Primer and probe combination, kit, method for identifying A1 and A2 type beta-casein genotype dairy cows and application of A1 and A2 type beta-casein genotype dairy cows

Abstract

The invention relates to a primer and probe combination, a kit, a method for identifying A1 and A2 type beta-casein genotype dairy cows and application of the A1 and A2 type beta-casein genotype dairy cows. In the combination of the primer and the probe, the sequence of the primer is as follows: a forward primer: 5 '-gcaccaccacagggtgga-3'; the reverse primer is 5 '-CTTGCCCACACAGTCTCT-3', and the reverse primer is 5 '-CTTGCCCACACAGTCTT-3'; the probe comprises an A1 type probe and an A2 type probe, the sequence of the A1 type probe is as shown in SEQ ID NO: 3, and the sequence of the A2 type probe is as shown in SEQ ID NO: 4. The dual fluorescent PCR technology is adopted, dual fluorescent PCR detection is conducted on the beta-casein CNS2 gene segment, efficient and accurate identification of A1 type beta-casein cows and A2 type beta-casein cows can be achieved, and the method is suitable for the fields of cow breeding and dairy product detection.

Description

Technical Field

[0001] This invention relates to primer and probe combinations, kits, and methods and applications for identifying A1 and A2 type β-casein genotypes in dairy cows, belonging to the field of biotechnology. Background Technology

[0002] Milk is an important source of protein for humans, and producing high-quality dairy products is the future direction of the dairy industry. In recent years, A1 and A2 types of β-casein in dairy products have gradually attracted significant attention from producers, breeding companies, researchers, and consumers, giving rise to new trends in the dairy market. Dairy producers in many countries worldwide have begun producing healthier A2-type milk, which has gained market favor. Efficient, rapid, and accurate methods for detecting A1 and A2 β-casein genotypes in dairy cows are fundamental to the targeted breeding of high-quality dairy cattle breeds and are crucial for the sustainable and healthy development of the dairy industry.

[0003] Milk proteins are mainly casein and whey protein. Casein accounts for about 80% of milk protein and includes four types: αS1, αS2, β-casein, and κ-casein. β-casein accounts for about 30% of the total protein, and the gene encoding bovine β-casein is CSN2. Early dairy cows contained only A2 β-casein and are considered the natural prototype of β-casein. Through long-term genetic transmission, crossbreeding with different dairy breeds, and improved breeding, there are now 13 genetic variants of casein, including A1, A2, A3, A4, B, C, D, E, F, H1, H2, I, and G. Among them, the most common in cattle are the A1 and A2 variants, and milk comes from three main genotypes: A1, A1A2, and A2. The difference between the A1 and A2 alleles lies in the mutation at amino acid position 67 (proline for A2 and histidine for A1). Histidine (A1 variant) causes the first 7 amino acid residues to break, releasing the biologically active β-tyrosine-7 (βCM-7), while the A2 variant does not produce βCM-7. In 2021, Wang Dan et al., in their study "Genetic Polymorphism of Bovine Milk β-Casein and A2 Type Dairy Products" published in the journal *China Dairy Industry*, suggested that βCM-7 may be associated with the occurrence of non-communicable diseases in some infants, such as type 1 diabetes, respiratory dysfunction, cardiovascular disease, digestive system diseases, immune dysfunction, schizophrenia, autism, and sudden infant death syndrome. Furthermore, A2 type dairy cows not only produce milk that is more beneficial to health, but also have higher milk yield, protein content, and lower fat percentage. Therefore, targeted breeding and establishment of A2 type dairy cow populations is a direction for the development of the dairy industry. Currently, methods for identifying and detecting A1 and A2 types include PCR, PCR-RFLP, multiplex PCR, sequencing, and electrophoresis. Chinese invention patent applications CN105925717A, CN105219839A, and CN105018582A disclose methods for detecting β-casein genotypes. However, these methods require prior PCR amplification using multiplex PCR primers or specific primers, restriction enzyme digestion of the amplified fragments, and observation of bands via agarose gel electrophoresis or sequencing. These methods suffer from high costs, high false-positive rates, long processing times, and the need for subsequent sequencing confirmation. Therefore, developing an accurate method for identifying and detecting the A1 and A2 β-casein genotypes in dairy cows is of great significance for the targeted breeding of dairy cows that possess both high yields and a genetic basis exclusively for A2-type β-casein. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide a primer and probe combination, a kit, and a method and application for identifying A1 and A2 type β-casein genotypes in dairy cows, so as to achieve accurate identification of A1 and A2 type β-casein genotypes.

