Quantitative real-time PCR (qPCR) detection chemistries affect enumeration of the Dehalococcoides 16S rRNA gene in groundwater

Quantitative real-time PCR (qPCR) commonly uses the fluorogenic 5′ nuclease (TaqMan) and SYBR Green I (SG) detection chemistries to enumerate biomarker genes. Dehalococcoides (Dhc) are keystone bacteria for the detoxification of chlorinated ethenes, and the Dhc 16S ribosomal RNA (rRNA) gene serves as a biomarker for monitoring reductive dechlorination in contaminated aquifers. qPCR enumeration of Dhc biomarker genes using the TaqMan or SG approach with the same primer set yielded linear calibration curves over a seven orders of magnitude range with similar amplification efficiencies. The TaqMan assay discriminates specific from nonspecific amplification observed at low template concentrations with the SG assay, and had a 10-fold lower limit of detection of ~3 copies per assay. When applied to Dhc pure cultures and Dhc-containing consortia, both detection methods enumerated Dhc biomarker genes with differences not exceeding 3-fold. Greater variability was observed with groundwater samples, and the SG chemistry produced false-positive results or yielded up to 6-fold higher biomarker gene abundances compared to the TaqMan method. In most cases, the apparent error associated with SG detection resulted from quantification of nonspecific amplification products and was more pronounced with groundwater samples that had low biomarker concentrations or contained PCR inhibitors. Correction of the apparent error using post-amplification melting curve analysis produced 2 to 21-fold lower abundance estimates; however, gel electrophoretic analysis of amplicons demonstrated that melting curve analysis was insufficient to recognize all nonspecific amplification. Upon exclusion of nonspecific amplification products identified by combined melting curve and electrophoretic amplicon analyses, the SG method produced false-negative results compared to the TaqMan method. To achieve sensitive and accurate quantification of Dhc biomarker genes in environmental samples (e.g., groundwater) and avoid erroneous conclusions, the analysis should rely on TaqMan detection chemistry, unless additional analyses validate the results obtained with the SG approach.

► qPCR detection chemistries affect Dehalococcoides biomarker gene enumeration.
► SYBR Green-based assays can produce false positive and false negative results.
► SYBR Green assays require post-amplification analysis for result validation
► TaqMan assays are recommended for enumeration of target genes in environmental samples.