<i>Aspergillus</i> in Italian Pistachios: Characterization and Detection of Major Aflatoxigenic Species With a Loop‐Mediated Isothermal Amplification Assay

Aflatoxigenic <i>Aspergillus</i> species are major postharvest contaminants of pistachio nuts, producing harmful aflatoxins that are strictly regulated by most countries. Therefore, their accurate detection is crucial for effective control strategies. Loop-mediated isothermal amplification (LAMP) is a simple molecular technique that uses 4-6 primers to achieve high sensitivity and specificity through a thermostatic amplification reaction. This study aimed to isolate <i>Aspergillus</i> spp. (section <i>Flavi</i>) from pistachios sampled in Italy and to determine their capacity to produce Aflatoxin B1 (AFB1). It revealed a high occurrence of <i>A. flavus</i>, followed by <i>A. parasiticus</i>. Furthermore, all isolates identified as these species exhibited AFB1 production, reaching 10107.8 and 1448.2 ng/mL for <i>A. flavus</i> PGA95 and <i>A. parasiticus</i> PGA72, respectively. Based on these results, a LAMP assay was developed to detect these species by targeting the <i>nor1</i> gene, involved in the aflatoxin production pathway. The assay demonstrated a sensitivity of 0.03 pg of DNA per reaction and high specificity in detecting only targeted species. To simplify the assay and enhance its applicability, primers were air-dried at 30°C to preserve stability and eliminate the need for repeated reagent preparation. Dehydrated primers maintained their stability for up to 45 days of storage even at 35°C. Additionally, the LAMP assay successfully detected fungi directly on pistachios at contamination levels as low as 0.5 spores/g. This study identifies prevalent aflatoxigenic <i>Aspergillus</i> species in Italian pistachios and introduces LAMP as a rapid, sensitive, and specific detection tool.