Method for detecting cattle horn-free gene through HRM (High Resolution Melting)
The detection of the hornless gene PC of cattle through HRM technology solves the problems of long detection time and high cost in traditional methods, and achieves a fast, accurate and economical detection effect, which is suitable for large-scale sample detection.
Patent Information
- Application Number
- CN202510034900.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-08
- Publication Date
- 2025-05-06
AI Technical Summary
The existing cattle hornless gene detection methods have problems such as long test results, expensive, and require a variety of advanced equipment and test personnel.
High-resolution melting curve (HRM) technology is used to design specific detection primers and reaction systems to achieve accurate detection of bovine hornless gene PCs. The method includes DNA extraction, primer design and formulation, HRM reaction system preparation and operation, and analysis and comparison of HRM melting curves.
HRM technology significantly shortens the detection time and usually obtains results within 1 to 1.5 hours. It is easy to operate and is suitable for large-scale sample testing. It has high sensitivity and accuracy, reducing costs and equipment needs.
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Figure CN119932199A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of gene detection, and in particular to a method for detecting a bovine hornless gene through HRM. Background Art
[0002] Horns are a unique tissue structure of cattle, belonging to the category of horns, and are a quality trait controlled by multiple genes. Currently, four genetic loci for the hornless phenotype of cattle have been identified, namely, CelticPOLLED (P C )、Friesian POLLED(P F )、Mongol ian POLLED(P M ) and Guarani POLLED (P G ) mutations are all located in a relatively concentrated area of bovine chromosome 1. These four variants are not located on any known genes, lncRNAs (long non-coding RNAs) or miRNAs. It is speculated that they regulate gene expression by affecting DNA regulatory factors (such as enhancers).
[0003] Medugorac et al. first discovered P in European cattle breeds C Mutation, P C Located between the IFNAR2 and OLIG1 genes, it is proposed that the mutation is a 202bp fragment that replaces the 10bp insertion-deletion in the original sequence. The mutation occurs between genes and does not affect the coding sequence and splicing site. It mainly controls the horn traits of beef and dairy cattle. It can regulate the horn phenotype by affecting the expression of the OLIG2 gene and lncRNA (LOC100848368 and LOC112447133) in the horn bud tissue. Gene editing knocked out the 10bp sequence in the original sequence, and the horns still grew, suggesting that the hornless phenotype is more strongly correlated with the 212bp base repeat. In recent years, researchers have detected and gene-edited the mutation sites of the hornless trait of different breeds of cattle and cultivated some new hornless lines. Tan et al. used TALEN technology to obtain gene-edited Holstein fetal fibroblast-positive cells based on the 202bp (1705843-1706045bp) repeated sequence on chromosome 1 of hornless Angus cattle. Carlson then used somatic cell nuclear transfer technology to obtain gene-edited Holstein dairy cows. All gene-edited cows were hornless. Alexey used CRISPR / Cas9 technology to gene-edit Holstein cattle somatic cells and introduced P 202ID The mutation site was found and finally the hornless calves were obtained. Shang Songyang et al. conducted P202ID and P 219ID Mutation site detection revealed that 93.96% of hornless individuals carried P202ID locus, among which polled Charolais cattle all carry P 202ID locus, no individuals homozygous for the mutation were found. These studies all indicate that P C Mutations are key genes that affect the phenotypic traits of cattle horns.
[0004] The following are the conventional methods for detecting mutation sites of the bovine hornless gene:
[0005] (1) Polymerase chain reaction (PCR) method
[0006] This is a commonly used molecular biology technique that designs specific primers to amplify gene mutation sites related to the hornless trait in cattle, and then detects them through gel electrophoresis and other methods.
[0007] (2) Restriction fragment length polymorphism (RFLP) method
[0008] The PCR product was digested with restriction endonucleases, and the genotype was determined based on the fragment length after electrophoresis.
[0009] (3) KASP technology
[0010] Competitive allele-specific PCR technology is a microfluidic chip-based SNP detection system that can quickly diagnose specific mutation sites of the bovine hornless gene. This technology can achieve efficient, rapid and accurate detection of the hornless gene and is suitable for large-scale detection applications.
