CN114672575B - Kit for rapidly detecting hybridized pilose antler based on RAA amplification - Google Patents

Abstract

The invention relates to a primer for detecting hybridized pilose antler based on an RAA method and a kit containing the primer, wherein the primer comprises an upstream primer 014F2, a red deer specific downstream primer 014R2 and a red deer specific downstream primer 014S2, biotin is marked at the 5 '-end of the upstream primer, digoxin is marked at the 5' -end of the downstream primer; the kit comprises the primer, a reaction reagent and a colloidal gold detection test strip. The invention designs the allele-specific amplification primer based on the SNP locus, establishes the SNP typing detection method of the colloidal gold test strip based on the allele-specific recombinase-mediated isothermal amplification, and provides a simple and effective scientific means for identifying the hybrid deer of the Hua-ma.

Description

A rapid detection kit for hybrid antler based on RAA amplification

Technical Field

The invention belongs to the field of molecular biology, and in particular relates to a hybrid antler identification method based on RAA amplification.

Background technique

China is the world's third largest deer-breeding country, mainly producing antlers. Generally speaking, antlers are harvested stably from 3-year-old sika deer and red deer, once in July, and twice in April and September each year. The weight of two-bar antlers does not exceed 120g, and the weight of three-branch antlers does not exceed 400g. Long-term low production has limited the production of antlers. In recent years, with the integration of the world economy and the globalization of the deer market, countries that mainly raise deer for meat have introduced a large number of antlers and their by-products into the Chinese market, which has brought great pressure to China's deer farming industry. In order to improve the international competitive advantage of antlers, increase the breeding efficiency of deer, and improve the production performance of deer and the output of antlers, hybrid deer have also emerged. Hybridization methods can be divided into three types according to the sources of parents: 1) Hybrid offspring of sika deer and red deer (sika-red hybrid), the maternal sources include: Northeast sika deer (Cervusnippon hortulorum), Changbai Mountain sika deer (Cervusnippon), Japanese sika deer (Cervusnippon nippon), etc., and the paternal sources include: Northeast red deer (Cervuselaphusxanthopygus), Tianshan red deer (Cervus elaphussongaricus), Tarim red deer (Cervuselaphus yarkandensis). 2) Hybrid offspring of red deer and red deer (horse-horse hybrid), the maternal sources include Northeast red deer (Cervus elaphusxanthopygus), Tianshan red deer (Cervus elaphussongaricus), Gansu red deer (Cervus elaphuskansuensis), etc., and the paternal sources include: Tianshan red deer (Cervus elaphussongaricus), Tarim red deer (Cervus elaphusyarkandensis) and Altai red deer (Cervuselaphussibericus), etc. 3) Hybrid offspring of sambar deer and red deer or sika deer, the maternal source is sambar deer (Cervus unicolor), and the paternal sources include: Xinjiang red deer (Cervus elaphus), European red deer (Cervus elaphus) and sika deer (Cervusnippon). In interspecific hybridization, the hybridization of sika deer and red deer can make the F1 generation show extremely significant and complex hybrid advantages, and can also significantly reduce the cost of breeding. For velvet antlers, hybridization of sika deer and red deer can expand the source of medicine, solve the problem of expanding the supply and demand of medicine, and is conducive to the utilization and development of velvet antler medicine. However, there are few qualitative analyses, quality evaluations, pharmacological effects, and safety evaluations of hybrid velvet antlers from the perspective of traditional Chinese medicine and drug analysis. It is unclear whether the quality and efficacy of hybrid velvet antlers will be affected when they are used.

For consumers, it is difficult to directly identify the specific antler species of most antler products with the naked eye. Unscrupulous merchants may use non-medicinal antlers or low-value red deer antlers to replace high-value sika deer antlers with mislabeled products, and obtain huge economic benefits through the price difference between different antlers. In addition, unlabeled antler products also provide certain opportunities for counterfeiting antler products. The emergence of hybrid antlers has further exacerbated the confusion in the antler market. Since hybrid antlers are not clearly defined as medicinal antlers in the Chinese Pharmacopoeia, whether hybrid antlers can be sold as medicinal antlers remains to be discussed.

