DNA extraction procedures and validation parameters of a real-time PCR method to control milk containing only A2 β-casein

Jiménez Montenegro, Lucía; Mendizábal Aizpuru, José Antonio; Alfonso Ruiz, Leopoldo

DNA extraction procedures and validation parameters of a real-time PCR method to control milk containing only A2 β-casein

dc.contributor.author	Jiménez Montenegro, Lucía
dc.contributor.author	Mendizábal Aizpuru, José Antonio
dc.contributor.author	Alfonso Ruiz, Leopoldo
dc.contributor.department	Institute on Innovation and Sustainable Development in Food Chain - ISFOOD	en
dc.contributor.funder	Gobierno de Navarra / Nafarroako Gobernua	es
dc.contributor.funder	Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa	es
dc.date.accessioned	2023-01-31T11:43:26Z
dc.date.available	2023-01-31T11:43:26Z
dc.date.issued	2022
dc.date.updated	2023-01-31T10:47:02Z
dc.description.abstract	Bovine milk mainly contains two types of β-casein: A1 and A2 variants. In recent years, a new variety of cows’ milk has emerged in the dairy sector called “A2 milk”. This novel product is characterised by the absence of A1 β-casein, which has been associated with possible gastrointestinal discomfort due to β-casomorphin-7 (BCM-7) release during gastrointestinal digestion. In this context, methods to verify the A1 allele absence in A2 milk are required as a quality control in the A2 milk commercialisation. Therefore, the aim of the present study was to develop a locked nucleic acid (LNA) probe-based duplex real-time PCR (qPCR) assay for A1 allele detection in A2 milk samples. Firstly, four DNA isolation methods from milk somatic cells were optimised and evaluated. The results suggests that the commercial kit NucleoSpin Tissue was the most suitable method in terms of DNA quality and amplificability for downstream applications. Then, optimisation and validation of the qPCR assay were carried out. For both A1 and A2 alleles, the absolute limits of detection of this qPCR assay were 7.3 DNA copies/reaction (2 x 10−5 ng DNA) and 30.4 DNA copies/reaction (0.1 ng DNA) at a 95% confidence level with synthetic reference DNA samples and heterozygous genotyped DNA sample, respectively. The relative limits of detection were 2% (15 copies) and 5% (152 copies) for the A1 allele in A2 samples at 95% confidence with synthetic reference and genotyped DNA samples, respectively. The qPCR assay was robust, with intra- and inter-assay variability below 4.3%, and specific, differentiating between A1 and A2 alleles with 100% genotyping accuracy. In conclusion, this cost-effective and fast method could be used to discriminate A1 allele in A2 samples and, consequently, to verify the A1 allele absence in “A2 milk” by screening commercial products on the market.	en
dc.description.sponsorship	This research was conducted in collaboration with the Rural Development Program (RDP), which is co-financed by FEDER funds and Government of Navarre (grant number 210190016 ) and Spanish Ministry of Science and Innovation (grant number PID2019-110752RB-I00). Open Access funding provided by Universidad Pública de Navarra (UPNA).	en
dc.format.mimetype	application/pdf	en
dc.format.mimetype	application/msword	en
dc.identifier.citation	Jiménez-Montenegro, L., Mendizabal, J. A., Alfonso, L., & Urrutia, O. (2022). DNA extraction procedures and validation parameters of a real-time PCR method to control milk containing only A2 β-casein. Food Control, 142, 109259. https://doi.org/10.1016/j.foodcont.2022.109259	en
dc.identifier.doi	10.1016/j.foodcont.2022.109259
dc.identifier.issn	0956-7135
dc.identifier.uri	https://academica-e.unavarra.es/handle/2454/44630
dc.language.iso	eng	en
dc.publisher	Elsevier	en
dc.relation.ispartof	Food Control 142 (2022) 109259	en
dc.relation.projectID	info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020/PID2019-110752RB-I00/ES/
dc.relation.publisherversion	https://doi.org/10.1016/j.foodcont.2022.109259
dc.rights	© 2022 The Authors. This is an open access article under the CC BY-NC-ND license	en
dc.rights.accessRights	info:eu-repo/semantics/openAccess
dc.rights.uri	https://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subject	A2 milk	en
dc.subject	Bovine	en
dc.subject	Real-time PCR	en
dc.subject	Validation	en
dc.subject	β-casein	en
dc.title	DNA extraction procedures and validation parameters of a real-time PCR method to control milk containing only A2 β-casein	en
dc.type	info:eu-repo/semantics/article
dc.type.version	info:eu-repo/semantics/publishedVersion
dspace.entity.type	Publication
relation.isAuthorOfPublication	55000216-532d-43a7-88fd-49a27bab1845
relation.isAuthorOfPublication	ee903831-0350-48ee-9595-de9942e3a89b
relation.isAuthorOfPublication.latestForDiscovery	55000216-532d-43a7-88fd-49a27bab1845