Multiplex amplification kit for simultaneous amplification of 20 horse STR loci and its application

Abstract

The present invention discloses a composite amplification kit for simultaneously amplifying 20 horse STR loci and its application. The kit comprises specific amplification primers for amplifying the following 20 STR loci; wherein the 20 STR loci are: HTG6, HTG7, COR22, CA425, HTG4, COR82, LEX54, COR69, AHT5, HMS3, HMS1, HMS6, HMS7, COR58, HTG10, ASB17, VHL20, HMS2, ASB2, and LEX34. Compared with the prior art, the present invention has the following advantages: (1) the kit can simultaneously amplify and detect 20 horse STR loci in a single tube, making it the kit with the most detection loci among similar products; (2) the selected loci are distributed on different chromosomes as much as possible, and there is no genetic linkage between them, thereby improving the overall recognition ability of the kit; (3) the specific amplification primers do not interfere with each other, no non-specific peaks are generated, and the kit has the characteristics of strong specificity, high resolution, accurate typing, high sensitivity, etc., and can fully meet the various needs of horse paternity testing, individual identification, sex determination, etc.

Description

Technical Field

[0001] The present invention belongs to the technical field of forensic genetics and relates to a method for horse paternity testing and individual identification, in particular to a composite amplification kit for simultaneously amplifying 20 horse STR loci and its application. Background Art

[0002] Horses are classified in animal taxonomy as follows: Vertebrata, Mammalia, Perissodactyla, Equidae, Equus, Equus caballus. They are herbivorous animals. Two subspecies exist: the domestic horse and the Przewalski's horse. At the end of 2011, my country's horse population was approximately 6.77 million, ranking second in the world. There are 29 local breeds, 13 cultivated breeds, and over 10 introduced breeds with established breeding populations, resulting in a rich and diverse horse population. However, over two-thirds of these breeds are in decline, endangered, or on the verge of extinction. my country's horse population is currently at a critical stage of conservation and transition to non-draft use. Horses are primarily distributed in Sichuan Province, Xinjiang Uyghur Autonomous Region, and Inner Mongolia Autonomous Region. Inner Mongolia Autonomous Region had a population of 638,300 in 2018, ranking third nationwide.

[0003] Short tandem repeat (STR) loci, as second-generation genetic markers following restriction fragment length polymorphism (RFLP), have been widely used in forensic evidence research and identification since the 1980s and 1990s, and are currently one of the most widely used genetic markers. Compared to other genetic markers, STR loci are smaller and easier to amplify, making them more suitable for trace and degraded specimens. Furthermore, multiple STR loci can be amplified simultaneously, offering advantages such as rapidity, efficiency, accuracy, sensitivity, and high information content. Consequently, they are widely used in forensic evidence identification and paternity testing. Research reports have also shown that this technology can be used for equine paternity testing and individual identification.

[0004] Currently, the only commercially available test kit for equine STRs on the market is the Thermo Scientific Bovine Genotypes Panel 3.1 from AB. This four-color kit includes 17 STR loci (AHT4, AHT5, ASB2, ASB17, ASB23, CA425, HMS1, HMS2, HMS3, HMS6, HMS7, HTG4, HTG6, HTG7, HTG10, LEX3, and VHL20). According to the user manual, these loci are selected from equine microsatellite loci recommended by FAO-ISAG and are suitable for horse breeds. However, because these loci are located only on autosomes and are present in a limited number, the possibility of encountering two individuals with identical alleles is present. In cases where two individuals have identical genotypes, kits with a limited number of loci will be unable to distinguish between the two individuals. Summary of the Invention

[0005] Technical Problem to Be Solved: To overcome the shortcomings of existing technologies, the present invention provides a multiplex amplification kit and its application for simultaneously amplifying 20 equine STR loci in a single tube, while maximally arranging the loci so that each locus is distributed on different chromosomes and has no genetic linkage. This ensures that primers do not interfere with each other during the amplification process, eliminating the generation of non-specific peaks. This allows for equine paternity testing, individual identification, and sex determination.

[0006] Technical solution: A multiplex amplification kit for simultaneously amplifying 20 horse STR loci, the kit comprising specific amplification primers for amplifying the following 20 STR loci; wherein the 20 STR loci are: HTG6, HTG7, COR22, CA425, HTG4, COR82, LEX54, COR69, AHT5, HMS3, HMS1, HMS6, HMS7, COR58, HTG10, ASB17, VHL20, HMS2, ASB2, and LEX34.

