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. 2021 Jul 21;10(8):1684.
doi: 10.3390/foods10081684.

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

Marianela Quintrel  1   2 , Felipe Jilberto  1   3 , Matías Sepúlveda  1   4 , María Elisa Marín  5 , David Véliz  1   6 , Cristián Araneda  1   7 , María Angélica Larraín  1   5
Affiliations

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

Marianela Quintrel et al. Foods. .

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.

Keywords: Mytilus; PCR; high-resolution melting; species identification; validation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Workflow for the selection of a reduced single nucleotide polymorphism (SNP) panel for species identification in Mytilus genus. Each box represents a stage with the remaining number of SNPs. Filtering criteria were summarized on the right side of each stage. In green is the number of loci added; in red is the number of loci removed. Minor allele frequency (MAF).
Figure 2
Figure 2
Example of PCR-HRM curves for the L7 SNP with alleles C/T. Each curve corresponds to one of the three possible genotypes. In purple (―) control curve C/C, in blue (―) control curve (T/T), in green (―) control curve (C/T) and in grey (―) query samples. (a) Normalized melting curves; (b) difference melting curves, using control curve for genotype T/T as reference.
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References

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