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: 6825569
• 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/6825569
• 关键词: bacterial genetics; bacterial proteins; bacteriolysis; genotype; mass spectrometry; matrix assisted laser desorption ionization; 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 or housekeeping 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) and Surface-Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry (SELDI-ToF MS). Last year we explored using SELDI for identification of selected gram-positive and gram-negative bacteria. This year, with the multi-department Clinical Center purchase of the MALDI-ToF MS system, we have expanded our studies to use this system. Preliminary studies indicate that unique, reproducible profiles could be obtained for Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis. The advantage of the MALDI system is that whole cells could be analyzed, reducing the variability of bacterial lysis which was required with the SELDI system. The difficulty with the MALDI system is the complexity of the profiles. We believe this can be resolved with the recent purchase of software for data analysis.

细菌的鉴定传统上是通过形态特征和表型测试来进行的。利用其特征形态和一些选定的表型测试，可以快速（例如不到一小时）识别一些常见微生物（例如金黄色葡萄球菌、肺炎链球菌、大肠杆菌）。然而，大多数生物体的识别速度很慢，需要数小时到数天才能得到明确的答案。最近，基因组学的使用（例如对核糖体 RNA 基因或管家基因进行测序）已被证明是一种有用的工具。尽管对特定基因进行测序是一种强大的区分工具，但目前的方法需要一天或多天才能获得结果。基因测序的一个逻辑延伸是使用基因产物进行细菌鉴定。结构基因序列的显着变化将导致蛋白质产物的变化。在过去 5 年中，使用基质辅助激光解吸/电离飞行时间质谱 (MALDI-ToF MS) 和表面增强激光解吸/电离飞行时间质谱 (SELDI-ToF MS) 进行了细菌蛋白质分析和鉴定的初步工作。去年，我们探索使用 SELDI 来鉴定选定的革兰氏阳性和革兰氏阴性细菌。今年，随着多科室临床中心购买MALDI-ToF MS系统，我们扩大了研究范围以使用该系统。初步研究表明，可以获得大肠杆菌、铜绿假单胞菌、金黄色葡萄球菌和粪肠球菌的独特、可重复的谱。 MALDI 系统的优点是可以分析整个细胞，减少 SELDI 系统所需的细菌裂解的变异性。 MALDI 系统的困难在于轮廓的复杂性。我们相信这个问题可以通过最近购买数据分析软件来解决。