Development of a Stepwise Molecular Workflow for the Detection of Undeclared Botanical Species in Commercial Oregano Products

Authentication of commercial oregano products remains challenging because processing may hinder the identification of undeclared botanical species. In this study, a stepwise molecular approach integrating DNA mini-barcoding, species-specific real-time PCR, and loop-mediated isothermal amplification (LAMP) was developed for the detection of undeclared botanical components in commercial oregano products. Eighty-five commercial oregano products were initially screened by DNA mini-barcoding targeting the plastidial trnL region. Sequence analysis identified Myrtus communis, Olea europaea, and Convolvulus althaeoides among the detected botanical species. Based on the identification of M. communis, species-specific real-time PCR and LAMP assays were developed and applied for its targeted detection, for which a species-specific molecular assay in oregano products has not previously been reported. M. communis was detected in 31 samples by real-time PCR and in 42 samples by LAMP, whereas 18 samples were identified as positive by DNA mini-barcoding among the subset subjected to cloning and sequencing. Sample-level comparison showed partial concordance among the three approaches, supporting their complementary use for M. communis detection. The LAMP assay showed specific amplification of M. communis, with a detection limit of 1 pg of target genomic DNA and consistent detection down to a 0.01% matrix-spiking level in oregano tissue. Overall, the proposed approach combines broad DNA-based screening with targeted molecular detection, while the newly developed trnL-based LAMP assay provides a rapid tool for the detection of undeclared M. communis in processed oregano products.