Specific and effective detection of anammox bacteria using PCR primers targeting the 16S rRNA gene and functional genes

Anaerobic ammonium-oxidizing (anammox) bacteria play an important role in the nitrogen cycle by coupling ammonium and nitrite to produce dinitrogen gas (N2). Polymerase chain reaction (PCR) is a fast, simple, and sensitive method that is widely used to assess the diversity, abundance, and activity of the slow-growing bacteria. In this review, we summarize and evaluate the wide variety of PCR primers targeting the 16S rRNA gene and functional genes (hzo, nir, and hzs) of anammox bacteria for their effectiveness and efficiencies in detecting this group of bacteria in different sample types. Furthermore, the efficiencies of different universal high-throughput sequencing 16S rRNA gene primers in anammox bacteria investigations were also evaluated to provide a reference for primer selection. Based on our in silico evaluation results, none of the 16S rRNA gene primers could recover all of the known anammox bacteria, but multiple hzo and hzs gene primers could accomplish this task. However, uncertain copies (1-3 copies) of hzo genes were identified in the genomes, and the hydrazine oxidation reaction catalyzed by hydrazine oxidoreductases (HZOs) can also be catalyzed by other hydroxylamine oxidoreductases (HAOs) in anammox bacteria, which can potentially result in large deviations in hzo-based qPCR and RT-qPCR analyses and results. Therefore, the use of optimal primers targeting unique hzs genes are recommended, although the efficiencies of these newly designed primers need further verification in practical applications. This article provides comprehensive information for the effective and specific detection of anammox bacteria using specific primers targeting the 16S rRNA gene and functional genes and serves as a basis for future high-quality primer design.