207
were further hybridized with new probes: B-LSPP for discriminating the
genus
Legionella
and LPN-B to discriminate
L. pneumophila
from others
Legionella
species (except
L. londonensis
that has a sequence identical to that
of the probe for
L. pneumophila
).
Actually, the amplification of the 16S rRNA gene is facilitated by the
development of some commercial kits that were already standardized and
effective in the selective amplification for
Legionella
. The gene that encode a
surface membrane protein of 24kDa is another gene that allows identification
of
Legionella
species, this protein is found in virulent strains that can replicate
inside alveolar macrophages by inhibiting phagosome-lysosome fusion
(LINDSAY
et al
., 1994). This gene was previously identified and sequenced
by Engelberg
et al
. (1989) as a enhancer for macrophages infection (mip),
and the sequence variation allowedDNAmanipulation techniques, including
PCR, to be applied for the recognition of
L. pneumophila
,
L. micdadei
and
L. bozemanii
. The detection of
Legionella
by amplification of the mip gene
was adapted for real time PCR quantification with (Ballard
et al
., 2000) or
without (Rantakokko-Jalava and Jalava, 2001) hybridization. This approach
was effective for such purpose and specificity for
L. pneumophila
.
The rpoB gene encoding the β-subunit of RNA polymerase has also been
used as a tool for identification of
Legionella
and other bacterial species. The
group of researchers Koa
et al
. (2003) used PCR-RFLP specific for
Legionella
and PCR-RFLP specific for
L. pneumophila
using the rpoB gene as a simple
and effective method which does not require expensive equipment or
specialized technique to identify and distinguish
Legionella
species. For this
purpose, after amplification, the fragment of 347 bp generated was separately
cleaved with restriction enzymes HaeIII, AluI, CfoI, PstI and MaeII allowing
the distinction of
Legionella
species.
