MS diagnostic bacterial identification library

MS诊断细菌鉴定文库

• 批准号: 8722128
• 负责人: Robert K Ernst
• 金额: $ 27.74万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8722128
• 关键词: Amino Acid Sequence; Antibiotics; Bacteria; Bacterial Infections; Bar Codes; Biological; Biological Assay; Bronchoalveolar Lavage Fluid; Cell Wall; Chemicals; Clinical; Data; Data Set; Development; Diagnosis; Diagnostic; Diagnostic Procedure; Failure; Fingerprint; Future; Generations; Growth; Hour; Individual; Infection; Intellectual Property; Laboratories; Libraries; Life; Lipids; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Membrane; Membrane Lipids; Methodology; Methods; Microbe; Morbidity - disease rate; Mycoses; Nucleic Acids; Organism; Outcome; Pathogen detection; Patients; Pattern; Peptide Sequence Determination; Phenotype; Physicians; Preparation; Protein Analysis; Proteins; Protocols documentation; Recovery; Resistance profile; Resistance to infection; Ribosomal Proteins; Sample Size; Sampling; Serum; Solid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Speed; Structure; Technology; Time; Translating; United States Food and Drug Administration; antimicrobial peptide; base; clinically relevant; cost; culture plates; design; fundamental research; fungus; genome sequencing; improved; interest; microbial; microwave electromagnetic radiation; mortality; novel; novel diagnostics; pathogen; public health relevance; research and development; treatment planning; vaccine development; wound

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.

项目总结 需要快速和准确的病原体检测和识别，以使医生能够做出反应和反应 适当地应对可能危及生命的感染。随着治疗危及生命的感染的需求增加 更快地，在几小时而不是几天内，使用最先进的技术来缩短时间来改进分析， 诊断样本量和每份化验成本都是必需的。当前食品和药物管理局(FDA)批准 临床实验室病原体检测方法包括生物培养、核酸扩增、 核糖体蛋白序列特征，或基因组测序。总而言之，这些方法会受到 这是时间密集的，需要扩大临床获得的材料，而且通常非常昂贵和 诊断实验室支持人员的负担。 基质辅助激光解吸电离飞行时间质谱仪的研究进展 TOF-MS)为病原体鉴定提供了一种准确、精确的方法。这个 该方法使用直接从培养板上的菌落中挑选的含有高丰度蛋白质的提取物作为 单个物种的化学条形码，用于检测核糖体蛋白组成的差异 ~90%的准确率。为了提高使用MALDI-TOF-MS快速诊断细菌感染的能力，这 提案将开发、提炼和利用超小型脂类纯化方法来提取 来自革兰氏阳性和阴性细菌以及真菌的高丰度脂质。基本的、高丰度的 脂类存在于这些微生物的所有细胞膜中，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， 可以用来通过质谱分析来鉴定病原体。我们的初步数据表明，这些脂质 结构是独特的，可用作新的化学条形码，用于物种形成和/或亚物种形成 细菌和真菌感染以及对部分抗生素和抗菌肽的耐药性模式。 在一种快速提取方法之后，脂类将通过质谱学进行分析，得到的光谱将 用来生成来自各种临床疾病的脂类“指纹”的质谱库 相关病原体背景。当结合起来时，蛋白质和脂肪表型的分析将提供&GT； 从各种样本中识别病原体的准确率为99%，例如固体介质、血清、 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做的治疗计划 应转化为改善结果，如更快的恢复时间，减少并发症，以及 降低感染患者的发病率和死亡率。