Four PCR primers necessary for the detection of periplasmic nitrate reductase genes in all groups of Proteobacteria and in environmental DNA

Abstract

Generic primers are available for detecting bacterial genes required for almost every reaction of the biological nitrogen cycle, the one notable exception being napA (gene for the molybdoprotein of the periplasmic nitrate reductase) encoding periplasmic nitrate reductases. Using an iterative approach, we report the first successful design of three forward oligonucleotide primers and one reverse primer that, in three separate PCRs, can amplify napA DNA from all five groups of Proteobacteria. All 140 napA sequences currently listed in the NCBI (National Center for Biotechnology Information) database are predicted to be amplified by one or more of these primer pairs. We demonstrate that two pairs of these primers also amplify PCR products of the predicted sizes from DNA isolated from human faeces, confirming their ability to direct the amplification of napA fragments from mixed populations. Analysis of the resulting amplicons by high-throughput sequencing will enable a good estimate to be made of both the range and relative abundance of nitrate-reducing bacteria in any community, subject only to any unavoidable bias inherent in a PCR approach to molecular characterization of a highly diverse target.