A primer set and kit for identifying the product breed of female deer based on SNP mutation detection technology and a method for identifying the same

Abstract

The application belongs to the technical field of species identification, and provides a primer set and kit for identifying the species source of female deer products based on SNP mutation detection technology and an identification method thereof, which comprises an upstream outer primer F68, an upstream inner primer F403w1, a downstream inner primer R450m6 and a downstream outer primer R615, the nucleotide sequence of the upstream outer primer F68 is shown as SEQ ID NO:1, the nucleotide sequence of the upstream inner primer F403w1 is shown as SEQ ID NO:2, the nucleotide sequence of the downstream inner primer R450m6 is shown as SEQ ID NO:3, and the nucleotide sequence of the downstream outer primer R615 is shown as SEQ ID NO:4. The above primer set can be used to specifically detect the authenticity of female deer products and whether they are hybrid varieties, and the parental origin thereof can be determined, thereby providing a detection technology for authentic identification of deer products and identification of hybrid varieties.

Description

Technical Field

[0001] This invention relates to the field of species identification technology, and in particular to a primer set and kit for identifying the germplasm of female deer products based on SNP mutation detection technology, as well as the identification method thereof. Background Technology

[0002] Deer antlers have significant medicinal value and are considered precious animal-derived medicinal materials. Among these, only sika deer and red deer are considered the authentic sources of medicinal deer products. Sika deer and red deer both belong to the order Artiodactyla, family Cervidae, and genus Cervidae. Their divergence occurred relatively recently, and their evolutionary relationship remains inconclusive. Current animal hybridization breeding techniques primarily utilize sika deer. The hybrid deer (male sika deer, female sika deer) has demonstrated extremely high economic benefits in antler production, making hybrid deer an important source of income in the deer industry. However, both traditional Chinese medicine and modern instrumental analysis indicate that hybrid deer products have lower or significantly lower medicinal value than purebred sika deer, and their market prices also differ considerably. In recent years, hybrid deer products have gradually entered the market, with some even being sold as purebred sika deer products for high profits. Current testing of deer products mainly focuses on antlers, largely based on DNA analysis techniques. These techniques are generally sufficient for identifying the authenticity of antlers and their parentage. However, with the increase in the number of hybrid deer herds and the higher slaughter rate, hybrid deer products such as venison, blood, heart, kidney, tendons, and tails are gradually entering the market. The sex of these products cannot be determined from their appearance, and the parentage of female hybrid deer remains unresolved.

[0003] Single nucleotide polymorphisms (SNPs) are third-generation molecular markers widely used in genetic diversity analysis of biological populations and marker-assisted selection. SNP sites are ubiquitous in the human and animal genomes and are DNA sequence polymorphisms caused by variations in a single nucleotide. Species-specific SNP sites can be used for species identification. There are various methods for detecting SNP sites. PCR-based detection techniques include restriction fragment length polymorphism (PCR-RFLP), single strand conformation polymorphism (SSCP), direct PCR analysis, allele-specific amplification, and quantitative real-time PCR. These techniques have different advantages and disadvantages for SNP detection. For example, the use of restriction enzymes results in a cumbersome detection process, long processing time, and high cost. Sequencing technology increases both time and economic costs, making it inconvenient for routine detection. Four-primer amplification inhibited mutation PCR is a method developed at the level of ordinary PCR for detecting and classifying various SNP sites. It designs two detection inner primers with opposite extension directions and two control outer primers for each SNP site. By using the presence, absence and size of the amplification products of the four primers, different genotypes can be directly distinguished on the electrophoretic pattern. It has been applied in the fields of medicine and economic animal breeding. However, there is currently no method to identify the germplasm and parentage of female deer products using SNP analysis technology. Summary of the Invention

[0004] The purpose of this invention is to provide a primer set and kit for identifying the germplasm of female deer products based on SNP mutation detection technology, as well as the identification method thereof. The primer set can specifically detect the authenticity of female deer products and whether they are hybrid varieties, and can determine their parentage, thus providing detection technology for the authenticity identification and hybrid variety identification of female deer products.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a primer set for identifying the germplasm of female deer products based on SNP mutation detection technology, comprising an upstream outer primer F68, an upstream inner primer F403w1, a downstream inner primer R450m6, and a downstream outer primer R615. The nucleotide sequence of the upstream outer primer F68 is shown in SEQ ID NO: 1, the nucleotide sequence of the upstream inner primer F403w1 is shown in SEQ ID NO: 2, the nucleotide sequence of the downstream inner primer R450m6 is shown in SEQ ID NO: 3, and the nucleotide sequence of the downstream outer primer R615 is shown in SEQ ID NO: 4. Details are as follows:

