Method for rapidly detecting allelic genotype of A1 / A2-beta-casein of dairy cow

Abstract

The invention discloses a method for rapidly detecting allelic genotypes of A1 / A2-beta-casein of a dairy cow. The method comprises the following steps: by taking genome DNA of a dairy cow to be detected as a template, carrying out PCR amplification on the genome of the dairy cow to be detected by using a specific primer, adding an electrophoretic indicator 10 * loading Buffer into a PCR product reaction system for dyeing, dyeing a PCR product, carrying out agarose gel electrophoresis with the concentration of 1%, observing a result in a gel imaging system, and photographing, and determining the allelic genotype of the A1 / A2-beta-casein of the dairy cow according to an image fragment in a gel imaging system. Experiments prove that a fragment with the size of 296bp is amplified by adding the A1 primer, which indicates that the sample contains the A1 gene; if a fragment with the size of 296bp is amplified by adding an A2 primer, the sample contains the A2 gene; a fragment with the size of 296bp is amplified through the primer A1, and a fragment with the size of 296bp is amplified through the primer A2, which indicates that the sample contains the genes A1 and A2. The method provided by the invention can provide a detection method which is high in efficiency and simple and convenient to operate for early-stage seed selection and molecular breeding of the dairy cow, can improve the breeding and propagation speed of the A2 beta-casein genotype dairy cow, and has wide application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a detection technology for rapidly detecting the A1 / A2-β-casein allele genotypes of dairy cows. Background Art

[0002] In recent years, with the rapid development of the economic society, the living standards of the vast majority of the people have been continuously improved. Milk is rich in high-quality protein, and the nutrients it provides are easily absorbed and utilized by the human body. However, some studies have found that different types of milk will hydrolyze Beta-Casomorphin-7 (BCM7) during the process of digestion and absorption, and Beta-Casomorphin-7 will exacerbate lactose intolerance, intestinal dysfunction, and gastrointestinal inflammation (NI Xiaoqin, LAI Weihua. Research progress on detection methods of bovine casein [J]. Food Science, 2014, 35(3): 290-294.). The proteins in milk are divided into whey protein and casein, and the content of casein accounts for about 80% of its total amount (KUMAR A, KUMAR S, SINGH R V, et al. Investigation of genetic polymorphism at beta-casein A1 / A2 loci and association analysis with production & reproduction traits in Vrindavani crossbred cows [J]. Anim Biotechnol, 2021, 2021: 1917423.). Casein is divided into beta-casein, alpha1-casein, alpha2-casein, and kappa-casein (G MOATSOU, A HATZINAKI, G P SATHAS, et al. Detection of caprine casein in ovine Halloumi cheese [J]. International Dairy Journal, 2004, 14(3)). Currently, it is known that the 67th amino acid of beta-casein plays a key role in the hydrolysis of milk protein to produce Beta-Casomorphin-7 (KAMINSKI S. Co-occurrence of kappa-casein B and beta-casein A1 alleles in Holstein bulls [J]. Appl Anim Sci, 2021, 37(4): 122-125.). The difference in the A1 and A2-beta-casein allele genotypes of dairy cows leads to a difference in the 67th amino acid of beta-casein in the milk they produce. The milk produced by A1 genotype dairy cows is called A1 milk. The 67th amino acid of beta-casein in A1 milk is histidine, which can produce Beta-Casomorphin-7 after hydrolysis; the milk produced by A2 genotype dairy cows is A2 milk. The 67th amino acid of beta-casein in A2 milk is proline, and no Beta-Casomorphin-7 is produced after hydrolysis.Compared with A1 milk, A2 milk is more beneficial to health, thus stimulating the increasing demand for A2 milk sources in the market. Therefore, differentiating A2 genotype cows by detecting the A1 / A2-β-casein allele genotype through gene detection is currently the most economical and effective method. Therefore, the detection technology of A1 / A2-β-casein allele genotype by this method provides a new rapid detection method for resource-limited areas.

