Técnicas de genotipagem de rebanhos leiteiros com polimorfismos no gene da β-caseína: potencial causador da alergia à proteína do leite de vaca (APLV)

Abstract

The search for innovation in milk production is an increasingly relevant guideline at the national level. In the last decade, milk producers have been investing on a food improvement to boost the market and reach new audiences: the production of milk containing A2-type β-casein, potentially less allergenic and with higher added value. β-casein is a protein encoded by a gene with at least 13 variants, where A1 and A2 variants are the most abundant. Milk from animals that own the A1 variant is related to allergenic effects in humans, with reports of diarrhea, flatulence, hives, mucosal inflammation and bleeding, symptoms directly associated with cow's milk protein allergy (CMPA). On the other hand, animals homozygous for the A2 variant (A2A2) produce potentially less allergenic milk, since the production of β-casomorphin-7 as by-products is reduced. Thus, the present work, carried out in partnership with the company TaqMol Diagnosticos, sought to develop a test based on PCR genotyping techniques with the objective of improving the management of animals with β-casein A2 variant for a milk production with higher added value in the Northeast region. For this, two PCR-based techniques were compared to determine the feasibility of applying a precise, sensitive, reliable and fast molecular test that is commercially attractive for milk producers and generates socioeconomic benefits for the region. The methodologies of PCR-RFLP and Real Time PCR (qPCR) were approached in the study. The samples were obtained through the company TaqMol Diagnosticos and had the DNA extracted from a commercial kit. Both for PCR-RFLP and for Real Time PCR, specific primers owned by the company were used for β-casein gene amplification. PCR products were digested with HinfI enzyme, providing different patterns when analyzed by agarose gel electrophoresis: 25% showed patterns referring to A2A2-type, 62,5% A1A2 and 12,5% inconclusive results. Sanger sequencing was performed in order to confirm the genotyping previously analyzed, revealing the low compatibility of the band patterns obtained. The Real-Time PCR approach was successful for the amplification of β-casein fragment, proving to be viable and promising for the identification of its variants in the bovine genome.