Duplex PCR Detection and Differentiation of Insect DNA Tenebrio molitor and Zophobas morio in Various Types of Food

Edible insects, particularly <i>Tenebrio molitor</i> (Linnaeus) (mealworm) and <i>Zophobas morio</i> (Fabricius) (superworm), have drawn increasing attention as alternative protein sources. This study aims to develop an accurate molecular detection method for <i>T. molitor</i>, an EU-approved food species, and to differentiate it from <i>Z. morio</i>, which remains unapproved for human consumption in the EU. The process enables precise and sensitive identification methods by optimizing singleplex and duplex PCR techniques targeting 16S rRNA and COI gene regions. The DNA of <i>T. molitor</i> was detected in various food matrices, including pastries, chocolate, and porridge, while avoiding cross-reactivity with <i>Z. morio</i>, <i>Gryllus asimilis</i>, and <i>Locusta migratoria</i>. The detection limit for both singleplex and duplex PCR was 10 pg of DNA, ensuring robustness against inhibitory effects from complex food matrices. The developed approach ensures reliable detection and compliance with EU regulations regarding insect-based foods, providing a critical tool for food authentication and preventing adulteration. The key advancements of this approach lie in its improved specificity and sensitivity, allowing for the ability to detect complex food matrices. An applied perspective was evaluated using real commercial food products.