期刊
SOIL BIOLOGY & BIOCHEMISTRY
卷 106, 期 -, 页码 109-118出版社
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.soilbio.2016.12.011
关键词
PCR; Inhibition; Dilution; DNA extraction; Microbe; Humic acid
类别
资金
- National Natural Science Foundation of China [31500409, 31401755]
- Applied Basic Research Project in Yunnan Province [2015FD026, 2012FD029]
- Yunnan Innovative Research Teams [2012HC007]
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.
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