Melt-curve-multiplex-haplotype-specific-PCR, a valuable tool for biological studies: Application in congeneric species discrimination assay

• Melt-Curve-MHS-PCR (MC-MHS-PCR) was applied in congeneric species discrimination assay.
• MC-MHS-PCR combines Multiplex-Haplotype-Specific-PCR (MHS-PCR) with Melt-Curve analysis.
• Guidelines concerning the design of MC-MHS-PCR primer-sets are provided to potential users.
• MC-MHS-PCR is recommended as method of choice for genetic/molecular identification.

The aim of the present study was the development of a genetic discrimination method named Melt-Curve-MHS-PCR (MC-MHS-PCR). In order to explore the advantages, limitations and designing aspects of this method, we have used the conventional (electrophoretic) MHS-PCR as a reference, and designed species-discrimination assays for three fishes of the genus Spicara: Spicara smaris, Spicara maena and Spicara flexuosa. These small-size fishes exhibit marked phenotypic diversity that hinders their discrimination. Partial 16S rRNA gene sequences were obtained from twelve Mediterranean Spicara individuals and were used for the designing of two primer-sets that were subsequently tested on Spicara specimens from six sampling locations of Eastern Mediterranean. All examined samples were correctly assigned by both conventional and melt-curve MHS-PCR, proving that the MC-MHS-PCR is as reliable and discriminatory as the MHS-PCR. Moreover, the MC-MHS-PCR is very rapid, easily implemented, straightforward, and minimizes the risk of sample mix-up and laboratory contamination. These features render the method suitable for large-scale discrimination tests, particularly for studies that include small number of species.