Single nucleotide polymorphism (SNP) technology is the most basic form of variation in genomic DNA. It uses one or several bases as molecular markers to achieve species identification through base differences at a certain site in the organism. Currently, SNP site molecular markers are widely used in the development of functional marker genes for plants and animals, the construction of genetic maps, variety identification, species evolution and other research. Since SNP markers can not only achieve species identification and individual identification in the traceability system, but also reveal the composition of individual gene varieties, they are gradually being applied to the characterization of food authenticity. The application of SNP-based molecular marker detection technology in species identification has been very extensive, but research on deer antler species identification is relatively small.

In recent years, the supply and demand of velvet antlers have been tight. In order to meet the demand for velvet antler products and increase velvet antler production, hybrid breeding has gradually emerged. Hybridization technology is used to cultivate deer species with excellent characteristics to expand the source of velvet antlers. Increasing the production of velvet antlers has also become a method to expand the production of velvet antlers. Although hybridization technology has promoted the development of the velvet antler industry, it has also added difficulties to the supervision of the velvet antler market. In addition, since hybrid velvet antlers are not included in the scope of medicinal velvet antlers specified in the "Chinese Pharmacopoeia", their medicinal value and safety need to be further evaluated. The currently commonly used DNA barcode method for species identification is based on the identification of mitochondrial genes, but mitochondria are maternally inherited, and hybrid velvet antlers cannot be identified only through gene fragments on mitochondria.

Colloidal gold immunochromatography is to fix specific antigens or antibodies on the NC membrane in the form of strips, and adsorb colloidal gold labeled reagents on the binding pad. When the sample to be tested is added to the sample pad at one end of the test strip, the colloidal gold labeled reagents on the binding pad are dissolved through chromatography and react with each other. When it moves to the fixed antigen or antibody area, the conjugate of the test object and the gold labeled reagent undergoes antigen-antibody binding, which makes the colloidal gold trapped and gathered on the detection band. The color development result can be observed by the naked eye. This detection method has high sensitivity, low cost, simple and fast operation.

Summary of the invention

The purpose of the present invention is to provide SNP primers for detecting hybrid deer, which are further designed into a kit, have the advantages of rapidity, sensitivity, and ease of operation, can be applied to on-site identification, and are of great significance to the identification of hybrid deer antlers.

To achieve the above objectives, this application adopts the following technical solutions:

The whole genome sequence of red deer (GCA_002197005.1) was downloaded from NCBI as a reference genome, and the genome data of 100 sika deer and 68 red deer were downloaded from BIOproject (PRJNA355630). DNA was extracted from 32 sika deer and 21 red deer, and the genome sequences of 53 individuals were obtained by second-generation sequencing. The genome sequences of the above 221 individuals were compared with the whole genome sequence of red deer on NCBI to find SNP difference sites. The SNP sites were screened by combining the sequences of 29 sika deer and red deer closely related species downloaded from NCBI. Finally, SNP primers were designed according to the two SNP sites obtained. Each group of SNP primers (3 primers) was composed of two pairs of specific primers for sika deer and red deer respectively. The primers were verified by AS-PCR gel electrophoresis specificity, and modifications were added to the 5′ end of the selected primers. The two pairs of primers were used to perform RAA amplification on antler samples with known species information. Finally, the colloidal gold test strips were used for detection to evaluate the effectiveness of the primers in hybrid antler identification.

The first aspect of the present invention provides a primer for detecting hybrid antler based on the RAA method, and the sequence of the primer is as follows:

Upstream primer 014F2: 5′-ATAAGTAGCAACAAACTGGTAAAGAGCAA-3′ (SEQ ID NO. 1);

Red deer specific downstream primer 014R2: 5′-TTAAGGAGCTTGAGTGGGTTACATACACCG-3′ (SEQ ID NO. 2);

Sika deer-specific downstream primer 014S2: 5′-TTAAGGAGCTTGAGTGGGTTACATACAGTA-3′ (SEQ ID NO. 3).