[0003] There are currently many methods for detecting the A1 / A2-β-casein allele genotype. For example, the PCR-RFLP method is widely used to detect the A1 / A2-β-casein allele genotype (PANDEY A, THAKUR M S, PANDEY Y, et al. Polymorphism of beta(beta)casein gene and their association with milk production traits in Malvi and Nimari breeds of cattle[J]. Indian J Anim Res, 2020, 54(5): 647-650.). However, the primers of this method need to have restriction enzyme sites, and it is difficult to find suitable endonucleases. This method takes a long time, has cumbersome operations, and has high technical requirements for testers. Other methods such as TaqMan probe method, rhAmp technology, and HRM analysis method have disadvantages such as high detection cost, high difficulty, low efficiency, and complicated operation process. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for detecting the A1 / A2-β-casein allele genotype with specificity, good repeatability, fewer detection steps, short time consumption, high efficiency, and low detection cost, which can quickly, accurately, and efficiently detect the A1 / A2-β-casein allele genotype.

[0005] In view of the problems mentioned in the background technology, the technical solution adopted by the present invention is as follows:

[0006] ①. DNA template preparation: Obtain the blood of the cow to be detected and extract DNA;

[0007] ②. Specific primer design and synthesis: The primer sequences are as follows:

[0008] Common forward primer F1: 5’-TGAAGAAAGTGGGTTAATGAGAAATCCT-3’

[0009] A1 reverse primer R1: 5’-TTTGTGGGAGGCTGTTAT-3’;

[0010] A2 reverse primer R2: 5’-TTTGTGGGAGGCTGTTAG-3’;

[0011] ③. Establishment of PCR reaction system: Perform PCR amplification on the DNA obtained in step ① and the primers designed in step ②;

[0012] ④. Staining with electrophoresis indicator 10*loading Buffer: Add electrophoresis indicator 10*loading Buffer to the reaction system in step ③ for staining to stain the PCR products.

[0013] ⑤. Result judgment: Through agarose gel electrophoresis with a concentration of 1% (voltage 120V, electrophoresis for 30 min), observe the results and take pictures in a gel imaging system; Amplifying a fragment with a size of 296 bp by adding primer A1 indicates that the sample contains the A1 gene; Amplifying a fragment with a size of 296 bp by adding primer A2 indicates that the sample contains the A2 gene; Amplifying a fragment with a size of 296 bp both by primer A1 and by primer A2 indicates that the sample contains both A1 and A2 genes.

[0014] A method for rapidly detecting the A1 / A2-β-casein allele genotypes of dairy cows, including the collection of dairy cow samples, the preparation of DNA templates, the design and synthesis of specific primers, the establishment of a PCR reaction system, staining with electrophoresis indicator 10*loading Buffer, and result judgment. The specific steps are as follows:

[0015] ①. Collection of dairy cow samples: Randomly collect dairy cow individuals in a dairy farm in Shihezi, Xinjiang. Draw 5 - 10 ml of blood samples from different individuals respectively, place them in cryopreservation tubes, label them, and store them in an environment of -80°C.

[0016] ②. Preparation of DNA templates: Take each of the above - collected samples, use a genomic DNA extraction kit to extract the DNA of the samples, and perform the extraction steps according to the instructions. The extracted DNA is detected by agarose gel electrophoresis to determine its purity, and it is stored in an environment of -20°C;

[0017] ③. Design and synthesis of specific primers: According to relevant research findings, the A1 / A2 allele mutation changes the codons on messenger RNA (mRNA), resulting in differences in the β-casein synthesized after the expression of the A1 gene and the A2 gene at the 67th amino acid. Based on the published cow gene sequence, the primer design software Primer 6.0 was used to design PCR primers. The 3' ends of the specific primers were respectively matched with the bases "T" and "G" for the A1 / A2-β-casein alleles, enabling the normal amplification of the A1 and A2 specific primers on the corresponding A1 and A2 genes, but unable to amplify on the other gene due to the mismatched bases at the 3' end. In other words, for the SNP locus of the A1 / A2 genotype, the last base at the 3' end of the downstream primer is "A" and "C". For dairy cow individuals carrying the A1 gene, it will be normally amplified with the base "T" matching the A1-β-casein allele; conversely, there is a mismatch between the specific primer and the gene of wild-type dairy cow individuals and it cannot be amplified. Therefore, the specific primers designed for the mutant SNP only amplify the matching fragments and do not amplify the mismatched fragments. The primer sequences are as follows:

