Molecular Analysis of Mycobacteria in Cutting Fluids

描述(申请人提供)：微生物污染物，包括 金属加工液中的非结核分枝杆菌(NTM)已被 与职业性呼吸道疾病有关。我们更广泛的目标是 开发和应用实用的基于DNA的方法进行实时检测， MWF中分枝杆菌的定量和鉴定 商业MWFS中的流行菌株及其生存能力。具体目标 (I)开发基于聚合酶链式反应(PCR)的检测方案 水基MWF中NTM的实时检测与定量；到聚合酶链式反应 不同商业化水基MWFS配方的现场样品筛选 对NTM进行菌株特异性鉴定，然后使用 分子分型方法；(Iii)研究已鉴定的NTM的生存能力 MWF中的菌株进行定量聚合酶链式反应。一种基于分枝杆菌属特异性的聚合酶链式反应 将优化方法用于水基MWF中NTM的实时检测。这个 方法将作为开发定量聚合酶链式反应的基础 现场MWF样品中NTM的定量。将从MWF分离出NTM菌株 在所有行业和选定工厂中使用的样本 过敏性肺炎病例。这些分离物将在 通过16S核糖体基因扩增序列测定物种水平和菌株水平 基于使用脉冲场凝胶电泳(PFGE)或 多态DNA快速扩增-聚合酶链式反应技术。的效果 菌株类型和杀菌剂负荷对MWF Will中选定菌株活力的影响 在模拟和现场条件下使用定量 聚合酶链式反应。由此产生的信息将有助于Nora的目标，通过提供 微生物暴露评估的实用方法和数据库的开发 与MWF暴露相关的干预策略。

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.