[0005] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: Primer and probe combinations used to identify A1 and A2 β-casein genotype dairy cows, wherein the primer sequences are as follows: Forward primer: 5'-ggcaccaccacaggggtttgag-3' (SEQ ID NO:1); Reverse primer: 5'-CTTTGCCCAGACACAGTCTCT-3' (SEQ ID NO:2); The probes include an A1-type probe (a probe specific to A1 type) and an A2-type probe (a probe specific to A2 type), the sequence of the A1-type probe is shown in SEQ ID NO:3, and the sequence of the A2-type probe is shown in SEQ ID NO:4.

[0006] The sequence of SEQ ID NO:3 is: 5'-(HEX)cCaTcC(A)tAaCagcCT(BHQ1)-3'; the sequence of SEQ ID NO:4 is: 5'-(FAM)gCcCaTCc(C)TaAcAg(BHQ1)-3'. In these sequences, uppercase letters indicate nucleotides modified with locked nucleic acids (LNA), and lowercase letters indicate unmodified nucleotides. Nucleotides A and C in parentheses are complementary to T and G at the SNP sites of the CNS2 gene, respectively.

[0007] The A1 type probe has a 5' end labeled with the fluorescent reporter group HEX and a 3' end labeled with the quencher group BHQ1; the A2 type probe has a 5' end labeled with the fluorescent reporter group FAM and a 3' end labeled with the quencher group BHQ1.

[0008] Based on the same inventive concept, the present invention also provides: a kit comprising a PCR reaction solution, a primer-probe mixture, a positive control, and a negative control, wherein the primer-probe mixture comprises the primer and probe combination as described above.

[0009] Optionally, the PCR reaction solution is THUNDERBIRD Probe qPCR Mix.

[0010] Optionally, the positive control includes a plasmid vector containing the sequence shown in SEQ ID NO:5 and a plasmid vector containing the sequence shown in SEQ ID NO:6, and the negative control is TE-Buffer.

[0011] The sequence of SEQ ID NO:5 is: ggcaccaccacaggggtttgagtaagaggagggatgttttgtgggaggCTgttaTggatgggcccagggaagggatagaCTagagaCTgtgtCTgggcaaag.

[0012] The sequence of SEQ ID NO:6 is: ggcaccaccacaggggtttgagtaagaggagggatgttttgtgggaggCTgttaGggatgggcccagggaagggatagaCTagagaCTgtgtCTgggcaaag.

[0013] The kit of this invention uses dual fluorescent PCR technology to detect the β-casein CNS2 gene fragment using dual fluorescent PCR, which can achieve simple, rapid, efficient and accurate identification of A1 and A2 type β-casein dairy cows, and can be applied to the fields of dairy cow breeding and dairy product testing.

[0014] Based on the same inventive concept, this invention also provides: a method for identifying A1 and A2 type β-casein genotype dairy cows, comprising the following steps: S1. Extract DNA from the cow to be identified to obtain sample DNA; S2. Perform fluorescent PCR amplification on the sample DNA obtained in S1 using the kit described above; S3. Obtain and analyze the results.

[0015] Optionally, in S1, DNA is extracted from one or more of the following: milk, blood, and hair follicle roots of the cow to be identified.

[0016] Optionally, in S2, the conditions for the fluorescent PCR amplification cycle reaction are: 95℃ pre-denaturation for 60s; 95℃ denaturation for 10s; 60℃ annealing for 40s; and collection of FAM and HEX fluorescence signals at 60℃, wherein denaturation and annealing are performed for 45 cycles.

[0017] Optionally, in S2, the reaction system for fluorescent PCR amplification is as follows: take 12.5 μL of PCR reaction solution and 7.5 μL of primer-probe mixture, vortex to mix, centrifuge briefly, take 20 μL of the above mixture and place it in a PCR reaction tube, then take 5 μL of sample DNA, 5 μL of negative control and 5 μL of positive control and add them to different PCR reaction tubes, cap the tubes, centrifuge briefly, and immediately perform PCR amplification cycle reaction.

