A re-evaluation of dilution for eliminating PCR inhibition in soil DNA samples

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.

从土壤样本中共同提取的抑制剂会影响实时定量聚合酶链式反应（qPCR）对目标微生物基因的准确定量分析。稀释DNA提取物是一种常用的降低抑制水平的方法。然而，稀释法的应用大多是凭经验的，需要进一步阐述。在此，我们提出一个稀释模型，重新评估稀释作为一种消除qPCR抑制的方法。我们发现，对于大多数所研究的土壤，未稀释或轻微稀释（例如10倍）的DNA提取物会导致qPCR抑制。然而，过度稀释（例如200倍或400倍）会导致对定量基因拷贝数的高估。只有在适度的稀释范围内，qPCR抑制才能被有效消除，这在我们提出的稀释模型中得到了很好的体现。对qPCR抑制进行预测试以确定提取的DNA样本的适当稀释范围，有助于土壤中核酸的准确定量。（C）2016爱思唯尔有限公司。保留所有权利。