P-22
Identification and quantification of ochratoxin synthesizing fungi on cereals
using real time PCR. Ochratoxin A (OTA) is a mycotoxin of considerable concern for human health
and is said to have carcinogenic, nephrotoxic and genotoxic properties.
Penicillium verrucosum and Aspergillus ochraceus have been
reported to be the moulds with the potential for OTA synthesis. Since 50-80% of
average consumer OTA intake is derived from cereals, it is of obvious importance
to analyse barley and malt for contamination by relevant fungi. To overcome
time-consuming incubation methods and subsequent microscopic identification of
OTA-producing fungi, we aimed to set up a PCR-based protocol. After the
purification of DNA from a total of 50 fungal isolates representing species from
different genera, we performed RAPD analyses using different random primers. One
primer resulted in the amplification of a polymorphic product only when A.
ochraceus DNA was used as template. The RAPD marker could be converted into
a robust PCR marker. A similar approach was used to generate a PCR primer pair
which allows the identification of Penicillium species. Although only
P. verrucosum is described as an OTA producer, it was necessary to
develop a more general PCR marker because nomenclature within the
Penicillia (toxigenic vs non-toxigenic species) is very unclear. After
inoculation of barley seeds with cultures of A. ochraceus and P.
verrucosum, respectively, we isolated fungal DNA from samples taken after
incubation at different points of time. Using the primers described, we could
identify the fungi exclusively in flours from those seeds inoculated before.
Additionally the degree of contamination could be quantified by using the
LightCycler(TM) system. This kind of real time PCR enabled us to monitor the
propagation of the fungi during the course of time. A PCR-based system for
detection and quantification of OTA-producing moulds on barley or malt samples
has a couple of advantages over conventional methods. 1) DNA preparation and PCR
take less than 4 hours. 2) The identification of relevant fungi does not require
microbiological expertise. 3) Quantification based on flour is more accurate
than counting of contaminated seeds. 4) Analysing fungal DNA instead of the
mycotoxins themselves enables the potential for OTA synthesis to be detected at
a later stage of processing. 5) Simultaneous PCR detection of other species,
like Fusarium, can easily be performed.
Michael Voetz, born in 1964, received a diploma in Biology from the
University of Cologne in 1991. In 1994 he worked in a gene mapping project at
Oregon State University. He earned a PhD in Plant Molecular Biology from the
University of Cologne/Max-Planck-Institute for Breeding Research in 1995. From
1995-2000 he was scientific collaborator at the Research Department of the
Weissheimer Malzfabrik in Andernach. Since 2000 he is head of the
biotechnology/PCR laboratory at the Research Institute for Raw-Materials within
VLB in Berlin.
MICHAEL VOETZ and Frank Rath. Research and Teaching Institute of Brewing,
Berlin, Germany.