Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for the Rapid Detection of Toxigenic Aspergillus flavus and A. carbonarius in Nuts

<i>Aspergillus</i> species create major postharvest problems due to the food losses caused by their mere presence and the hazardous mycotoxins they produce, such as aflatoxin B1 (AFB1) and ochratoxin A (OTA). These mycotoxins are mainly produced by <i>A. flavus</i> and <i>A. carbonarius</i>, respectively. In this study, we developed a rapid detection method for the two aforementioned species based on loop-mediated isothermal amplification (LAMP). The primers were designed to target genes belonging to the mycotoxin clusters <i>pks</i> and <i>aflT</i> for <i>A. carbonarius</i> and <i>A. flavus</i>, respectively. Result visualization was carried out in real time via the detection of fluorescent signals. The method developed showed high sensitivity and specificity, with detection limits of 0.3 and 0.03 pg/reaction of purified DNA of <i>A. carbonarius</i> and <i>A. flavus</i>, respectively. The assays were further implemented on inoculated nuts, including pistachios and almonds, after one-step crude DNA extraction. These tests revealed a detection level of 0.5 spore/g that shows the effectiveness of LAMP as a rapid method for detecting potentially toxigenic <i>Aspergillus</i> spp. directly in food. The validation of the assays included tests on a larger scale that further confirmed their sensitivity and specificity, as well as enabling the production of ready-to-use LAMP prototype kits. These kits are easy to use and aim to simplify the screening of food samples in order to monitor the presence of specific <i>Aspergillus</i> contaminations.