Design and assessment of species-level qPCR primers targeting comammox

Published PCR primers targeting the ammonia monooxygenase gene (amoA) were applied to samples from activated sludge systems operated with low dissolved oxygen (DO) to quantify total and clade-levelNitrospirathat perform complete ammonium oxidation (comammox); however, we found these existing primers resulted in significant artifact-associated non-target amplification. This not only overestimated comammoxamoAcopies but also resulted in numerous false positive detections in the environmental samples tested, as confirmed by gel electrophoresis. Therefore, to more accurately quantify known comammox, we designed specific and sensitive primers targeting three candidate species:Candidatus(Ca.) Nitrospira nitrosa,Ca.N. inopinata, andCa.N. nitrificans. The primers were tested withamoAtemplates of these candidate species, and used to quantify comammox at the species level in low DO activated sludge systems. We found that comammox related toCa.N. nitrosa were present and abundant in the majority of samples from low DO bioreactors and were not detected in samples from a high DO system. In addition, the greatest abundance ofCa.N. nitrosa was found in bioreactors operated with a long solids retention time.Ca.N. inopinata andCa.N. nitrificans were only detected sporadically in these samples, indicating a minor role of these comammox in nitrification under low DO conditions.Abstract art