[0007] F68 (SEQ ID NO: 1): 5'-GCTGATGCTACACACACGGATG-3';

[0008] F403w1 (SEQ ID NO: 2): 5'-GTCTTGACGAGTGAATCCATACATAG-3';

[0009] R450m6 (SEQ ID NO: 3): 5'-TCAAGGTGTCCAATGTCAGGCA-3';

[0010] R615 (SEQ ID NO: 4): 5'-CTCACAGTTGCCTTTGTCATCCT-3'.

[0011] The present invention also provides a kit for identifying the germplasm of female deer products based on SNP mutation detection technology, including the above-mentioned primer set and detection reagent.

[0012] Preferably, the detection reagent includes Taq enzyme, 10× buffer, dNTP mixture, MgCl2 and ddH2O.

[0013] Preferably, the final concentrations of the upstream outer primer F68, the upstream inner primer F403w1, the downstream inner primer R450m6, and the downstream outer primer R615 in the PCR system are independently 0.25 μmol / L, 0.25 μmol / L, 0.4 μmol / L, and 0.25 μmol / L.

[0014] This invention also provides a method for identifying the germplasm of female deer products using the above-mentioned primer set or the above-mentioned kit, comprising the following steps:

[0015] (1) Extract genomic DNA from the female deer products to be tested;

[0016] (2) Using the genomic DNA extracted in step (1) as a template, PCR amplification was performed using the above primer set to obtain the amplification product;

[0017] (3) Perform electrophoretic detection on the amplification products obtained in step (2); when the amplification products have only two electrophoretic bands of 549bp and 213bp, they are determined to be purebred sika deer; when the amplification products have only two electrophoretic bands of 549bp and 383bp, they are determined to be purebred red deer; when the amplification products have only three electrophoretic bands of 549bp, 213bp and 383bp, they are determined to be hybrid offspring of red deer; when the amplification products have only one electrophoretic band of 549bp, they are determined to be deer breeds other than red deer and sika deer.

[0018] Preferably, the female deer products to be tested in step (1) include venison, deer blood, deer heart, deer kidney, deer tendon, deer tail, deer placenta and deer placenta powder.

[0019] Preferably, the PCR amplification reaction system described in step (2), in 20 μL, comprises the following components: 10× buffer 2.0 μL, 25 mmol / L dNTP mixture 1.6 μL, 25 mmol / L MgCl2 2.0 μL, 10 μmol / L upstream outer primer F68 0.5 μL, 10 μmol / L upstream inner primer F403w1 0.5 μL, 10 μmol / L downstream inner primer R450m6 0.8 μL, 10 μmol / L downstream outer primer R615 0.5 μL, 5 U / μL Taq enzyme 0.4 μL, template DNA 3.0 μL, and ddH2O 8.7 μL.

[0020] Preferably, the PCR amplification program in step (2) is as follows: pre-denaturation at 95℃ for 5-10 min; denaturation at 94℃ for 30-50 s, annealing at 58-65℃ for 35-60 s, extension at 72℃ for 40-300 s, and denaturation, annealing, and extension are performed for 30-36 cycles; final extension at 72℃ for 5-12 min.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] This invention provides a method for identifying the hybrid offspring of female sika deer, filling the current technological gap in the identification of hybrid deer products, especially hybrid female deer germplasm. It represents a significant supplement and breakthrough to current deer product authenticity detection technologies. Based on a common PCR and electrophoresis detection platform, this invention meets the equipment requirements of all molecular detection laboratories, facilitating detection applications. This invention can effectively identify products that may originate from female deer, excluding antlers and deer penis. The method is simple, efficient, and low-cost, providing technical support for food and drug regulatory departments, improving the regulatory level of the traditional Chinese medicine market, and contributing to market stability. Attached Figure Description

[0023] Figure 1 The results are for ZFX gene amplification, where M is DNA Marker C (100-1200bp), lanes 1-2 are sika deer, lanes 3-4 are red deer, and lanes 5-6 are sika deer × red deer hybrid offspring.

[0024] Figure 2 The sequencing results of different genotypes at the X428 locus of the ZFX gene are as follows: GG represents sika deer, TT represents red deer, and GT represents the offspring of a sika deer × red deer hybrid.

