The nirK1F and nirK5R primers [9] are the most
widely used tools to survey diversity of the NirK denitrifying
bacteria in environmental samples. The nirK1F
primer binds to a region of the nirK gene that has an
insert of three bases in some sequences of denitrifying
bacteria (Accession. Nos. AJ002516, U62291,
AF339044–AF339048 and AB076606). This insert may
be the explanation for the poor amplification of this
particular primer. R€osch et al. [13] used a forward primer
that targets the same area as primer nirK1F, but is a
few bases longer. This primer faces the same problem as
nirK1F. Yan et al. [15] as well as Liu et al. [22] used a
modified version of the F1aCu primer together with a
modification of primer nirK3R [9]. The nirK3R primer
was unsuccessful both in the original study and in the
present re-evaluation, and the small number of conserved
bases within the sequences may be the explanation.
The Cunir3 primer designed by Casciotti andWard
[12] targets a highly conserved region encoding for a
copper-binding site, but unfortunately there are only a
few sequences available in this region. If they are not
representative, this would explain the poor results from
the amplification of denitrifying strains in the present
study. There are only 12 complete nirK gene sequences
in the databases.