Preferably, the 5' end of the upstream primer is labeled with biotin, and the 5' end of the downstream primer is labeled with digoxigenin.

Preferably, the hybrid antler is a hybrid antler of red deer and sika deer.

The second aspect of the present invention provides a kit for detecting hybrid velvet antlers based on the RAA method, the kit comprising the above-mentioned primers, reaction reagents and colloidal gold detection test strips.

Preferably, the detection line T of the colloidal gold test strip is embedded with streptavidin, the quality control line C is embedded with rabbit anti-sheep secondary antibody, and the colloidal gold is bound to the sheep anti-digoxigenin antibody.

Preferably, the final concentration of the upstream primer and the downstream primer in the RAA kit is 10 μM.

Preferably, the reaction system (50 μL) is as follows: 25 μL hydrolysis buffer, 3 μL 10 μM upstream primer, 3 μL 10 μM downstream primer, 10.5 μL deionized water, 6 μL DNA template, and 2.5 μL magnesium acetate.

Preferably, the hybrid antler is a hybrid antler of a sika deer and a red deer.

The third aspect of the present invention provides the use of the above primers or kit in preparing a reagent for detecting hybrid velvet antlers.

Preferably, the hybrid antler is a hybrid antler of a sika deer and a red deer.

The fourth aspect of the present invention provides a method for detecting hybrid velvet antlers using the above-mentioned kit, comprising the following steps:

(1) Sample DNA extraction: Pulp antlers were crushed and DNA was extracted;

(2) RAA amplification: using the extracted antler DNA as a template, amplification was performed using the RAA kit;

(3) Colloidal gold test strip detection: The RAA amplified product is diluted and added to the sample pad of the colloidal gold test strip for detection. When the C line is present, the T line has a band indicating a positive result, and the T line has no band indicating a negative result.

Preferably, the 50 μL reaction system of the kit in step (2) is as follows: 25 μL hydrolysis buffer, 3 μL 10 μM upstream primer, 3 μL 10 μM downstream primer, 10.5 μL deionized water, 6 μL DNA template, and 2.5 μL magnesium acetate.

Preferably, during the RAA amplification in step (2), the reaction temperature is 37° C.-41° C., and the reaction time is 20-40 min.

More preferably, during the RAA amplification in step (2), the reaction temperature is 37° C. and the reaction time is 30 min.

Preferably, the hybrid antler is a hybrid antler of a sika deer and a red deer.

The beneficial effects of the present invention are:

1. Aiming at the difficulty of identifying the hybrid deer of spotted deer with conventional mitochondrial DNA barcode, the present invention compares the whole genome sequences of sika deer, red deer and other closely related deer species, finds out the specific SNP sites that can be used to distinguish the sika deer and red deer, designs allele specific-PCR (AS-PCR) primers based on the SNP sites, establishes a colloidal gold test strip SNP typing detection method based on allele specific recombinase mediated isothermal amplification (AS-RAA), and provides a simple and effective scientific means for the identification of hybrid deer of spotted deer.

2. The present invention designs specific SNP primers based on recombinase-mediated isothermal nucleic acid amplification technology, and establishes a rapid detection kit for hybrid antler based on RAA, which can complete the amplification reaction at 37°C in 30 minutes. Combined with the colloidal gold test strip detection technology, it can quickly perform on-site detection of antler.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a PCR electrophoresis diagram of the Tm gradient specificity verification of Ce0141 primers (Note: M: DL2000 molecular weight standard; A: Red deer specific primer 014F1/R1; B: Sika deer specific primer 014F1/S1; a1: Red deer antler, 52°C; a2: Red deer antler, 54°C;

a3: antler of red deer, 56℃; a4: antler of red deer, 58℃; a5: antler of red deer, 60℃; a6: antler of red deer, 62℃; c1: antler of sika deer, 52℃; c2: antler of sika deer, 54℃; c3: antler of sika deer, 56℃; c4: antler of sika deer, 58℃; c5: antler of sika deer, 60℃; c6: antler of sika deer, 62℃; b: blank control);

