Validation of a tRNA-Glu-cytochrome b key for the molecular identification of 12 hake species (Merluccius spp.) and Atlantic Cod (Gadus morhua) using PCR-RFLPs, FINS, and BLAST

The goal of this study was to develop a diagnostic key for hake meat to solve the limitations of previous identification methodologies, mainly related to the high degradation of the DNA recovered from processed foods. We describe the development of two molecular tools based on polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphisms of the cytochrome b gene, respectively, to identify DNA from 12 hake species in commercial products. The first assay is an exclusion test consisting of the PCR amplification of a 122 bp fragment using nested primers interspecifically conserved in Merluccius spp. and in Gadus morhua. This 122 bp amplicon, being the shortest one so far designed for hake DNA, is a useful traceability tool for highly degraded samples because its sequence contains enough interspecific diagnostic variation to identify 10 hake species and cod and has been successfully amplified from most commercial products so far tested. The second identification key follows a positive outcome of the exclusion test and consists of the PCR amplification of a 464-465 bp fragment and its digestion with three restriction enzymes whose targets map at interspecifically nonconserved sites of the cytochrome b. The key presented here has passed through a rigorous methodological calibration including its testing for genus specificity, its validation on a large number of authenticated sample types from each species range, and its implementation with a maximum likelihood method for the assignment of unknown samples. Together, these two procedures constitute the most complete molecular key so far developed for Merluccius spp., which is optimal for routine identification of hakes in large commercial samples at a reasonable cost-time ratio.