209
between viable and non-viable cells (dead cells). A new approach for
detecting viable for DNA intercalating dyes (Ethidium Bromide MonoAzide,
EMA or Propidium Monoazide, PMA) in combination with qPCR cells has
been proposed in the last ten years. This technique allows the molecular
amplification of target DNA from cultivable and viable cells, but prevents the
PCRamplificationof DNA fromnon-viable cells. EMAselectively bindsDNA
in cells with compromised membranes intact cell membrane as a barrier for
the dye. Once inside a cell not feasible, the EMA azide group allows the DNA
crosslinking after exposure to strong visible light. The photolysis of EMA
takes the azide group into a highly reactive nitrene group that binds DNA. In
this connected state, the DNA is not amplified by PCR, whereas DNA from
non-viable cells remain attached to the EMAmolecules, and therefore can be
amplified and quantified (MANSI
et al
. 2014).
Still, the detection of
Legionella
by culture method should be used in
all analyzes. Today more accurate to confirm the identification of the strain
or serotype is performed by DNA sequencing and comparison / alignment
of the sequence obtained with those present in public database genomic
data (Figure 1). Building a phenetic tree (Figure 2) allows the confirmation
of how similar or dissimilar an organism is in relation to others enabling
epidemiological studies and origin of the infectious agent.
Diederen
et al
. (2007) reported that, although PCR-based 16S rRNA
gene had a good performance on an assessment in vitro
Legionella
presence,
a PCR-based on mip genes was more sensitive, showing that this is a
technique is reliable, sensitive and highly specific and suitable for diagnosis
of
L. pneumophila
in respiratory samples. The combination of a urinary
antigen test and PCR analysis of mip gene show more patients diagnosed
with Legionnaires´ disease compared with the use of urinary antigen alone.
