Bioterrorism Rapid Response and Advanced Technology (BRRAT) Laboratory, Laboratory Preparedness and Response Branch (LPRB), Division of Preparedness and Emerging Infections (DPEI), Centers for Disease Control and Prevention (CDC), Atlanta, GA 30333, USA.
Diagn Microbiol Infect Dis. 2011 Jul;70(3):299-306. doi: 10.1016/j.diagmicrobio.2011.02.010. Epub 2011 May 4.
This study evaluated commercial automated and manual DNA extraction methods for the isolation of Francisella tularensis DNA suitable for real-time polymerase chain reaction (PCR) analysis from cell suspensions and spiked cotton, foam, and polyester swabs. Two automated methods, the MagNA Pure Compact and the QIAcube, were compared to 4 manual methods, the IT 1-2-3 DNA sample purification kit, the MasterPure Complete DNA and RNA purification kit, the QIAamp DNA blood mini kit, and the UltraClean Microbial DNA isolation kit. The methods were compared using 6 F. tularensis strains representing the 2 subspecies which cause the majority of reported cases of tularemia in humans. Cell viability testing of the DNA extracts showed that all 6 extraction methods efficiently inactivated F. tularensis at concentrations of ≤10⁶ CFU/mL. Real-time PCR analysis using a multitarget 5' nuclease assay for F. tularensis revealed that the PCR sensitivity was equivalent using DNA extracted by the 2 automated methods and the manual MasterPure and QIAamp methods. These 4 methods resulted in significantly better levels of detection from bacterial suspensions and performed equivalently for spiked swab samples than the remaining 2. This study identifies optimal DNA extraction methods for processing swab specimens for the subsequent detection of F. tularensis DNA using real-time PCR assays. Furthermore, the results provide diagnostic laboratories with the option to select from 2 automated DNA extraction methods as suitable alternatives to manual methods for the isolation of DNA from F. tularensis.
本研究评估了商用自动化和手动 DNA 提取方法，以从细胞悬浮液和涂抹拭子中提取适合实时聚合酶链反应 (PCR) 分析的土拉弗朗西斯菌 (Francisella tularensis) DNA。将两种自动化方法（MagNA Pure Compact 和 QIAcube）与 4 种手动方法（IT 1-2-3 DNA 样品纯化试剂盒、MasterPure Complete DNA 和 RNA 纯化试剂盒、QIAamp DNA 血液迷你试剂盒和 UltraClean 微生物 DNA 分离试剂盒）进行比较。使用代表引起人类大多数报告的土拉弗朗西斯菌亚种的 6 株 F. tularensis 菌株比较了这些方法。DNA 提取物的细胞活力测试表明，所有 6 种提取方法均能有效灭活浓度≤10⁶ CFU/mL 的 F. tularensis。使用针对 F. tularensis 的多重 5' 核酸酶检测实时 PCR 分析表明，使用 2 种自动化方法和手动 MasterPure 和 QIAamp 方法提取的 DNA 的 PCR 灵敏度相当。这些 4 种方法从细菌悬浮液中的检测水平明显更高，并且与其他 2 种方法相比，对涂抹拭子样本的检测效果相当。本研究确定了最佳的 DNA 提取方法，用于处理涂抹拭子样本，以便随后使用实时 PCR 检测 F. tularensis DNA。此外，这些结果为诊断实验室提供了选择，他们可以从 2 种自动化 DNA 提取方法中选择适合手动方法的替代方法，用于从 F. tularensis 中分离 DNA。
