Development and Validation of a TaqMan Real-Time PCR Assay for Species-Level Authentication of Locusta migratoria in Commercial Food and Feed Matrices

Locusta migratoria (Orthoptera: Acrididae) is one the insect species authorised in the European Union for food and feed applications, including as a novel food under Regulation (EU) 2021/1975, and as a processed animal protein (PAP) for aquaculture, poultry, and pig feed under Regulations (EU) 2021/1372 and 2021/1925. Given its nutritional value and growing relevance in the edible insect and feed sectors, reliable species-level authentication tools are needed to ensure correct labelling and regulatory compliance.

In this work, a species-specific TaqMan real-time PCR assay targeting the mitochondrial cytochrome c oxidase subunit I (COI) gene of L. migratoria (GenBank: KM384849.1) was developed and validated for its detection and quantification in complex food and feed matrices. Specific primers and TaqMan probe were designed, and DNA was extracted using the NucleoSpin® Food Kit. Although in silico analysis revealed no cross-reactivity, non-specific amplification was observed for Oxya yezoensis during cross-reactivity testing. To address this challenge, three Locked Nucleic Acid (LNA) modifications were incorporated into the forward primer, successfully eliminating this cross-reactivity. Specificity was confirmed against 86 non-target matrices, including 49 insect species, 13 seafood species, 10 cereals/seeds/legumes, 8 spices/herbs, and 6 fruits/vegetables matrices, with no false-positive amplification. The LOD was 0.0001 ng of L. migratoria genomic DNA. A calibration curve constructed from binary mixtures of L. migratoria flour in wheat flour at 50%, 30%, 20%, 15%, 10% and 5% w/w yielded a PCR efficiency of 96.11%, R² of 0.998, and LOQ of 1% (w/w). Method trueness, evaluated at four concentration levels, 25%, 7.5% 2.5% and 1% (w/w), yielded percentage errors ranging from -26.22 to 10.78% and coefficients of variation below 0.54%.

The validated assay was applied to 47 commercial insect-labelled products, confirming L. migratoria DNA in four products, declaring this species and detecting it in four unlabelled products (33.83<Cq<38.15), indicating potential cross-contamination or mislabelling. Overall, this assay provides a sensitive and specific tool for L. migratoria authentication in food and feed products