[0025] Figure 3The results of genotyping at the X428 site of the ZFX gene were obtained by four-primer mismatch PCR. In this study, M is DNA Marker C (100-1200 bp), lane 1 is the negative control, lane 2 is the reindeer, lanes 3-4 are the sika deer, lanes 5-6 are the red deer, and lanes 7-8 are the offspring of the sika deer × red deer hybrid.

[0026] Figure 4 To identify commercially available deer products using the method of this invention, M represents DNA Marker C (100-1200 bp), lanes 1-4 are red deer, lanes 5-6 are sika deer × red deer hybrids, lanes 7-9 are sika deer, lanes 10-11 and 13 are red deer, lane 12 is a sika deer × red deer hybrid, lanes 14-18 and 20, 24 are sika deer, lane 19 is a sika deer × red deer hybrid, lanes 21 and 23 are reindeer, and lane 22 is red deer. Detailed Implementation

[0027] This invention provides a primer set, kit, and method for identifying the genetic origin of female deer products based on SNP mutation detection technology. In mammals, sex is mainly determined by the X and Y sex chromosomes, with XX and XY chromosome combinations representing female and male, respectively. The sex-determining gene SRY is a unique gene located on the Y chromosome and is commonly used for animal sex identification. In addition, genes that can distinguish between X and Y sex chromosomes include the zinc finger protein gene ZFX / Y, the enamel gene AMELX / Y, the SRY-box inclusion protein Sox9 gene, and the steroidogenic factor SF1 gene. The zinc finger protein gene ZFX / Y exists on both the X and Y chromosomes, but they are not homologous regions. These two genes on the X and Y chromosomes have inherited sex and species-specific information during evolution, exhibiting sequence differences. The ZFX / Y gene shows greater variation than the ZFX gene, while the ZFX gene is considered to be inactivated by the X chromosome and is more conserved during evolution, thus better representing species specificity. Female gametes (XX) are inherited from the X chromosomes of both parents and carry the specific ZFX gene sequences of zinc finger proteins from different parents. Therefore, the ZFX genes of red deer and sika deer can be analyzed and compared to find species-specific single nucleotide polymorphism (SNP) sites in the sequence differences and establish corresponding detection methods for the parental origin of hybrid female deer, so as to trace the parental origin of female sika deer × red deer hybrid offspring.

[0028] This invention provides a primer set for identifying the germplasm of female deer products based on SNP mutation detection technology, comprising an upstream outer primer F68, an upstream inner primer F403w1, a downstream inner primer R450m6, and a downstream outer primer R615. The nucleotide sequence of the upstream outer primer F68 is shown in SEQ ID NO: 1, the nucleotide sequence of the upstream inner primer F403w1 is shown in SEQ ID NO: 2, the nucleotide sequence of the downstream inner primer R450m6 is shown in SEQ ID NO: 3, and the nucleotide sequence of the downstream outer primer R615 is shown in SEQ ID NO: 4. Details are as follows:

[0029] F68 (SEQ ID NO: 1): 5'-GCTGATGCTACACACACGGATG-3';

[0030] F403w1 (SEQ ID NO: 2): 5'-GTCTTGACGAGTGAATCCATACATAG-3';

[0031] R450m6 (SEQ ID NO: 3): 5'-TCAAGGTGTCCAATGTCAGGCA-3';

[0032] R615 (SEQ ID NO: 4): 5'-CTCACAGTTGCCTTTGTCATCCT-3'.

[0033] The present invention also provides a kit for identifying the germplasm of female deer products based on SNP mutation detection technology, including the above-mentioned primer set and detection reagent.

[0034] In this invention, the detection reagents include Taq enzyme, 10× buffer, dNTP mixture, MgCl2 and ddH2O.

[0035] In this invention, the final concentrations of the upstream outer primer F68, the upstream inner primer F403w1, the downstream inner primer R450m6, and the downstream outer primer R615 in the PCR system are independently 0.25 μmol / L, 0.25 μmol / L, 0.4 μmol / L, and 0.25 μmol / L.

[0036] This invention also provides a method for identifying the germplasm of female deer products using the above-mentioned primer set or the above-mentioned kit, comprising the following steps:

[0037] (1) Extract genomic DNA from the female deer products to be tested;

[0038] (2) Using the genomic DNA extracted in step (1) as a template, PCR amplification was performed using the above primer set to obtain the amplification product;

[0039] (3) Perform electrophoretic detection on the amplification products obtained in step (2); when the amplification products have only two electrophoretic bands of 549bp and 213bp, they are determined to be purebred sika deer; when the amplification products have only two electrophoretic bands of 549bp and 383bp, they are determined to be purebred red deer; when the amplification products have only three electrophoretic bands of 549bp, 213bp and 383bp, they are determined to be hybrid offspring of red deer; when the amplification products have only one electrophoretic band of 549bp, they are determined to be deer breeds other than red deer and sika deer.

