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

 描述(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明显较高，最常见的原因是有效的抗菌药治疗出现延误。尤其是对于血液感染，需要快速识别病原体和 耐药谱是至关重要的，因为使用微生物敏感的抗生素治疗是必要的，而且是时间关键的。事实上，在所有医院死亡中，有多达一半与脓毒症有关。临床医生通常至少要到24小时才能收到病原体的药敏信息 抽样后数小时，因为依赖基于培养的诊断方法。近年来，由碳青霉烯类耐药肠杆菌科(CRE)引起的血液感染问题日益严重。迫切需要一种快速诊断方法来检测和确定这些病原菌的耐药性。尽管基于聚合酶链式反应的分析是快速、特异的，而且易于进行多重分析，但它们在很大程度上未能在血液样本中发挥作用。我们的工业合作伙伴大盆公司已经开发了一种用于培养血液中病原体检测的完全一次性卡盒系统。我们建议通过开发一种多路、非扩增、非培养、基于核酸的检测和鉴定多药耐药病原体的新型集成光流控设备来对该平台进行重大改进。细菌将通过错流过滤直接从血液样本中浓缩，然后被输送到裂解和DNA剪切室。含有感兴趣基因的目标DNA将通过杂交被捕获在固体底物上。分子信标将与捕获的核酸上的特定目标杂交。这些络合物将被释放，并由能够检测单分子荧光的先进光流体学系统检测到特定的信标。我们将在血液中细菌含量低至10cfu/ml的一小时内进行鉴定。最初，重点将是检测和鉴定直接从血液样本中分离出来的Cre。将鉴定KPC、NDM、VIM和IMP碳青霉烯酶基因，同时检测肺炎克雷伯菌、大肠埃希菌和肠杆菌的特异性标志物。这个平台很容易扩展到其他病原体及其相关的抗生素耐药基因。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.