[0011] (4) Capillary electrophoresis
[0012] This is a technology that separates DNA fragments through capillary electrophoresis. It can be used to detect mutation sites in the cattle hornless gene, but it is relatively expensive and requires specialized equipment.
[0013] (5) Direct sequencing
[0014] The mutation site of the hornless gene is determined by directly sequencing the DNA sample. This method can obtain detailed sequence information, but it is costly and complex to operate.
[0015] Compared with HRM technology, traditional detection methods have many shortcomings, specifically:
[0016] The polymerase chain reaction (PCR) method requires subsequent processing steps such as gel electrophoresis, which is time-consuming and requires high primer specificity, limiting the ability to detect unknown mutations;
[0017] The restriction fragment length polymorphism (RFLP) method relies on specific enzyme cutting sites and requires PCR amplification, enzyme cutting and electrophoresis analysis. The process is cumbersome and only applicable to known mutations.
[0018] Although KASP technology has the ability to rapidly detect SNPs, the equipment and reagents are expensive and limited by the specific conditions of microfluidic chips;
[0019] Capillary electrophoresis requires special equipment, is costly and complex to operate, and is difficult to meet high-throughput requirements. Although direct sequencing methods (such as Sanger and NGS) provide accurate sequence information, they are costly, complex to operate, and have relatively slow detection speeds, making them unsuitable for large-scale screening.
[0020] Therefore, HRM technology is significantly superior to the above traditional methods in terms of time efficiency, cost-effectiveness and ease of operation. Summary of the invention
[0021] The purpose of the present invention is to provide a method for HRM detection of cattle hornless gene, so as to solve the problems that the existing traditional detection methods have a long detection time, high cost and require more advanced equipment and test personnel.
[0022] In order to solve the above technical problems, the present invention provides the following technical solutions:
[0023] A detection primer set for HRM detection of cattle hornless gene, the detection primer set comprising an upstream primer and a downstream primer, the nucleotide sequence of the primers being:
[0024] Upstream primer: 5′-TCAAGAAGGCGGCACTATCT-3′;
[0025] Downstream primer: 5′-TGATAAACTGACCCTCTGCCTATA-3′.
[0026] A method for detecting the hornless gene of cattle by HRM, specifically comprising:
[0027] Step 1: Extract DNA from the sample to be tested;
[0028] Step 2: Design and prepare upstream primers and downstream primers;
[0029] Step 3: Prepare HRM reaction system;
[0030] Step 4: Run the HRM reaction program to perform HRM reaction analysis and obtain the HRM melting curve;
[0031] Step 5: Compare the HRM melting curve with the HRM standard curve to determine whether the sample being tested has corners.
[0032] As a limitation of the present invention, step 1 is specifically:
[0033] The blood of the sample to be tested is collected using a blood sample collection card. After it is completely dried in a natural shade, a hole is punched in the blood sample collection card with the blood of the sample to be tested to obtain a blood sample to be tested. The blood sample to be tested is added to a 0.02M NaOH solution, and incubated at 60°C for 30 minutes. After 30 minutes, the liquid is discarded, and 1×TE is added, and the solution is allowed to stand for 5 minutes. After 5 minutes, the liquid is discarded, and the solution is dried naturally to obtain the DNA of the sample to be tested.
[0034] As a limitation of the present invention, the HRM reaction system includes 2×Taq PCR Master Mix premix, DNA of a sample to be tested, an upstream primer, a downstream primer, LC Green saturated fluorescent dye, and deionized water.
[0035] As a limitation of the present invention, each 25 μL HRM reaction system includes: 12.5 μL 2×Taq PCR Master Mix premix, 1 μL template DNA, 0.5 μL 10 umol / L upstream primer, 0.5 μL 10 umol / L downstream primer, 0.5 μL LC Green saturated fluorescent dye, and 10 μL deionized water.
[0036] As a limitation of the present invention, the HRM reaction procedure includes: pre-denaturation at 95°C for 10 minutes, amplification after the pre-denaturation, amplification at 95°C for 10 seconds, amplification at 40°C for 10 seconds, amplification at 72°C for 10 seconds, and a total of 40 cycles of amplification; the HRM analysis procedure includes: running at 95°C for 5 minutes and running at 40°C for 1 minute to form a hybrid; and collecting fluorescence signals.