[0007] Preferably, the sequences of the specific amplification primers are as follows: HTG6, SEQ ID NOs: 1-2; HTG7, SEQ ID NOs: 3-4; COR22, SEQ ID NOs: 5-6; CA425, SEQ ID NOs: 7-8; HTG4, SEQ ID NOs: 9-10; COR82, SEQ ID NOs: 11-12; LEX54, SEQ ID NOs: 13-14; COR69, SEQ ID NOs: 15-16; AHT5, SEQ ID NOs: 17-18; HMS3, SEQ ID NOs: 19-20; HMS1, SEQ ID NOs: 21-22; HMS6, SEQ ID NOs: 23-24; HMS7, SEQ ID NOs: 25-26; COR58, SEQ ID NOs: 27-28; HTG10, SEQ ID NOs: 29-30; ASB17, SEQ ID NOs: 31-32; VHL20, SEQ ID NOs: NO:33-34; HMS2, SEQ ID NO:35-36; ASB2, SEQ ID NO:37-38; LEX34, SEQ ID NO:39-40.

[0008] Preferably, the concentrations of the specific amplification primers are as follows: HTG6, 0.2 μM; HTG7, 0.15 μM; COR22, 0.15 μM; CA425, 0.2 μM; HTG4, 0.25 μM; COR82, 0.25 μM; LEX54, 0.25 μM; COR69, 0.25 μM; AHT5, 0.25 μM; HMS3, 0.2 μM; HMS1, 0.15 μM; HMS6, 0.2 μM; HMS7, 0.25 μM; COR58, 0.25 μM; HTG10, 0.25 μM; ASB17, 0.25 μM; VHL20, 0.2 μM; HMS2, 0.2 μM; ASB2, 0.25 μM; LEX34, 0.2 μM. Specific information of the specific amplification primers is shown in the following table:

[0009]

[0010]

[0011] Preferably, the specific amplification primers are divided into 5 groups, HTG6, HTG7, COR22, CA425 are group 1, HTG4, COR82, LEX54, COR69 are group 2, AHT5, HMS3, HMS1, HMS6 are group 3, HMS7, COR58, HTG10, ASB17 are group 4, and VHL20, HMS2, ASB2, LEX34 are group 5.

[0012] Preferably, the specific amplification primers are labeled with any one of the fluorescent dyes FAM, HEX, SUM, LYN, and PUR, and the fluorescent dyes used in the five sets of primers are different, and the internal standard is labeled with orange fluorescent SIZ.

[0013] Preferably, the 5' end of at least one of the specific amplification primers for each locus is fluorescently labeled.

[0014] Preferably, the kit comprises: allelic typing markers for 20 loci, a reaction mixture, a hot-start Taq enzyme, horse genomic DNA, sdH2O, and a fluorescent molecular weight internal standard.

[0015] Preferably, the reaction mixture comprises: MgCl2 8mM, Tris-HCl 150mM, KCl 150mM, dNTPs 7.5mM, and BSA 2g / L; wherein BSA is an albumin in bovine serum, which has a protective effect on the enzyme, can prevent enzyme decomposition and nonspecific adsorption, and can reduce enzyme denaturation.

[0016] Application of any of the above-mentioned multiplex amplification kits for simultaneously amplifying 20 horse STR loci in horse paternity testing or individual identification.

[0017] Beneficial effects: (1) The kit of the present invention can simultaneously amplify and detect 20 horse STR loci in a single tube, making it the kit with the most detection loci among similar products; (2) The selected loci are distributed on different chromosomes as much as possible, and there is no genetic linkage between them, thereby improving the overall recognition ability of the kit; (3) The specific amplification primers do not interfere with each other, and no non-specific peaks are generated. The kit has the characteristics of strong specificity, high resolution, accurate typing, high sensitivity, etc., and can fully meet the various needs of horse paternity testing, individual identification, sex discrimination, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a schematic diagram of the gene locus arrangement of the present invention;

[0019] Figure 2 This is the typing diagram of the actual sample 1 of the present invention.

