Detection kit for detecting genetic relationship and individual recognition of pigs by using fluorescent PCR capillary electrophoresis technology and application of detection kit for detecting genetic relationship and individual recognition of pigs by using fluorescent PCR capillary electrophoresis technology

Abstract

The invention provides a detection kit for detecting the genetic relationship of pigs and identifying individuals by using a fluorescent PCR capillary electrophoresis technology and application of the detection kit, and relates to the technical field of biology. According to the invention, STR loci published by ISAG and STR loci with high polymorphism obtained through high-throughput sequencing data analysis are combined and screened for the first time to obtain a group of STR loci suitable for pig genetic relationship and / or individual identification. STR loci are optimized to improve the specificity of the primers, no linkage effect exists between the STR loci and the primers, typing is rich, compatibility is good, and the detection result is accurate and reliable; the operation method is simple, and the detection time is short; a plurality of sites can be detected at one time, and the cost is low; the sensitivity is high, and accurate typing can be achieved when the DNA template amount is as low as 2 ng / mu L; the detection kit provided by the invention has good specificity and stability, no non-specificity is generated in multiple experiments, and the signal intensity is stable.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a detection kit for detecting pig kinship and individual identification by using fluorescent PCR capillary electrophoresis technology and its application. Background Art

[0002] Molecular markers are genetic markers based on nucleotide sequence variation within genetic material between individuals. They are a direct reflection of genetic polymorphism at the DNA level and can be widely used in research fields such as genetic breeding, genome mapping, gene localization, species kinship identification, gene bank construction, and gene cloning. Microsatellite markers, also known as simple sequence repeats (STRs), are evenly distributed in the genome of eukaryotic organisms and consist of tandem repeat fragments of 1-6 nucleotides. Due to the differences in STR repeat motifs and the number of repeats, they are highly variable between individuals and are abundant in number. They are widely used in genetic hybrid breeding, drawing chromosome genetic maps, and other fields. Compared with other molecular markers (RFLP, VNTR, RAPD, AFLP, SNP, etc.), STR markers have the characteristics of high polymorphism information content, co-dominant inheritance, simple technology, good repeatability, and strong specificity.

[0003] With the rapid development of molecular genetics, molecular biotechnology, quantitative genetics and computer technology, the theory and technical methods of livestock and poultry breeding are also constantly improving. In modern breeding, especially with the application of artificial insemination technology and animal models in livestock and poultry genetic assessment, accurate pedigree records are particularly important to use the phenotypic values ​​of various relative information.

[0004] However, in the production of frozen semen, insemination, feeding management and performance testing, errors in pedigree records make it impossible to accurately confirm the blood relationship of some individuals, so that individual genetic assessment cannot be accurately carried out, which seriously affects the development of breeding work. Many domestic scholars have estimated that the error rate of pedigree records is about 8% to 20%. The losses caused are related to the progress obtained by selection, and it also affects the accuracy of genetic map construction and QTL positioning. Therefore, it is particularly necessary to conduct effective paternity testing on pigs to improve the accuracy of their genetic evaluation.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] One of the objects of the present invention is to provide a primer composition for detecting pig kinship and / or individual identification, so as to at least solve one of the technical problems existing in the prior art.

[0007] A second object of the present invention is to provide a reagent for detecting pig relatedness and / or individual identification.

[0008] A third object of the present invention is to provide a kit for detecting pig relatedness and / or individual identification.

[0009] A fourth object of the present invention is to provide a method for detecting pig relatedness and / or individual identification.

[0010] A fifth object of the present invention is to provide the application of the above-mentioned parrot composition, reagent, kit or method.