[0040] In this invention, genomic DNA is first extracted from the female deer product to be tested, preferably using an animal tissue DNA extraction kit.

[0041] In this invention, the female deer products to be tested include venison, deer blood, deer heart, deer kidney, deer tendon, deer tail, deer placenta, and deer placenta powder.

[0042] In this invention, after obtaining the genomic DNA of the female deer product to be tested, the DNA quality is detected, thus enabling DNA quality detection D... 260 / 280 The value should be between 1.8 and 2.0, preferably 1.9.

[0043] In this invention, it is preferable to perform sex identification before identifying the female deer products to be tested. Deer products identified as male are identified using mitochondrial genes and Y chromosome-specific genes to determine the species and hybrid parentage. Deer products identified as female continue with subsequent testing steps.

[0044] In this invention, the extracted genomic DNA is used as a template, and the primer set described above is used for PCR amplification to obtain the amplification product.

[0045] In this invention, the PCR amplification reaction system, in 20 μL, comprises the following components: 10× buffer 2.0 μL, 25 mmol / L dNTP mixture 1.6 μL, 25 mmol / L MgCl2 2.0 μL, 10 μmol / L upstream outer primer F68 0.5 μL, 10 μmol / L upstream inner primer F403w1 0.5 μL, 10 μmol / L downstream inner primer R450m6 0.8 μL, 10 μmol / L downstream outer primer R615 0.5 μL, 5 U / μL Taq enzyme 0.4 μL, template DNA 3.0 μL, and ddH2O 8.7 μL.

[0046] In this invention, the PCR amplification program is as follows: pre-denaturation at 95℃ for 5-10 min; denaturation at 94℃ for 30-50 s, annealing at 58-65℃ for 35-60 s, extension at 72℃ for 40-300 s, with 30-36 cycles of denaturation, annealing, and extension; and final extension at 72℃ for 5-12 min. A preferred PCR amplification program is as follows: pre-denaturation at 95℃ for 5 min; denaturation at 94℃ for 35 s, annealing at 61℃ for 40 s, extension at 72℃ for 1 min, with 35 cycles of denaturation, annealing, and extension; and final extension at 72℃ for 10 min.

[0047] In this invention, the amplification product obtained in step (2) is subjected to electrophoretic detection. The initial concentration of agarose during electrophoretic detection is 1.5 to 2.5 wt%, preferably 1.7 to 2.3 wt%, and more preferably 1.9 to 2.1 wt%. Preferably, the agarose gel used for electrophoretic detection also contains one ten-thousandth of the nucleic acid dye Gelred.

[0048] In this invention, after electrophoresis detection, when the amplification product has only two electrophoretic bands of 549bp and 213bp, it is determined to be a purebred sika deer; when the amplification product has only two electrophoretic bands of 549bp and 383bp, it is determined to be a purebred red deer; when the amplification product has only three electrophoretic bands of 549bp, 213bp and 383bp, it is determined to be a hybrid offspring of red deer; when the amplification product has only one electrophoretic band of 549bp, it is determined to be a deer breed other than red deer and sika deer.

[0049] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0050] Example 1

[0051] This embodiment provides ZFX gene sequence-specific loci for sika deer, red deer, and their hybrid offspring. The specific steps are as follows:

[0052] (1) Five blood samples were collected from sika deer, red deer, and their hybrid offspring (all samples included male and female individuals with complete pedigree records from the breeding farm and confirmed by DNA barcoding). Genomic DNA was extracted according to the instructions of the animal tissue kit (purchased from Hangzhou Xinjing Biotechnology Co., Ltd.). Primers were designed based on the red deer ZFX gene sequence (KP 257294.1) in GenBank and compared with the ZFX gene sequence on the X chromosome in the whole genome sequence of red deer (CM008041.1:c153615887-153602265). The primer sequences are as follows:

[0053] Upstream primer ZFX1F (SEQ ID NO: 5): 5′-CCACAAGAACCAAACTCATT-3′;

[0054] Downstream primer ZFX1R (SEQ ID NO: 6): 5′-TTGATAACTTCAGGGCAAG-3′.

