Molecular Analysis of Mycobacteria in Cutting Fluids

切削液中分枝杆菌的分子分析

• 批准号: 6437155
• 负责人: Jagjit S Yadav
• 金额: $ 22.95万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2004-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6437155
• 关键词: Mycobacterium; allergic pneumonitis; environmental exposure; genetic strain; metal working fluid; method development; occupational hazard; polymerase chain reaction

DESCRIPTION (provided by applicant): Microbial contaminants including nontuberculous mycobacteria (NTM) in metalworking fluids (MWFS) have been implicated in occupational respiratory illnesses. Our broader goals are to develop and apply practical DNA-based approaches for real-time detection, quantitation, and identification of mycobacteria in MWF for understanding the prevalent strains and their survivability in commercial MWFS. The specific aims are (i) to develop polymerase chain reaction (PCR)-based protocols for real-time detection and quantitation of NTM in water-based MWF; (ii). to PCR screen field samples of different commercial formulations of water-based MWFs for NTM followed by strain-specific identification of the NTM isolates using molecular typing methods; (iii) to investigate viability of the identified NTM strains in MWF using quantitative PCR. A Mycobacterium genus-specific PCR-based method will be optimized for real-time detection of NTM in water-based MWF. The method will be used as a basis for developing a quantitative PCR protocol for quantitation of NTM in field MWF samples. NTM strains will be isolated from MWF samples in use across the industries and in selected plants linked with hypersensitivity pneumonitis cases. The isolates will be identified at the species level by 16S ribosomal gene amplicon sequencing and at the strain level based on genome fingerprinting using pulsed field gel electrophoresis (PFGE) or rapid amplification of polymorphic DNA-PCR (RAPD-PCR) technique. Effect of strain type and biocide load on viability of the selected strains in MWF will be investigated in simulated as well as field conditions using quantitative PCR. The resulting information will help NORA's objectives by providing practical methods for microbial exposure assessment and database for developing intervention strategies related to MWF exposures.

描述（由申请人提供）：微生物污染物，包括 金属加工液 (MWFS) 中的非结核分枝杆菌 (NTM) 与职业性呼吸系统疾病有关。我们更广泛的目标是 开发并应用基于 DNA 的实用方法进行实时检测， MWF 中分枝杆菌的定量和鉴定，以了解 流行菌株及其在商业 MWFS 中的生存能力。具体目标 (i) 开发基于聚合酶链式反应 (PCR) 的方案 水基金属加工液中NTM的实时检测和定量； (二).至PCR 筛选水基金属加工液不同商业配方的现场样品 对于 NTM，然后使用 NTM 分离株进行菌株特异性鉴定 分子分型方法； (iii) 调查已确定的非关税措施的可行性 使用定量 PCR 检测 MWF 中的菌株。基于分枝杆菌属特异性 PCR 的 该方法将针对水基金属加工液中 NTM 的实时检测进行优化。这 方法将用作开发定量 PCR 方案的基础 现场 MWF 样品中 NTM 的定量。 NTM菌株将从MWF中分离出来 跨行业和选定工厂使用的样本 过敏性肺炎病例。分离株将在 通过 16S 核糖体基因扩增子测序和菌株水平进行物种水平 基于使用脉冲场凝胶电泳 (PFGE) 的基因组指纹分析或 快速扩增多态性DNA-PCR（RAPD-PCR）技术。效果 菌株类型和杀菌剂用量对 MWF 中选定菌株活力的影响 使用定量方法在模拟和现场条件下进行研究 PCR。由此产生的信息将通过提供以下信息来帮助 NORA 实现目标： 微生物暴露评估的实用方法和开发数据库 与小额信贷批发基金暴露相关的干预策略。