[0011] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0012] The first aspect of the present invention provides a primer combination for detecting pig kinship and / or individual identification, characterized in that the primer combination consists of primer pairs for amplifying the following 21 STR loci: SW72, SW830, SW122, SW632, NC_010448.4: 30771560-30771774, NC_010444.4: 79465902-79466147, SW2410, SW857, S0090, S0026, NC_010461.5: 1251686 17-125168844, NC_010448.4:133201992-133202223, SW24, SW951, SW787, S0068, NC_010446.5:111894542-111894769, NC_010456.5:2142411-2142640, NC_010445.4:30015724-30015971, NC_010445.4:61466571-61466802 and IGF1, among which:

[0013] The upstream primer SW72-F for amplifying SW72, the nucleotide sequence of which is shown in SEQ ID No. 1;

[0014] The downstream primer SW72-R for amplifying SW72 has a nucleotide sequence as shown in SEQ ID No. 2;

[0015] The upstream primer SW830-F for amplifying SW830 has a nucleotide sequence as shown in SEQ ID No. 3;

[0016] The downstream primer SW830-R for amplifying SW830 has a nucleotide sequence as shown in SEQ ID No. 4;

[0017] The upstream primer SW122-F for amplifying SW122 has a nucleotide sequence as shown in SEQ ID No. 5;

[0018] The downstream primer SW122-R for amplifying SW122 has a nucleotide sequence as shown in SEQ ID No.6;

[0019] The upstream primer SW632-F for amplifying SW632 has a nucleotide sequence as shown in SEQ ID No. 7;

[0020] The downstream primer SW632-R for amplifying SW632 has a nucleotide sequence as shown in SEQ ID No. 8;

[0021] The upstream primer NC_010448.4:30771560-30771774-F for amplifying NC_010448.4:30771560-30771774, the nucleotide sequence of which is shown in SEQ ID No.9;

[0022] The downstream primer NC_010448.4:30771560-30771774-R for amplifying NC_010448.4:30771560-30771774, the nucleotide sequence of which is shown in SEQ ID No.10;

[0023] The upstream primer NC_010444.4:79465902-79466147-F for amplifying NC_010444.4:79465902-79466147, the nucleotide sequence of which is shown in SEQ ID No.11;

[0024] The downstream primer NC_010444.4:79465902-79466147-R for amplifying NC_010444.4:79465902-79466147, the nucleotide sequence of which is shown in SEQ ID No.12;

[0025] The upstream primer SW2410-F for amplifying SW2410 has a nucleotide sequence as shown in SEQ ID No. 13;

[0026] The downstream primer SW2410-R for amplifying SW2410 has a nucleotide sequence as shown in SEQ ID No. 14;

[0027] The upstream primer SW857-F for amplifying SW857, the nucleotide sequence of which is shown in SEQ ID No. 15;

[0028] The downstream primer SW857-R for amplifying SW857 has a nucleotide sequence as shown in SEQ ID No. 16;

[0029] The upstream primer S0090-F for amplifying S0090 has a nucleotide sequence as shown in SEQ ID No. 17;

[0030] The downstream primer S0090-R for amplifying S0090 has a nucleotide sequence as shown in SEQ ID No. 18;

[0031] The upstream primer S0026-F for amplifying S0026 has a nucleotide sequence as shown in SEQ ID No. 19;

[0032] The downstream primer S0026-R for amplifying S0026 has a nucleotide sequence as shown in SEQ ID No. 20;

[0033] The upstream primer NC_010461.5:125168617-125168844-F for amplifying NC_010461.5:125168617-125168844, the nucleotide sequence of which is shown in SEQ ID No.21;

[0034] The downstream primer NC_010461.5:125168617-125168844-R for amplifying NC_010461.5:125168617-125168844, the nucleotide sequence of which is shown in SEQ ID No.22;

[0035] The upstream primer NC_010448.4:133201992-133202223-F for amplifying NC_010448.4:133201992-133202223, the nucleotide sequence of which is shown in SEQ ID No.23;

[0036] The downstream primer NC_010448.4:133201992-133202223-R for amplifying NC_010448.4:133201992-133202223, the nucleotide sequence of which is shown in SEQ ID No.24;

[0037] The upstream primer SW24-F for amplifying SW24, the nucleotide sequence of which is shown in SEQ ID No. 25;

[0038] The downstream primer SW24-R for amplifying SW24 has a nucleotide sequence as shown in SEQ ID No. 26;

[0039] The upstream primer SW951-F for amplifying SW951, the nucleotide sequence of which is shown in SEQ ID No. 27;

[0040] The downstream primer SW951-R for amplifying SW951 has a nucleotide sequence as shown in SEQ ID No. 28;