[0018] Common forward primer F1: 5’-TGAAGAAAGTGGGTTAATGAGAAATCCT-3’

[0019] A1 reverse primer R1: 5’-TTTGTGGGAGGCTGTTAT-3’;

[0020] A2 reverse primer R2: 5’-TTTGTGGGAGGCTGTTAG-3’;

[0021] The above primers are of appropriate length, and it is difficult to form dimers and hairpin structures between primers. They do not have complementary sequences within themselves and are difficult to form hairpin structures on their own. Moreover, the primer initiation efficiency at the mismatch site is extremely low, and they can be tightly complementary to the template sequence, with high amplification efficiency and high sensitivity;

[0022] ④. Establishment of the PCR reaction system: Using the prepared DNA as a template for PCR amplification, the total volume of each PCR reaction is 20 μL, containing 0.1 μL of TaKaRa Ex Taq (5 U / μl), 2 μL of 10×PCR Buffer (Mg 2+ free), 1.2 μL of MgCl2 (25 mM), 1.6 μL of dNTP Mixture (2.5 mM each), 0.5 μL of each 0.1 nM primer, 13.1 μL of ddH2O, and 1 μL of DNA template. PCR reaction parameters: Pre-denaturation at 95°C for 4 min, denaturation at 98°C for 10 s, annealing at 54°C for 10 s, extension at 72°C for 20 s, 35 cycles, and finally extension at 72°C for 5 min;

[0023] ⑤. Electrophoresis indicator 10×loading Buffer staining: After the PCR reaction is completed, add loading Buffer electrophoresis indicator with a final concentration of 10× to the reaction system to stain the PCR products. After mixing evenly, immediately perform agarose gel electrophoresis with a concentration of 1%;

[0024] ⑥. Result judgment: Perform agarose gel electrophoresis with a concentration of 1% (voltage 120V, electrophoresis for 30 min), observe the results and take pictures in the gel imaging system; if a fragment with a size of 296 bp is amplified by adding primer A1, it indicates that the sample contains the A1 gene; if a fragment with a size of 296 bp is amplified by adding primer A2, it indicates that the sample contains the A2 gene; if a fragment with a size of 296 bp is amplified both by primer A1 and primer A2, it indicates that the sample contains both A1 and A2 genes.

[0025] Compared with the prior art, the detection method of the present invention has good specificity and repeatability, fewer detection steps, short time consumption, high efficiency, low detection cost, and the detection results conform to the "gold standard" of Sanger sequencing for detecting the A1 / A2-β-casein allele genotypes in dairy cows. However, this method has low detection cost, is simple and fast, and has high application value and promotion prospects.

[0026] Preferably, allele-specific PCR (AS-PCR) can obtain results within 2 hours. Therefore, compared with other methods, it has the characteristics of accuracy, specificity, simplicity, and rapidity. First, different specific primers are designed using SNPs sites to distinguish different genotype detection methods. Specific primers are designed at the mutation sites of the target fragment. The 3' ends of the specific primers are respectively matched with the bases "T" and "G" for the A1 / A2-β-casein alleles, so that the A1 and A2 specific primers can be normally amplified on the corresponding A1 and A2 genes, but cannot be amplified on the other gene due to the effect of the mismatched base at the 3' end. By designing artificial mismatched primers, the specificity and sensitivity of the detection are improved, the demand for instrument equipment is reduced, the cost is low, and it is easy to operate, providing a new rapid detection method for resource-limited areas.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] 1) The detection method of the present invention has good specificity and repeatability, fewer detection steps, short time consumption, high efficiency, low detection cost, can quickly and efficiently detect the A1 and A2 genes, and has high application value and promotion prospects;

[0029] 2) The detection method of the present invention designs primers using the artificial mismatch method. The 3' ends of the specific primers are respectively matched with the bases "T" and "G" for the A1 / A2-β-casein alleles, enabling the A1 and A2 specific primers to amplify normally on the corresponding A1 and A2 genes, but unable to amplify on the other gene due to the effect of the mismatched bases at the 3' end, thus improving the specificity of amplification.

