Two new qPCR assays for detecting and quantifying the Aspergillus flavus and Aspergillus parasiticus clades in maize kernels

Abstract

The fungi of Aspergillus section Flavi mostly produce carcinogenic mycotoxins -- aflatoxins (AFs) -- of two types: types B and G (AFBs and AFGs). AF are highly hazardous for human and animal health. Their levels in food and feed are therefore highly regulated, with a low acceptable limit for AF content. In France, global warming has led to the detection of AFs in maize harvests since 2015. Mycoflora analyses have identified two species, A. flavus (producing AFBs) and A. parasiticus (producing both AFBs and AFGs), as responsible for this AF contamination. However, mycoflora analysis is a time-consuming method that cannot readily be applied to large numbers of samples. We propose here an alternative clade-specific functional TaqMan(R) qPCR method based on the calmodulin gene for distinguishing between the A. flavus clade (AfC) and the A. parasiticus clade (ApC). We applied this method to 553 maize samples collected in three different harvest years (2018 to 2020). Both clades were detected in about 40% of the samples tested. As expected, we observed significant positive correlations between AFBs and AfC DNA (R{superscript 2} =0.708), and between AFGs and ApC DNA (R{superscript 2} =0.885). This method will be useful for the rapid, simple and cheap characterisation of maize grain contamination with Aspergillus section Flavi. This method will make it possible to study the relationship between agroclimatic conditions, AF content and species prevalence, to facilitate the anticipation of AF risks due to global warming in France.