Rapid detection of aflatoxin producing fungi in food by real-time quantitative loop-mediated isothermal amplification

Aflatoxins represent a serious risk for human and animal health. They are mainly produced by Aspergillus flavus and Aspergillus parasiticus but also by Aspergillus nomius. Three species specific turbidimeter based real-time LAMP (loop-mediated isothermal amplification) assays were developed to quantify the three species individually in conidial solutions and to define contamination levels in samples of shelled Brazil nuts, maize, and peanuts. Standard curves relating spore numbers to time to threshold (T_t) values were set up for each of the species. Assays had detection limits of 10, 100 and 100 conidia per reaction of A.flavus, A.parasiticus, and A.nomius, respectively. Analysis of contaminated sample materials revealed that the A.nomius specific real-time LAMP assay detected a minimum of 10conidia/g in Brazil nuts while assays specific for A.flavus and A.parasiticus had detection limits of 10²conidia/g and 10⁵conidia/g, respectively in peanut samples as well as 10⁴conidia/g and 10⁴conidia/g, respectively in samples of maize. The real-time LAMP assays developed here appear to be promising tools for the prediction of potential aflatoxigenic risk at an early stage and in all critical control points of the food and feed production chain.