Figure 2 is a Tm gradient specificity verification PCR electrophoresis diagram of Ce0142 primer (Note: M: DL2000 molecular weight standard; Aa1: red deer specific primer 014F2/R2, red deer antler, 54°C; Aa2: red deer specific primer 014F2/R2, red deer antler, 58°C; Aa3: red deer specific primer 014F2/R2, red deer antler, 62°C; Aa4: red deer specific primer 014F2/R2, red deer antler, 66°C; Ac1: red deer specific primer 014F2/R2, sika deer antler, 54°C; Ac2: red deer specific primer 014F2/R2, sika deer antler, 58°C; Ac3: red deer specific primer 014F2/R2, sika deer antler, 62°C; Ac4: red deer specific primer 014F2 /R2, sika deer antler, 66℃; Ba1: sika deer specific primer 014F2/S2, red deer antler, 54℃; Ba2: sika deer specific primer 014F2/S2, red deer antler, 58℃; Ba3: sika deer specific primer 014F2/S2, red deer antler, 62℃; Ba4: sika deer specific primer 014F2/S2, red deer antler, 66℃; Bc1: sika deer specific primer 014F2/S2, sika deer antler, 54℃; Bc2: sika deer specific primer 014F2/S2, sika deer antler, 58℃; Bc3: sika deer specific primer 014F2/S2, sika deer antler, 62℃; Bc4: sika deer specific primer 014F2/S2, sika deer antler, 66℃; b: blank control);

Figure 3 is a PCR electrophoresis diagram of the Tm gradient specificity verification of Ce1741 primers (Note: M: DL2000 molecular weight standard; Aa1: red deer specific primer 174F1/R1, red deer antler, 60°C; Aa2: red deer specific primer 174F1/R1, red deer antler, 64°C; Aa3: red deer specific primer 174F1/R1, red deer antler, 68°C; Ac1: red deer specific primer 174F1/R1, sika deer antler, 60°C; Ac2: red deer specific primer 174F1/R1, sika deer antler, 64°C; Ac3: red deer specific primer 174F1/R1 1/R1, sika deer antler, 68℃; Ba1: sika deer specific primer 174F1/S1, red deer antler, 60℃; Ba2: sika deer specific primer 174F1/S1, red deer antler, 64℃; Ba3: sika deer specific primer 174F1/S1, red deer antler, 68℃; Bc1: sika deer specific primer 174F1/S1, sika deer antler, 60℃; Bc2: sika deer specific primer 174F1/S1, sika deer antler, 64℃; Bc3: sika deer specific primer 174F1/S1, sika deer antler, 68℃; b: blank control);

Figure 4 is a PCR electrophoresis diagram of the Tm gradient specificity verification of Ce1745 primers (Note: M: DL2000 molecular weight standard; Aa1: red deer specific primer 174F5/R5, red deer antler, 62°C; Aa2: red deer specific primer 174F5/R5, red deer antler, 64°C; Aa3: red deer specific primer 174F5/R5, red deer antler, 66°C; Aa4: red deer specific primer 174F5/R5, red deer antler, 68°C; Aa5 : Red deer specific primer 174F5/R5, red deer antler, 70℃; Ac1: Red deer specific primer 174F5/R5, sika deer antler, 62℃; Ac2: Red deer specific primer 174F5/R5, sika deer antler, 64℃; Ac3: Red deer specific primer 174F5/R5, sika deer antler, 66℃; Ac4: Red deer specific primer 174F5/R5, sika deer antler, 68℃; Ac5: Red deer specific primer 174F5/ R5, sika deer antler, 70℃; Ba1: sika deer specific primer 174F5/S5, red deer antler, 62℃; Ba2: sika deer specific primer 174F5/S5, red deer antler, 64℃; Ba3: sika deer specific primer 174F5/S5, red deer antler, 66℃; Ba4: sika deer specific primer 174F5/S5, red deer antler, 68℃; Ba5: sika deer specific primer 174F5/S5, red deer antler, 70℃; Bc1: sika deer specific primer 174F5/S5, sika deer antler, 62°C; Bc2: sika deer specific primer 174F5/S5, sika deer antler, 64°C; Bc3: sika deer specific primer 174F5/S5, sika deer antler, 66°C; Bc4: sika deer specific primer 174F5/S5, sika deer antler, 68°C; Bc5: sika deer specific primer 174F5/S5, sika deer antler, 70°C; b: blank control);

