Detection of transcripts of the aflatoxin genes aflD, af10, and aflP by reverse transcription-polymerase chain reaction allows differentiation of aflatoxin-producing and non-producing isolates of Aspergillus flavus and Aspergillus parasiticus

The aim of this study was to test the suitability of the RT-PCR (reverse transcription-polymerase chain reaction) technique to differentiate aflatoxin-producing from aflatoxin-non-producing strains of Aspergillus flavus and Aspergillus parasiticus. Total RNAs of 13 strains grown under inducing yeast extract-sucrose (YES) and non-inducing yeast extract-peptone (YEP) media, respectively, were analyzed by using specific primers based on the conserved regions of nine structural genes (aflD, aflG, aflH, aflI, aflK, aflM, aflO, aflP and aflQ) and two regulatory genes aflS and aflR of the aflatoxin B1 biosynthetic pathway. Transcription was confirmed by the expression of the beta-tubulin gene. The expression of the majority aflatoxin biosynthetic, genes including aflR and aflS of all strains varied with regard to the aflatoxin-producing ability and the growth conditions. Nonetheless, we found that the expression profile of the three genes aflD, aflO, and aflP was consistently correlated with a strain's ability to produce aflatoxins or not in YES as well as the inability to produce aflatoxins in YEP. The devised RT-PCR profiling method reflects aflatoxin concentrations ranging from 0.1 to 60 mug/ml of the culture filtrates as determined by fluorescence HPLC. The results are discussed in relation to the suitability of RT-PCR as well as cDNA-based array techniques in diagnostic laboratory settings where individual isolates are being tested for potential toxin production to identify toxigenic isolates of Aspergillus species. (C) 2004 Elsevier B.V. All rights reserved.