[0041] The upstream primer SW787-F for amplifying SW787, the nucleotide sequence of which is shown in SEQ ID No. 29;

[0042] The downstream primer SW787-R for amplifying SW787 has a nucleotide sequence as shown in SEQ ID No. 30;

[0043] The upstream primer S0068-F for amplifying S0068 has a nucleotide sequence as shown in SEQ ID No. 31;

[0044] The downstream primer S0068-R for amplifying S0068 has a nucleotide sequence as shown in SEQ ID No. 32;

[0045] The upstream primer NC_010446.5:111894542-111894769-F for amplifying NC_010446.5:111894542-111894769, the nucleotide sequence of which is shown in SEQ ID No.33;

[0046] The downstream primer NC_010446.5:111894542-111894769-R for amplifying NC_010446.5:111894542-111894769, the nucleotide sequence of which is shown in SEQ ID No.34;

[0047] The upstream primer NC_010456.5:2142411-2142640-F for amplifying NC_010456.5:2142411-2142640, the nucleotide sequence of which is shown in SEQ ID No.35;

[0048] The downstream primer NC_010456.5:2142411-2142640-R for amplifying NC_010456.5:2142411-2142640, the nucleotide sequence of which is shown in SEQ ID No.36;

[0049] The upstream primer NC_010445.4:30015724-30015971-F for amplifying NC_010445.4:30015724-30015971, the nucleotide sequence of which is shown in SEQ ID No.37;

[0050] The downstream primer NC_010445.4:30015724-30015971-R for amplifying NC_010445.4:30015724-30015971, the nucleotide sequence of which is shown in SEQ ID No.38;

[0051] The upstream primer NC_010445.4:61466571-61466802-F for amplifying NC_010445.4:61466571-61466802, the nucleotide sequence of which is shown in SEQ ID No.39;

[0052] The downstream primer NC_010445.4:61466571-61466802-R for amplifying NC_010445.4:61466571-61466802, the nucleotide sequence of which is shown in SEQ ID No.40;

[0053] The upstream primer IGF1-F for amplifying IGF1 has a nucleotide sequence as shown in SEQ ID No.41;

[0054] The downstream primer IGF1-R for amplifying IGF1 has a nucleotide sequence as shown in SEQ ID No.42.

[0055] Further, the primer composition is divided into four groups according to the STR loci, and the primer composition consists of amplification primers for the first group of STR loci, amplification primers for the second group of STR loci, amplification primers for the third group of STR loci, and amplification primers for the fourth group of STR loci;

[0056] The first group of STR loci included: SW72, SW830, SW122, SW632, NC_010448.4: 30771560-30771774, and NC_010444.4: 79465902-79466147;

[0057] The second group of STR loci included: SW2410, SW857, S0090, S0026, NC_010461.5:125168617-125168844, and NC_010448.4:133201992-133202223;

[0058] The third group of STR loci includes: SW24, SW951, SW787 and S0068;

[0059] The fourth group of STR loci included: NC_010446.5:111894542-111894769, NC_010456.5:2142411-2142640, NC_010445.4:30015724-30015971, NC_010445.4:61466571-61466802, and IGF1;

[0060] The fluorescent dye labels used by the amplification primers of the first group of STR sites, the amplification primers of the second group of STR sites, the amplification primers of the third group of STR sites and the amplification primers of the fourth group of STR sites are different from each other.

[0061] Specifically, the fluorescent dye marker used by the amplification primers of the first group of STR sites is FAM; the fluorescent dye marker used by the amplification primers of the second group of STR sites is HEX; the fluorescent dye marker used by the amplification primers of the third group of STR sites is TAMRA; and the fluorescent dye marker used by the amplification primers of the fourth group of STR sites is ROX.

[0062] According to a second aspect of the present invention, there is provided a reagent for detecting pig relatedness and / or individual identification, comprising the above-mentioned primer combination.

[0063] Further, the reagent may be a solution;

[0064] In the reagent, the final concentration of the amplification primers of the first group of STR sites is 0.2 μmol / L; the final concentration of the amplification primers of the second group of STR sites is 0.15 μmol / L; the final concentration of the amplification primers of the third group of STR sites is 0.25 μmol / L; and the final concentration of the amplification primers of the fourth group of STR sites is 0.18 μmol / L.

