Identificazione di specie nel settore lattiero-caseario con tecniche molecolari. Parte II: SNuPE (Single Nucleotide Primer Extension)

Species identification has recently assumed an important role in the dairy industry because of two new problems: the prevention of food allergies, due to an increment of allergic diseases; and the detection of food substitutions, that can widely reduce the food value. In this work the AA report the set up of innovative methods allowing to obtain a clear and reliable identification of the four principal species that interest the dairy industry (Bos, Ovis, Capra, Bubalus) in processed dairy products. The method is based on the comparative analysis of 63 sequences of the cytb gene which is traditionally used for species identification, in order to identify two regions whose sequences were used to design a set of universal primers which were subsequently utilized in the PCR amplification of a part of the cytb gene from pure dairy products. In this second part the AA show the set up of a method for species identification based on the detection of species-specific single nucleotide polymorphisms (SNPs) of the mitochondrial cytb gene. The technique is called SNuPE (Single Nucleotide Primer Extension). A primer strictly close to a characterising point mutation is used in a primer extension reaction, where only one labelled species-specific nucleotide will be incorporated and it will be subsequently detected with a capillary electrophoresis system. The incorporated nucleotide determinates which species is being analysed.