Identificazione di specie nel settore lattiero-caseario con tecniche molecolari: Parte 1a: Terminal-RFLP (T-RFLP)

Species identification has recently assumed an important role because of the incresing of the number of people that suffer of allergic diseases and because of the increasing of food substitutions that cause damage to consumers. In this work the AA report the set up of an innovative method -highly sensitive and easily applicable to dairy products - based on capillary electrophoresis: Terminal-RFLP technique (T-RFLP). This method required the comparative analysis of 63 available cytb sequences of the four main species that interest the dairy industry (Bos, Ovis, Capra, Bubalus), 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 cytbgene fragment from pure DNA. The PCR step was carried out using the reverse primer labelled with the fluorochrome HEX A set of three restriction enzymes was selected on the basis of sequence data analysis, in order to generate species-specific terminal labelled restriction fragments. Finally, the capillary electro phoresis system provided a digital output which allowed an accurate and sensitive detection of the only terminal species-specific restriction fragment that contains the labelled reverse primer.