Toward rapid testing for molecular authentication: Novel method for multianalyte identification of olive, sunflower, soy, sesame and corn DNA by visual biosensing

Background: Product authentication ensures consumer safety by verifying composition. Two main strategies exist: (i) instrumental methods (NMR, FTIR, Raman, chromatography, mass spectrometry) with chemometrics to create metabolite fingerprints, (ii) DNA-based molecular authentication. DNA methods are robust against environmental or harvest variations. They start with PCR amplification, followed by capillary electrophoresis with laser-induced fluorescence for high-resolution DNA fragment separation. Additional tools, including high-resolution melting analysis and flow cytometry, further confirm DNA amplification. There is a need for methodological platforms in the simple rapid test format amenable to field testing for molecular authentication. Results: Unlike existing methods that require costly equipment and specialized personnel, we present a novel method combining multiplex conventional PCR with direct multiplex visual identification of five species by a dry-reagent strip biosensor. Olive, sunflower, soybean, sesame, and corn DNA are differentiated during PCR, as amplification occurs only when the corresponding species is present. PCR products are applied directly to the biosensor eliminating the need for post-PCR enzymic reactions, purification, pretreatment, or heat denaturation, thereby reducing assay time and user steps. The biosensor delivers results within 15–20 min, detecting olive DNA adulteration with as low as 5 % sesame, soybean, and sunflower, and 10 % corn. Measurement variability is below 5 %. Significance: The proposed method integrates multiplex conventional PCR and direct multiplex biosensor, addressing the challenge of multiplex adulteration detection in a rapid, field-deployable format. It offers unique advantages of simplicity, portability, and low cost, with detection by the naked eye eliminating the need for expensive instrumentation. The multiplexing capability allows simultaneous identification of several potential adulterants, making the method broadly applicable for molecular authenticity assessment of various products.