Fish parvalbumin gene: Detection and quantification by universal primers for forensic application

The substitution of illegal fish species poses economic, health and environmental risks. Identifying fish species, particularly closely related ones lacking external features, is a challenge. This study introduces an alternative approach using nuclear gene markers for positive detection, identification, and quantification of mitochondrial gene markers common in existing assays. We developed a universal primer targeting the parvalbumin (pvalb) beta gene, a common fish allergen with an amplicon size of 117 bp long. The developed fish-specific primer assay was tested on a diverse panel of 54 fish species, including commonly consumed species such as salmon, carp, and cod, along with non-fish species such as Buffalo, chicken and pork, and vegetables such as wheat, celery, and mint. The assay consistently identified the fish pvalb gene, demonstrating our assay's high specificity and effectiveness in detecting this gene in a variety of fish species. The assay exhibited a low limit of detection (LOD) of 5 pg, detecting trace DNA quantities. The assay also effectively quantified parvalbumin with good efficiency and linearity. These findings highlight pvalb as a reliable forensic tool for the identification of fish species. Implementing nuclear gene markers mitigates fish mislabelling and fraud, reducing economic and environmental impacts.