Department of Microbiological Diagnostics, Unit of Gastrointestinal Infection, Statens Serum Institut, 2300 S Copenhagen, Denmark.
Diagn Microbiol Infect Dis. 2011 Mar;69(3):240-4. doi: 10.1016/j.diagmicrobio.2010.09.023.
In this study, 5 different commercial DNA extraction systems were tested on a stool sample collection containing 81 clinical stool specimens that were culture-positive for diarrheagenic Escherichia coli, Campylobacter jejuni, Salmonella enterica, or Clostridium difficile. The purified DNAs were analyzed by polymerase chain reaction (PCR) directed toward the relevant organisms. The results showed that conventional PCR combined with the extraction systems BioRobot EZ1 (Qiagen, Hilden, Germany), Bugs'n Beads (Genpoint, Oslo, Norway), ChargeSwitch (Invitrogen, Paisley, UK), QIAamp Stool Mini Kit (Qiagen), and 2 protocols (generic and Specific A) for EasyMag (BioMérieux, Marcy I'Etoile, France) were able to identify 89%, 62%, 85%, 88%, 85%, and 91%, respectively, of the pathogens originally identified by conventional culture-based methods. When TaqMan PCR was combined with the EasyMag Specific A protocol, 99% of the samples were correctly identified. The results demonstrate that the extraction efficiencies can vary significantly among different extraction systems, careful optimization may have a significant positive effect, and the use of sensitive and specific detection methods like TaqMan PCR is an ideal choice for this type of analysis.
在这项研究中,对含有 81 份临床粪便标本的粪便样本采集物进行了 5 种不同的商业 DNA 提取系统测试,这些标本均为致泻性大肠杆菌、空肠弯曲菌、沙门氏菌或艰难梭菌的培养阳性。将纯化的 DNA 通过聚合酶链反应(PCR)分析针对相关生物体。结果表明,常规 PCR 结合提取系统 BioRobot EZ1(德国基森的 Qiagen)、Bugs'n Beads(挪威奥斯陆的 Genpoint)、ChargeSwitch(英国佩斯利的 Invitrogen)、QIAamp Stool Mini Kit(德国基森的 Qiagen)和 2 种 EasyMag 协议(通用和特定 A)(法国马西-埃托勒的 BioMérieux)分别能够识别最初通过常规基于培养的方法识别的病原体的 89%、62%、85%、88%、85%和 91%。当将 TaqMan PCR 与 EasyMag Specific A 方案结合使用时,99%的样本得到了正确识别。结果表明,不同提取系统之间的提取效率可能存在显著差异,仔细优化可能会产生显著的积极影响,并且使用敏感和特异性检测方法,如 TaqMan PCR,是此类分析的理想选择。