Reaction- and sample-specific inhibition affect standardization of qPCR assays of soil bacterial communities

Quantitative PCR (qPCR) is a popular technique used to quantify target sequences from DNA isolated from soil, but PCR inhibition makes it difficult to estimate gene copy number. Here, we evaluated the extent to which inhibition associated with reaction conditions and sample-specific properties influence the linear range of amplification, and the efficiency and sensitivity of qPCR assays of three bacterial gene targets. We adopted a sample pool approach and exploited the mathematical basis of qPCR to correct for sample-specific effects on amplification. Results revealed that qPCR efficiency and sensitivity were dependent on all conditions tested. In addition, the effect of annealing temperature and SYBR green PCR kit was target-specific, suggesting that the sample pool approach is appropriate for evaluating the quality of new primers. Likewise, the efficiency and sensitivity of qPCR amplification was sample-specific and is likely a result of site and date-specific co-extractants. When relativized against calculations based on plasmid curves alone, reaction-specific and sample-specific inhibition influenced calculations of gene copy number. To account for these differences, we present a brief protocol for soil samples that will facilitate comparison of future datasets.