A re-evaluation of dilution for eliminating PCR inhibition in soil DNA samples
A re-evaluation of dilution for eliminating PCR inhibition in soil DNA samples
复制标题
重新评估土壤 DNA 样品中消除 PCR 抑制作用的稀释度
DOI：
10.1016/j.soilbio.2016.12.011
复制
发表时间：
2017-03-01
影响因子：
9.7
通讯作者：
Tian, Kun
中科院分区：
文献类型：
--
作者：
Wang, Hang;Qi, Jinfeng;Tian, Kun
实时定量PCR（qPCR）分析的目标微生物基因的准确定量是妥协的共提取抑制剂从土壤样品的存在。稀释DNA提取物是降低抑制水平的常用方法。但是，稀释法的应用大多是经验性的，需要进一步阐述。在这里，我们提出了一种稀释模型，以重新评估稀释作为消除qPCR抑制的方法。我们发现没有稀释或轻微稀释的DNA提取物（例如，10倍）导致大多数研究土壤的ciPCR抑制。然而，过度稀释（例如，200-或400倍）导致对定量的基因CDPy数目的高估。只有在中等稀释范围内才能有效消除qPCR抑制，这已经被我们提出的稀释模型很好地捕获。用于确定提取的DNA样品的适当稀释范围的qPCR抑制预测试有助于准确定量土壤中的核酸。(C)2016爱思唯尔有限公司版权所有
Quantitative real-time PCR (qPCR) analysis for accurate quantification of targeted microbial genes is compromised by the presence of co-extracted inhibitors from soil samples. Dilution of DNA extracts is a commonly-used method to reduce levels of inhibition. However, the applications of dilution method are mostly empirical, and need to be further elaborated. Here, we propose a dilution model to re-evaluate dilution as a method to eliminate qPCR inhibition. We found that DNA extracts without dilution or with a minor dilution (e.g., 10-fold) resulted in ciPCR inhibition for most of studied soils. However, excessive dilution (e.g., 200- or 400-fold) caused an overestimation of the quantified gene cdpy numbers. Only under a moderate dilution range could qPCR inhibition be efficiehtly eliminated, which has been well captured by our proposed dilution model. The pre-testing of qPCR inhibition for determining the appropriate dilution range for extracted DNA samples aids accurate quantification of nucleic acids in soils. (C) 2016 Elsevier Ltd. All rights reserved.