[0065] According to a third aspect of the present invention, there is provided a kit for detecting pig relatedness and / or individual identification, the kit comprising the above-mentioned primer composition or reagent.

[0066] The kit can simultaneously amplify 21 pig STR loci in a single tube, mainly including: SW72, SW830, SW122, SW632, NC_010448.4: 30771560-30771774, NC_010444.4: 79465902-79466147, SW2410, SW857, S0090, S0026, NC_010461.5: 125168617-125168844, NC_010 448.4:133201992-133202223, SW24, SW951, SW787, S0068, NC_010446.5:111894542-111894769, NC_010456.5:2142411-2142640, NC_010445.4:30015724-30015971, NC_010445.4:61466571-61466802 and IGF1.

[0067] Furthermore, the kit may also include Taq DNA polymerase, dNTP, PCR buffer, Mg 2+ One or more of .

[0068] The various reagent components of the kit may be present in separate containers, or may be pre-combined in whole or in part into a reagent mixture.

[0069] In some specific embodiments, the kit includes a 10 μL PCR amplification reaction system, including 4 μL 2.5×Master Mix, 1 μL of each primer mixture, 4 μL nuclease-free pure water and 1 μL DNA template.

[0070] According to a fourth aspect of the present invention, there is provided a method for detecting pig relatedness and / or individual identification, comprising the following steps:

[0071] 1) Extract DNA from the sample to be tested;

[0072] 2) using the DNA as a template and using the above primer combination, reagent or kit to perform PCR amplification to obtain an amplified product;

[0073] 3) Detection by capillary electrophoresis;

[0074] 4) Data analysis.

[0075] Fluorescence PCR capillary electrophoresis technology is used to detect pig STR loci information. Through one-tube amplification, 21 STR loci can be detected simultaneously. Compared with existing technologies, the detection method is simple, fast, low-cost, and highly accurate.

[0076] Furthermore, step 4) is to obtain 21 STR typing pattern information through Genemapper6.0 software analysis.

[0077] In the above method, the PCR reaction procedure may include:

[0078] Step 1: pre-denaturation at 95°C for 5 minutes;

[0079] Step 2: denaturation at 95°C for 10 seconds, annealing and extension at 58°C for 2 minutes, and cycle step 2 30 times;

[0080] Step 3: 60℃ extension for 45 minutes;

[0081] Step 4: Store at 4°C.

[0082] In addition, according to the fifth aspect of the present invention, there is also provided the use of the above primer composition, reagent, kit or method in any of the following:

[0083] (a) pig individual identification or products for preparing pig individual identification;

[0084] (b) pig parentage identification or products for preparing pig parentage identification;

[0085] (c) Pig population genetics analysis or the preparation of products for pig population genetics analysis;

[0086] (d) pig DNA related case tests or products for preparing pig DNA related case tests;

[0087] (e) Construction of a pig DNA database or products for the construction of a pig DNA database.

[0088] Furthermore, the pig may be a Large White pig, a Landrace pig, a Duroc pig, a Chinese Min pig, a wild boar, or the like.

[0089] Compared with the prior art, the present invention has the following beneficial effects:

[0090] The present invention combines the STR loci published by ISAG with the highly polymorphic STR loci obtained by high-throughput sequencing data analysis for the first time to obtain a group of STR loci suitable for pig kinship and / or individual identification. The present invention also optimizes the STR loci to improve the specificity of the primers, there is no linkage effect between each other, the typing is rich and the compatibility is good, so that the detection results are accurate and reliable; the operation method is simple, the detection time is short; multiple loci can be detected at one time, and the cost is low; the sensitivity is high, and the DNA template amount as low as 2ng / μL can be accurately typed; and the detection kit described in the present invention has good specificity and stability, no non-specific generation in multiple experiments, and stable signal strength; it is applicable to a variety of test materials, such as blood, oral swabs, cells, tissues, semen, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0092] Figure 1 A result diagram of a conventional sample DNA test provided by an embodiment of the present invention;

[0093] Figure 2a This is one of the test results of the kit repeatability test provided by the embodiment of the present invention;

[0094] Figure 2b This is one of the test results of the kit repeatability test provided by the embodiment of the present invention;

[0095] Figure 2c This is one of the results of the repeatability test of the kit provided in the embodiment of the present invention. DETAILED DESCRIPTION

[0096] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0097] Generally, the nomenclature and its technology used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally carried out according to conventional methods as well-known in the art, and as described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as generally achieved in the art, or as described herein. The nomenclature and its laboratory procedures and technology used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0098] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than 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.

