Tehran University of Medical Sciences Iranian Journal of Allergy, Asthma and Immunology 1735-1502 0 0 2026 09 13 1 14 Mina Mohammad-rezaei Department of Immunology, Immunology Research Center, School of Medicine, Iran University of Medical Sciences, Tehran, Iran Mina Noroozbeygi Department of Immunology, Immunology Research Center, School of Medicine, Iran University of Medical Sciences, Tehran, Iran Amir gisouei Alimentary Tract Research Center, Clinical Sciences Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Salar khazeni fard Department of Medicine Sciences, QoMS.C, Islamic Azad University, Qom, Iran Majid Khoshmirsafa Department of Immunology, Immunology Research Center, School of Medicine, Iran University of Medical Sciences, Tehran, Iran Mohammad-Ali Assarehzadegan Department of Immunology, Immunology Research Center, School of Medicine, Iran University of Medical Sciences, Tehran, Iran 2026 05 04 2026 06 13 Peanut allergy is a major global health concern, and undeclared peanut residues in processed foods pose serious risks to sensitized individuals. Reliable analytical methods are essential for accurate labeling and consumer protection, particularly because high-heat processing can cause protein denaturation and DNA fragmentation. This study assessed the applicability and analytical reliability of a short-amplicon duplex quantitative polymerase chain reaction (qPCR) system targeting the Ara h 1 gene across diverse peanut genotypes, thermally processed samples, and commercial food matrices. Genomic DNA was extracted from raw peanuts representing 11 varieties, roasted and autoclaved samples, and commercial foods using a modified cetyltrimethylammonium bromide (CTAB)-based protocol. The duplex qPCR assay targeted the Ara h 1 gene and incorporated 18S rRNA as an internal amplification control on the Rotor-Gene Q platform. The assay detected all peanut varieties consistently, with a limit of detection (LOD) of 100 pg of DNA. The short amplicon (<150 bp) enabled reliable detection even after severe thermal processing (autoclaving at 121°C for 15 min). Validation across commercial food products demonstrated that the 18S rRNA internal control effectively prevented false-negative results in inhibitor-rich matrices, such as chocolate and cocoa-based foods. These findings confirm that Ara h 1 is a stable and robust molecular target for detecting trace peanut residues in both raw and processed food products, supporting regulatory compliance and food safety for allergic consumers. https://ijaai.tums.ac.ir/index.php/ijaai/article/view/4822 https://ijaai.tums.ac.ir/index.php/ijaai/article/download/4822/2423