[0055] The ZFX gene sequence of sika deer, red deer, and their hybrid offspring was amplified by PCR. The basal PCR reaction volume was 20 μL, and the reaction program was as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 40 s, 72℃ extension for 2 min, for 35 cycles of denaturation, annealing, and extension, followed by a final extension at 72℃ for 10 min. The positive PCR product was approximately 730 bp. The results are shown in the figure. Figure 1 After 1.5 wt% agarose gel electrophoresis, positive products were sent to Hangzhou Qingke Biotechnology Co., Ltd. for bidirectional sequencing. The matching of sequencing peaks and sequences was checked, and the sequences were edited, assembled, and compared using Editseq software. MegAlign software was used to compare the sequences obtained from sika deer, red deer, and hybrid deer. The results showed that the sequences of the five sika deer individuals were completely identical, and the sequences of the five red deer individuals were also completely identical. However, there was a specific difference between the two species: a SNP site (GT) at X428. This site showed heterozygosity in the hybrid offspring, consistent with the sequencing results of the hybrid deer. Sika deer were homozygous for the GG genotype, red deer were homozygous for the TT genotype, and the female hybrid offspring of sika deer and red deer were heterozygous for the GT genotype. Figure 2 As shown.

[0056] This invention establishes a PCR detection technique for a four-primer amplification-restricted mutant system based on the X428 mutation site. A positive control primer is also introduced to form a dual PCR system. When designing the inner primers, the 3' ends of both the upstream and downstream inner primers are positioned at the X428 site, pairing with two different genotypes respectively. Simultaneously, a pair of reference outer primers are designed at the same conserved sequence position shared by sika deer and red deer. To improve primer specificity, a CA weak mismatch is introduced at the penultimate position of the 3' end of the upstream inner primer for primer optimization. Similarly, a GT weak mismatch is introduced at the penultimate position of the 3' end of the downstream inner primer, and a GG weak mismatch is introduced at the 5th position of the 5' end of the primers. The designed primer sequences are shown in Table 1.

[0057] Table 1 Primer sequences

[0058]

[0059] Note: Underlined bases indicate the introduction of mismatched bases.

[0060] The genotyping results of the zinc finger protein ZFX gene at the X428 locus in female red deer, sika deer, and dappled horse hybrid females using the above primer set are shown below. Figure 3 .

[0061] Example 2

[0062] This embodiment provides a method for identifying the germplasm of female deer products based on SNP mutation detection technology, including the following steps:

[0063] (1) Genomic DNA of the deer products to be tested was extracted using an animal tissue DNA extraction kit (purchased from Hangzhou Xinjing Biotechnology Co., Ltd.), and the DNA quality was tested.

[0064] (2) Using the genomic DNA extracted in step (1) as a template, PCR amplification was performed using species sex identification primers to obtain amplification products. Species sex identification was performed in accordance with the literature (Ennis S, Gallagher TF. A PCR-based sex-determination assay in cattle based on the bovine amelogenin locus[J]. Animal Genetics, 1994, 25(6):425.).

[0065] (3) Using the genomic DNA of the female deer product extracted in step (2) as a template, PCR amplification was performed using the following primer set to obtain the amplification product.

[0066] F68 (SEQ ID NO: 1): 5'-GCTGATGCTACACACACGGATG-3';

[0067] F403w1 (SEQ ID NO: 2): 5'-GTCTTGACGAGTGAATCCATACATAG-3';

[0068] R450m6 (SEQ ID NO: 3): 5'-TCAAGGTGTCCAATGTCAGGCA-3';

[0069] R615 (SEQ ID NO: 4): 5'-CTCACAGTTGCCTTTGTCATCCT-3'.

[0070] The PCR amplification reaction system, in 20 μL, includes the following components: 2.0 μL of 10× buffer, 1.6 μL of 25 mmol / L dNTP mixture, 2.0 μL of 25 mmol / L MgCl2, 0.5 μL of 10 μmol / L upstream outer primer F68, 0.5 μL of 10 μmol / L upstream inner primer F403w1, 0.8 μL of 10 μmol / L downstream inner primer R450m6, 0.5 μL of 10 μmol / L downstream outer primer R615, 0.4 μL of 5 U / μL Taq enzyme, 3.0 μL of template DNA, and 8.7 μL of ddH2O.

[0071] The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 35 s, 61℃ annealing for 40 s, 72℃ extension for 1 min, with 35 cycles of denaturation, annealing, and extension; and a final extension at 72℃ for 10 min.

