Quantification of anaerobic ammonium-oxidizing bacteria in enrichment cultures by quantitative competitive PCR

Abstract

The anaerobic ammonium-oxidizing (ANAMMOX) bacteria were enriched from a sequencing batch biofilm reactor (SBBR). A quantitative competitive polymerase chain reaction (QC-PCR) system was successfully developed to detect and quantify ANAMMOX bacteria in environmental samples. For QC-PCR system, PCR primer sets targeting 16S ribosomal RNA genes of ANAMMOX bacteria were designed and used. The quantification range of this system was 4 orders of magnitude, from 10(3) to 10(6) copies per PCR, corresponding to the detection limit of 300 target copies per mL. A 312-bp internal standard was constructed, which showed very similar amplification efficiency with the target amxC fragment (349 bp) over 4 orders of magnitude (10(3)-10(6)). The linear regressions were obtained with R2 of 0.9824 for 10(3) copies, 0.9882 for 10(4) copies, 0.9857 for 10(5) copies and 0.9899 for 10(6) copies, respectively. Using this method, ANAMMOX bacteria were quantified in a shortcut nitrification/denitrification-anammox system which was set for piggery wastewater treatment.