[0018] Optionally, in S3, if the positive control shows two typical S-shaped amplification curves (FAM, HEX) and the CT value is ≤30, and the negative control shows no amplification curve and no CT value, then the test is valid; if the sample DNA's FAM fluorescence channel shows a typical amplification curve and the CT value is <40, while the sample DNA's HEX fluorescence channel shows no amplification curve and the CT value is >45, then the cow's β-casein genotype is A2; if the sample DNA's HEX fluorescence channel shows a typical amplification curve and the CT value is <40, while the sample DNA's FAM fluorescence channel shows no amplification curve and the CT value is >45, then the cow's β-casein genotype is A1; if both the sample DNA's FAM and HEX fluorescence channels show typical amplification curves and the CT value is <40, then the cow's β-casein genotype is A1A2 heterozygous.

[0019] The kit described above is used in the identification of A1 and A2 type β-casein dairy cows, dairy cow genetic breeding, dairy product safety testing, and / or the development of functional dairy products.

[0020] This invention designs primers and probes for the CSN2 genotype encoding A1 and A2 β-casein, and finally selects a pair of primers and probes with excellent performance for use in this invention.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention targets the conserved fragment of the gene CSN2 that produces β-casein, and designs a common primer for A1 and A2. Combined with A1 and A2 specific probes, it can avoid cross-reaction and has the characteristics of high specificity and high sensitivity. It can also detect a large number of samples at the same time, providing a new identification and detection technology for dairy cow genetic breeding, dairy product safety testing and functional dairy product development.

[0022] (2) The present invention also has the advantages of being fast and efficient, and can complete the detection and obtain the results within 2 hours; the identification cost is low and simple, and no sequencing or complex equipment is required; dual-channel detection is adopted, and synchronous identification and detection are achieved through HEX (A1) and FAM (A2) fluorescence signals. Attached Figure Description

[0023] Figure 1 This is the amplification curve of the β-casein A1 gene detected by fluorescent PCR (a typical amplification curve appears in the HEX fluorescent channel).

[0024] Figure 2 This is the amplification curve of the β-casein A2 gene detected by fluorescent PCR (typical amplification curve appears in the FAM fluorescent channel).

[0025] Figure 3The amplification curves for the β-casein A1A2 heterozygous fluorescent PCR detection are shown (typical amplification curves are observed in both the HEX and FAM fluorescent channels).

[0026] Figure 4 This is the standard curve for A1 type fluorescent PCR detection.

[0027] Figure 5 This is the standard curve for A2 type fluorescent PCR detection. Detailed Implementation

[0028] The present invention will be described in detail below with reference to embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its content. Experimental methods in the following embodiments without specific conditions are generally performed under conventional conditions or according to the conditions recommended by the reagent manufacturer; the materials and reagents used in the following embodiments are commercially available unless otherwise specified; the primers and probes used are synthesized by a biotechnology company.

[0029] Example 1: Establishment of a dual-fluorescent PCR detection method for A1 and A2 type β-casein in dairy cows 1. Primer and probe design Based on the bovine β-casein encoding gene (CSN2) sequence published in NCBI GenBank, this invention uses PrimerExpress 5.0 software for sequence analysis and alignment, selects different sites to design primers and probes, and after analysis and pairing experiments, obtains the common primer pair of SEQ ID No. 1 and SEQ ID No. 2, as well as the A1 type probe SEQ ID No. 3 and the A2 type probe SEQ ID No. 4.

[0030] Primers were designed based on a 102 bp nucleotide sequence from the bovine β-casein encoding gene (CSN2 gene): Forward primer: 5′-ggcaccaccacaggggtttgag-3′ (SEQ ID No. 1); Reverse primer: 5′-CTTTGCCCAGACACAGTCTCT-3′ (SEQ ID No. 2).

[0031] The dual-labeled LNA hybridization probe is designed as follows, complementary to the reverse DNA strand and spanning a mutation site (SNP): A1 probe: 5'-(HEX)cCaTcC(A)tAaCagcCT(BHQ1)-3', 5' end labeled with the fluorescent reporter group HEX, 3' end labeled with the quencher group BHQ1; A2 probe: 5′-(FAM)gCcCaTCc(C)TaAcAg (BHQ1)-3′, with the 5' end labeled with the fluorescent reporter group FAM and the 3' end labeled with the quencher group BHQ1.

[0032] To distinguish between A1 and A2 type genes during fluorescent PCR amplification, locked nucleic acid (LNA) nucleotides are used to improve probe binding specificity and stability and to accurately differentiate between A1 and A2. Uppercase letters in the sequence represent locked nucleic acid chemical modifications; lowercase letters represent ordinary DNA nucleotides; and nucleotides in parentheses represent nucleotides labeled as complementary to the target SNP.