[0072] (4) The amplification product obtained in step (3) is detected by agarose gel electrophoresis. The initial concentration of agarose during the agarose gel electrophoresis is 1.5 wt%, and the agarose gel also contains 0.01% nucleic acid dye Gelred.

[0073] (5) When the amplification product has only two electrophoretic bands of 549bp and 213bp, it is determined to be a purebred sika deer; when the amplification product has only two electrophoretic bands of 549bp and 383bp, it is determined to be a purebred red deer; when the amplification product has only three electrophoretic bands of 549bp, 213bp and 383bp, it is determined to be a hybrid offspring of red deer; when the amplification product has only one electrophoretic band of 549bp, it is determined to be a deer breed other than red deer and sika deer.

[0074] Example 3

[0075] This embodiment provides a method for identifying the germplasm of female deer products based on SNP mutation detection technology, including the following steps:

[0076] (1) Genomic DNA of the deer products to be tested was extracted using an animal tissue DNA extraction kit (purchased from Hangzhou Xinjing Biotechnology Co., Ltd.), and sex was determined using the method for identifying species sex in Example 2. The female deer products obtained were then further identified.

[0077] (2) Using the genomic DNA of the female deer product extracted above as a template, PCR amplification was performed using the following primer set to obtain the amplification product.

[0078] F68 (SEQ ID NO: 1): 5'-GCTGATGCTACACACACGGATG-3';

[0079] F403w1 (SEQ ID NO: 2): 5'-GTCTTGACGAGTGAATCCATACATAG-3';

[0080] R450m6 (SEQ ID NO: 3): 5'-TCAAGGTGTCCAATGTCAGGCA-3';

[0081] R615 (SEQ ID NO: 4): 5'-CTCACAGTTGCCTTTGTCATCCT-3'.

[0082] The PCR amplification reaction system, in 20 μL, includes the following components: 2.0 μL of 10× buffer, 1.6 μL of 25 mmol / L dNTP mixture, 2.0 μL of 25 mmol / L MgCl2, 0.5 μL of 10 μmol / L upstream outer primer F68, 0.5 μL of 10 μmol / L upstream inner primer F403w1, 0.8 μL of 10 μmol / L downstream inner primer R450m6, 0.5 μL of 10 μmol / L downstream outer primer R615, 0.4 μL of 5 U / μL Taq enzyme, 3.0 μL of template DNA, and 8.7 μL of ddH2O.

[0083] The PCR amplification program was as follows: 95℃ pre-denaturation for 6 min; 94℃ denaturation for 30 s, 60℃ annealing for 40 s, 72℃ extension for 2 min, and 30 cycles of denaturation, annealing, and extension; and a final extension at 72℃ for 8 min.

[0084] (3) The amplification products obtained above are detected by agarose gel electrophoresis. The initial concentration of agarose during the agarose gel electrophoresis is 2wt%, and the agarose gel also contains 0.01% nucleic acid dye Gelred.

[0085] (4) When the amplification product has only two electrophoretic bands of 549bp and 213bp, it is determined to be a purebred sika deer; when the amplification product has only two electrophoretic bands of 549bp and 383bp, it is determined to be a purebred red deer; when the amplification product has only three electrophoretic bands of 549bp, 213bp and 383bp, it is determined to be a hybrid offspring of red deer; when the amplification product has only one electrophoretic band of 549bp, it is determined to be a deer breed other than red deer and sika deer.

[0086] Example 4

[0087] This embodiment provides a method for identifying the germplasm of female deer products based on SNP mutation detection technology, including the following steps:

[0088] (1) Genomic DNA of the deer products to be tested was extracted using an animal tissue DNA extraction kit (purchased from Hangzhou Xinjing Biotechnology Co., Ltd.), and sex was determined using the method for identifying species sex in Example 2. The female deer products obtained were then further identified.

[0089] (2) Using the genomic DNA of the female deer product extracted above as a template, PCR amplification was performed using the following primer set to obtain the amplification product.

[0090] F68 (SEQ ID NO: 1): 5'-GCTGATGCTACACACACGGATG-3';

[0091] F403w1 (SEQ ID NO: 2): 5'-GTCTTGACGAGTGAATCCATACATAG-3';

[0092] R450m6 (SEQ ID NO: 3): 5'-TCAAGGTGTCCAATGTCAGGCA-3';

[0093] R615 (SEQ ID NO: 4): 5'-CTCACAGTTGCCTTTGTCATCCT-3'.

