in wheat by Detection of Aspergillus flavus PCR ,_,_.„ • .-.--... ---.... - ..... 1 - ·, .-- - . ·.-...,, .- .-- ROLF GEISEN*, SVEN MULFINGER AND LUDWIG NIESSEN Bundesforschungsanstalt für Ernährung, 76131 Karlsruhe, Germany Lehrstuhl für Technische Mikrobiologie· Technische Universität München, D-85350 Freising, Gennany Received September 4, 1998; accepted February 28, 1999. Summary A PCR method specific for fungi producing aflatoxins based on aflatoxin biosynthetic genes has been applied to detectAspergillus flavus in wheat. Two methods for isolating DNA were used. Both gave consistent results, however with the more sophisticated method the occurrence of artificial bands was reduced. A positive PCR signal was achieved with samples containing about 10 4 cfu/ g of A. flavus. In the same samples aflatoxin could be detected by TLC. With noninfected samples the PCR gave no signal. Keywords: aflatoxin, Aspergillus flavus, PCR, wheat, aflatoxin biosynthetic genes 1. Introduction The aflatoxins, potent toxic and carcinogenic secondary metabolites, are produced mainly by Aspergillus Jlavus and A. parasiticus. These species can occur in a variety of foods, especially in warmer geographical regions. They have been isolated from pea- nuts, maize, wheat, rice, cottonseed, figs and nuts (Bullerman, 1979; Ellis et al., 1991). Conventional methods for detecting and identifying these species are time consuming and require expertise in fungal taxonomy. For quality control of food commodities rapid objective detection methods are needed. Pitt et al. (1983) described Aspergillus flavus and parasiticus agar (AFPA), which is an indicator medium for A. parasiticus and A. flavus, but does not differentiale these species. Other methods, including the immuno- logical latex agglutination assay are also not specific enough to differentiate them. Cross reaction occurs even with some species of Penicillium (de Ruiter et al., 1993). Molecular methods such as PCR have the advantage of being specific if a unique target sequence can be identified. PCR methods for the detection of species producing aflatoxins have been described (Geisen, 1996, Shapira et al., 1996). The application of PCR to food and feed samples can be ham.pered by inhibitory compounds, which are isolated along with theDNA(Rossenetal.,1992).ForthisreasonthePCRextractionmethodmustbeadapted to each food commodity (Lantz et al., 1994). The purpose of this study was to test the application of the PCR approach for the detection of fungi producing aflato":ins in wheat. *Corresponding author: Dr R. Geisen, Bundesforschungsanstalt für Ernährung, Haid- und Neustr. 9, 76131 Karlsruhe, Germany.e-mail:rolf.geisen@bfe.uni-karlsruhe.de J Food Mycol 19jlr,}(4):211-218 211 © 1998 Aspen Publishers, Inc. .,_..., 1 „,_