[0037] As a limitation of the present invention, the temperature when collecting the fluorescent signal is 70° C. to 95° C., and the frequency is 25 times per second.
[0038] As a limitation of the present invention, the preparation method of the HRM standard curve is:
[0039] Step 1: Extract DNA from samples with horns and samples without horns;
[0040] Step 2: Design and prepare upstream primers and downstream primers;
[0041] Step 3: preparing an HRM reaction system containing DNA of a sample with corners and an HRM reaction system containing DNA of a sample without corners;
[0042] Step 4: Run the HRM reaction program to perform HRM reaction analysis to obtain the HRM melting curve of the sample with corners and the HRM melting curve of the sample without corners;
[0043] Step 5: Put the HRM melting curve of the sample with horns and the HRM melting curve of the sample without horns together to create the HRM standard curve.
[0044] Compared with the prior art, the present invention has the following beneficial effects:
[0045] The present invention adopts high-resolution melting curve (High-Resolution Melting Analysis, HRM) technology to achieve the bovine hornless gene P C Accurate detection. HRM technology is a real-time PCR-based analytical method that can identify different genotypes by monitoring the fluorescence changes of DNA during heating. This method has high sensitivity and specificity and can effectively distinguish the differences of single nucleotides, thus providing a novel and efficient solution for the detection of cattle hornless genes.
[0046] The HRM detection technology proposed in the present invention does not require additional subsequent processing steps, such as electrophoresis analysis, thereby significantly shortening the overall time from reaction system configuration to result acquisition, which can usually be controlled within 1 to 1.5 hours. In addition, compared with other traditional detection methods, the HRM detection technology proposed in the present invention is simple to operate and suitable for large-scale sample detection, which can effectively meet the needs of modern aquaculture for rapid and accurate gene detection. BRIEF DESCRIPTION OF THE DRAWINGS
[0047] Figure 1 The HRM method for detecting P in Example 1 C HRM standard curve profile of the gene;
[0048] Figure 2 It is the HRM melting curve spectrum of the sample to be tested in Example 2;
[0049] Figure 3 This is the HRM melting curve of the sample to be tested in Example 3. DETAILED DESCRIPTION
[0050] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0051] 2×Taq PCR Master Mix (containing 0.1U Tag Polymerase / μL, 500μM dNTPeach, 20mM Tris-HCl (pH=8.3), 100mM KCI, 3mM MgCl2), 1×TE (containing 10mM Tris and 1mM EDTA, pH=8.0), and blood sample collection card (38mm×100mm) are all commercially available.
[0052] Example 1: Establishing a detection P C HRM standard curve for genes
[0053] Step 1: Extract DNA
[0054] Use a blood collector to collect 3 mL of blood from the caudal vein of a calf with horns, and drop or stick the collected blood on a blood sample collection card. After completely drying in a natural shade, store it at room temperature away from light. Use a 2.0 mm DNA manual punch sampler to punch holes in the blood sample collection card with caudal vein blood. Place the punched sample in a 200 μL PCR centrifuge tube, add 200 μL of 0.02 M NaOH solution, cover the tube tightly, and incubate at 60°C for 30 minutes. After 30 minutes, open the PCR centrifuge tube cap and discard the liquid in the PCR centrifuge tube. Add 200 μL 1×TE to the PCR centrifuge tube, cover the tube tightly, let it stand for 5 minutes, open the PCR centrifuge tube cap after 5 minutes, discard the liquid in the PCR centrifuge tube, and dry it naturally for 20 minutes to obtain the DNA of the horned sample. Use the same method to extract the DNA of the hornless sample from the calf without horns.
[0055] Step 2: Design and prepare primers
[0056] The detection primer set includes an upstream primer and a downstream primer, and the primer nucleotide sequence is:
[0057] Upstream primer: 5′-TCAAGAAGGCGGCACTATCT-3′;
[0058] Downstream primer: 5′-TGATAAACTGACCCTCTGCCTATA-3′.
[0059] Dilute the upstream and downstream primers to 10 M with sterile deionized water and store at 4°C for a short term and at -20°C for a long term.