[0099] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0100] Example 1 Screening of STR loci and design of primers

[0101] In the present invention, part of the STR loci are selected from the STR loci published by ISAG, and part of the STR loci are highly polymorphic STR loci obtained through high-throughput sequencing data analysis.

[0102] The STR loci published by ISAG include: SW72, SW830, SW632, SW2410, SW857, S0090, S0026, SW24, SW122, SW951, SW787, S0068, and IGF1.

[0103] STR loci obtained from high-throughput sequencing data analysis include: NC_010448.4:30771560-30771774, NC_010444.4:79465902-79466147, NC_010461.5:125168617-125168844, NC_010448.4:1332019 92-133202223, NC_010446.5:111894542-111894769, NC_010456.5:2142411-2 142640, NC_010445.4:30015724-30015971, NC_010445.4:61466571-61466802.

[0104] According to the above, 21 porcine STR loci (STR sites) were finally selected as target sites for primer design. The primers were designed using Primer5 software, and the specificity of the primers was compared using the NCBI website. The primer fragment size was designed within the range of 75-450bp, and the Tm value was controlled at (60±2)°C. 21 pairs of primers were selected for single amplification of porcine genomic DNA, and capillary electrophoresis was used to check the amplification efficiency of each pair of primers and whether there were non-specific fragments. If low efficiency or non-specific fragments occur, it is necessary to replace the primers or optimize the PCR amplification conditions until the efficiency of the 21 pairs of primers is basically the same and no non-specific fragments are generated.

[0105] The primer sequence information obtained by screening is as follows:

[0106]

[0107]

[0108] Example 2 Primer grouping and reaction system primer concentration optimization

[0109] The 21 pairs of primers in Example 1 are divided into 4 groups of fluorescent markers according to product size and expected interference between primers. The fluorescent dye marker used by the amplification primers of the first group of STR loci is FAM, and the 6 loci are as follows: SW72, SW830, SW122, SW632, NC_010448.4: 30771560-30771774, NC_010444.4: 79465902-79466147; the fluorescent dye marker used by the amplification primers of the second group of STR loci is HEX, and the 6 loci are as follows: SW2410, SW857, S0090, S0026, NC_010461.5: 125168617-125168844, NC _010448.4:133201992-133202223; the fluorescent dye marker used by the amplification primers of the third group of STR loci is TAMRA, and the 4 loci are as follows: SW24, SW951, SW787, and S0068; the fluorescent dye marker used by the amplification primers of the fourth group of STR loci is ROX, and the 5 loci are as follows: NC_010446.5:111894542-111894769, NC_010456.5:2142411-2142640, NC_010445.4:30015724-30015971, NC_010445.4:61466571-61466802, and IGF1.

[0110] According to the above grouping, after completing the single primer screening and design work, the single primers are mixed according to the above grouping and composite amplification is performed to adjust the primer concentration. In the initial composite amplification, equal molar numbers of primers should be added to the reaction system, and then the concentration ratio of each pair of primers is adjusted according to the reaction results; finally, the final primer concentration is determined through a large number of experiments.

[0111] Determine the final concentration of primers in the amplification system as follows:

[0112]

[0113] Example 3 Construction of fluorescent STR composite amplification system and reaction program

[0114] According to the composite primers prepared in Example 2, the addition amount of PCR buffer, primer mixture, total DNA and H2O in the reaction system was determined through multiple experiments.

[0115]

[0116] Optimize the PCR program, explore the annealing temperature, extension temperature and time suitable for the composite system, and determine the optimal program:

[0117] Step 1: pre-denaturation at 95°C for 5 minutes;

[0118] Step 2: denaturation at 95°C for 10 seconds, annealing and extension at 58°C for 2 minutes, and cycle step 2 30 times;

[0119] Step 3: 60℃ extension for 45 minutes;

[0120] Step 4: Store at 4°C.

