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

Abstract

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 Mytilus 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 M. chilensis, M. edulis, M. galloprovincialis and M. trossulus 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, p < 0.001). In conclusion, the method was suitable for the intended use and to be applied in the seafood industry.

High-Resolution Melting (HRM) Analysis for Rapid Molecular Identification of Sparidae Species in the Greek Fish Market

“…HRM analysis has previously been used for fish species identification in Gadidae [ 33 , 45 ], hake [ 66 ], Takifugu pufferfish [ 31 ], Macrourus [ 32 ], sharks [ 67 ], pangasius [ 68 ] and sepias [ 69 ]. It has been validated for mussels [ 70 ] and salmonids [ 71 ], for which it has been applied to market surveys, showing the potential of a common approach for seafood authentication throughout the supply chain. HRM is carried out in a single reaction in one apparatus, and a large number of samples (95 max) can be analyzed within three hours, as post-PCR melt curve profiles are rapidly processed by the analysis software.…”

“…HRM is carried out in a single reaction in one apparatus, and a large number of samples (95 max) can be analyzed within three hours, as post-PCR melt curve profiles are rapidly processed by the analysis software. The advantages of being applied on samples when no morphological identification is possible (i.e., filleted or juvenile fish) or for those that are cooked and canned [ 70 ] rapidly in real-time observation of the results in a cost-effective manner, eliminating the necessity of large-number sequencing reactions, are obvious. Moreover, using a single piece of equipment for analysis offers the advantage of enabling the technique to be established in multiple control laboratories [ 71 ] across the supply chain.…”

Comparison of two commercial methods for smooth-shelled mussels (Mytilus spp.) species identification

“…The higher the complexity of a method, the more time it consumes, affecting the practicability required in routine analysis. Consequently, using a small panel of informative SNPs is a good option to address the presence of hybrids ( Quintrel et al, 2021 ). For example, all individuals used in this work were previously genotyped with a 49 SNP panel without any evidence of being hybrids.…”

“…Species identification was performed through clearly distinguishable melting curves, for extended protocol see Jilberto et al (2017) . The validation of the HRM-PAPM analysis was published in Quintrel et al (2021) , and a validation summary is included in Table S2 .…”

SNP genotyping revealed a hybrid zone between Mytilus chilensis and M. platensis in southern South America (the Strait of Magellan, Isla Grande de Tierra del Fuego and the Falkland Islands)

“…However, these mono-locus markers have produced ambiguous results when analysed separately, especially in samples collected from hybrid zones (Kijewski et al, 2006(Kijewski et al, , 2019Riginos et al, 2002;Zbawicka et al, 2010). The development of single nucleotide polymorphism (SNP) markers for Mytilus species (Araneda et al, 2016;Zbawicka et al, 2012Zbawicka et al, , 2014 allowed the successful use of multi-locus SNPs panels for SI in this genus (Gardner et al, 2016;Quintrel et al, 2021;Wenne et al, 2016Wenne et al, , 2020Wilson et al, 2018;Zbawicka et al, 2018Zbawicka et al, , 2019.…”