[0033] The A1 amplification target gene fragment sequence (SEQ ID NO:5) information is as follows: ggcaccaccacaggggtttgagtaagaggagggatgttttgtggggggCTgttaTggatgggcccagggaagggatagaCTagagaCTgtgtCTgggcaaag (SEQ ID NO: 5).

[0034] The A2 amplification target gene fragment sequence (SEQ ID NO:6) information is as follows: ggcaccaccacaggggtttgagtaagaggagggatgttttgtggggggCTgttaGggatgggcccagggaagggatagaCTagagaCTgtgtCTgggcaaag (SEQ ID NO: 6).

[0035] Following conventional methods for constructing recombinant plasmids, recombinant plasmid vector pUC57-CSN2-A1 containing an A1-type amplified target gene fragment sequence (SEQ ID NO:5) and recombinant plasmid vector pUC57-CSN2-A2 containing an A2-type amplified target gene fragment sequence (SEQ ID NO:6) were constructed.

[0036] 2. Optimization of detection method conditions Optimization of the fluorescent PCR reaction system and conditions mainly includes optimization of the reaction program and the reaction system itself. The reaction program includes the number of cycles and annealing temperature. Optimization of the reaction system mainly includes optimization of the final concentrations of primers and probes, and optimization of template concentration. Using pUC57-CSN2-A1 and pUC57-CSN2-A2 plasmid DNA as templates, the matrix method was used to determine the concentrations of primers and probes in the dual fluorescent PCR reaction system, as well as the optimal reaction conditions.

[0037] Using the optimized reaction program, recombinant plasmid DNA from pUC57-CSN2-A1 and pUC57-CSN2-A2 was used as template for fluorescent PCR. The final probe concentration was fixed at 0.25 μmol / L. Primer concentrations of 0.30 μmol / L, 0.35 μmol / L, 0.40 μmol / L, 0.45 μmol / L, and 0.50 μmol / L were used for pairing experiments. The primer concentration with the lowest average CT value between the two channels was selected as the most suitable concentration (see Table 1). Experimental results showed that the most suitable final primer concentration was 0.40 μmol / L, at which point the amplification efficiency was highest.

[0038] Table 1. Average CT values ​​of dual channels under different primer concentrations.

[0039] Using the optimized reaction program, recombinant plasmid DNA from pUC57-CSN2-A1 and pUC57-CSN2-A2 was used as template for fluorescent PCR. The final primer concentration was fixed at 0.40 μmol / L, and probe concentrations of 0.10 μmol / L, 0.15 μmol / L, 0.20 μmol / L, 0.25 μmol / L, and 0.30 μmol / L were used for pairing experiments. The probe concentration with the lowest average CT value between the two channels was selected as the most suitable concentration (see Table 2). The experimental results showed that the amplification efficiency was highest when the probe concentration was 0.20 μmol / L.

[0040] Table 2. Results of dual-channel average CT values ​​under different probe final concentration conditions.

[0041] Recombinant plasmid DNA from pUC57-CSN2-A1 and pUC57-CSN2-A2 was used as positive template for fluorescent PCR. A 1 / 10000 dilution of the positive template with TE-Buffer was used as a weak positive template, with TE-Buffer serving as a negative control. PCR reactions were performed in a dual-mode quantitative PCR system with varying cycling parameters, ranging from 35 to 45 cycles. Additionally, the annealing temperature was varied to 58℃, 60℃, and 62℃. The fluorescence signal intensity and CT value of the amplification curves were analyzed, and the cycling parameters with the lowest average CT value across the two channels were selected as the most suitable cycling conditions (see Table 3). Experimental results showed that the optimal results were obtained with 95℃ pre-denaturation for 90 s, followed by 95℃ for 10 s and 60℃ for 40 s, for 45 cycles.

