«ALG-CASEIN A1/A2 MAX» reagent kit
β‑casein is a protein consisting of a chain of 224 amino acids, which accounts for about 30% of all milk proteins. There are two major variants of β‑casein: A1 and A2. Variant A1 differs from A2 by only one amino acid. As a result, dairy products containing A1 or A2 β‑casein are digested differently. As a result of β‑casein metabolism «opioid peptide» BCM‑7 appears and about 25% of people are sensitive to this component. Some people who believe they have milk intolerance may be sensitive to A1 β‑casein. Consumption of milk with A2 β‑casein reduces acute gastrointestinal symptoms of milk intolerance, while regular milk with A1 β‑casein reduces lactase activity and enhances gastrointestinal symptoms.
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The formation of a genetically safe group of highly productive dairy cattle herd is one of the successful approaches in modern animal husbandry to increase the productivity and A2 milk yield without A1 impurities. This approach will not only guarantee the “healthy” A2 milk production, but also completely displace and eliminate the production of A1 milk within 10 years.
There are 3 types of cows
A1/A2 status is determined in cows and bulls by a pair of genes on the 6th chromosome, namely, polymorphic variants of the β‑casein gene β‑CNA1 and β‑CNA2, respectively. Each cow and bull carries two copies of this gene, and two main variants are distinguished – the A1 and A2 β‑casein alleles. Regardless of the breed, a herd of cattle is formed by three types of animals: A2A2 homozygous genotype, A1A1 homozygous genotype and A1A2 heterozygous genotype.
Figures 1 and 2 show the frequency of alleles and genotypes occurrence in various breeds.
To form a prosperous group of high-yielding dairy herd of cattle, it is advisable to select and sort animals and to form a separate herd of only homozygous type A2A2 cows and bulls, which give only A2 milk, gradually increasing the number of such animals and completely excluding the production of A1 milk.
At the current stage, expensive sequencing methods or time-consuming approaches using gel electrophoresis are mainly used for genotyping and certification of cattle. Unlike other cow genotyping technologies based on β‑casein variants, real-time PCR analysis is the fastest, most convenient and affordable method that allows sensitive and specific detection of A1 and/or A2 DNA in samples. This approach is implemented in the «ALG-CASEIN A1/A2 MAX».
«ALG-CASEIN A1/A2 MAX» – complete reagent kit, includes reagents for all stages of the study: samples prelysis, DNA extraction and polymerase chain reaction (PCR). The reagent kit is designed to study 96 samples.
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We express our gratitude for the joint development and validation of studies on animal samples to the employees of the Center for Genetics and Reproduction (a branch of Algimed Techno LLC). Services for genotyping of cattle to identify A1 and A2 animals can be ordered on the website – https://labgen.by/.
- Low cost;
- A turnkey solution that includes reagents for all stages of PCR analysis;
- Time input reduction (automation of DNA extraction stage);
- Fast, reliable and highly sensitive result;
- Easy result analysis;
- Analysis of A1 and A2 alleles percentage in whole milk.
The test principle is based on DNA extraction from material samples and subsequent amplification of a specific DNA site with allele-specific probes. The accumulation of specific amplification products is recorded by measuring the intensity of the fluorescent signal in real time using the FAM and HEX channels in real time.
The ALG-CASEIN A1/A2 MAX reagent kit includes all the necessary components for real-time PCR: positive, negative and internal control, water, buffers and enzymes.
- CASMAX‑1
- CASMAX‑2
ALG-CASEIN A1/A2 MAX reagent kit (release form 1, 96 reactions):
Part 1
- Magnetic Sorbent 1,1 ml × 2 pcs;
- Prelysis Buffer 52,5 ml × 1 pcs;
- Sorbing Buffer 21 ml × 1 pcs;
- Lysing Buffer 44 ml × 1 pcs;
- Wash Buffer No. 1 50 ml × 1 pcs;
- Wash Buffer No. 2 50 ml × 1 pcs;
- Elution Buffer 12 ml × 1 pcs;
- Dilution Buffer 12 ml × 1 pcs.
Part 2
- А1А2 Casein PCR Buffer (2х) 1,25 ml × 1 pcs;
- PCR Water 1,25 ml × 1 pcs;
- А1А1 Positive Control Sample 100 μl × 1 pcs;
- А1А2 Positive Control Sample 100 μl × 1 pcs;
- А2А2 Positive Control Sample 100 μl × 1 pcs;
- Negative Control Sample 100 μl × 1 pcs.
ALG-CASEIN A1/A2 MAX reagent kit (release form 2, 96 reactions):
Part 1
- Magnetic Sorbent 1,1 ml × 1 pcs;
- Prelysis Buffer 52,5 ml × 1 pcs;
- Sorbing Buffer 21 ml × 1 pcs;
- Lysing Buffer 96 x 0,4 ml × 1 pcs;
- Wash Buffer No. 1 96 x 0,7 ml × 1 pcs;
- Wash Buffer No. 2 96 x 0,7 ml × 1 pcs;
- Elution Buffer 96 x 0,1 ml × 1 pcs;
- Dilution Buffer 12 ml × 1 pcs.
Part 2
- А1А2 Casein PCR Buffer (2х) 1,25 ml × 1 pcs;
- PCR Water 1,25 ml × 1 pcs;
- А1А1 Positive Control Sample 100 μl × 1 pcs;
- А1А2 Positive Control Sample 100 μl × 1 pcs;
- А2А2 Positive Control Sample 100 μl × 1 pcs;
- Negative Control Sample 250 μl × 1 pcs.
Research material
- Bos taurus tissue samples (blood, cartilage tissue);
- Whole milk;
- Bos taurus semen.
Time of the research
- DNA extraction: from 30 minutes;
- PCR analysis:
- time for manual manipulations – 5 – 15 min;
- PCR protocol time – 71 min;
- total time for analysis – 90 min.
Detection threshold:
- 7,5 genomic copies per reaction;
- detection of up to 1 individual carrier of the A1 allele out of 50 in pooled milk samples.
Shelf life: 12 months.