An accurate high-resolution melting method to genotype bovine β-casein
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1 3 Eur Food Res Technol (2014) 238:295–298 DOI 10.1007/s00217-013-2101-z ORIGINAL PAPER An accurate high‑resolution melting method to genotype bovine β‑casein Luis J. Royo · Ana del Cerro · Fernando Vicente · Alfonso Carballal · Begoña de la Roza‑Delgado Received: 3 June 2013 / Revised: 21 August 2013 / Accepted: 21 September 2013 / Published online: 11 October 2013 © Springer-Verlag Berlin Heidelberg 2013 Introduction Dairy farms and products are involved in an acute crisis, and the traditional aspects related to quantity and qual- ity cannot guarantee a sufficient profit for farmers. Due to these reasons dairy farms are suffering important changes in their structure and productive strategy, but also new aspects about quality and safety of milk products have arisen. Some of these aspects to be considered are trace- ability, certification to guarantee the exclusive use of con- ventional food and total absence of drugs and contaminants (organic milk), the modification of fatty acids profile in milk, and the reduction in or elimination of allergenic or dangerous compounds from milk and milk products. Casein represents about 80 % of total protein in cows’ milk, about 30–35 % of which is β-casein [1]. Within β-casein, there are a number of variants which are geneti- cally determined, being A1, A2, and B the most common variants. A1 and, to a lesser extent, B have been impli- cated in the literature in relation to human disease [2–4]. Many β-casein genetic variants have been described (see for example [5]), being only A1, A2, and B universally distributed with a frequency high enough to be taken into consideration [6, 7]. DNA mutations that define these three variants are located in exon VII of the gene [8]. A1 and B β-casein have a histidine at position 67 that allows an enzymatic cleavage to occur releasing a 7 amino acid called ‘β casomorphin 7’ (βCM-7) [2]. The A2 variant has a proline at position 67 so that βCM-7 is not released. βCM-7 has opioid properties and has a wide variety of potential effects in the body, including immune suppres- sant activity. These may be important in any potential etiological effects of A1 β-casein on disease processes, although other mechanisms are also possible, and more research is needed. Abstract In the present study, we used high-resolution melting analysis to simultaneously identify the two muta- tions that taken together give us the genotype of the most common bovine β-casein variants. Allelic variants are detected in real time, and no post-PCR manipulations are required, therefore limiting costs and possible car- ryover contamination. Data can be copied to a Microsoft Excel spreadsheet for semiautomatic determination of the genotype. The method was validated using different DNA sources, in 122 samples from 12 different bovine popula- tions, obtaining the first report on bovine β-casein geno- types for some of them. Keywords HRM · β-Casein · Bovine milk · Milk quality Electronic supplementary material The online version of this article (doi:10.1007/s00217-013-2101-z) contains supplementary material, which is available to authorized users. L. J. Royo (*) · F. Vicente · A. Carballal · B. de la Roza-Delgado Department of Nutrition, Grasslands and Forages, SERIDA, PO Box 13, 33300 Villaviciosa, Asturias, Spain e-mail: [email protected] F. Vicente e-mail: [email protected] A. Carballal e-mail: [email protected] B. de la Roza-Delgado e-mail: [email protected] A. del Cerro Department of Animal Health, CBA-SERIDA, 33394 Gijón, Asturias, Spain e-mail: [email protected]
Transcript of An accurate high-resolution melting method to genotype bovine β-casein
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Eur Food Res Technol (2014) 238:295–298DOI 10.1007/s00217-013-2101-z
ORIgInal PaPER
An accurate high‑resolution melting method to genotype bovine β‑casein
Luis J. Royo · Ana del Cerro · Fernando Vicente · Alfonso Carballal · Begoña de la Roza‑Delgado
Received: 3 June 2013 / Revised: 21 august 2013 / accepted: 21 September 2013 / Published online: 11 October 2013 © Springer-Verlag Berlin Heidelberg 2013
Introduction
Dairy farms and products are involved in an acute crisis, and the traditional aspects related to quantity and qual-ity cannot guarantee a sufficient profit for farmers. Due to these reasons dairy farms are suffering important changes in their structure and productive strategy, but also new aspects about quality and safety of milk products have arisen. Some of these aspects to be considered are trace-ability, certification to guarantee the exclusive use of con-ventional food and total absence of drugs and contaminants (organic milk), the modification of fatty acids profile in milk, and the reduction in or elimination of allergenic or dangerous compounds from milk and milk products.
Casein represents about 80 % of total protein in cows’ milk, about 30–35 % of which is β-casein [1]. Within β-casein, there are a number of variants which are geneti-cally determined, being a1, a2, and B the most common variants. a1 and, to a lesser extent, B have been impli-cated in the literature in relation to human disease [2–4]. Many β-casein genetic variants have been described (see for example [5]), being only a1, a2, and B universally distributed with a frequency high enough to be taken into consideration [6, 7]. Dna mutations that define these three variants are located in exon VII of the gene [8]. a1 and B β-casein have a histidine at position 67 that allows an enzymatic cleavage to occur releasing a 7 amino acid called ‘β casomorphin 7’ (βCM-7) [2]. The a2 variant has a proline at position 67 so that βCM-7 is not released. βCM-7 has opioid properties and has a wide variety of potential effects in the body, including immune suppres-sant activity. These may be important in any potential etiological effects of a1 β-casein on disease processes, although other mechanisms are also possible, and more research is needed.