[0042] Table 3. Optimization of Reaction Conditions and Cyclic Parameters

[0043] Primer and probe concentrations were screened using a matrix method. In a 25 μL fluorescent PCR reaction system, the following were included: 12.5 μL PCR premix: 1 μL each of forward and reverse primers (final concentration 400 nmol / L), 0.5 μL probe (final concentration 200 nmol / L), 5 μL DEPC water, and 5 μL nucleic acid template. The fluorescent PCR reaction conditions were: 95℃ pre-denaturation for 90 s; 95℃ for 10 s, 60℃ for 40 s (collecting HEX and FAM fluorescence), for 45 cycles. The positive control showed two typical S-shaped amplification curves (FAM, HEX) with a CT value ≤ 30; the negative control showed no amplification curve and no CT value, indicating a valid test. The sample showed a typical amplification curve in the FAM fluorescence channel with a CT value < 40, while the HEX fluorescence channel showed no amplification curve with a CT value > 45, indicating an A2 type gene. The sample showed a typical amplification curve in the HEX fluorescence channel with a CT value < 40, while the FAM fluorescence channel showed no amplification curve with a CT value > 45, indicating an A1 type gene. The sample showed typical amplification curves in both the FAM and HEX fluorescence channels with a CT value < 40, indicating an A1A2 heterozygous gene.

[0044] 3. Methods for extracting genomic DNA from milk samples Transfer 1 ml of the solution to a milk centrifuge tube and centrifuge at 3000 rpm for 10 min at 4°C to collect somatic cells from the milk. Discard the supernatant. Resuspend the cells in one volume of ice-cold PBS and centrifuge again, discarding the supernatant. Carefully resuspend the cells in ice-cold PBS and centrifuge to collect the cells. Discard the supernatant and resuspend the cells in 1 ml of TE (pH 8.0), transferring the resuspended cells to a 2 ml centrifuge tube. Add 1 / 3 volume of lysis buffer and incubate at 37°C for 1 h. Add proteinase K (20 mg / ml) to a final concentration of 100 µg / ml and mix thoroughly. Incubate at 50°C for 3 h, shaking occasionally. Cool the solution to room temperature, add an equal volume of equilibrated phenol, and vortex gently for 10 min to gently mix the two phases. Centrifuge at 6500 rpm for 15 min to separate the two phases. Transfer the aqueous phase to another centrifuge tube using a pipette. Extract twice with phenol and collect the aqueous phase. Add 0.2 volumes of 1 mol / L ammonium acetate and 2 volumes of anhydrous ethanol, mix thoroughly, and centrifuge at 6500 rpm for 5 min at room temperature. Collect the precipitate. Wash the DNA precipitate twice with 70% ethanol, and centrifuge to collect the DNA. Aspirate as much residual ethanol as possible, and place the DNA precipitate in an open tube at room temperature until the ethanol has completely evaporated. Dissolve the DNA in 100 μL TE buffer and store the DNA solution at -20°C for later use.

[0045] 4. Methods for extracting genomic DNA from bovine tail hair follicle samples Hair follicle samples plucked from the cow's tail were placed in a 0.75% NaCl solution. 0.5cm of the hair follicle was cut from the root and placed into sterile 1.5mL centrifuge tubes, 10-20 follicles per tube. 1mL of DNA extraction buffer (10mM Tris-Cl, pH 8.0, 15mM NaCl, 10mM EDTA, pH 8.0, 0.4% SDS) was added. Proteinase K was then added to a final concentration of 120ug / mL. The mixture was incubated at 55℃ with shaking for 3 hours. 0.2mL of TE buffer was added and mixed for 10 minutes. Then, 200µl of a mixture of phenol, chloroform, and isoamyl alcohol (volume ratio 25:24:1) was added. The centrifuge tubes were slowly inverted for approximately 10 minutes. Transfer the upper aqueous phase to a centrifuge tube using a pipette tip, add an equal volume of ice-cold isocyanate, incubate at 0°C for 10 min, centrifuge at 4500 rpm for 10 min, rinse the precipitate twice with 70% ethanol, air dry, dissolve in TE buffer, and store at -20°C for later use.

[0046] 5. Methods for extracting DNA from cow blood Transfer 500 μL of anticoagulated blood sample into a 1.5 mL EP tube. Add 445 μL of TE buffer, 25 μL of proteinase K (10 mg / mL), and 25 μL of SDS (10%) to the tube, and mix well. Incubate overnight at 55°C with shaking. Add 500 μL of Tris-saturated phenol and invert for 10 min. Centrifuge at 12000 rpm for 10 min, and carefully transfer the upper aqueous phase to a new EP tube. Avoid touching or aspirating the middle layer proteins to prevent severely affecting DNA quality and subsequent reactions. Add an equal volume of a mixture of phenol, chloroform, and isoamyl alcohol (volume ratio: 25:24:1) to the aspirated upper aqueous phase, and invert for 10 min. Centrifuge at 12000 rpm for 10 min, and carefully transfer the upper aqueous phase to a new EP tube. Add an equal volume of a mixture of chloroform and isoamyl alcohol (volume ratio: 24:1), mix, and invert for 10 min. Centrifuge at 10,000 rpm for 10 min, and carefully aspirate the supernatant into a new EP tube. Add 2 volumes of anhydrous ethanol (pre-cooled to -20°C), and vortex horizontally for 3 min. Centrifuge at 10,000 rpm for 10 min; a white DNA precipitate will be visible at the bottom of the tube. Resuspend and wash the DNA precipitate with 1 mL of 70% ethanol. Centrifuge at 8,000 rpm for 5 min, discard the ethanol, and invert the EP tube onto filter paper until the ethanol has completely evaporated. Add 100 μL of TE buffer, and incubate at 55°C until the DNA is completely dissolved. Store at -20°C for later use.

