Optofluidic Platform

 描述（由申请人提供）：抗生素耐药性已成为一个主要的公共卫生威胁。感染耐药病原体的患者的发病率和死亡率显著更高，这通常是由于有效的抗微生物治疗的施用延迟。特别是对于血流感染，需要快速鉴定病原体和 耐药性是至关重要的，因为用生物体敏感的抗生素治疗是必要的，而且时间紧迫。事实上，脓毒症涉及到所有医院死亡的一半。病原体的药物敏感性信息通常在至少24小时之前不会被临床医生接收到。 由于依赖于基于培养的诊断方法，最近，由碳青霉烯类耐药肠杆菌科（CRE）引起的血流感染已变得越来越成问题。迫切需要一种用于检测和测定这些病原体的抗性的快速诊断测定法。尽管基于PCR的测定快速、特异且易于多重化，但它们在很大程度上未能在血液样品中执行。我们的工业合作伙伴Great Basin Corporation开发了一种用于培养血液中病原体检测的完全一次性检测盒系统。我们建议通过开发一种多路复用的、非扩增的、非培养的、基于核酸的检测方法来对该平台进行重大改进，该方法使用新型集成光流体装置来检测和鉴定多药耐药病原体。通过错流过滤直接从血液样品中浓缩细菌，然后将其输送到裂解和DNA剪切室。通过杂交将含有目的基因的靶DNA捕获在固体基质上。分子信标将与捕获的核酸上的特定靶标杂交。这些复合物将被释放，并通过能够检测单分子荧光的先进光流体系统检测特定的信标。我们将证明在一小时内识别血液中的细菌，其水平低至10 CFU/mL。最初，重点将是检测和表征直接从血液样本中分离的CRE。将沿着同时检测肺炎克雷伯菌、大肠埃希菌和肠杆菌属的特异性标志物，鉴别KPC、NDM、Vim和IMP碳青霉烯酶基因。该平台易于扩展到其他病原体及其相关的抗生素抗性基因。这项技术有可能显著缩短诊断时间并改善临床结果。

 DESCRIPTION (provided by applicant): Antibiotic resistance has emerged as a major public health threat. Patients infected with drug- resistant pathogens suffer significantly higher rates o morbidity and mortality, most often due to delays in the administration of effective antimicrobial therapies. In particular for bloodstream infections, the need to rapidly identify both pathogen and resistance profile is crucial, as treatment with antibiotics to which the organism is sensitive is essential and time-critical. Indeed, sepsis is involved in up to half of all hospital deaths. Drug susceptibility information for a pathogen is typically not received by clinicians until at least 24 hours post-sampling, because of reliance on culture-based diagnostic methods. Recently, bloodstream infections caused by carbapenem-resistant Enterobacteriaceae (CRE) have become increasingly problematic. A rapid diagnostic assay for the detection and resistance determination of these pathogens is urgently needed. Although PCR-based assays are rapid, specific, and amenable to multiplexing, they have largely failed to perform in blood samples. Our industrial partner, Great Basin Corporation, has developed a fully disposable cartridge system for pathogen detection in cultured blood. We propose major improvements to this platform through the development of a multiplexed, non-amplified, non-cultured, nucleic acid-based assay for the detection and identification of multidrug resistant pathogens using a novel integrated optofluidic device. Bacteria will be concentrated directly from a blood sample by cross-flow filtration, and then delivered to a lysis and DNA-shearing chamber. Target DNAs containing the genes of interest will be captured on a solid substrate by hybridization. Molecular beacons will be hybridized to specific targets on the captured nucleic acids. These complexes will be released and specific beacons detected by an advanced optofluidics system capable of detecting single molecule fluorescence. We will demonstrate identification within one hour of bacteria in blood at levels as low as 10 CFU/mL. Initially, the focus will be to detect and characterize CRE isolated directly from a blood sample. The KPC, NDM, VIM, and IMP carbapenemase genes will be identified along with the simultaneous detection of specific markers for Klebsiella pneumoniae, Escherichia coli, and Enterobacter species. This platform is readily expandable to additional pathogens and their relevant antibiotic resistance genes. This technology has the potential to significantly reduce time to diagnosis and improve clinical outcomes.