Development and evaluation of a multiplex PCR assay for simultaneous detection of major mycotoxigenic fungi from cereals

The aim of the present study was to develop a multiplex PCR (mPCR) assay for the simultaneous detection of five major metabolic pathway genes viz. aflr (Aflatoxin), pks (Ochratoxin A), tri5 (Trichothecene), pks13 (Zearalenone) and fum13 (Fumonisin), producing Aspergillus, Penicillium and Fusarium species. The mPCR assay with competitive internal amplification control to eliminate false negative results employing specific primers for each of the above mentioned five genes was optimized and validated using standard strains. The standardized mPCR assay detected all five major mycotoxin metabolic genes along with artificially inoculated maize seeds with mycotoxigenic Fusarium, Penicillium and Aspergillus spores. The detection limit of this mPCR assay was 1 x 10(3) spores per gram of artificially inoculated samples upon 48 h of incubation at room temperature. When the developed mPCR assay was applied on to 177 contaminated maize, paddy and sorghum, many of the samples (100 out of 177) were contaminated with either one or more mycotoxins. The mPCR results were further evaluated with high performance liquid chromatography and in general both the methods provided unequivocal results. The developed mPCR based assay was found to be rapid, cost effective and user friendly and can be used for diagnosis of major mycotoxigenic fungi.