Reverse Line Blot Daignostic Test for Agents of Sepsis

描述（由申请人提供）：败血症在美国每年夺去多达120，000人的生命，是住院患者死亡的主要原因，估计每年花费170亿美元用于医疗保健。然而，目前还没有直接的诊断测试。 血液培养是常规使用，但通常需要24-48小时的结果，并在相当大的比例的情况下是阴性的。病原微生物的鉴定对于有效的诊断和治疗至关重要。 将抗菌剂与感染因子相匹配可显著提高成功治疗和结局的可能性。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取代培养。 本项目的目的是开发一种用于败血症多种细菌和真菌因子的反向线印迹试验。 在反向线印迹（RLE）方法中，单独的探针以离散条带附着于膜。然后将提取的细菌DNA与膜一起孵育，允许互补序列与相应的探针杂交。该方法的优点在于：（1）可以通过探针矩阵完成生物体的分型，其中杂交独立于每个探针发生;（2）源样品可以在杂交之前通过PCR扩增;（3）矩阵是宏观的，容易分析而不需要昂贵的仪器，并且适合于分析数十至数百个探针杂交。申请人打算通过将其转化为流通膜形式来进一步改进该方法，以显著减少杂交时间。在所提出的方法中，细菌DNA将直接从血液样品中提取，以消除与血液培养相关的延迟，并使用在原核物种中广泛保守的伴侣蛋白60基因（Cpn 60或HSP 60）的区域的通用简并引物进行PCR扩增。Cpn 60已被证明含有可用作鉴定细菌物种的探针的物种特异性序列。 最终产品将是用于鉴定脓毒症的细菌和真菌因子的诊断检测试剂盒，包括流通杂交单元、含有针对多种病原体的Cpn 60衍生探针的反向线印迹膜以及血液样本中存在的病原体中Cpn 60序列的PCR扩增所需的引物。

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.