Microbial Identification Using Surface Enhanced Laser De

使用表面增强激光德进行微生物鉴定

• 批准号: 6675254
• 负责人: Patrick R Murray
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6675254
• 关键词: bacterial genetics; bacterial proteins; bacteriolysis; genotype; mass spectrometry; method development; microorganism classification; nucleic acid sequence; phenotype; protein binding; protein purification; proteomics; tissue /cell culture

Identification of bacteria has traditionally been by morphologic features and phenotypic testing. Identification of some common organisms (e.g., Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli) can be rapid (e.g., less than one hour), exploiting their characteristic morphology and a few selected phenotypic tests. However, the identification of most organisms is slow, requiring hours to days for a definitive answer. More recently the use of genomics such as sequencing ribosomal RNA genes has proved to be a useful tool. Although sequencing specific genes is a powerful discriminatory tool, the current methodology requires one or more days before a result is available. A logical extension of sequencing genes is to use the gene products for bacterial identification. Significant variations in a structural gene sequence would result in variations in the protein product. In the last 5 years, preliminary work in the analysis of bacterial proteins for identification has been performed using Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-ToF MS). These studies demonstrated that whole microorganisms can provide distinct and reproducible mass spectra of proteins. The advantage of this approach is the results are available in minutes. However, MALDI-ToF MS produces extremely complex spectra of low molecular weight proteins, which may be unsuitable for applications in the clinical microbiology laboratory. During the past 6 months, we have explored using an alternative method, Surface-Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry (SELDI-ToF MS). The advantage of this technique is ease of use, relative low cost for the instrumentation and consumable supplies, and the use of "protein chips" for selective capture of proteins from the bacterial cell lysate. The protein chips are available in a variety of chromatographic surfaces that allow binding proteins with different affinities (e.g., cationic, anionic, and metal affinity). Bacterial lysates can be exposed to one or more protein chips and then the bound proteins analyzed by SELDI-ToF MS. In our preliminary experiments we have (1) defined the conditions required for lysing both gram-positive and gram-negative bacteria, and (2) identified the appropriate protein chips for analysis. We believe we have completed the first phase of the experiments and are currently assessing the reproducibility of the protein spectra and the discriminatory power of the profiles. If these results appear to be satisfactory, we will expand the study to include a large number of well-characterized, clinically significant organisms selected from our culture collection and the American Type Cell Culture (ATCC) collection. Current work on another project (CL010326-01 DLM) has revealed that phenotypic identification of culture collection isolates (including the ATCC collection) is not precise. That is, genomic studies have demonstrated that a number of collection isolates are misidentified by phenotypic tests. Therefore, the identification of all isolates used in these studies will be by comprehensive phenotypic and genotypic methods.

细菌的鉴定传统上是通过形态特征和表型试验。一些常见微生物(例如金黄色葡萄球菌、肺炎链球菌、大肠杆菌)的鉴定可以快速(例如，不到一小时)，利用它们的特征形态和一些选定的表型试验。然而，大多数生物体的鉴定速度很慢，需要几个小时到几天的时间才能得到明确的答案。最近，基因组学的使用，如核糖体RNA基因测序，已被证明是一个有用的工具。虽然对特定基因进行测序是一种强大的鉴别工具，但目前的方法需要一天或几天才能得出结果。基因测序的一个合乎逻辑的延伸是使用基因产物进行细菌鉴定。结构基因序列的显著变化会导致蛋白质产物的变化。在过去的5年里，利用基质辅助激光解吸/电离飞行时间质谱仪(MALDI-ToF MS)对用于鉴定的细菌蛋白质的分析进行了初步工作。这些研究表明，整个微生物可以提供清晰和可重现的蛋白质质谱图。这种方法的优点是几分钟内就能得到结果。然而，MALDI-ToF MS产生了极其复杂的低分子蛋白质谱，这可能不适合在临床微生物学实验室中应用。在过去的6个月里，我们探索了使用另一种方法-表面增强激光解吸/电离飞行时间质谱仪(SELDI-ToF MS)。这项技术的优点是易于使用，仪器和消耗品的成本相对较低，并使用“蛋白质芯片”从细菌细胞裂解液中选择性地捕获蛋白质。蛋白质芯片可用于各种层析表面，允许结合具有不同亲和力(例如，阳离子、阴离子和金属亲和力)的蛋白质。细菌裂解物可以接触一个或多个蛋白质芯片，然后用SELDI-ToF MS分析结合蛋白。在我们的初步实验中，我们(1)定义了裂解革兰氏阳性和革兰氏阴性细菌所需的条件，(2)确定了适当的蛋白质芯片进行分析。我们相信我们已经完成了第一阶段的实验，目前正在评估蛋白质光谱的重复性和图谱的辨别能力。如果这些结果看起来令人满意，我们将扩大研究范围，包括从我们的培养物收藏和美国模式细胞培养(ATCC)收藏中挑选的大量具有良好特征、具有临床意义的生物。另一个项目(CL010326-01DLM)的当前工作表明，培养收集分离物(包括ATCC收集)的表型鉴定不准确。也就是说，基因组研究已经证明，一些收集分离物被表型测试错误识别。因此，这些研究中使用的所有分离株的鉴定将通过综合的表型和基因分型方法进行。