Development and application of real-time PCR for quantification of specific ammonia-oxidizing bacteria in activated sludge of sewage treatment systems

In this study, four real-time polymerase chain reaction (PCR) primer sets were developed for the 16S rRNA genes of specific ammonia-oxidizing bacteria (AOB) found in activated sludge of sewage treatment systems. The primer sets target two of several sequence types of the Nitrosomonas oligotropha cluster, members within the Nitrosomonas communis cluster, and all members of the Nitrosomonas europaea-Nitrosococcus mobilis cluster. The detection limit of each primer set was in the range of 3x10(1)-6x10(2) genes reaction(-1). Reliable quantification of the target AOB DNA was obtained when the target AOB DNA comprised more than 0.1% of total AOB DNA in the sample. The application of the primer sets to samples taken from five sewage treatment systems showed that, in all systems, the majority of the AOB population was comprised of one sequence type of the N. oligotropha cluster (3.9 +/- 1.5x10(9)-1.7 +/- 0.5x10(10) cell l(-1)) and, in most systems, followed by members within the N. communis cluster (2.8 +/- 0.3x10(9)-1.0 +/- 0.1x10(10) cell l(-1)) or/and another sequence type of the N. oligotropha cluster (1.5 +/- 0.6x10(8)-5.5 +/- 0.5x10(8) cell l(-1)). N. europaea-N. mobilis cluster arose solely in small numbers (4.9 +/- 0.8x10(8) cell l(-1)) in one system. Real-time PCR-amplified products obtained from genomic DNA extracted from samples were verified using clone library, and it revealed that only the target AOB DNA were PCR amplified, without amplification of the nontarget sequences.