Hansen Wendy L J, Bruggeman Cathrien A, Wolffs Petra F G
Department of Medical Microbiology, Maastricht University Medical Center, Maastricht, The Netherlands.
Methods Mol Biol. 2013;943:81-90. doi: 10.1007/978-1-60327-353-4_4.
Molecular diagnostics is an increasing popular approach for the direct detection and identification of pathogenic bacteria in clinical samples. Conventional culture techniques are time-consuming and therefore causing a delay in the diagnosis of the patient. Alternative techniques based on nucleic acid amplification offer a shorter turn-around-time and the ability to identify fastidious and non-cultivable organisms. However, molecular detection of bacteria in blood, by for example PCR, RT-PCR, or sequencing of the 16S rDNA genes is often complicated by the presence of PCR-inhibitory compounds. Here we describe several different methods for the extraction of bacterial DNA from whole blood samples. The methods differ regarding costs, hands-on time as well as regarding sensitivity. In combination with a model PCR the detection limits that can be reached using the different methods range from 1,000 to 50 cfu/ml.
分子诊断是一种在临床样本中直接检测和鉴定病原菌的越来越流行的方法。传统培养技术耗时，因此会导致患者诊断延迟。基于核酸扩增的替代技术提供了更短的周转时间以及鉴定苛求菌和不可培养微生物的能力。然而，例如通过PCR、RT-PCR或16S rDNA基因测序在血液中进行细菌分子检测常常因存在PCR抑制性化合物而变得复杂。在此我们描述了几种从全血样本中提取细菌DNA的不同方法。这些方法在成本、实际操作时间以及灵敏度方面存在差异。与模型PCR相结合，使用不同方法可达到的检测限范围为1000至50 cfu/ml。