[0020] Figure 3 This is the actual sample 2 typing diagram of the present invention. DETAILED DESCRIPTION

[0021] The following examples further illustrate the present invention but are not to be construed as limiting the present invention. Modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the present invention are intended to fall within the scope of the present invention. Unless otherwise specified, the techniques used in the examples are conventional means well known to those skilled in the art.

[0022] Example 1

[0023] A multiplex amplification kit for simultaneously amplifying 20 horse STR loci, specifically including:

[0024] 1. Gene locus screening

[0025] Based on the requirements of high polymorphism, unlinked markers, and easy genotyping, as well as the actual primer test results and locus arrangement requirements, 20 chromosomal STR loci were screened: HTG6, HTG7, COR22, CA425, HTG4, COR82, LEX54, COR69, AHT5, HMS3, HMS1, HMS6, HMS7, COR58, HTG10, ASB17, VHL20, HMS2, ASB2, and LEX34.

[0026] 2. Locus Arrangement

[0027] Based on the above 20 loci, a unique locus arrangement and chemical fluorescent dye labeling method were designed: HTG6, HTG7, COR22, CA425 are the first group, and the fluorescent dye marker is FAM; HTG4, COR82, LEX54, COR69 are the second group, and the fluorescent dye marker is HEX; AHT5, HMS3, HMS1, HMS6 are the third group, and the fluorescent dye marker is SUM; HMS7, COR58, HTG10, ASB17 are the fourth group, and the fluorescent dye marker is LYN; VHL20, HMS2, ASB2, LEX34 are the fifth group, and the fluorescent dye marker is PUR. The locus arrangement is as follows Figure 1 .

[0028] 3. Specific primer design and establishment of multiplex amplification conditions

[0029] Download the locus sequence using the UCSC or NCBI website according to the locus name or chromosome location; secondly, design primers based on the sequences on both sides of the repeat unit of each locus.

[0030] (1) Specific primer design

[0031] When designing specific primers, the best combination of design software is Premier and Oligo, with Premier performing automatic searches and Oligo performing analysis and evaluation. The base distribution of specific primers should be random, the Tm values ​​should be similar, the GC content should be between 40% and 60%, and there should be no complementary sequences within the primers themselves or between primers. At the same time, the specificity of primer amplification must also be ensured. Use Primer-BLAST software to compare and analyze the designed primers in the NCBI database, and fully consider the specificity of the 3' end of the primer, because high sequence homology at the 3' end of the primer can easily lead to incorrect initiation.

[0032] As the number of primers in the multiplex amplification system increases, the mutual interference between specific primers for different loci becomes more and more serious, and the dynamics of the reaction system becomes more and more complex. Therefore, it is necessary to design a large number of primer sequences for complex testing to ultimately ensure the amplification specificity and efficiency of the kit.

[0033] (2) Establishment of multiplex amplification conditions

[0034] First, the single amplification conditions of the 20 loci were optimized. On the basis of successfully establishing the amplification conditions for a single locus, the PCR reaction conditions for the multiplex amplification of the 20 loci were studied. Through a large number of repeated experiments, various parameters in the multiplex amplification were determined, including cycle parameters, annealing temperature, buffer ionic strength, enzyme amount, changes in the multiplex amplification reaction volume, and template DNA amount, so that the amplification products reached the requirements of balance and specificity. A multiplex amplification system was established to amplify 20 loci or sites simultaneously. The final specific primer sequences and concentrations are shown in Table 1.

[0035] Table 1 Specific primer sequences and concentrations

[0036]

[0037]

[0038]

[0039] The specific amplification primers are divided into five groups: HTG6, HTG7, COR22, and CA425 in Group 1; HTG4, COR82, LEX54, and COR69 in Group 2; AHT5, HMS3, HMS1, and HMS6 in Group 3; HMS7, COR58, HTG10, and ASB17 in Group 4; and VHL20, HMS2, ASB2, and LEX34 in Group 5. The specific amplification primers are labeled with any one of the fluorescent dyes FAM, HEX, SUM, LYN, and PUR, and the five groups of primers use different fluorescent dyes. An orange fluorescent SIZ marker is used as the internal standard. The 5' end of at least one of the specific amplification primers for each locus is fluorescently labeled.

