[Comparative analysis of primer-based PCR strategies for characterizing ammonia-oxidizing bacterial communities in...

PCR-based techniques are commonly used to characterize ammonia-oxidizing bacterial (AOB) communities, but are subjected to the bias induced by PCR primers. In this study, the sensitivity and specificity of two pairs of primer were assessed based on a group of lake sediment samples. Compared to the low sensitivity of the CTO primer, the betaAMO primer produced intensive single bands in all samples tested. However, the sequences retrieved from one of the samples by betaAMO did not cluster with Nitrosomonadales, to which all beta-AOB affiliate. In contrast, all sequences amplified with CTO fell within the Nitrosomonas europaea/"Nitrosococcus mobilis" lineage. Furthermore, the products of four approaches were subjected to denaturing gradient gel electrophoresis (DGGE), and the patterns of the nested strategies with betaAMO followed CTO or the universal primer of 16S rRNA gene followed CTO were highly similar to that of CTO amplification. These findings suggest that betaAMO is not competent for charactering the beta-AOB communities due to the low specificity. The nested approaches with both selective and specific primers are the choice because they can amplify effectively and retrieve community composition of beta-AOB in lake sediments.