Improved real-time PCR estimation of gene copy number in soil extracts using an artificial reference
Improved real-time PCR estimation of gene copy number in soil extracts using an artificial reference
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DOI：
10.1016/j.mimet.2012.07.010
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发表时间：
2012-10-01
影响因子：
2.2
通讯作者：
Roberts, D. M.
中科院分区：
文献类型：
--
作者：
Daniell, T. J.;Davidson, J.;Roberts, D. M.
聚合酶链式反应(聚合酶链式反应)技术的应用已经从一种定性技术发展到包括实时聚合酶链式反应在内的强大的定量技术，这些技术在许多环境中经常用于核酸的检测和量化，包括基于培养的技术不适用的群落分析。实时荧光聚合酶链式反应的许多应用涉及绝对定量，这容易受到DNA提取过程中的损失或共提取化合物造成的抑制而造成的不准确的影响。我们在此提出了一种改进的方法，包括在核酸提取之前增加一个人工内标物。该标准是通过对大肠杆菌模板进行原位突变而产生的，以确保它既不会与用于定量的细菌引物进行扩增，又足够短，以最大限度地减少对其他分析的可能干扰。通过相对丰度估计基因目标拷贝，这种方法既考虑了提取过程中的丢失，也考虑了抑制效应。我们提出了一种新的应用相对实时聚合酶链式反应的方法，使用内标作为参考，可以准确地估计各种土壤中的细菌总数，并通过比较两种方法对相同土壤的酯连接脂肪酸分析，证明了其相对于绝对定量的改进。(C)2012爱思唯尔B.V.保留所有权利。
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. (C) 2012 Elsevier B.V. All rights reserved.