[0047] The concentration of genomic DNA is determined using a micro spectrophotometer. Generally, an OD260 / 280 ratio between 1.6 and 1.8 is sufficient to meet experimental requirements.

[0048] Example 2: Kit preparation and usage 1. Preparation of primer-probe mixture Primer dilution: The forward primer (SEQ ID NO:1) and reverse primer (SEQ ID NO:2) were diluted to 10 µM using TE buffer with low EDTA pH 8.0 (Shanghai Sangon Biotech, catalog number B541019).

[0049] Fluorescent probe dilution: Dilute the A1 type probe: 5'-(HEX)cCaTcC(A)tAaCagcCT(BHQ1)-3' (SEQ ID NO:3) and the A2 type probe: 5'-(FAM)gCcCaTCc(C)TaAcAg(BHQ1)-3' (SEQ ID No. 4) to a concentration of 20 µM using low-EDTA pH 8.0 TE buffer (Shanghai Sangon Biotech, catalog number B541019). Primers and probes can also be diluted with DEPC water, but the low-EDTA TE buffer provides a weakly alkaline environment, which is beneficial for the preservation of oligonucleotides.

[0050] Mix the following proportions: (1) 53 µl of diluted 10 µM forward primer; (2) 53 µl of diluted 10 µM reverse primer; (3) 18.6 µl each of diluted 10 µM A1 and A2 probes; (4) 254.4 µl of DEPC water or sterile deionized water. Then, dispense the mixture into brown tubes at 400 µL / tube to prepare the primer-probe mixture.

[0051] 2. Preparation and testing of dual-fluorescence PCR reaction solution The commercially available THUNDERBIRD Probe qPCR Mix, brand name Toyobo; catalog number QPS-101; specification 5mL. Dispense 650ul / tube of fluorescent PCR reaction solution in a laminar flow hood and store at -20℃.

[0052] 3. Preparation of positive and negative controls The bacterial cultures containing the constructed pUC57-CSN2-A1 and pUC57-CSN2-A2 recombinant plasmid vectors were amplified. Plasmids were extracted and their concentrations determined. The purified plasmids were serially diluted 10-fold and detected by fluorescent PCR. The dilution corresponding to a CT value range of 20 was selected for further dilution. Equal volumes of the pUC57-CSN2-A1 and pUC57-CSN2-A2 recombinant plasmids were mixed and aliquoted into 100µL tubes as the positive control. The negative control was TE-Buffer, aliquoted into 100µL tubes. Storage was at -20℃.

[0053] 4. Assembly of the reagent kit: Pack the kit according to the components shown in Table 4.

[0054] Table 4

[0055] 5. Kit Usage Nucleic acid extraction: Genomic DNA is extracted from bovine blood samples, bovine tail hair follicle samples, or milk samples using the methods described above to obtain sample DNA. Alternatively, commercially available nucleic acid extraction reagents (kits) can be used to extract nucleic acid DNA from the samples.

[0056] Preparation and addition of dual-fluorescence PCR reagents: Take (n+1)×12.5μL of PCR reaction solution and (n+1)×7.5μL of primer-probe mixture (n is the number of reaction tubes), vortex to mix for a few seconds, and then centrifuge briefly. Place 20μL of the above mixture into separate PCR reaction tubes. Then, add 5μL of sample DNA, 5μL of negative control, and 5μL of positive control to separate PCR reaction tubes, cap the tubes, centrifuge briefly, and immediately perform the PCR amplification reaction.

[0057] The PCR amplification reaction was performed under the following parameters as shown in Table 5.

[0058] Table 5

[0059] Results Analysis and Result Determination (1) Baseline and threshold setting: The baseline can usually be set automatically by the instrument. The principle for setting the threshold is that the threshold line should just exceed the highest point of the fluorescence curve of the negative control.