[0030] 3) The detection method of the present invention stains the PCR amplification products with the electrophoresis indicator 10*loading Buffer. After adding the dye, it can be immediately subjected to 1% agarose gel electrophoresis (voltage 120V, electrophoresis for 30 min), and the results are observed and photographed in the gel imaging system; when the A1 primer is added and a fragment of 296 bp in size is amplified, it indicates that the sample contains the A1 gene; when the A2 primer is added and a fragment of 296 bp in size is amplified, it indicates that the sample contains the A2 gene; when a fragment of 296 bp in size is amplified both by the A1 primer and the A2 primer, it indicates that the sample contains both the A1 and A2 genes. Brief Description of the Drawings

[0031] Figure 1 、 2 is a test picture obtained by using the method of the present invention to detect the A1 and A2 gene samples of dairy cows, that is, the detection of the accuracy of the method of the present invention.

[0032] Figure 3 is a test picture obtained by using the method of the present invention to detect the gradient-diluted dairy cow genomic samples, that is, the detection of the sensitivity of the method of the present invention. The sensitivity of the method of the present invention is 0.05 ng / μL.

[0033] Figure 4 、 5 、6 is the sequencing peak map obtained by sending the PCR products detected by the method of the present invention to Sangon Biotech Co., Ltd. to further verify the accuracy of the method of the present invention.

[0034] Note: Figure 1 、 2 is the result map of the amplification product using the specific primer in Figure 1 M: 100 bp maker; 01, 02: dairy cow No. 31; 03, 04: dairy cow No. 15. Among them, dairy cow No. 31 is of A1 type and dairy cow No. 15 is of A2 type. Figure 2M: 100 bpmaker; 01, 02: Dairy cow No. 02; 03, 04: Dairy cow No. 06; 05, 06: Dairy cow No. 07; 07, 08: Dairy cow No. 15; 09, 10: Dairy cow No. 28; 11, 12: Dairy cow No. 31. Among them, dairy cows No. 02, 06, 07, and 28 are of A1A2 type, and dairy cow No. 15 is of A2 type. Figure 3 Sensitivity detection result graph of the amplified product using specific primers, where 1, 2, 3, 4, 5 are 50 ng / μL, 5 ng / μL, 0.5 ng / μL, 0.05 ng / μL, 0.005 ng / μL respectively.

[0035] Figure 4 , 5 , 6 is the sequencing peak graph of Sangon Biotech Co., Ltd., where Figure 4 is dairy cow No. 06, Figure 5 is dairy cow No. 15, Figure 6 is dairy cow No. 31. Specific implementation mode

[0036] The present invention will be further described in detail below in combination with specific implementation modes. The given examples are only for clarifying the present invention and not for limiting the scope of the present invention.

[0037] Based on the dairy cow gene sequence, the inventor designed specific primers for this gene and combined this PCR method with agarose gel electrophoresis to quickly and accurately detect the A1 / A2-β-casein allele genotypes of dairy cows, and completed the invention on this basis.

[0038] A method for quickly detecting the A1 / A2-β-casein allele genotypes of dairy cows, comprising the following steps:

[0039] 1. Collection of dairy cow samples and preparation of DNA templates

[0040] Randomly collect dairy cow individuals in a dairy farm in Shihezi, Xinjiang. Draw 5 - 10 ml blood samples from different individuals respectively, place them in anticoagulation tubes and label them, and then store them in an environment of -20°C; use a genomic DNA extraction kit to extract the DNA of the samples, and the extraction steps are carried out according to the instructions. The extracted DNA is detected by agarose gel electrophoresis to determine its purity, and then stored in an environment of -20°C.

