Aspergillus in Italian Pistachios: Characterization and Detection of Major Aflatoxigenic Species With a Loop-Mediated Isothermal Amplification Assay

Aflatoxigenic Aspergillus 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 Aspergillus spp. (section Flavi) from pistachios sampled in Italy and to determine their capacity to produce Aflatoxin B1 (AFB1). It revealed a high occurrence of A. flavus, followed by A. parasiticus. Furthermore, all isolates identified as these species exhibited AFB1 production, reaching 10107.8 and 1448.2 ng/mL for A. flavus PGA95 and A. parasiticus PGA72, respectively. Based on these results, a LAMP assay was developed to detect these species by targeting the nor1 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 degrees 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 degrees 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 Aspergillus species in Italian pistachios and introduces LAMP as a rapid, sensitive, and specific detection tool.