[0040] The kit includes: allelic markers for 20 loci, reaction mixture, hot-start Taq enzyme, horse genomic DNA, sdH2O, and a fluorescent molecular weight internal standard. The specific system is shown in Table 2:

[0041] Table 2 Kit amplification system

[0042] Kit component name volume Reaction mixture 4.0μL Horse genomic DNA XμL content is 0.125-1ng Mixture of specific primers 2.0 μL <![CDATA[sdH2O]]> Make up to 10.0 μL

[0043] This kit is only for amplification of extracted samples. Equine genomic DNA refers to equine genomic DNA extracts. The concentration of the resulting DNA extract varies depending on the sample and extraction method. The addition amount (X) is a variable value. Adjust the amount of equine genomic DNA based on the concentration of the extracted DNA extract to ensure accurate typing results using a DNA concentration of 0.125-1 ng. The maximum addition amount (X) for equine genomic DNA is 2 μL.

[0044] The reaction mixture includes: MgCl2 8mM, Tris-HCl 150mM, KCl 150mM, dNTPs 7.5mM, and BSA 2g / L; wherein BSA is an albumin in bovine serum, which has a protective effect on the enzyme, can prevent the enzyme from decomposition and nonspecific adsorption, and can reduce the denaturation of the enzyme.

[0045] Example 2

[0046] Example 1 Application of the Multiplex Amplification Kit in Horse Paternity Testing or Individual Identification. The application steps are as follows:

[0047] 1. Prepare PCR amplification system;

[0048] 2. Amplification Thermal Cycling

[0049] (1) Place the PCR amplification tube on a thermal cycler;

[0050] (2) Select the PCR amplification conditions recommended in Table 3 for PCR amplification;

[0051] (3) The amplified product should be stored away from light;

[0052] Table 3

[0053]

[0054] 3. Fluorescence detection of amplified products on a genetic analyzer

[0055] The loading mixture was composed of deionized formamide and the molecular weight internal standard AGCU Marker SIZ-500 in the system: (0.5 μL AGCU Marker SIZ-500) × (number of injections) + (12 μL deionized formamide) × (number of injections). AGCU Marker SIZ-500 was purchased from Wuxi Sino-German Meilian Biotechnology Co., Ltd.

[0056] 12.5 μL of the loading mixture was mixed with 1 μL of amplified product or 20-locus allelic typing standard Allelic Ladder (Wuxi Zhongde Meilian Biotechnology Co., Ltd.), avoiding the generation of bubbles, denatured at 95°C for 3 min, ice-bathed for 3 min, and then subjected to electrophoresis detection on a genetic analyzer as soon as possible;

[0057] 4. Type analysis

[0058] The data collected by the genetic analyzer in step 3 were analyzed using the fragment analysis software GeneMapper ID-X, and electrophoresis was performed using multi-channel or single-channel capillary electrophoresis.

[0059] In this example, sample 1 was horse meat powder purchased from the National Bureau of Metrology, from which horse genomic DNA was extracted using the Chelex method. Sample 2 was suspected horse meat counterfeited as donkey meat, submitted for inspection by the Wuxi Food and Drug Administration, from which horse genomic DNA was extracted using a magnetic bead extraction kit.

[0060] The results showed that the present invention was used to perform typing tests on the above-mentioned horse samples. The present invention can effectively distinguish individual horses and plays a significant role in individual horse identification.