Abstract In the present study, we used high-resolution melting analysis to simultaneously identify the two muta-tions that taken together give us the genotype of the most common bovine β-casein variants. allelic variants are detected in real time, and no post-PCR manipulations are required, therefore limiting costs and possible car-ryover contamination. Data can be copied to a Microsoft Excel spreadsheet for semiautomatic determination of the genotype. The method was validated using different Dna sources, in 122 samples from 12 different bovine popula-tions, obtaining the first report on bovine β-casein geno-types for some of them.
Keywords HRM · β-Casein · Bovine milk · Milk quality
Electronic supplementary material The online version of this article (doi:10.1007/s00217-013-2101-z) contains supplementary material, which is available to authorized users.
l. J. Royo (*) · F. Vicente · a. Carballal · B. de la Roza-Delgado Department of nutrition, grasslands and Forages, SERIDa, PO Box 13, 33300 Villaviciosa, asturias, Spaine-mail: [email protected]
F. Vicente e-mail: [email protected]
a. Carballal e-mail: [email protected]
B. de la Roza-Delgado e-mail: [email protected]
a. del Cerro Department of animal Health, CBa-SERIDa, 33394 gijón, asturias, Spaine-mail: [email protected]
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Early nutrition has important health consequences for both childhood and adulthood. Cows’ milk is a basic food for infants and children and a common food for adults in Western countries. The health conditions that have been postulated to be affected by the a1/a2 β-casein content of milk are important and are reasonably common. There-fore, if the composition of milk is a causative or protective factor for these conditions, then it has major public health implications.
High-resolution melting is a post-PCR method that allows detection of subtle sequence polymorphisms by monitoring the melting behavior of PCR amplicons using a highly saturating fluorescent dye [9]. although different methods have been proposed to genotype milk proteins [6, 7, 10, 11], in this paper, we propose the use of high-resolu-tion melting (HRM) analysis as a new close-tube, inexpen-sive, fast, and sensitive method for genotyping large num-ber of samples.
Materials and methods
Sampling and Dna isolation
Dna from 122 individuals belonging to 12 bovine differ-ent populations was isolated by standard methods [12].
Sampling includes whole blood in EDTa, frozen meat, and hair follicle (see Table 1).
Real-time PCR and high-resolution melting analysis design
Two single-nucleotide polymorphisms (SnP) differenti-ate among a1, a2, and B bovine β-casein variants (see for example Figure 1 in [6]). Primers were designed, based on the sequence with genBank accession number EF628290, to have high HRM efficiency. To do that, we developed two independent PCRs to generate amplicons of 117 and 71 bp in size (Table 2) and to avoid having more than one SnP per amplicon. The PCR assays were performed using the Step One Real-Time PCR System (applied Biosystems, Madrid, Spain) in a total volume of 15 μl containing 1× MeltDoc-tor HRM™ Master mix, 0.25 μM of each primer, and 25–50 ng of Dna. The real-time PCR program consisted of 40 amplification cycles consisting in two steps: denaturation for 20 s at 95 °C and annealing–elongation for 40 s at 60 °C. The fluorescent signals were detected in real time during the annealing–elongation step. Each assay included five samples of known genotypes representing all combinations of the polymorphisms tested as positive controls (a2a2, a1B, a2B, a1a2, a1a1) and one no-template control (nTC). no sample was found to have the BB genotype. Melting curves shapes were analyzed for each amplicon by the StepOne HRM anal-ysis software (applied Biosystem, Madrid, Spain) and visual inspection. a semiautomatic procedure was implemented to obtain the genotype of each individual, based on both High Resolution Melt Software v3.0.1 (applied Biosystems) and an Excel Macro available on request (Supplementary infor-mation). genotypes of unknown samples were considered reliable only if the results for all reference samples were cor-rect, and nTC gives no result.
Method validation
One individual of each different melting curves shapes, as well as six others randomly chosen, was sequenced. To do so, the twelve individuals were amplified by using primers located the most upstream and downstream in the sequence (primers SnP1-F and SnP2-R in Table 2). Both strands of the amplicon were then sequenced by using the BigDye Terminator v3.1 cycle Sequencing Kit (applied Biosystem, Madrid, Spain).
