PCR-ELISA for the semiquantitative detection of Nile perch (Lates niloticus) in sterilized fish muscle mixtures

A PCR-ELISA technique was developed for the semiquantitative detection of Nile perch (Lates niloticus) in experimentally sterilized fish muscle mixtures. Specific oligonucleotides derived from the 5S rDNA gene of Nile perch were selected. A forward primer, together with a reverse digoxigenin-labeled primer, permitted the amplification of specific 185 bp DNA fragments showing DNA intensities proportional to the contents of Nile perch muscle tissue in the fish mixtures. A biotinylated probe immobilized onto streptavidin-coated microplates was used to capture the digoxigenin-labeled fragments that were detected with peroxidase antidigoxigenin conjugate. Subsequent enzymatic conversion of substrate gave distinct absorbance differences when assaying fish binary mixtures containing different percentages of Nile perch muscle.