[0094] The PCR amplification reaction system, in 20 μL, includes the following components: 2.0 μL of 10× buffer, 1.6 μL of 25 mmol / L dNTP mixture, 2.0 μL of 25 mmol / L MgCl2, 0.5 μL of 10 μmol / L upstream outer primer F68, 0.5 μL of 10 μmol / L upstream inner primer F403w1, 0.8 μL of 10 μmol / L downstream inner primer R450m6, 0.5 μL of 10 μmol / L downstream outer primer R615, 0.4 μL of 5 U / μL Taq enzyme, 3.0 μL of template DNA, and 8.7 μL of ddH2O.

[0095] The PCR amplification program was as follows: 95℃ pre-denaturation for 8 min; 94℃ denaturation for 45 s, 62℃ annealing for 60 s, 72℃ extension for 1.5 min, with 35 cycles of denaturation, annealing, and extension; and a final extension at 72℃ for 12 min.

[0096] (3) The amplification products obtained above are detected by agarose gel electrophoresis. The initial concentration of agarose during the agarose gel electrophoresis is 2.5 wt%, and the agarose gel also contains 0.01% nucleic acid dye Gelred.

[0097] (4) When the amplification product has only two electrophoretic bands of 549bp and 213bp, it is determined to be a purebred sika deer; when the amplification product has only two electrophoretic bands of 549bp and 383bp, it is determined to be a purebred red deer; when the amplification product has only three electrophoretic bands of 549bp, 213bp and 383bp, it is determined to be a hybrid offspring of red deer; when the amplification product has only one electrophoretic band of 549bp, it is determined to be a deer breed other than red deer and sika deer.

[0098] Experimental Example 1

[0099] Using the detection method for identifying the germplasm components of hybrid deer established in this invention, combined with DNA barcoding (Wu Y, Yang Y, Liu M, Wang B, Wang Y, Wang H. Applying COI Barcode to Identify Animal Origin of Food. J Food Sci. 2019, 84(6):1256-1265), 40 commercially available deer products were used to identify their authenticity and germplasm. The detection process for 24 of these products is as follows:

[0100] (1) Genomic DNA of the female deer products to be tested was extracted using an animal tissue DNA extraction kit (purchased from Hangzhou Xinjing Biotechnology Co., Ltd.).

[0101] (2) Using the genomic DNA extracted in step (1) as a template, PCR amplification was performed using the following DNA barcode primer set to obtain the amplification product:

[0102] Upstream primer 1 (SEQ ID NO: 7): 5'-TGTAAAACGACGGCCAGTCGACTAATCATAAAGATATCGGCAC-3';

[0103] Upstream primer 2 (SEQ ID NO: 8): 5'-TGTAAAACGACGGCCAGTCAACCAACCACAAAGACATTGGCAC-3';

[0104] Downstream primer 1 (SEQ ID NO: 9): 5'-CAGGAAACAGCTATGACACTTCAGGGTGACCGAAGAATCAGAA-3';

[0105] Downstream primer 2 (SEQ ID NO: 10): 5'-CAGGAAACAGCTATGACACCTCAGGGTGTCCGAARAAYCARAA-3'.

[0106] The PCR amplification reaction system, in 20 μL, includes the following components: 2.0 μL of 10× buffer, 1.6 μL of 25 mmol / L dNTP mixture, 1.6 μL of 25 mmol / L MgCl2, 10.4 μL of 10 μmol / L upstream primer, 20.4 μL of 10 μmol / L upstream primer, 10.4 μL of 10 μmol / L downstream primer, 20.4 μL of 10 μmol / L downstream primer, 0.4 μL of 5 U / μL Taq enzyme, 3.0 μL (50 ng) template DNA, and 9.8 μL of ddH2O.

[0107] The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 40 s, 50℃ annealing for 30 s, 72℃ extension for 1 min, and 30 cycles of denaturation, annealing, and extension; and a final extension at 72℃ for 10 min.

[0108] (3) The amplification product obtained in step (2) is detected by agarose gel electrophoresis. The initial concentration of agarose during the agarose gel electrophoresis is 1.5 wt%, and the agarose gel also contains 0.01% nucleic acid dye Gelred.

[0109] (4) When the amplification product produces a bright 700bp electrophoresis band, the PCR product is sent to Hangzhou Qingke Biotechnology Co., Ltd. for bidirectional sequencing. The sequencing results are compared with the BLAST species database.

