Kloos et al. [18] screened for nosZ in different Azospirillum
strains and other plant growth-promoting rhizobacteria
with the primers nosZ-F and nosZ-R. The
same primers were later used to investigate nosZ in an
acid forest soil [13] and meadow soil [29]. In our evaluation,
the forward primer, nosZ-F, was combined with
the reverse primers nosZ-R, Nos1773R [16] and
nosZ1622R, with satisfactory results, but the combination
nosZ-F:nosZ1622R gave even better results.
Nos661F and Nos1773R were the first primers targeting
nosZ [16], and were based on just a few sequences. They
were primarily used to amplify nosZ from marine sediments,
but were recently used to survey nosZ in native
and cultivated soil [26]. Nogales et al. [17] modified these
primers (nosZ661b and nosZ1773b) to be more degenerate.
However, none of the sets were successful in the
present re-evaluation. Cheneby et al. [24] constructed
primers (nosLb and nosRb) for amplification of nosZ
genes in two agricultural soils. The primers were not
efficient in our study as shown by amplification of the
correct gene fragment from only nine of the pure cultures.
The two primers (nosZf and nosZr) designed by
Delorme et al. [41] amplify a 1433-bp fragment and have
been used to specifically study genetic diversity in fluorescent
pseudomonads in soil. This fragment is too long
for DGGE analysis but nosZf could be combined with
the nos661F target site to generate a 250-bp fragment.
However, since this re-evaluation has shown that
Nos661F is not suitable, nosZf was excluded from the
evaluation. Otherwise, the primer looks promising with
many conserved bases within the sequences. The nosZr
is located at the very end of the nosZ gene, from which
about 15 sequences are available. These sequences do
not appear to be very conserved and, therefore, it cannot
serve as a general nosZ primer.