The nirS, nirK and nosZ sequences presently available
from the GenBank database were aligned, and conserved
regions that could provide suitable primer target
sites were re-assessed. For the nirS gene, 11 forward and
five reverse primers were tested. For the nirK gene, three
forward and three reverse primers were evaluated for
PCR amplification of gene fragments, while five forward
and six reverse primers were tested for nosZ (Table 3,
Fig. 1). All possible primer combinations for nirK and
nosZ, except those including nosLb and nosRb, were
evaluated against the denitrifying strains, the non-denitrifying
strains and the environmental samples. For nirS
merely combinations yielding amplicons >400 bp were
tested. The reason for this was to reduce the number of
combinations and to only work with primers that amplify
fragments with enough information to be useful in
environmental studies. In the case of minor variations in
the primer sequence, e.g., modifications of F1acd (Table
3), only the result from the best combination is shown in
Table 1. The oligonucleotides were purchased from Invitrogen
(Carlsbad, CA, USA).