[0110] (5) The identification results obtained using the identification method of the present invention are as follows: Figure 4 As shown in Table 2, because DNA barcoding uses mitochondrial DNA genes to identify the genetic origin of deer products, it can only identify hybrid offspring as sika deer or red deer. Figure 4 As shown in Table 2, this invention can identify parental lineage by detecting the X428 site of the specific ZFX gene in sika deer and red deer. It has advantages such as simplicity, convenience, and meeting the instrument platform requirements of most testing units. It is an important supplement to deer product identification and traceability technology, fills the gap in parental lineage traceability detection technology for female hybrid deer, and provides technical support for the identification of female hybrid offspring of sika deer, which is of great significance.

[0111] Table 2. Identification Results

[0112]

[0113]

[0114] Note: "-" indicates different batches of the same sample.

[0115] As can be seen from the above embodiments and experimental examples, the present invention provides a primer set and kit for identifying the germplasm of female deer products based on SNP mutation detection technology, as well as the identification method thereof. The primer set can specifically detect the authenticity of female deer products and whether they are hybrid varieties, and can determine their parental origin, providing detection technology for the authenticity identification of female deer products and the identification of hybrid varieties.

[0116] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A primer set for identifying the germplasm of female deer products based on SNP mutation detection technology, characterized in that, It includes an upstream outer primer F68, an upstream inner primer F403w1, a downstream inner primer R450m6, and a downstream outer primer R615. The nucleotide sequence of the upstream outer primer F68 is shown in SEQ ID NO: 1, the nucleotide sequence of the upstream inner primer F403w1 is shown in SEQ ID NO: 2, the nucleotide sequence of the downstream inner primer R450m6 is shown in SEQ ID NO: 3, and the nucleotide sequence of the downstream outer primer R615 is shown in SEQ ID NO:

4.

2. A kit for identifying the germplasm of female deer products based on SNP mutation detection technology, characterized in that, Includes the primer set and detection reagent as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The detection reagents include Taq enzyme, 10× buffer, dNTP mixture, MgCl2 and ddH2O.

4. The reagent kit according to claim 2, characterized in that, The final concentrations of the upstream outer primer F68, upstream inner primer F403w1, downstream inner primer R450m6, and downstream outer primer R615 in the PCR system were independently 0.25 μmol / L, 0.25 μmol / L, 0.4 μmol / L, and 0.25 μmol / L.

5. A method for identifying the germplasm of female deer products using the primer set of claim 1 or the kit of claim 2, characterized in that, Includes the following steps: (1) Extract genomic DNA from the female deer products to be tested; (2) Using the genomic DNA extracted in step (1) as a template, PCR amplification was performed using the primer set described in claim 1 to obtain the amplification product; (3) Perform electrophoretic detection on the amplification products obtained in step (2); when the amplification products have only two electrophoretic bands of 549bp and 213bp, they are determined to be purebred sika deer; when the amplification products have only two electrophoretic bands of 549bp and 383bp, they are determined to be purebred red deer; when the amplification products have only three electrophoretic bands of 549bp, 213bp and 383bp, they are determined to be hybrid offspring of red deer; when the amplification products have only one electrophoretic band of 549bp, they are determined to be deer breeds other than red deer and sika deer.

6. The method for identifying the germplasm of female deer products according to claim 5, characterized in that, The female deer products to be tested in step (1) include venison, deer blood, deer heart, deer kidney, deer tendon, deer tail, deer placenta and deer placenta powder.

7. The method for identifying the germplasm of female deer products according to claim 5, characterized in that, The PCR amplification reaction system described in step (2), in 20 μL, includes the following components: 2.0 μL of 10× buffer, 1.6 μL of 25 mmol / L dNTP mixture, 2.0 μL of 25 mmol / L MgCl2, 0.5 μL of 10 μmol / L upstream outer primer F68, 0.5 μL of 10 μmol / L upstream inner primer F403w1, 0.8 μL of 10 μmol / L downstream inner primer R450m6, 0.5 μL of 10 μmol / L downstream outer primer R615, 0.4 μL of 5 U / μL Taq enzyme, 3.0 μL of template DNA, and 8.7 μL of ddH2O.

8. The method for identifying the germplasm of female deer products according to claim 5, characterized in that, The PCR amplification program in step (2) is as follows: pre-denaturation at 95℃ for 5-10 min; denaturation at 94℃ for 30-50 s, annealing at 58-65℃ for 35-60 s, extension at 72℃ for 40-300 s, and denaturation, annealing, and extension are performed for 30-36 cycles; final extension at 72℃ for 5-12 min.

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