Reverse Line Blot Daignostic Test for Agents of Sepsis

败血症因子的反向线印迹诊断测试

• 批准号: 7000041
• 负责人: Andrew E. Levin
• 金额: $ 46.56万
• 依托单位: IMMUNETICS, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7000041
• 关键词: bacterial disease; blood disorder diagnosis; blood tests; clinical research; cooperative study; diagnosis design /evaluation; diagnosis quality /standard; human subject; microorganism classification; mycosis; nucleic acid hybridization; nucleic acid probes; polymerase chain reaction; rapid diagnosis; septicemia

DESCRIPTION (provided by applicant): Sepsis claims up to 120,000 lives per year in the U.S., is a leading cause of death in hospitalized patients, and is estimated to cost $17 billion annually in healthcare. Yet, at present there is no straightforward diagnostic test available. Blood cultures are in routine use, yet typically require 24-48 hours for results and are negative in a substantial percentage of cases. Identification of the causative organism(s) is critical to effective diagnosis and treatment. Matching the antimicrobial to the infectious agent significantly enhances the probability of successful treatment and outcome. In particular, multiplexed detection methods which would enable simultaneous probing for a range of organisms may potentially substitute for culture. The aim of this project is development of a Reverse Line Blot assay for multiple bacterial and fungal agents of sepsis. In the Reverse Line Blot (RLE) approach, separate probes are attached to a membrane in discrete bands. Extracted bacterial DNA is then incubated with the membrane permitting complementary sequences to hybridize with the corresponding probes. The advantages of this method are that (1) typing of organisms can be accomplished via a matrix of probes, with hybridization occurring independently to each probe; (2) the source sample can be amplified by PCR prior to hybridization; (3) the matrix is macroscopic, easily analyzed without expensive instrumentation, and is suitable for analysis of tens to hundreds of probe hybridizations. Applicants intend to further improve this methodology by converting it to a flow-through membrane format, in order to significantly reduce hybridization times. In the proposed approach, bacterial DNA will be extracted directly from a blood sample to eliminate the delay associated with blood culture, and subject to PCR amplification using universal degenerate primers for a region of the chaperonin 60 gene (Cpn60 or HSP60) widely conserved among prokaryotic species. Cpn60 has been shown to contain species-specific sequences useful as probes for identification of bacterial species. The final product will be a diagnostic test kit for identification of bacterial and fungal agents of sepsis, comprising the flow-through hybridization unit, a Reverse Line Blot membrane containing Cpn60-derived probes to multiple pathogens, and primers necessary for PCR amplification of the Cpn60 sequence in pathogens present in blood samples.

描述(申请人提供)：败血症在美国每年夺走多达12万人的生命，是住院患者死亡的主要原因，估计每年在医疗保健方面花费170亿美元。然而，目前还没有直接的诊断测试可用。血培养是常规使用，但通常需要24-48小时才能得到结果，在相当大比例的病例中呈阴性。确定病原体(S)是有效诊断和治疗的关键。将抗菌剂与感染剂配对显著提高了成功治疗和结果的可能性。特别是，能够同时探测一系列生物体的多重检测方法可能会潜在地取代培养。 本项目的目的是建立一种检测脓毒症的多种细菌和真菌制剂的反向线粒斑法。在反转线印迹(RLE)方法中，独立的探针以离散的条带附着在膜上。提取的细菌DNA随后与膜孵育，允许互补序列与相应的探针杂交。这种方法的优点是：(1)生物的分型可以通过一组探针完成，每个探针的杂交是独立进行的；(2)来源样本可以在杂交前通过PCR进行扩增；(3)基质是宏观的，无需昂贵的仪器就可以进行分析，适用于数十到数百种探针杂交的分析。申请者打算进一步改进这一方法，将其转换为流通膜形式，以便显著减少杂交时间。在所提出的方法中，将直接从血液样本中提取细菌DNA，以消除与血液培养相关的延迟，并使用通用简并引物对原核生物中广泛保守的伴侣蛋白60基因(Cpn60或HSP60)的区域进行PCR扩增。Cpn60已被证明含有物种特异的序列，可用作细菌物种鉴定的探针。最终产品将是一种用于鉴定脓毒症细菌和真菌病原体的诊断检测试剂盒，其中包括流动杂交单元、包含针对多种病原体的Cpn60衍生探针的反向Line Blot膜，以及用于对血液样本中存在的病原体的Cpn60序列进行聚合酶链式反应(PCR)扩增所需的引物。