Exploiting 16S rRNA gene for the detection and quantification of fish as a potential allergenic food: A comparison of two real-time PCR approaches

•Universal primers targeting the 16S rRNA gene were designed for fish detection.•Two real-time PCR systems based on the EvaGreen dye and a TaqMan probe were compared.•Sensitivities of 0.01 pg of fish DNA and 0.0001% of fish in béchamel were reached.•The probe system was successfully validated in terms of precision and trueness.•A high level of mislabelling for fish as allergen was verified in foodstuffs. Fish is one of the most common allergenic foods that should be accurately labelled to protect the health of allergic consumers. In this work, two real-time PCR systems based on the EvaGreen dye and a TaqMan probe are proposed and compared. New primers were designed to target the 16S rRNA gene, as a universal maker for fish detection, with fully demonstrated specificity for a wide range of fish species. Both systems showed similar absolute sensitivities, down to 0.01 pg of fish DNA, and adequate real-time PCR performance parameters. The probe system showed higher relative sensitivity and dynamic range (0.0001–50%) than the EvaGreen (0.05–50%). They were both precise, but trueness was compromised at the highest tested level with the EvaGreen assay. Therefore, both systems were successful, although the probe one exhibited the best performance. Its application to verify labelling compliance of foodstuffs suggested a high level of mislabelling and/or fraudulent practices.