Table 1 number of samples per breed and Dna source used in this work
Breed/population Whole blood
Frozen meat
Hair follicle
Total
asturiana de los Valles 7 7
avileña 8 8
Berrenda en negro 10 10
Cárdena 10 10
asturiana de la Montaña 3 3
Crossbreed 11 11
Holstein 38 1 4 43
limousin 2
Retinta 10 10
Rubia gallega 2 2
Toro de lidia 10 10
Tudanca 6 6
Total 86 32 4 122
Table 2 Primer sequence as well as amplicon sizes (for each primer pair) for the diagnostics of the two SnP included in this protocol
SnP Primer Sequence 5′–3′ amplicon size (bp)
1 SnP-1 F CTagTCTaTCCCTTCCCTgg 117
R ggagaCTCCCaTTaCTTCaggCTg
2 SnP-2 F CTaaaTaTCCagTTgagCCCTTTaCTg 71
R aaggTgCagaTTTTCaaCaT
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Results and discussion
This method allows the simultaneous identification of the three most common variants of the bovine β-casein, and its accuracy has been demonstrated on a sample of 122
individuals (Table 3). The correlation between the geno-types of 12 sequenced samples and our method was 100 %, then allowing us to use them as positive controls in subse-quent analysis. The obtained aligned melt curves and dif-ference plot curves are shown in Fig. 1. For PCR1, three
Table 3 allelic and genotypic frequencies of the bovine β-casein locus
Breed/Population N a1 a2 B a1/a1 a1/a2 a1/B a2/a2 a2/B
asturiana de los Valles
7 0.57 0.36 0.07 0.43 0.29 0.00 0.14 0.14
avileña 8 0.31 0.69 0 0.25 0.13 0.00 0.63 0.00
Berrendo en negro 10 0.05 0.95 0 0.00 0.10 0.00 0.90 0.00
Cárdena 10 0.15 0.8 0.05 0.00 0.20 0.10 0.70 0.00
asturiana de la Montaña
3 0 0.83 0.17 0.00 0.00 0.00 0.67 0.33
Crossbreed 11 0.55 0.36 0.09 0.45 0.09 0.09 0.27 0.09
Holstein 43 0.34 0.62 0.04 0.09 0.44 0.05 0.37 0.05
limousin 2 0.25 0.5 0.25 0.00 0.00 0.50 0.50 0.00
Retinta 10 0.2 0.75 0.05 0.00 0.40 0.00 0.50 0.10
Rubia gallega 2 0 1 0 0.00 0.00 0.00 1.00 0.00
Toro de lidia 10 0.35 0.65 0 0.20 0.30 0.00 0.50 0.00
Tudanca 6 0.25 0.75 0 0.17 0.17 0.00 0.67 0.00
Total 122 0.25 0.69 0.06 0.13 0.18 0.06 0.57 0.06
Fig. 1 aligned melt curves and difference plot: a In SnP1, three dif-ferent shapes corresponding to three different genotypes are detected in the aligned melt curve (left) and the difference plot (right): blue line: C/C; red line: a/a; gold line: C/a. b In SnP2, two different
shapes corresponding to two different genotypes are detected in the aligned melt curve (left) and the difference plot (right): red line: C/C; blue line: C/g
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possible genotypes were found, corresponding with both homozygous and the heterozygote. For PCR2, only two dif-ferent genotypes were found, due to the absence of the B/B genotype in our sample.
The most frequent allele in the whole database was the a2 (0.69), being the less frequent one the B (0.06). The wild-type a2 allele is the most frequent in all analyzed breed, except for asturiana de los Valles and the cross-breed groups where a1 allele is more frequent one. allele B is the less frequent one in all analyzed populations, being absent in some of them. Regarding genotypes, we found five of the six possible genotypes, being the homozygous a2/a2 the most frequent one in almost all the breeds (with the exception of asturiana de los Valles, Holstein, and the crossbreed groups) and B/B absent (Table 3).
Despite the low number of individuals analyzed in some breeds, this work is the first attempt to estimate the fre-quency and distribution of bovine β-casein in some Iberian beef cattle breeds. Most of these breeds had the wild-type a2 allele in high frequency (Berrendo en negro, Cárdena, Retinta), avileña and Toro de lidia showed a moderate fre-quency of a1 allele, and only in asturiana de los Valles, the a1 allele is the most frequent. This fact can reflect the intro-gression suffered by this breed during the first half of the twentieth century [13] and agrees with Martin-Burriel et al. [14] that consider asturiana de los Valles as an admixed breed. However, other works analyzing a higher number of samples found that also in asturiana de los Valles a2 allele is the most frequent one [7].
The assay described here is a good alternative method for routine typing of a low or middle number of polymor-phisms, as is the case for most frequent β-casein variants, although it is absolutely necessary to include positive con-trols as well as 1 no-template control in every assay. Fur-thermore, the assay is robust enough to be performed with Dna purified from different sources of templates, includ-ing hair roots. allelic variants are detected in real time, and with no post-PCR manipulations, reducing time, costs, and possible carryover contamination.
In conclusion, we have developed and validated a HRM assay for robust, reliable, and reproducible genotyping of bovine β-casein gene in a fast, simple, accurate, and cost-effective way.
Acknowledgments This work was supported by the CEnIT SEnI-FOOD Spanish project. authors thank Rocío Pelayo (Universidad de Córdoba, Spain) and Mª Josefa garcía Espina (SERIDa) for their kind support and help with sampling.
Conflict of interest none.
Compliance with Ethics Requirements This article does not con-tain any studies with human or animal subjects.
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