[0060] (2) Quality control: The positive control shows two typical S-shaped amplification curves (FAM, HEX) and the CT value is ≤30; the negative control has no amplification curve and no CT value, which can be considered as valid test.

[0061] (3) Result determination: If the sample shows a typical amplification curve in the FAM fluorescence channel and the CT value is <40, while the HEX fluorescence channel shows no amplification curve and the CT value is >45, it is judged as A2 type gene; if the sample shows a typical amplification curve in the HEX fluorescence channel and the CT value is <40, while the FAM fluorescence channel shows no amplification curve and the CT value is >45, it is judged as A1 type gene; if both the FAM fluorescence channel and the HEX fluorescence channel show typical amplification curves and the CT value is <40, it is judged as A1A2 heterozygous gene.

[0062] Example 3: Reagent kit specificity test Tail hair follicle samples from cows with known genotypes A1, A2, and A1A2 β-casein were used as the detection subjects. The kit from Example 2 was used for testing, and the experimental results are as follows: See [link to example]. Figure 1In the A1 type sample, only the HEX fluorescence channel showed a typical amplification curve with a CT value of 15.12, while the FAM fluorescence channel showed no amplification curve and a CT value > 45; see [link to relevant documentation]. Figure 2 In the A2 type sample, only the FAM fluorescence channel showed a typical amplification curve with a CT value of 22.09, while the HEX fluorescence channel showed no amplification curve and a CT value > 45; see [link to relevant documentation]. Figure 3 In the A1A2 type samples, typical amplification curves were observed in both the FAM and HEX fluorescence channels, with CT values ​​of 15.16 and 13.98, respectively, both less than 40. The experiments demonstrate that the primer pairs, specific fluorescent probes, fluorescent PCR reaction system, and detection method used in the kit of this invention exhibit good specificity.

[0063] Example 4: Reagent Kit Sensitivity Test DNA from an equal mixture of pUC57-CSN2-A1 and pUC57-CSN2-A2 recombinant plasmids was used as a template for fluorescent PCR. The plasmid concentration after mixing was measured to be 104.20 µg / mL. After conversion to copy number, the plasmid was serially diluted with TE buffer to a concentration of 1.5 × 10⁻⁶. 7 copies / mL, 1.5 × 10 6 copies / mL, 1.5 × 10 5 copies / mL, 1.5 × 10 4 copies / mL, 1.5 × 10 3 copies / mL, 1.5 × 10 2 copies / mL, 1.5 × 10 1 copies / mL, 1.5 × 10 0 Using copies / mL as a template, the kit described in Example 2 was used for fluorescent PCR to test the sensitivity. The results showed that the kit could detect 1.5 × 10⁻⁶ copies / mL. 2 Copies / mL, see Table 6.

[0064] Table 6 Sensitivity Test Results

[0065] The A1 standard positive control (preparation method as described in Example 2, the difference being that the pUC57-CSN2-A2 recombinant plasmid vector was not added) was serially diluted 10-fold to obtain 10 -4 ~10 -10 Seven dilutions were used as templates for fluorescent PCR, with three replicates for each dilution. A standard curve was plotted using ABI 7500 software V2.0.6. See [link / reference]. Figure 4 The standard curve regression equation is: y = -3.247x + 41.129, R0 2=0.980, showing a good linear relationship, with a slope of -3.247, indicating that the amplification efficiency of A1 is good.

[0066] The A2 standard positive control (preparation method as described in Example 2, the difference being that the pUC57-CSN2-A1 recombinant plasmid vector was not added) was serially diluted 10-fold to obtain 10 -4 ~10 -10 Seven dilutions were used as templates for fluorescent PCR, with three replicates for each dilution. A standard curve was plotted using ABI 7500 software V2.0.6. See [link / reference]. Figure 5 The standard curve regression equation is: y = -3.368x + 46.452, R0 2 =0.981, showing a good linear relationship, with a slope of -3.368, indicating that the amplification efficiency of A2 is good.

