European Society of Mycobacteriology - Instituto Nacional de Saúde ...

PP-50
DEVELOPMENT OF REAL-TIME PCR ASSAY FOR
QUANTIFICATION OF MYCOBACTERIA IN SURFACE WATERS
Lucas, Francoise 1, Radomski, Nicolas 1, Cambau, Emmanuelle 2, Moulin, Laurent 3, Haenn, Sophie 3, Moilleron, Regis 1
1 - Leesu, University Paris-Est
2 - CNRMYC, CHU Saint-Louis de Paris
3 - Etude Biologie, Crecep
Most non-tuberculous mycobacteria (NTM) are saprophytes living in natural environments. However, some species
are opportunistic pathogens involved in various human diseases. An increase in incidence of mycobacteriosis has been
recognized worldwide, probably linked to changes in water use, population vulnerability. One important question arising
from the increasing occurrence of NTM diseases is the origin of these pathogens. Several cases showed that water
play a significant role in the transmission of NTM. Indeed NTM can be found in various aquatic ecosystems, and also in
water distribution systems. However their number and diversity in environmental water bodies and in wastewaters are
often underscored by the actual detection methods and no standard protocol exist. Cultural studies are time consuming
and poorly specific for NTM growth. Few quantitative PCR have been developed for NTM species. However, these PCR
methods have only been applied to clinical samples and may not be adapted for environmental samples. The aim of this
study is to develop a real-time PCR assay to quantify NTM in surface water samples. First the specificity and sensitivity
towards NTM of several published target genes, such as 16S rRNA, rpoB, hsp65, ITS and gyrA and gyrB have been
evaluated using the algorithm BLAST. This first screening resulted in the selection of 8 primer pairs, which specificity and
sensitivity were empirically evaluated by DNA amplification of 50 microbial isolates from the river Seine (France), which
belong to Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes and Mycetes. This strain library was completed
with 25 NTM and other 8 Actinobacteria strains from national collections. This second step of screening resulted in the
selection of two highly specific primer pairs targeting gyrB and rrs genes, showing respectively 94,83% et 93,10 % of
specificity. The efficiency of the real time PCR was evaluated for both primer pairs using the strain libraries.
122 ESM 2009