[0060] Step 3: Prepare HRM reaction system
[0061] Add 12.5 μL of 2×Taq PCR Master Mix, 1 μL of DNA of horn samples, 0.5 μL of 10 umol / L upstream primer and 0.5 μL of 10 umol / L downstream primer prepared in the previous step, 0.5 μL of LC Green saturated fluorescent dye, and 10 μL of deionized water into the eight-tube strip and mix well to prepare the HRM reaction system of horn samples. Then, use the same method to prepare the HRM reaction system of non-horn samples.
[0062] Step 4: Run the HRM reaction program
[0063] After the HRM reaction system was prepared, the reaction program was run on the LightCycler 480 platform to perform HRM data analysis on the HRM reaction system. The reaction conditions of the HRM reaction system were as follows: 95°C pre-denaturation for 10 min; amplification program: 95°C for 10 s, 40°C for 10 s, 72°C for 10 s, for a total of 40 cycles; HRM analysis program: 95°C for 5 min; 40°C for 1 min to form a hybrid; fluorescence signal collection from 70°C to 95°C, 25 signals per second. After collection, cool to 40°C, and perform HRM data analysis using the LightCycler 480 built-in software.
[0064] Step 5: Create an HRM standard curve
[0065] The HRM data were analyzed using the LightCycler 480 software to generate HRM melting curves for samples with corners and samples without corners. Stable melting curves were selected by observing the shapes of HRM melting curves, and the two stable melting curves were put together to establish a stable melting curve. Figure 1 By comparing the HRM standard curve shown with the sample being tested, it can be determined whether the sample being tested has corners.
[0066] Example 2: A method for detecting the hornless gene in cattle by HRM
[0067] Step 1: Extraction of DNA from the sample to be tested
[0068] Use a blood collector to collect 3 mL of blood from the caudal vein of the calf to be tested and drop or stick the collected blood on a blood sample collection card. After completely drying in a natural shade, store it at room temperature away from light. Use a 2.0 mm DNA manual punch sampler to punch holes in the blood sample collection card with caudal vein blood. Place the sample obtained by punching in a 200 μL PCR centrifuge tube, add 200 μL of 0.02M NaOH solution, cover the tube tightly, and incubate at 60°C for 30 minutes. After 30 minutes, open the PCR centrifuge tube cover, discard the liquid in the PCR centrifuge tube, add 200 μL 1×TE to the PCR centrifuge tube, cover the tube tightly, let it stand for 5 minutes, open the PCR centrifuge tube cover after 5 minutes, discard the liquid in the PCR centrifuge tube, and dry it naturally for 20 minutes to obtain the DNA of the sample to be tested.
[0069] Step 2: Design and prepare primers
[0070] The detection primer set includes an upstream primer and a downstream primer, and the primer nucleotide sequence is:
[0071] Upstream primer: 5′-TCAAGAAGGCGGCACTATCT-3′;
[0072] Downstream primer: 5′-TGATAAACTGACCCTCTGCCTATA-3′.
[0073] Dilute the upstream and downstream primers to 10 M with sterile deionized water and store at 4°C for a short term and at -20°C for a long term.
[0074] Step 3: Prepare HRM reaction system
[0075] Add 12.5 μL of 2×Taq PCR Master Mix, 1 μL of template DNA, 0.5 μL of each of the 10 umol / L upstream and downstream primers prepared in the previous step, 0.5 μL of LC Green saturated fluorescent dye, and 10 μL of deionized water into the eight-tube strip, mix well, and prepare the HRM reaction system.
[0076] Step 4: Run the HRM reaction program
[0077] After the HRM reaction system was prepared, the reaction program was run on the LightCycler 480 platform to perform HRM data analysis on the HRM reaction system. The reaction conditions of the HRM reaction system were as follows: 95°C pre-denaturation for 10 minutes, amplification after the pre-denaturation, 95°C amplification for 10 seconds, 40°C amplification for 10 seconds, 72°C amplification for 10 seconds, and a total of 40 cycles of amplification; the HRM analysis program included: 95°C for 5 minutes, 40°C for 1 minute to form a hybrid; fluorescence signal collection from 70°C to 95°C, 25 signals per second. After collection, it was cooled to 40°C, and the HRM data analysis was performed using the LightCycler 480 built-in software.
