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

使用集成光流控平台对多重耐药病原体进行多重、非扩增、基于核酸的鉴定

• 批准号: 9441612
• 负责人: Aaron R. Hawkins
• 金额: $ 98.85万
• 依托单位: BRIGHAM YOUNG UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2020-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9441612
• 关键词: Antibiotic Resistance; Antibiotics; Bacteremia; Bacteria; Bacterial Infections; Biological Assay; Blood; Blood Circulation; Blood Tests; Blood specimen; Cells; Cessation of life; Clinical; Complex; Cytolysis; DNA; Dangerousness; Detection; Development; Devices; Diagnosis; Diagnostic Procedure; Diagnostic tests; Enterobacter; Enterobacteriaceae; Enzymes; Escherichia coli; FDA approved; Filtration; Fluorescence; Genes; Goals; Health; Healthcare; Hospitalization; Hospitals; Hour; Human; Hybrids; Industrialization; Infection; Klebsiella pneumonia bacterium; Label; Lead; Length; Medical; Methods; Microfluidics; Molecular; Morbidity - disease rate; Nucleic Acids; Organism; Outcome; Pathogen detection; Patients; Pharmaceutical Preparations; Predisposition; Prevalence; Process; Public Health; Reproducibility; Resistance; Resistance profile; Resistance to infection; Sampling; Sepsis; Solid; System; Technology; Testing; Time; Treatment Cost; Whole Blood; antimicrobial; bacterial resistance; base; blood filtration; carbapenem-resistant Enterobacteriaceae; carbapenemase; design; diagnostic assay; drug resistant bacteria; drug resistant pathogen; effective therapy; fluorophore; global health; improved; inhibitor/antagonist; interest; mortality; multi-drug resistant pathogen; multiplex detection; novel; pathogen; public health relevance; resistance gene; single molecule; success; treatment strategy

 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.

 描述（由适用提供）：抗生素耐药性已成为主要的公共卫生威胁。感染抗药性病原体的患者的发病率和死亡率明显更高，这通常是由于延迟了有效的抗菌治疗。特别是对于血液感染，需要快速识别病原体和 耐药性特征至关重要，因为用抗生素治疗生物敏感的抗生素是必不可少的且至关重要的。确实，败血症涉及所有医院死亡的一半。临床医生通常不会收到病原体的药物敏感性信息，直到至少24 采样后小时，由于对基于培养的诊断方法的缓解。最近，抗碳青霉烯肠杆菌科（CRE）引起的血液感染变得越来越有问题。迫切需要对这些病原体的检测和耐药性测定的快速诊断测定。尽管基于PCR的测定是快速，特异性的，并且可以适合多重化，但是它们在血液样本中基本上未能进行。我们的工业伙伴大盆地公司（Great Basin Corporation）开发了一种完全处置的墨盒系统，用于培养的血液中的病原体检测。我们通过开发一种多重，非培养的，基于核酸的核酸测定法，使用一种新型的集成的光氟烯设备来检测和鉴定多发性耐药病原体，从而对该平台提出了重大改进。细菌将通过跨流过滤直接从血液样本中浓缩，然后输送到裂解和DNA剪切室。包含感兴趣基因的靶DNA将通过杂交在固体底物上捕获。分子信标将与捕获的核酸的特定靶标杂交。这些复合物将被释放，并通过能够检测单分子荧光的高级光荧光系统检测到特定的信标。我们将在细菌的血液中以低至10 CFU/mL的水平来证明鉴定。最初，重点是检测和表征直接从血液样本中分离出来的CRE。将确定KPC，NDM，VIM和IMP碳酸碳纤维酶基因，并简单地检测到肺炎，大肠杆菌和肠杆菌的特定标记物。该平台很容易扩展到其他病原体及其相关的抗生素抗性基因。这项技术有可能大大减少诊断时间并改善临床结果。