Ecological Sciences, The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK.
J Microbiol Methods. 2012 Oct;91(1):38-44. doi: 10.1016/j.mimet.2012.07.010. Epub 2012 Jul 17.
Application of polymerase chain reaction (PCR) techniques has developed significantly from a qualitative technology to include powerful quantitative technologies, including real-time PCR, which are regularly used for detection and quantification of nucleic acids in many settings, including community analysis where culture-based techniques are not suitable. Many applications of real-time PCR involve absolute quantification which is susceptible to inaccuracies caused by losses during DNA extraction or inhibition caused by co-extracted compounds. We present here an improvement to this approach involving the addition of an artificial internal standard, prior to nucleic acid extraction. The standard was generated by in-situ mutagenesis from an E. coli template to ensure it both did not amplify with bacterial primers used for quantification and was short enough to minimise possible interference with other analyses. By estimating gene target copies by relative abundance, this approach accounts for both loss during extraction and inhibition effects. We present a novel application of relative real time PCR, using the internal standard as a reference, allowing accurate estimation of total bacterial populations both within and across a wide range of soils and demonstrate its improvement over absolute quantification by comparison of both approaches to ester linked fatty acid analysis of the same soils.
聚合酶链反应 (PCR) 技术的应用已经从定性技术显著发展为包括实时 PCR 在内的强大定量技术，该技术经常用于许多环境中核酸的检测和定量，包括不适合基于培养的技术的社区分析。实时 PCR 的许多应用涉及绝对定量，这种定量容易受到 DNA 提取过程中损失或共提取化合物抑制引起的不准确的影响。我们在这里介绍了一种改进方法，即在核酸提取之前添加人工内部标准。该标准是通过大肠杆菌模板的原位突变产生的，以确保它既不会与用于定量的细菌引物扩增，又足够短以最小化可能对其他分析的干扰。通过相对丰度估计基因靶标拷贝数，该方法考虑了提取过程中的损失和抑制作用。我们提出了相对实时 PCR 的一种新应用，将内部标准作为参考，允许在广泛的土壤范围内准确估计总细菌种群，并通过比较两种方法对相同土壤的酯键脂肪酸分析，证明其优于绝对定量。
