206
of the chromosomal DNA of
L. pneumophila
with restriction enzymes EcoR1
and Hind III and the fragments cloned into a plasmid and subsequently by
Southern analysis, hybridized in positive samples. Bacteria of other genera
tested did not generate positive signs in these conditions.
Inorder to improve thedetectionof
Legionella
, Jonas
et al
. (1995)designed
primers specific for 16S rRNA gene sequence of
L. pneumophila
ATCC 33152
(primers JFP and JRP) that may be used for the PCR detection.These primers
after amplification generated a 350 bp PCR fragment, which was hybridized
with DNA obtained from different bacterial species and showed that this
strategy is specific to members of the genus
Legionella
. Miyamoto
et al
.
(1997) presented a similar work using nested PCR. For the first step of PCR,
the LEG-225 and LEG-858 primers were used to amplify a 654 bp fragment
of the 16S rRNA gene. In the second PCR step, the first primer (LEG-225)
was substituted for GAL and GAL-448-858, generating a 430 bp fragment of
the 16S rDNA gene. Then a primer labeled with digoxigenin was used as a
primer probe that is complementary to positions 630-649 in 16S rRNA gene.
Could
et al
. (2000) tested the same primers developed by Jonas
et al
.
(1995), and confirmed not only 31 positive cultures as previously described
but also 12 from 181 samples that were diagnosed as negative cultures. The
DNA fragments amplified from these cultures, which resulted in positive
PCR, were sequenced allowing the confirmation that all (31 + 12 samples)
belonged to the genus
Legionella
.
Van Der Zee
et al
. (2002b) designed another strategy based on the PCR
technique. For this purpose a set of primers were designed from the sequence
of the gene encoding the 16S rRNA, allowing the amplification of a specific
200 bp fragment specific for the genus
Legionella
.The PCR products obtained
