Abstract
We designed novel quantitative real-time polymerase chain reaction (qPCR) primers for the polyphosphate kinase 1 (ppk1) gene, targeting eight individual “Candidatus Accumulibacter” (referred to as Accumulibacter) clades. An evaluation of primer sets was conducted regarding the coverage, specificity, and PCR efficiency. (i) All primer sets were designed to cover all available sequences of the target clade. (ii) The phylogenetic analysis of the sequences retrieved from the qPCR products by each primer set demonstrated a high level of specificity. (iii) All calibration curves presented high PCR efficiencies in the range of 85–112% (R2 = 0.962–0.998). In addition, the possible interference of non-target amplicons was individually examined using the qPCR assay for 13 Accumulibacter clades, which were either undetected or showed negligible detection. With the primers designed by other research groups, a highly selective and sensitive qPCR-based method was developed to quantify all Accumulibacter clades, with the exception of Clade IE, in one assay, which enables more comprehensive insights into the community dynamics. The applicability to environmental samples was demonstrated by profiling the Accumulibacter clades in activated sludge samples of nine full-scale wastewater treatment plants.
Highlights
We designed novel quantitative real-time polymerase chain reaction primers for the polyphosphate kinase 1 gene, targeting eight individual “Candidatus Accumulibacter” clades
The 16S ribosomal RNA method was used as the primary approach to identify the occurrence and phylogeny of Accumulibacter in Enhanced biological phosphorus removal (EBPR) systems, until their inability to partition Accumulibacter into ecotypes, owing to their high similarities, was proven
A recent study has revealed that the total Accumulibacter lineage in 10 of 18 WWTPs, as determined by former primer sets for the ppk[1] gene[6], accounted for less than 50% of the total Accumulibacter determined by the Accumulibacter 16S ribosomal RNA (rRNA) gene[8]
Summary
We designed novel quantitative real-time polymerase chain reaction (qPCR) primers for the polyphosphate kinase 1 (ppk1) gene, targeting eight individual “Candidatus Accumulibacter” (referred to as Accumulibacter) clades. (i) All primer sets were designed to cover all available sequences of the target clade. (ii) The phylogenetic analysis of the sequences retrieved from the qPCR products by each primer set demonstrated a high level of specificity. Quantitative real-time PCR (qPCR) primers that target Accumulibacter Type I and Clades IIA, IIB, IIC and IID, were designed and verified to differentiate divergence among Accumulibacter ecotypes[6]. In 2008, Peterson et al.[7] subdivided the Accumulibacter Type I into five clades and discovered Clades IIE, IIF and IIG of Type II, based on the phylogenetic distance of the ppk[1] gene. The first reason for this result was that Clade IB was not covered by the primer set Acc-ppk1-763f www.nature.com/scientificreports/
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