Development, validation and field application of a novel species-specific PCR assay for wahoo (Acanthocybium...

Acanthocybium solandri, commonly known as wahoo, is a commercially important fish with high demand in both national and international seafood markets. Because of its delicacy, consumer preference and high market value, wahoo is frequently adulterated/substitution with low-value fish. To facilitate authentication of wahoo products and prevent seafood fraud, a species-specific PCR assay was developed using newly designed primers.

The newly developed species-specific PCR assay amplifies the mitochondrial genome D-loop region of the wahoo species and not that of the other fish species. The specificity test revealed that this assay amplified only the wahoo species, and not the other 13 species. The sensitivity test detected a minimum threshold of 1 ng μL^-1. The reproducibility test amplified the wahoo fish collected from six different geographical locations. The robustness test proved that this assay could amplify wahoo DNA from samples processed using different methods. In-house validation successfully identified the three wahoo samples mixed with 12 other samples. Field validation of 96 wahoo products revealed 17% adulteration in Indian markets.

The developed assay provides a sensitive, specific, reproducible and robust molecular approach for the authentication of A. solandri. It enables the reliable detection of species substitution/adulteration in both raw and processed seafood products. Therefore, this assay has strong potential for application in seafood quality control, market surveillance, traceability and regulatory enforcement. © 2026 Society of Chemical Industry.