[0067] Example 5: Reproducibility Test of the Reagent Kit To verify the stability of the fluorescent PCR detection method, 10 -3 ~10 -9 Recombinant plasmid DNA diluted with different concentrations of pUC57-CSN2-A1 and pUC57-CSN2-A2 was used as the test sample and detected using the kit described in Example 2. Each sample and each concentration was tested in triplicate for inter-assay and intra-assay reproducibility experiments. The reproducibility of the method was assessed by Cv. The results showed that the intra-assay Cv ranged from 0.67% to 2.01%, and the inter-assay Cv ranged from 0.48% to 2.56%, with all Cv values ​​less than 3.00%. Specifically, 10... -9 The results of three repeated tests of the diluted test samples were consistent between and within batches, with no CT values ​​in any of them. This indicates that the kit has good repeatability and high stability (see Table 7).

[0068] Table 7 Results of Repeatability Tests

[0069] Example 6: Clinical Sample Application Validation Forty clinical samples were collected from Holstein cows of known genotypes: ten blood samples, ten milk samples, and twenty tail hair follicle samples. All samples came from a Holstein herd at a dairy farm in Jinan City, Shandong Province. The kit described in Example 2 was used for testing. Results showed that 28 samples were type A2, 9 samples were type A1, and 3 samples were type A1A2. Simultaneously, β-casein gene sequencing was performed on the sample DNA for confirmation. The test results indicate that the detection results using the kit of this invention have a 100% concordance rate with known genotypes, with no false positives or false negatives. This demonstrates that the kit and its detection method of this invention have high specificity and sensitivity, and are a fluorescent PCR kit that can accurately identify and detect A1 and A2 type β-casein in dairy cows.

[0070] The above embodiments should be understood as being used only to illustrate the present invention more clearly, and not to limit the scope of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope defined by the appended claims.

Claims

1. A primer and probe combination, characterized in that, The sequence of the primer is: Forward primer: 5'-ggcaccaccacaggggtttgag-3'; Reverse primer: 5'-CTTTGCCCAGACACAGTCTCT-3'; The probe comprises an A1 type probe and an A2 type probe, the sequence of the A1 type probe is shown as SEQ ID NO: 3, and the sequence of the A2 type probe is shown as SEQ ID NO:

4.

2. A kit characterized in that, The kit comprises a PCR reaction solution, a primer probe mixture, a positive control, and a negative control, and the primer probe mixture comprises the primer and the probe combination as claimed in claim 1.

3. The kit of claim 2, wherein The PCR reaction solution is THUNDERBIRD ProbeqPCR Mix.

4. The kit of claim 2, wherein The positive control comprises a plasmid vector containing a sequence as shown in SEQ ID NO: 5 and a plasmid vector containing a sequence as shown in SEQ ID NO: 6, and the negative control is TE-Buffer.

5. A method for identifying A1 and A2 type beta-casein genotype dairy cows, characterized by, The kit comprises the following steps: S1, extracting DNA of a dairy cow to be identified to obtain sample DNA; S2, performing fluorescent PCR amplification on the sample DNA obtained in S1 using the kit as claimed in any one of claims 2-4; S3, obtaining and analyzing the results.

6. The detection method according to claim 5, characterized in that, In S1, DNA is extracted from one or more of milk, blood, and the root of a cow tail hair follicle of the dairy cow to be identified.

7. The detection method according to claim 5, characterized in that, In S2, the conditions of the fluorescent PCR amplification cycle reaction are as follows: 95℃ pre-denaturation for 60s; 95℃ denaturation for 10s, 60℃ annealing for 40s, and 60℃ collection of FAM fluorescence signal and HEX fluorescence signal, wherein the denaturation and annealing are performed for 45 cycles.

8. The assay of any one of claims 5-7, wherein, In S3, when the positive control appears two typical S-shaped amplification curves and the CT value is ≤30, and the negative control has no amplification curve and no CT value, the test is valid; when the sample DNA appears a typical amplification curve in the FAM fluorescence channel and the CT value is <40, and the sample DNA has no amplification curve in the HEX fluorescence channel and the CT value is >45, the β-casein genotype of the dairy cow is A2 type gene; when the sample DNA appears a typical amplification curve in the HEX fluorescence channel and the CT value is <40, and the sample DNA has no amplification curve in the FAM fluorescence channel and the CT value is >45, the β-casein genotype of the dairy cow is A1 type gene; and when the sample DNA appears a typical amplification curve in both the FAM fluorescence channel and the HEX fluorescence channel and the CT value is <40, the β-casein genotype of the dairy cow is A1A2 hybrid type gene.

9. Use of the kit as claimed in any one of claims 2-4 in identifying A1 and A2 type β-casein dairy cows, dairy cow genetic breeding, dairy product safety detection, and / or functional dairy product development.

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