MS diagnostic bacterial identification library

项目摘要 需要快速准确的病原体检测和识别，以便医生做出反应和应对 适合于潜在的危及生命的感染。随着治疗危及生命的感染的需求增加， 更快地在数小时内而不是数天内，使用最先进的技术改进测定，减少时间， 需要诊断样本大小和每次测定的成本。当前食品药品监督管理局（FDA）批准 临床实验室中病原体检测的方法包括生物培养，核酸扩增， 核糖体蛋白质序列表征或基因组测序。总的来说，这些方法遭受 时间密集，需要放大临床获得的材料，并且通常非常昂贵， 这对于诊断实验室支持人员来说是沉重的负担。 基质辅助激光解吸电离飞行时间质谱（MALDI-MS）的研究进展 TOF-MS）使得能够开发用于病原体鉴定的准确且精确的方法。的 方法使用含有高丰度蛋白质的提取物，直接从培养板上的菌落中挑选， 用于单个物种的化学条形码，用于检测核糖体蛋白质组成的差异， ~90%的准确度。为了提高使用MALDI-TOF-MS快速诊断细菌感染的能力， 该提案将开发，完善和利用超小规模脂质纯化方法，用于提取 来自革兰氏阳性和阴性细菌以及真菌的高丰度脂质。必需的，高丰度 脂质存在于这些微生物的所有膜中，并且是一组高度多样化的分子。这种多样性 形成了我们假设的基础，即“基本的细菌和真菌脂质构成了一种化学条形码， 可用于通过质谱分析鉴定病原体”。我们的初步数据显示，这些脂质 结构是独特的，并且可以用作用于物种形成和/或亚物种形成的新化学条形码。 细菌和真菌感染以及对抗生素和抗微生物肽亚类的抗性模式。 在快速提取方法之后，将通过质谱法分析脂质，并且将得到的谱图 用于从各种临床上的脂质“指纹”产生质谱特征库， 相关病原体背景当结合时，蛋白质和脂质表型的分析将提供> 从固体培养基、血清等各种样品中鉴定病原体的准确率达99%， 支气管肺泡灌洗液和/或伤口流出物。最终，针对特定感染量身定制的治疗计划 应该转化为改善的结果，如更快的恢复时间，减少并发症， 降低感染患者的发病率和死亡率。

PROJECT SUMMARY Rapid and accurate pathogen detection and identification is needed to allow physicians to react and respond appropriately to potentially life threatening infections. With an increased need to treat life-threatening infections more rapidly, in hours instead of days, improved assays using state-of-the-art technologies that decrease time, diagnosis sample size, and cost per assay are needed. Current Food and Drug Administration (FDA)-approved methods for pathogen detection in a clinical laboratory include biological culture, nucleic acid amplification, ribosomal protein sequence characterization, or genome sequencing. Collectively, these methods suffer from being time intensive, requiring amplification of clinically obtained material, and are often significantly costly and burdensome for diagnostic laboratory support staff. Recent advances in matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI- TOF-MS) have enabled development of an accurate, and precise method for pathogen identification. The method uses extracts containing high abundance proteins, picked directly from colonies on culture plates as a chemical bar code for individual species to detect differences in the composition of ribosomal proteins with ~90% accuracy. To increase the ability to rapidly diagnose bacterial infections using MALDI-TOF-MS, this proposal will develop, refine, and utilize ultra-small scale lipid purification methodologies for the extraction of high abundance lipids from Gram-positive and -negative bacteria, as well as fungi. Essential, high abundance lipids are found in all membranes of these microbes and are a highly diverse set of molecules. This diversity forms the basis of our hypothesis that "essential bacterial and fungal lipids constitute a chemical barcode that can be used to identify pathogens by mass spectrometry profiling". Our preliminary data show that these lipid structures are unique and can be used as novel chemical barcodes for speciation and/or sub-speciation of bacterial and fungal infections and resistance patterns to a subset of antibiotic and antimicrobial peptides. Following a rapid extraction method, lipids will be analyzed by mass spectrometry and the resulting spectra will be used to generate a mass spectral signature library of lipid "fingerprints" from a wide variety of clinically relevant pathogen backgrounds. When combined, the analysis of the protein and lipid phenotype will provide > 99% accuracy in pathogen identification from a variety of samples, such as solid medium, serum, bronchoalveolar lavage fluid and/or wound effluent. Ultimately, treatment plans tailored to specific infections should translate to improved outcomes, such as faster recovery times, decreased complications, and decreased morbidity and mortality infected patients.