Codfish authentication by a fast Short Amplicon High Resolution Melting Analysis (SA-HRMA) method

In this work we had developed a one-tube fast High Resolution Melting Analysis (HRMA) method for the identification of G. morhua adulteration with Gadus macrocephalus and Gadus chalcogrammus. HRMA of the ca. 600 bp COI fragment initially assayed proved to be inefficient in species discrimination which lead to the development of a new approach targeting small fragments (ca. 100 bp) containing sequence variations between the three species. Seven small polymorphic regions were identified and specific primer pairs were de novo designed. One region proved to be highly efficient in the species discrimination in HRMA mode. The technique was applied in marketed processed samples showing that 2 out of 33 samples (6%) were mislabeled as G. morhua (identified as G. macrocephalus). PCR and SA-HRMA was performed in fast mode allowing very short analysis time (less than 1 h) enabling therefore the possibility of high sample throughput.