FIG5 is a Tm gradient specificity verification electrophoresis diagram of Ce1742 and Ce1743 primers (Note: M: DL2000 molecular weight standard; (1): Ce1742; (2): Ce1743; Aa1: Red deer specific primer 174F2/R2 (174F3/R3), Red deer antler, 50°C; Aa2: Red deer specific primer 174F2/R2 (174F3/R3), Red deer antler, 55°C; Aa3: Red deer specific primer 174F2/R2 (174F3/R3), Red deer antler, 60°C; Aa4: Red deer specific primer 174F2/R2 (174F3/R3), Red deer antler, Antler, 65℃; Aa5: Red deer specific primers 174F2/R2 (174F3/R3), Red deer antler, 70℃; Ac1: Red deer specific primers 174F2/R2 (174F3/R3), Sika deer antler, 50℃; Ac2: Red deer specific primers 174F2/R2 (174F3/R3), Sika deer antler, 55℃; Ac3: Red deer specific primers 174F2/R2 (174F3/R3), Sika deer antler, 60℃; Ac4: Red deer specific primers 174F2/R2 (174F3/R3), Sika deer antler, 65℃; Ac5: Red deer specific primers 174 F2/R2 (174F3/R3), sika deer antler, 70℃; Ba1: sika deer specific primers 174F2/S2 (174F3/S3), red deer antler, 50℃; Ba2: sika deer specific primers 174F2/S2 (174F3/S3), red deer antler, 55℃; Ba3: sika deer specific primers 174F2/S2 (174F3/S3), red deer antler, 60℃; Ba4: sika deer specific primers 174F2/S2 (174F3/S3), red deer antler, 65℃; Ba5: sika deer specific primers 174F2/S2 (174F3/S3), red deer antler Antler, 70℃; Bc1: sika deer specific primers 174F2/S2 (174F3/S3), sika deer antler, 50℃; Bc2: sika deer specific primers 174F2/S2 (174F3/S3), sika deer antler, 55℃; Bc3: sika deer specific primers 174F2/S2 (174F3/S3), sika deer antler, 60℃; Bc4: sika deer specific primers 174F2/S2 (174F3/S3), sika deer antler, 65℃; Bc5: sika deer specific primers 174F2/S2 (174F3/S3), sika deer antler, 70℃; b: blank control);

FIG6 is a PCR electrophoresis diagram of Tm gradient specificity verification of Ce1744 and Ce1746 primers (Note: M: DL2000 molecular weight standard; (1): Ce1744; (2): Ce1746; Aa1: red deer specific primers 174F4/R4 (174F6/R6), red deer antler, 55°C; Aa2: red deer specific primers 174F4/R4 (174F6/R6), red deer antler, 60°C; Aa3: red deer specific primers 174F4/R4 (174F6/R6) , red deer antler, 65℃; Aa4: red deer specific primer 174F4/R4 (174F6/R6), red deer antler, 70℃; Ac1: red deer specific primer 174F4/R4 (174F6/R6), sika deer antler, 55℃; Ac2: red deer specific primer 174F4/R4 (174F6/R6), sika deer antler, 60℃; Ac3: red deer specific primer 174F4/R4 (174F6/R6), sika deer antler, 65℃; Ac4: red deer specific primer 174F4/R4 (174F6/R6), sika deer antler, 70℃; Ba1: sika deer specific primer 174F4/S4 (174F6/S6), red deer antler, 55℃; Ba2: sika deer specific primer 174F4/S4 (174F6/S6), red deer antler, 60℃; Ba3: sika deer specific primer 174F4/S4 (174F6/S6), red deer antler, 65℃; Ba4: sika deer specific primer 174F4/S4 (174F6/S6), Red deer antler, 70℃; Bc1: Sika deer specific primers 174F4/S4 (174F6/S6), Sika deer antler, 55℃; Bc2: Sika deer specific primers 174F4/S4 (174F6/S6), Sika deer antler, 60℃; Bc3: Sika deer specific primers 174F4/S4 (174F6/S6), Sika deer antler, 65℃; Bc4: Sika deer specific primers 174F4/S4 (174F6/S6), Sika deer antler, 70℃; b: blank control);