[0061] Wuxi Food, Drug and Environmental Protection Samples Bureau of Metrology Horse Standards HTG6 112.9 / 114.8 100.2 / 115.2 HTG7 181.1 179.4 / 181.4 COR22 222.9 / 224.9 223.2 / 225.2 CA425 360 / 363.9 356.2 / 364 HTG4 129.5 / 131.5 129.8 COR82 182.2 / 187.8 182.5 / 184.4 LEX54 226.2 / 228.2 226.4 / 228.4 COR69 310.9 / 313.3 300.5 / 306.9 AHT5 106.1 / 108.1 91.5 / 106.2 HMS3 192.5 / 198.5 186.8 / 190.8 HMS1 238.2 / 240.1 242.2 / 244.2 HMS6 318.2 / 322.2 320.4 / 324.4 HMS7 136.2 / 145.2 138.8 / 143.2 COR58 215.2 / 217.1 213.6 / 219.4 HTG10 254.7 / 269.2 259.1 / 261.1 ASB17 355.5 / 373.7 372.2 / 377.8 VHL20 133.8 / 140.2 127.9 HMS2 216.2 / 218.2 214.6 / 229.9 ASB2 271.4 / 273.4 277.5 / 279.5 LEX34 340.2 / 342.1 347.5 Sequence Listing <110> Criminal Investigation Detachment of Hulunbuir Public Security Bureau Wuxi Sino-German Meilian Biotechnology Co., Ltd. <120> Multiplex amplification kit for simultaneous amplification of 20 horse STR loci and its application <160> 40 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty three <212> DNA <213> Artificial Sequence <400> 1 agcttcctgc ttggaggctg tga 23 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 tctgtgccct tccttgtgtc 20 <210> 3 <211> twenty three <212> DNA <213> Artificial Sequence <400> 3 ggcagtagct gaggtttggc aat 23 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <400> 4 ctacacggac tcttccacc 19 <210> 5 <211> twenty one <212> DNA <213> Artificial Sequence <400> 5 tcaaatgtgc caaacaatgc c 21 <210> 6 <211> twenty two <212> DNA <213> Artificial Sequence <400> 6 caaagggagc tatgagcctg aa 22 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <400> 7 gtggggatgg cagggttc 18 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <400> 8 caaagcccct gaccacgg 18 <210> 9 <211> twenty two <212> DNA <213> Artificial Sequence <400> 9 ctccctccct ccctctgttc tc 22 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 tccctccctc cctctgttct 20 <210> 11 <211> twenty three <212> DNA <213> Artificial Sequence <400> 11 tcaggaggaa gactcgcatg tgt 23 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <400> 12 acgggatctc gcagacttg 19 <210> 13 <211> twenty four <212> DNA <213> Artificial Sequence <400> 13 ctcaattttc tttggcctgg ataa 24 <210> 14 <211> twenty one <212> DNA <213> Artificial Sequence <400> 14 catattgcat gagccaattc c 21 <210> 15 <211> twenty one <212> DNA <213> Artificial Sequence <400> 15 aatgtccttt ggtggatgaa c 21 <210> 16 <211> 17 <212> DNA <213> Artificial Sequence <400> 16 atttcacgcg cctccct 17 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <400> 17 caaccagcca cggacacatc 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <400> 18 gtgcaggcta aggaggctca 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <400> 19 ttgctctaaa gccccaactc 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <400> 20 tgctaaaccc tccccatcct 20 <210> twenty one <211> twenty two <212> DNA <213> Artificial Sequence <400> twenty one ctttggcaaa cacaaaacaa gc 22 <210> twenty two <211> twenty two <212> DNA <213> Artificial Sequence <400> twenty two ctcagcctca ccatcactct tc 22 <210> twenty three <211> twenty three <212> DNA <213> Artificial Sequence <400> twenty three aacctatgac ctttttagga gtg 23 <210> twenty four <211> twenty one <212> DNA <213> Artificial Sequence <400> twenty four gtattcaacc attggcactt t 21 <210> 25 <211> twenty four <212> DNA <213> Artificial Sequence <400> 25 caggaaactc atgttgatac catc 24 <210> 26 <211> twenty four <212> DNA <213> Artificial Sequence <400> 26 cgtgacaaaa ttgcatagaa ctaa 24 <210> 27 <211> twenty four <212> DNA <213> Artificial Sequence <400> 27 acaacccacc aggctaagta gcca 24 <210> 28 <211> 19 <212> DNA <213> Artificial Sequence <400> 28 gggaaggacg atgagtgac 19 <210> 29 <211> twenty one <212> DNA <213> Artificial Sequence <400> 29 aggccagttt ggtcctttga a 21 <210> 30 <211> 19 <212> DNA <213> Artificial Sequence <400> 30 acaaatggcc aattcccgc 19 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <400> 31 gctaaccagg cagcagtcag 20 <210> 32 <211> 18 <212> DNA <213> Artificial Sequence <400> 32 gtatcgctga tggagggc 18 <210> 33 <211> twenty four <212> DNA <213> Artificial Sequence <400> 33 caagtcctct tacttgaaga ctag 24 <210> 34 <211> twenty three <212> DNA <213> Artificial Sequence <400> 34 aactcaggga gaatcttcct cag 23 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <400> 35 gaaagctgtg gtcaccctaa 20 <210> 36 <211> 18 <212> DNA <213> Artificial Sequence <400> 36 ggaagccact acacccct 18 <210> 37 <211> twenty one <212> DNA <213> Artificial Sequence <400> 37 cttatctctt tgcgcacttc c 21 <210> 38 <211> twenty one <212> DNA <213> Artificial Sequence <400> 38 aaggtcaacc tctcggctat t 21 <210> 39 <211> twenty two <212> DNA <213> Artificial Sequence <400> 39 gaagtggtag ggactaggtt gg 22 <210> 40 <211> 19 <212> DNA <213> Artificial Sequence <400> 40 catggcatct tctccgggt 19