[0078] Step 5: Determine the HRM reaction results
[0079] The HRM data were analyzed using the LightCycler 480 software to generate a stable, well-aggregated HRM melting curve, such as Figure 2 As shown, by comparing it with the HRM standard curve, it can be seen that it is highly similar to the HRM melting curve of the genotype showing hornlessness in the HRM standard curve. Therefore, it is judged that the calf to be tested is a hornless calf.
[0080] Example 3: A method for detecting the hornless gene in cattle by HRM
[0081] Step 1: Extraction of DNA from the sample to be tested
[0082] Use a blood collector to collect 3 mL of blood from the caudal vein of the calf to be tested and drop or stick the collected blood on a blood sample collection card. After completely drying in a natural shade, store it at room temperature away from light. Use a 2.0 mm DNA manual punch sampler to punch holes in the blood sample collection card with caudal vein blood. Place the sample obtained by punching in a 200 μL PCR centrifuge tube, add 200 μL of 0.02M NaOH solution, cover the tube tightly, and incubate at 60°C for 30 minutes. After 30 minutes, open the PCR centrifuge tube cover, discard the liquid in the PCR centrifuge tube, add 200 μL 1×TE to the PCR centrifuge tube, cover the tube tightly, let it stand for 5 minutes, open the PCR centrifuge tube cover after 5 minutes, discard the liquid in the PCR centrifuge tube, and dry it naturally for 20 minutes to obtain the DNA of the sample to be tested.
[0083] Step 2: Design and prepare primers
[0084] The detection primer set includes an upstream primer and a downstream primer, and the primer nucleotide sequence is:
[0085] Upstream primer: 5′-TCAAGAAGGCGGCACTATCT-3′;
[0086] Downstream primer: 5′-TGATAAACTGACCCTCTGCCTATA-3′.
[0087] Dilute the upstream and downstream primers to 10 M with sterile deionized water and store at 4°C for a short term and at -20°C for a long term.
[0088] Step 3: Prepare HRM reaction system
[0089] Add 12.5 μL of 2×Taq PCR Master Mix, 1 μL of template DNA, 0.5 μL of each of the 10 umol / L upstream and downstream primers prepared in the previous step, 0.5 μL of LC Green saturated fluorescent dye, and 10 μL of deionized water into the eight-tube strip, mix well, and prepare the HRM reaction system.
[0090] Step 4: Run the HRM reaction program
[0091] After the HRM reaction system was prepared, the reaction program was run on the LightCycler 480 platform to perform HRM data analysis on the HRM reaction system. The reaction conditions of the HRM reaction system were as follows: 95°C pre-denaturation for 10 minutes, amplification after the pre-denaturation, 95°C amplification for 10 seconds, 40°C amplification for 10 seconds, 72°C amplification for 10 seconds, and a total of 40 cycles of amplification; the HRM analysis program included: 95°C for 5 minutes, 40°C for 1 minute to form a hybrid; fluorescence signal collection from 70°C to 95°C, 25 signals per second. After collection, it was cooled to 40°C, and the HRM data analysis was performed using the LightCycler 480 built-in software.
[0092] Step 5: Determine the HRM reaction results
[0093] The HRM data were analyzed using the LightCycler 480 software to generate a stable, well-aggregated HRM melting curve, such as Figure 3 As shown, by comparing it with the HRM standard curve, it can be seen that it is highly similar to the HRM melting curve of the genotype in the HRM standard curve that is hornless. Therefore, it is judged that the calf to be tested is a horned calf.
[0094] The present invention realizes beef cattle P by high-resolution dissolution curve (HRM) analysis technology C Compared with the traditional PCR detection method, the detection of mutation sites has the following advantages:
[0095] 1. Ease of operation
[0096] Traditional PCR detection methods usually require multiple steps, including complex experimental operations and data analysis. HRM technology simplifies the operation steps by real-time monitoring of the melting process. It reduces the professional skills requirements for experimental personnel, makes the detection process more intuitive and easy to operate, and reduces the possibility of human error.
[0097] 2. Save time
[0098] After completing PCR amplification, HRM analysis can directly perform melting curve analysis without the need for additional subsequent processing steps, such as electrophoresis analysis (the complete PCR detection technology, including PCR system configuration, PCR reaction program, agarose gel preparation and electrophoresis analysis, takes about 3 to 4 hours in total). This feature significantly shortens the overall time from experimental system configuration to result analysis, which can usually be controlled within 1 to 1.5 hours, thereby significantly improving laboratory work efficiency, meeting the needs of rapid detection, and being able to adapt to the application scenarios of large-scale sample detection.
