Development of rapid and cost-effective multiplex PCR assays to differentiate catfish of the genus Brachyplatystoma (Pimelodidae–Siluriformes) sold in Brazil

The catfishes Brachyplatystoma filamentosum (Kumakuma), Brachyplatystoma vaillantii (Laulao catfish), and Brachyplatystoma rousseauxii (gilded catfish) are important fishery resources in Brazil, where they are sold both fresh and in the form of fillets or steaks. These species have morphological similarities, thus, they can be easily misidentified or substituted, especially after processed. Therefore, accurate, sensitive, and reliable methods are needed for the identification of these species to avoid commercial fraud. In the present study, we develop two multiplex PCR assays for the identification of the three catfish species. Each multiplex protocol combined three species-specific forward primers and a universal reverse primer to produce banding patterns able to discriminate the target species unequivocally. The length of the cytochrome C oxidase subunit I (COI) fragments was approximately 254 bp for B. rousseauxii, 405 bp for B. vaillantii, and 466 bp for B. filamentosum, while the control region (CR) assay produced fragments of approximately 290 bp for B. filamentosum, 451 bp for B. vaillantii, and 580 bp for B. rousseauxii. The protocols were sensitive enough to detect the target species at a DNA concentration of 1 ng/µL, with the exception of the CR of B. vaillantii, in which the fragment was only detectable at 10 ng/µL. Therefore, the multiplex assays developed in the present study were sensitive, accurate, efficient, rapid, and cost-effective for the unequivocal identification of the target species of Brachyplatystoma. They can be utilized by fish processing industries to certify their products, or by government agencies to authenticate products and prevent fraudulent commercial substitutions.