Development and Validation of a Multi-Locus PCR-HRM Method for Species Identification in <i>Mytilus</i> Genus with Food Authenticity Purposes

• Main Authors: Marianela Quintrel, Felipe Jilberto, Matías Sepúlveda, María Elisa Marín, David Véliz, Cristián Araneda, María Angélica Larraín
• Format: Article
• Language: English
• Published: MDPI AG 2021-07-01
• Series: Foods
• Subjects: <i>Mytilus</i>; species identification; validation; high-resolution melting; PCR
• Online Access: https://www.mdpi.com/2304-8158/10/8/1684

DNA-based methods using informative markers such as single nucleotide polymorphism (SNPs) are suitable for reliable species identification (SI) needed to enforce compliance with seafood labelling regulations (EU No.1379/2013). We developed a panel of 10 highly informative SNPs to be genotyped by PCR-High resolution melting (HRM) for SI in the <i>Mytilus</i> genus through in silico and in vitro stages. Its fitness for purpose and concordance were assessed by an internal validation process and by the transference to a second laboratory. The method was applicable to identify <i>M. chilensis, M. edulis, M. galloprovincialis</i> and <i>M. trossulus</i> mussels, fresh, frozen and canned with brine, oil and scallop sauce, but not in preserves containing acetic acid (wine vinegar) and tomato sauce. False-positive and negative rates were zero. Sensitivity, expressed as limit of detection (LOD), ranged between 5 and 8 ng/μL. The method was robust against small variations in DNA quality, annealing time and temperature, primer concentration, reaction volume and HRM kit. Reference materials and 220 samples were tested in an inter-laboratory assay obtaining an “almost perfect agreement” (κ = 0.925, <i>p</i> < 0.001). In conclusion, the method was suitable for the intended use and to be applied in the seafood industry.