[0121] Capillary electrophoresis was used to detect PCR amplification products, and the offline data were analyzed using Genemapper6.0 software to obtain 21 STR typing pattern information.

[0122] Example 4 Application of a kit for detecting pig relatedness and individual identification

[0123] 1. Collect pig blood and register it;

[0124] 2. Extract genomic DNA from pig blood;

[0125] DNA was prepared using the TianGen automated blood DNA extraction kit (magnetic bead method).

[0126] 3. Configure PCR reaction system

[0127] After each reaction reagent (2.5× Master Mix, primer mixture, nuclease-free pure water) was shaken and mixed, a PCR reaction mixture was prepared according to the volume ratio (except template), and 9 μL was dispensed into PCR reaction tubes. Finally, 1 μL of template was added to each reaction tube, and the next step was performed after centrifugation. The PCR reaction system and reaction procedure are as shown in Example 3.

[0128] 4. Capillary electrophoresis detection

[0129] Take 1 μL of amplified product (stock solution or diluted product), add 8.8 μL HIDI and 0.2 μL LIZ500, mix and let stand for a few minutes, denature at 95℃ for 3 minutes, immediately put in ice bath for 3 minutes, centrifuge and place on ABI3730 sequencer for testing. After the test is completed, use Genemapper6.0 software to interpret the typing results of each locus. Set the panel according to the size of the product fragment, and judge the typing of each locus based on the position of the peak and the fluorescence color to generate a map, see below Figure 1 The kit of the present invention was used to detect and obtain complete STR typing results, with sharp peaks and no site loss, which fully met the needs of individual recognition and identification.

[0130] Example 5 Repeatability Verification

[0131] The above kit was used to perform repeated amplification tests on 2 ng / μl samples, and the amplified products were detected using an ABI 3730 sequencer to test the repeatability of the kit. Through experimental data analysis, the peak types of each site in the three repetitions can be clearly detected and the typing is consistent, so it can be judged that the kit has good repeatability. The results are as follows Figure 2a~2c shown.

[0132] Example 6 Study on Applicable Models

[0133] The above kits were tested for amplification using BIO-RAD and ABI 9700 PCR amplification instruments, and the products were detected using ABI 3730 sequencer. Data analysis showed that the amplification efficiency of BIO-RAD was higher than that of ABI 9700. This showed that under the same conditions, the system could be amplified normally on both BIO-RAD and ABI 9700 PCR amplification instruments, and the performance was not affected by the amplification instruments. The amplified products could be clearly detected on ABI 3730, and the test results were consistent.

[0134] Example 7 Stability Study

[0135] The kit was stored at -20℃±5℃ and protected from light until frozen; thawed and frozen on the 3rd, 7th, 15th, and 30th days. The 8 test samples were amplified and tested by repeated freezing and thawing, and the test results were consistent. There was no abnormality in the test results after freezing and thawing on the 30th day.

