Alfonso Ruiz, Leopoldo

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https://academica-e.unavarra.es/handle/2454/48493

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Alfonso Ruiz

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Leopoldo

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Agronomía, Biotecnología y Alimentación

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IS-FOOD. Research Institute on Innovation & Sustainable Development in Food Chain

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0000-0002-5662-9997

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88522

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1974

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Author: Jiménez Montenegro, Lucía × Subject: Duplex qPCR assay ×

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    PublicationOpen Access
    Development of a duplex qPCR assay with locked nucleic acid probes for A, B and E kappa-casein variants detection
    (Springer Nature, 2022) Jiménez Montenegro, Lucía; Mendizábal Aizpuru, José Antonio; Alfonso Ruiz, Leopoldo; Azparren Domínguez, Leire; Urrutia Vera, Olaia; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura; Institute on Innovation and Sustainable Development in Food Chain - ISFOOD
    Milk proteins determine important milk technological characteristics. Among caseins, Ƙ-casein has been correlated with fat and protein content and cheese yield. Fourteen Ƙ-caseins variants have been described but the alleles A, B and E are the most important ones due to their frequency and/or influence on the technological aptitudes of milk. Therefore, in the present study two different duplex qPCR assays with locked nucleic acid probes (for positions 13104 and 13124 of the Ƙ-casein gene) were developed for the detection of A, B and E variants. Firstly, DNA isolation method from milk somatic cells and hair was optimised. The developed 13124-qPCR assay showed an increased sensitivity reaching up to 6.7 copies DNA copies/reaction at a 95% confidence level with A, B and E alleles reference samples. The 13104-qPCR assay reached up to 6.7 DNA copies/reaction for A allele reference sample and 67 DNA copies/reaction for B and E samples. Intra-assay variation results were below 6%. Applicability was determined using DNA samples from animals with known genotype for Ƙ-casein (AA, AB, BB, BE, AE, EE) and both assays were able to discriminate among the six genotypes with 100% accuracy. Thus, this qPCR method represents a sensitive and rapid option for the detection of Ƙ-casein alleles in both hair and milk samples.