[0041] 2. Design and synthesis of specific primers

[0042] According to relevant research findings, the A1 / A2 allele gene mutation changes the codons on messenger RNA (mRNA), resulting in differences in the 67th amino acid of β-casein synthesized after the expression of the A1 gene and the A2 gene. Based on the published dairy cow gene sequence, the primer design software Primer 6.0 was used to design PCR primers. The 3' ends of the specific primers were respectively matched with the bases "T" and "G" for the A1 / A2-β-casein allele genes, enabling the normal amplification of the A1 and A2 specific primers on the corresponding A1 and A2 genes, but unable to amplify on the other gene due to the effect of the mismatched bases at the 3' end. In other words, for the SNP locus of the A1 / A2 genotype, the last base at the 3' end of the downstream primer is "A" and "C". For dairy cow individuals carrying the A1 gene, it will be normally amplified with the base "T" matching the A1-β-casein allele gene; conversely, there is a mismatch between the specific primer and the gene of the wild-type dairy cow individual and it cannot be amplified. Therefore, the specific primers designed for the mutant SNP only amplify the matching fragments and do not amplify the mismatched fragments. The primer sequences are as follows:

[0043] Common forward primer F1: 5’-TGAAGAAAGTGGGTTAATGAGAAATCCT-3’

[0044] A1 reverse primer R1: 5’-TTTGTGGGAGGCTGTTAT-3’;

[0045] A2 reverse primer R2: 5’-TTTGTGGGAGGCTGTTAG-3’;

[0046] The above primers are of appropriate length, and it is difficult to form dimers and hairpin structures between the primers. They do not have complementary sequences within themselves and are difficult to form hairpin structures by themselves. Moreover, the primer initiation efficiency at the mismatched site is extremely low, and they can be closely complementary to the template sequence, with high amplification efficiency and high sensitivity.

[0047] 3. Establishment of the PCR reaction system: Using the prepared DNA as a template for PCR amplification, the total volume of each PCR reaction is 20 μL, containing 0.1 μL of TaKaRa Ex Taq (5 U / μl), 2 μL of 10×PCR Buffer (Mg 2+ free), 1.2 μL of MgCl2 (25 mM), 1.6 μL of dNTP Mixture (2.5 mM each), 0.5 μL of each 0.1 nM primer, 13.1 μL of ddH2O, and 1 μL of DNA template. PCR reaction parameters: pre-denaturation at 95°C for 4 min, denaturation at 98°C for 10 s, annealing at 54°C for 10 s, extension at 72°C for 20 s, 35 cycles, and finally extension at 72°C for 5 min;

[0048] 4. Electrophoresis indicator staining and result judgment

[0049] ①. Staining with electrophoresis indicator 10× loading Buffer: Add the loading Buffer nucleic acid dye with a final concentration of 10× to the reaction product to stain the PCR product.

[0050] ②. Result judgment

[0051] Perform agarose gel electrophoresis with a concentration of 1% (voltage 120V, electrophoresis for 30 min), observe the results in a gel imaging system and take pictures; if a fragment with a size of 296 bp is amplified by adding primer A1, it indicates that the sample contains the A1 gene; if a fragment with a size of 296 bp is amplified by adding primer A2, it indicates that the sample contains the A2 gene; if a fragment with a size of 296 bp is amplified both by primer A1 and primer A2, it indicates that the sample contains both A1 and A2 genes. This method has good specificity and repeatability, high sensitivity and accuracy, low detection cost, is simple and rapid, and has high application value and popularization prospect.

[0052] Detection of the accuracy and sensitivity of the method of the present invention

[0053] ①. Detection of the accuracy of the method of the present invention

[0054] Send the above samples to Sangon Biotech Co., Ltd. for sequencing to verify the results of gel electrophoresis. The results show that the results of gel electrophoresis verification and sequencing verification are completely consistent, and the accuracy rate of this detection method is 100%.

[0055] ②. Detection of the sensitivity of the method of the present invention

[0056] Select the DNA of the sequenced verified dairy cow A1 / A2-β-casein allele genotype samples, use specific primers to amplify the gradient-diluted DNA template, and detect the sensitivity of this method. The results show that this method can detect a concentration of 0.05 ng / μL.