Claims

1. A multiplex amplification kit for simultaneously amplifying 20 horse STR loci, characterized in that: The kit includes specific amplification primers for amplifying the following 20 STR loci; wherein the 20 STR loci are: HTG6, HTG7, COR22, CA425, HTG4, COR82, LEX54, COR69, AHT5, HMS3, HMS1, HMS6, HMS7, COR58, HTG10, ASB17, VHL20, HMS2, ASB2, LEX34; The sequences of the specific amplification primers are as follows: HTG6, SEQ ID NOs: 1-2; HTG7, SEQ ID NOs: 3-4; COR22, SEQ ID NOs: 5-6; CA425, SEQ ID NOs: 7-8; HTG4, SEQ ID NOs: 9-10; COR82, SEQ ID NOs: 11-12; LEX54, SEQ ID NOs: 13-14; COR69, SEQ ID NOs: 15-16; AHT5, SEQ ID NOs: 17-18; HMS3, SEQ ID NOs: 19-20; HMS1, SEQ ID NOs: 21-22; HMS6, SEQ ID NOs: 23-24; HMS7, SEQ ID NOs: 25-26; COR58, SEQ ID NOs: 27-28; HTG10, SEQ ID NOs: 29-30; ASB17, SEQ ID NOs: 31-32; VHL20, SEQ ID NOs: 33-34. NO:33-34; HMS2, SEQ ID NO:35-36; ASB2, SEQ ID NO:37-38; LEX34, SEQ ID NO:39-40; The specific amplification primers are divided into 5 groups, HTG6, HTG7, COR22, CA425 as group 1, HTG4, COR82, LEX54, COR69 as group 2, AHT5, HMS3, HMS1, HMS6 as group 3, HMS7, COR58, HTG10, ASB17 as group 4, and VHL20, HMS2, ASB2, LEX34 as group 5; The specific amplification primers are labeled with any one of the fluorescent dyes FAM, HEX, SUM, LYN, and PUR, and the fluorescent dyes used in the five primer groups are all different. The internal standard is labeled with orange fluorescent SIZ.

2. The multiplex amplification kit for simultaneously amplifying 20 horse STR loci according to claim 1, characterized in that: The concentrations of the specific amplification primers are as follows: HTG6, 0.2 μM; HTG7, 0.15 μM; COR22, 0.15 μM; CA425, 0.2 μM; HTG4, 0.25 μM; COR82, 0.25 μM; LEX54, 0.25 μM; COR69, 0.25 μM; AHT5, 0.25 μM; HMS3, 0.2 μM; HMS1, 0.15 μM; HMS6, 0.2 μM; HMS7, 0.25 μM; COR58, 0.25 μM; HTG10, 0.25 μM; ASB17, 0.25 μM; VHL20, 0.2 μM; HMS2, 0.2 μM; ASB2, 0.25 μM ;LEX34, 0.2μM.

3. The multiplex amplification kit for simultaneously amplifying 20 horse STR loci according to claim 1, characterized in that: At least one of the specific amplification primers for each locus is fluorescently labeled at its 5' end.

4. The multiplex amplification kit for simultaneously amplifying 20 horse STR loci according to claim 1, characterized in that: The kit includes: allele typing standards of 20 loci, reaction mixture, hot start Taq enzyme, horse genomic DNA, sdH2O and fluorescent molecular weight internal standard.

5. The multiplex amplification kit for simultaneously amplifying 20 horse STR loci according to claim 4, characterized in that: The reaction mixture includes: MgCl2 8mM, Tris-HCl 150mM, KCl 150mM, dNTPs 7.5mM, and BSA 2g / L.

6. Use of the multiplex amplification kit for simultaneously amplifying 20 horse STR loci according to any one of claims 1 to 5 in horse paternity testing or individual identification.

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