Real-time PCR quantification of Colletotrichum coccodes DNA in soils from bioherbicide field-release assays, with normalization for PCR inhibition

Abstract

A real-time polymerase chain reaction (PCR) assay was developed to quantify simultaneously the biocontrol agent Colletotrichum coccodes (DAOM 183088) and soil compounds inhibitory to PCR. The external control used in this assay was spiked at known concentrations in soil DNA extracts and amplified in real-time PCR with its own primer set. A comparison between the estimated quantities of the external control and the known quantities added to the extracts allowed an estimation of the PCR efficiency on a sample per sample basis in 18 extracts of soil DNA analyzed, originating from the bioherbicide field-release trials. All 18 extracts tested positive for the presence of inhibitory compounds, but with substantial variability in the magnitude of PCR efficiency (from 12% to 82%) from 1 g of soil sample to another, even when soil DNA extracts had been diluted. This variability demonstrates quantitatively the heterogeneity of soil with regards to content in PCR-inhibitory compounds. The differences in amplification efficiency were used to normalize the amounts of target C. coccodes DNA previously quantified from the same samples.