[0099] 3. High sensitivity and accuracy
[0100] HRM technology can identify tiny sequence differences, such as single nucleotide polymorphisms (SNPs), which means it has high sensitivity in mutation detection. It provides highly accurate test results, provides reliable technical support for genetic research and breeding of beef cattle, and reduces the risk of misdiagnosis.
[0101] In summary, the present invention proposes a method for detecting Pc mutation sites in beef cattle by observing high-resolution HRM melting curve technology, which has obvious advantages in ease of operation, time efficiency, detection sensitivity and economic benefits.
[0102] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.
Claims
1. A detection primer set for detecting the bovine hornless gene by HRM, characterized in that: The detection primer set includes an upstream primer and a downstream primer, and the primer nucleotide sequence is: Upstream primer: 5′-TCAAGAAGGCGGCACTATCT-3′; Downstream primer: 5′-TGATAAACTGACCCTCTGCCTATA-3′.
2. A method for detecting the hornless gene of cattle by HRM, characterized in that: Specifically: Step 1: Extract DNA from the sample to be tested; Step 2: Design and prepare upstream primers and downstream primers; Step 3: Prepare HRM reaction system; Step 4: Run the HRM reaction program to perform HRM reaction analysis and obtain the HRM melting curve; Step 5: Compare the HRM melting curve with the HRM standard curve to determine whether the sample being tested has corners.
3. The method for detecting the hornless gene of cattle by HRM according to claim 2, characterized in that: Step 1 is as follows: The blood of the sample to be tested is collected using a blood sample collection card. After it is completely dried in a natural shade, a hole is punched in the blood sample collection card with the blood of the sample to be tested to obtain a blood sample to be tested. The blood sample to be tested is added to a 0.02M NaOH solution, and incubated at 60°C for 30 minutes. After 30 minutes, the liquid is discarded, and 1×TE is added, and the solution is allowed to stand for 5 minutes. After 5 minutes, the liquid is discarded, and the solution is dried naturally to obtain the DNA of the sample to be tested.
4. The method for detecting the hornless gene of cattle by HRM according to claim 2, characterized in that: The HRM reaction system includes 2×Taq PCR Master Mix premix, DNA of the sample to be tested, upstream primers, downstream primers, LC Green saturated fluorescent dye, and deionized water.
5. The method for detecting the hornless gene of cattle by HRM according to claim 4, characterized in that: Each 25 μL HRM reaction system includes: 12.5 μL 2×Taq PCR Master Mix premix, 1 μL DNA of the sample to be tested, 0.5 μL 10 umol / L upstream primer, 0.5 μL 10 umol / L downstream primer, 0.5 μL LC Green saturated fluorescent dye, and 10 μL deionized water.
6. The method for detecting the hornless gene of cattle by HRM according to claim 2, characterized in that: The HRM reaction procedure includes: pre-denaturation at 95°C for 10 min, amplification after pre-denaturation, amplification at 95°C for 10 s, amplification at 40°C for 10 s, amplification at 72°C for 10 s, and a total of 40 cycles of amplification; the HRM analysis procedure includes: running at 95°C for 5 min and running at 40°C for 1 min to form a hybrid; and collecting fluorescence signals.
7. The method for detecting the bovine hornless gene by HRM according to claim 6, characterized in that: The temperature when collecting the fluorescence signal was 70°C to 95°C, and the frequency was 25 times per second.
8. The method for detecting the bovine hornless gene by HRM according to claim 2, characterized in that: The HRM standard curve was prepared as follows: Step 1: Extract DNA from samples with horns and samples without horns; Step 2: Design and prepare upstream primers and downstream primers; Step 3: preparing an HRM reaction system containing DNA of a sample with corners and an HRM reaction system containing DNA of a sample without corners; Step 4: Run the HRM reaction program to perform HRM reaction analysis to obtain the HRM melting curve of the sample with corners and the HRM melting curve of the sample without corners; Step 5: Put the HRM melting curve of the sample with horns and the HRM melting curve of the sample without horns together to create the HRM standard curve.