abstract book - Clostridia

DETECTION OF CLOSTRIDIUM BOTULINUM NEUROTOXIN GENES
(BONT) A, B, E AND F WITH REAL-TIME-PCR
U. Messelhäusser, R. Zucker, W. Kleih, C. Höller, U. Busch
Bavarian Health and Food Safety Authority, 85764 Oberschleißheim,
Germany
Intoxications with C. botulinum toxins belong also in Germany to the
rare occurring food-poisonings; the mortality, however, is very high. In
the last years the risk for the consumer´s health is mainly due to not
commercially produced food cans or salted bacon. Because of the
difficult and time-consuming cultural detection of C. botulinum, PCR
methods to screen for the toxin genes A, B, E and F, which are relevant
in human medicine, have been used increasingly during the last years.
These comprised mostly conventional PCR methods which are not so
efficient to screen large numbers of samples. Therefore we designed
for the C. botulinum-detection in our routine laboratory two probe-based
real-time-PCR-assays, one triplex-PCR-assay for the detection of
BONT A and B in combination with an Internal Amplification Control
(IAC) and one duplex-system for the detection of BONT E and F, both
based on the Taqman-technology. As IAC we used the pUC 19
plasmid. Both assays were tested for specifity and sensitivity with pure
cultures first and then with artificially contaminated food samples (meat,
fish and honey) after enrichment. The sensitivity with pure cultures was
10 cfu/ml for all four toxin genes. Food samples contaminated with
10 cfu/g, were reliably detected as positive with real-time-PCR after
enrichment. Subsequently we checked 147 routine samples with our
real-time-PCR-systems, the conventional PCR-system of Lindström et
al., 2002 and the cultural method in parallel. We examinated mainly
food samples (honey, carps, canned meat and vegetables), but also
animal feeding stuff and environmental samples. Six samples were
positive with the real-time-PCR and the convential PCR-system of
Lindström et al., 2002 (five samples for BONT B and one sample for
BONT E), but only five samples were positive with the cultural method.
The results of our examinations indicates, that the two real-time-PCRassays
are suitable in a routine laboratory for a fast screening for the
presence of Clostridium botulinum type A, B, E and F in food samples.
43