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

•When a DNA sample is diluted, inhibitory substances and target genes are co-diluted.•Insufficient dilution causes underestimation of gene copy numbers.•Excessive dilution causes overestimation of gene copy numbers.•Target genes with low copy numbers are more susceptible to inhibitors when diluted.•Inhibition test of soil DNA samples is essential for downstream analyses.

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 qPCR inhibition for most of studied soils. However, excessive dilution (e.g., 200- or 400-fold) caused an overestimation of the quantified gene copy numbers. Only under a moderate dilution range could qPCR inhibition be efficiently 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.