Figure 7 shows the test results of Ce0142 on 9 antler samples using RAA-colloidal gold test strips (Note: A: 014F2/R2; B: 014F2/S2; 1-3: pure red deer; 4-6: sika deer; 7-9: hybrid deer; b: blank control).

Detailed ways

The present invention provides a primer and a kit for detecting hybrid deer by using a colloidal gold test strip based on RAA amplification. The present invention is further described by way of examples, but the present invention is not limited to the following examples.

Experimental Example 1: Design of SNP primers for identifying hybrid velvet antlers

1. Primer design

The present invention designs primers according to the SNP sites obtained by screening, in order to identify hybrid velvet antlers. All primers are synthesized by Sangon Biotech (Shanghai) Co., Ltd.

Table 1 Information on SNP site AS-PCR primers

2. Analysis of test results

From the Tm gradient specificity verification PCR electrophoresis diagrams of different primers in Figures 1-6, it can be seen that the sika deer specific primer 014F1/R1 in primer Ce0141 has no obvious amplification at different temperatures (Figure 1), and the red deer specific primer 014F2/R2 and sika deer specific primer 014F2/S2 in primer Ce0142 are specific to red deer and sika deer, respectively (Figure 2). Primers Ce1741, Ce1742, Ce0143, Ce0144, Ce0145 and Ce0146 have no specificity for sika deer and red deer (Figures 3-6).

According to the SNP primer Ce0142 obtained by screening, the 5' end of the upstream primer is labeled with biotin, and the 5' end of the downstream primer is labeled with digoxigenin. All primers are synthesized by Sangon Biotech (Shanghai) Co., Ltd. The present invention designs three primers, including a universal primer and two specific primers, as shown in Table 2.

Table 2 Hybrid antler SNP primers

Primers name Sequence (5′-3′) 5′ modification Universal primer 014F2 ATAAGTAGCAACAAACTGGTAAAGAGCAA Bio Red deer specific primers 014R2 TTAAGGAGCTTGAGTGGGTTACATACACCG Dig Sika deer specific primers 014S2 TTAAGGAGCTTGAGTGGGTTACATACAGTA Dig

Experimental Example 2: Usage and Functional Verification of RAA Kit for Identifying Hybrid Deer Antlers

1. Sample DNA extraction

For the whole antler, rinse it three times with 75% ethanol, cut it into slices after washing, put it in an oven to dry, and then cut the dried antler into 1cm×1cm blocks; if it is a slice of dry antler, cut it directly into 1cm×1cm blocks. Put the processed block antler sample into the tissue grinder and grind it at a frequency of 30Hz for 60s. Transfer it to a centrifuge tube and store it at 4℃. Weigh 100mg of the fully ground antler tissue sample and extract DNA with the NucleoSpin FoodDNA extraction kit. Follow the instructions of the kit. For samples that are not easy to lyse, the lysis time can be extended by 30-60min.

