Development of the Quantitative PCR Method for Candidatus ‘Accumulibacter phosphatis’ and Its Application to Activated Sludge

Abstract

To quantify Candidatus ‘Accumulibacter phosphatis’ in activated sludge, quantitative PCR method was developed utilizing SYBR GREEN I and a specific primer set targeted on the 16S rRNA gene of Candidatus ‘Accumulibacter phosphatis’. Following optimization of PCR condition, specificity was evaluated based on the melting curve and the sequencing analysis of the PCR products with DNA extracted from activated sludge. Both the melting curve and the sequencing analysis of the PCR product showed that only the target DNA from Candidatus ‘Accumulibacter phosphatis’ was amplified. Standard curves with a series of tenfold dilution of the DNA from 16S rRNA gene fragment of Candidatus ‘Accumulibacter phosphatis’ gave R² values greater than 0.999. The minimum detection limit was 1.0×10³ copies per reaction. The amount of Candidatus ‘Accumulibacter phosphatis’ in laboratory-scale and full-scale activated sludge samples were quantified both by the quantitative PCR method and by the FISH method. The quantification results by these two methods agreed satisfactorily, with an R² value of 0.6871 showing a statistically significant correlation (p<0.001). Thus, we developed a rapid quantification method by using quantitative PCR for the quantification of Candidatus ‘Accumulibacter phosphatis’ in activated sludge.