Molecular Analysis of Mycobacteria in Cutting Fluids

描述（由申请方提供）：微生物污染物，包括 金属加工液（MWFS）中的非结核分枝杆菌（NTM）已被 与职业性呼吸道疾病有关我们更广泛的目标是 开发和应用基于DNA的实时检测方法， 定量和鉴定MWF中的分枝杆菌，以了解 流行菌株及其在商业MWFS中的存活率。具体目标 （i）开发基于聚合酶链反应（PCR）的方案， 实时检测和定量水基MWF中的NTM;（ii）.在pcr 筛选水基MWFs不同商业配方的现场样品 随后使用NTM分离株的菌株特异性鉴定 分子分型方法;（iii）研究已鉴定的NTM的活力 菌株的MWF中使用定量PCR。一种基于分枝杆菌属特异性PCR的 该方法将被优化用于实时检测水基MWF中的NTM。的 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 各行业和选定工厂中使用的样本， 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 基于使用脉冲场凝胶电泳（PFGE）的基因组指纹图谱，或 快速扩增多态性DNA-PCR（RAPD-PCR）技术。的影响 菌株类型和杀生物剂负荷对MWF中所选菌株活力的影响将 在模拟和现场条件下使用定量 pcr法由此产生的信息将有助于诺拉的目标， 微生物接触评估的实用方法和开发数据库 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.