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

- Real-time LAMP assays for aflatoxin producing fungi were set up and tested.
- Conidia of Aspergillus flavus, Aspergillus nomius, and Aspergillus parasiticus were quantified.
- Effect of background DNA on real-time LAMP reactions was analyzed.
- Contamination levels in spiked Brazil nuts, peanuts and maize were analyzed.
- Real-time LAMP assays proofed to be useful tools in food quality control.

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 (Tt) 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 10 conidia/g in Brazil nuts while assays specific for A. flavus and A. parasiticus had detection limits of 102 conidia/g and 105 conidia/g, respectively in peanut samples as well as 104 conidia/g and 104 conidia/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.