[0057] In the design and synthesis steps of the above specific primers, allele-specific PCR (AS-PCR) can obtain results within 2 hours. Therefore, compared with other methods, it has the characteristics of accuracy, specificity, simplicity and rapidity. First, different specific primers are designed using SNPs sites to distinguish different genotype detection methods. Specific primers are designed at the mutation sites of the target fragment. The 3' ends of the specific primers are respectively matched with the bases "T" and "G" for the A1 / A2-β-casein alleles, enabling the A1 and A2 specific primers to amplify normally on the corresponding A1 and A2 genes, but unable to amplify on the other gene due to the mismatch of the 3' end bases.

[0058] The embodiments described above have elaborated on the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the present invention. Any modifications, supplements, or substitutions in a similar manner within the principle scope of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for rapid detection of A1 / A2-β-casein allele genotypes in dairy cows, comprising the following steps: ①. DNA template preparation: Obtain the blood of the cow to be detected and extract DNA; ②. Specific primer design and synthesis: The primer sequences are respectively: Common forward primer F1: 5’-TGAAGAAAGTGGGTTAATGAGAAATCCT-3’ A1 reverse primer R1: 5’-TTTGTGGGAGGCTGTTAT-3’; A2 reverse primer R2: 5’-TTTGTGGGAGGCTGTTAG-3’; ③. Establishment of PCR reaction system: Place the DNA sample obtained in step ① into two PCR tubes and perform PCR amplification with the common primer and specific primers designed in step ②; ④. Staining with electrophoresis indicator 10*loading Buffer: Add electrophoresis indicator 10*loading Buffer to the reaction system in step ③ for staining the PCR products; ⑤. Perform agarose gel electrophoresis with a concentration of 1% (voltage 120V, electrophoresis for 30 min), observe the results and take pictures in the gel imaging system; Amplification of a fragment with a size of 296 bp by adding the A1 primer indicates that the sample contains the A1 gene; Amplification of a fragment with a size of 296 bp by adding the A2 primer indicates that the sample contains the A2 gene; Amplification of a fragment with a size of 296 bp by both the A1 primer and the A2 primer indicates that the sample contains both the A1 and A2 genes.

2. The method for rapidly detecting the A1 / A2-β-casein allele genotype of dairy cows according to claim 1, wherein: Collect dairy cow blood samples, place the sampled samples in anticoagulant tubes, label them, and store them in an environment at -20°C. Use a genomic DNA extraction kit to extract the DNA of the sampled samples. The extracted DNA is subjected to agarose gel electrophoresis to determine its purity and is stored in an environment at -20°C.

3. The method for rapidly detecting the A1 / A2-β-casein allele genotype of dairy cows according to claim 1, wherein: Using the prepared DNA as a template for PCR amplification, the total volume of each PCR reaction was 20 μL, containing 0.1 μL of TaKaRa Ex Taq (5 U / μL), 2 μL of 10×PCR Buffer (Mg 2+ free), 1.2 μL of MgCl2 (25 mM), 1.6 μL of dNTP Mixture (2.5 mM each), 0.5 μL of each 0.1 nM primer, 13.1 μL of ddH2O, and 1 μL of DNA template. The PCR reaction parameters were: pre-denaturation at 95°C for 4 min, denaturation at 98°C for 10 s, annealing at 54°C for 10 s, extension at 72°C for 20 s, 35 cycles, and finally extension at 72°C for 5 min.

4. A method for rapidly detecting the A1 / A2-β-casein allele genotype of dairy cows according to claim 1, characterized in that: Add loading Buffer electrophoresis indicator with a final concentration of 10× to the reaction system.

5. A method for rapidly detecting the A1 / A2-β-casein allele genotype of dairy cows according to claim 1, characterized in that: Perform agarose gel electrophoresis with a concentration of 1% (voltage 120V, electrophoresis for 30 min), observe the results and take pictures in the gel imaging system; Amplification of a fragment with a size of 296 bp by adding the A1 primer indicates that the sample contains the A1 gene; Amplification of a fragment with a size of 296 bp by adding the A2 primer indicates that the sample contains the A2 gene; Amplification of a fragment with a size of 296 bp by both the A1 primer and the A2 primer indicates that the sample contains both the A1 and A2 genes.

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