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

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 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 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. 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 A1 allele in A2 samples at a 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 A1 allele absence in “A2 milk” by screening commercial product on the market. • The LNA duplex qPCR assay developed could be used to verify A1 allele absence in “A2 milk”. • LNA probes reliably discriminated between A1 and A2 alleles. • The relative limits of detection reach up to 2% (15 copies) for A1 allele in A2 samples at a 95% confidence. • Milk is a challenging matrix for DNA isolation due to the presence of PCR inhibitors that leads to low DNA yield.