2. RAA amplification

The extracted antler DNA was used as a template and amplified using the RAA kit (Jiangsu Qitian Gene Biotechnology Co., Ltd.). The 50 μL reaction system of the kit was as follows: 25 μL hydrolysis buffer, 3 μL 10 μM upstream primer, 3 μL 10 μM downstream primer, 10.5 μL deionized water, 6 μL DNA template, 2.5 μL magnesium acetate, and heated at 37°C for 30 min.

3. Colloidal gold test strip detection

Dilute the RAA amplified product 5 times, add 70 μL of the diluted sample to the sample pad of the colloidal gold test strip, observe the color of the T line and C line band, and determine whether it is a hybrid antler of the spotted horse.

4. Results Analysis

As shown in FIG7 , the results show that for the pure red deer samples of sequence numbers 1-3, only the T line of 014F2/R2 has a color band, for the pure sika deer antler samples of sequence numbers 4-6, only the T line of 014F2/S2 has a color band, and for the hybrid antler samples of 7-9, both the T lines of 014F2/R2 and 014F2/S2 have bands. Therefore, the product amplified by the AS-RAA primers designed in the present invention can be used to distinguish the hybrid antler of sika deer.

Although specific embodiments of the present invention have been described, it will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the scope or spirit of the present invention. Therefore, the present invention is intended to cover all such changes and modifications that fall within the scope of the appended claims and their equivalents.

Sequence Listing

<110> China Institute of Inspection and Quarantine

<120> A rapid detection kit for hybrid antler based on RAA amplification

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gaggagccaa gatggcagag gaatagggca 30

Claims (9)

1. A primer for detecting hybrid velvet antler based on the RAA method, characterized in that the sequence of the primer is as follows: Upstream primer 014F2: 5ˊ-ATAAGTAGCAACAAACTGGTAAAGAGCAA-3ˊ; Red deer specific downstream primer 014R2: 5ˊ-TTAAGGAGCTTGAGTGGGTTACATACACCG-3ˊ; Sika deer-specific downstream primer 014S2: 5ˊ-TTAAGGAGCTTGAGTGGGTTACATACAGTA-3ˊ; The hybrid antler is a hybrid antler of red deer and sika deer. 2. The primer according to claim 1, characterized in that the 5' end of the upstream primer is labeled with biotin, and the 5' end of the downstream primer is labeled with digoxigenin. 3. A kit for detecting hybrid antlers based on the RAA method, characterized in that the kit comprises the primers described in claim 2 and a colloidal gold detection test strip; the hybrid antlers are hybrid antlers of red deer and sika deer. 4. The kit according to claim 3 is characterized in that the detection line T line of the colloidal gold test strip is embedded with streptavidin, the quality control line C line is embedded with rabbit anti-goat secondary antibody, and the colloidal gold is combined with goat anti-digoxin antibody. 5 . The kit according to claim 3 , wherein the final concentration of the upstream primer and the downstream primer in the RAA kit is 10 μM. 6. The kit according to claim 3, characterized in that the reaction system is as follows: 25 μL hydrolysis buffer, 3 μL 10 μM upstream primer, 3 μL 10 μM downstream primer, 10.5 μL deionized water, 6 μL DNA template, and 2.5 μL magnesium acetate. 7. Use of the primers according to any one of claims 1 to 2 or the kit according to any one of claims 3 to 6 in the preparation of a reagent for detecting hybrid antlers; the hybrid antlers are hybrid antlers of red deer and sika deer. 8. A method for detecting hybrid velvet antlers using the kit according to any one of claims 3 to 6, comprising the following steps: (1) Sample DNA extraction: crush the antlers and extract DNA; (2) RAA amplification: using the extracted antler DNA as a template, amplification was performed using the RAA kit; (3) Colloidal gold test strip detection: dilute the RAA amplified product and drop it onto the sample pad of the colloidal gold test strip for detection. When the C line is present, the T line has a band indicating a positive result, and the T line has no band indicating a negative result. The hybrid antler is a hybrid antler of red deer and sika deer. 9. The method according to claim 8, characterized in that during the RAA amplification in step (2), the reaction temperature is 37°C-41°C and the reaction time is 20-40 min.