Quantitative estimation of Dehalococcoides mccartyi at laboratory and field scale: Comparative study between CARD-FISH and Real Time PCR

Dehalococcoides mccartyi detectability in the field is a fundamental tool to assess the efficiency of natural attenuation or engineered bioremediation in chlorinated solvent-contaminated sites. This study reports on the direct comparison of quantitative data obtained by Real Time PCR (qPCR) and CAtalyzed Reporter Deposition-Fluorescence In situ Hybridization (CARD-FISH) over a wide range of Dehalococcoides concentrations (10–108 cells mL− 1) both in three independent 10-fold serial dilutions of a laboratory dechlorinating enrichment and in 49 groundwater samples from 6 different contaminated sites. Dehalococcoides enumeration by CARD-FISH yielded a linear curve in the analyzed concentration range which was consistent with the expected concentrations and showed good reproducibility in triplicate assays. Alternatively, qPCR did not allow for the discrimination of 16S rRNA gene concentrations lower than 103 gene copies mL− 1 either in the dechlorinating mixed culture or in field samples. Overall this study highlights the limits of qPCR quantification, especially in samples where low concentrations of this microorganism may be expected, and suggests the use of a confirmatory methodology under these particular conditions.

• CARD-FISH and qPCR were simultaneously applied to quantify Dehalococcoides mccartyi.
• The methods were applied on a PCE dechlorinating culture and field samples.
• Accuracy of CARD-FISH quantification over a large concentration range.
• qPCR yielded Dehalococcoides concentration lower than that obtained by CARD-FISH.
• qPCR failed to estimate 16SrRNA Dehalococcoides gene copies lower than 103 mL–1.