[0136] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer composition for detecting pig relatedness and / or individual identification, characterized in that: The primer combination consists of primer pairs for amplifying the following 21 STR loci: SW72, SW830, SW122, SW632, NC_010448.4: 30771560-30771774, NC_010444.4: 79465902-79466147, SW2410, SW857, S0090, S0026, NC_010461.5: 125168617-125168844, NC_01044 8.4:133201992-133202223, SW24, SW951, SW787, S0068, NC_010446.5:111894542-111894769, NC_010456.5:2142411-2142640, NC_010445.4:30015724-30015971, NC_010445.4:61466571-61466802 and IGF1, among which: The nucleotide sequences of the primer pair for amplifying SW72 are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively; The nucleotide sequences of the primer pair for amplifying SW830 are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively; The nucleotide sequences of the primer pair for amplifying SW122 are shown in SEQ ID No. 5 and SEQ ID No. 6, respectively; The nucleotide sequences of the primer pair for amplifying SW632 are shown in SEQ ID No. 7 and SEQ ID No. 8, respectively; The nucleotide sequences of the primer pair for amplifying NC_010448.4: 30771560-30771774 are shown in SEQ ID No. 9 and SEQ ID No. 10, respectively; The nucleotide sequences of the primer pair for amplifying NC_010444.4: 79465902-79466147 are shown in SEQ ID No. 11 and SEQ ID No. 12, respectively; The nucleotide sequences of the primer pair for amplifying SW2410 are shown in SEQ ID No. 13 and SEQ ID No. 14, respectively; The nucleotide sequences of the primer pair for amplifying SW857 are shown in SEQ ID No. 15 and SEQ ID No. 16, respectively; The nucleotide sequences of the primer pair for amplifying S0090 are shown in SEQ ID No. 17 and SEQ ID No. 18, respectively; The nucleotide sequences of the primer pair for amplifying S0026 are shown in SEQ ID No. 19 and SEQ ID No. 20, respectively; The nucleotide sequences of the primer pair for amplifying NC_010461.5: 125168617-125168844 are shown in SEQ ID No. 21 and SEQ ID No. 22, respectively; The nucleotide sequences of the primer pair for amplifying NC_010448.4: 133201992-133202223 are shown in SEQ ID No. 23 and SEQ ID No. 24, respectively; The nucleotide sequences of the primer pair for amplifying SW24 are shown in SEQ ID No. 25 and SEQ ID No. 26, respectively; The nucleotide sequences of the primer pair for amplifying SW951 are shown in SEQ ID No. 27 and SEQ ID No. 28, respectively; The nucleotide sequences of the primer pair for amplifying SW787 are shown in SEQ ID No. 29 and SEQ ID No. 30, respectively; The nucleotide sequences of the primer pair for amplifying S0068 are shown in SEQ ID No. 31 and SEQ ID No. 32, respectively; The nucleotide sequences of the primer pair for amplifying NC_010446.5: 111894542-111894769 are shown in SEQ ID No. 33 and SEQ ID No. 34, respectively; The nucleotide sequences of the primer pair for amplifying NC_010456.5: 2142411-2142640 are shown in SEQ ID No. 35 and SEQ ID No. 36, respectively; The nucleotide sequences of the primer pair for amplifying NC_010445.4: 30015724-30015971 are shown in SEQ ID No. 37 and SEQ ID No. 38, respectively; The nucleotide sequences of the primer pair for amplifying NC_010445.4: 61466571-61466802 are shown in SEQ ID No. 39 and SEQ ID No. 40, respectively; The nucleotide sequences of the primer pair for amplifying IGF1 are shown in SEQ ID No.41 and SEQ ID No.42, respectively.

2. The primer composition according to claim 1, characterized in that The primer combination is divided into four groups according to the STR loci, and the primer combination consists of amplification primers for the first group of STR loci, amplification primers for the second group of STR loci, amplification primers for the third group of STR loci, and amplification primers for the fourth group of STR loci; The first group of STR loci included: SW72, SW830, SW122, SW632, NC_010448.4: 30771560-30771774, and NC_010444.4: 79465902-79466147; The second group of STR loci included: SW2410, SW857, S0090, S0026, NC_010461.5:125168617-125168844, and NC_010448.4:133201992-133202223; The third group of STR loci includes: SW24, SW951, SW787 and S0068; The fourth group of STR loci included: NC_010446.5:111894542-111894769, NC_010456.5:2142411-2142640, NC_010445.4:30015724-30015971, NC_010445.4:61466571-61466802, and IGF1; The fluorescent dye labels used by the amplification primers of the first group of STR sites, the amplification primers of the second group of STR sites, the amplification primers of the third group of STR sites and the amplification primers of the fourth group of STR sites are different from each other.

3. The primer composition according to claim 2, characterized in that The fluorescent dye marker used by the amplification primers of the first group of STR loci is FAM; the fluorescent dye marker used by the amplification primers of the second group of STR loci is HEX; the fluorescent dye marker used by the amplification primers of the third group of STR loci is TAMRA; and the fluorescent dye marker used by the amplification primers of the fourth group of STR loci is ROX.

4. A reagent for detecting pig relatedness and / or individual identification, characterized in that: The reagent comprises the primer composition according to any one of claims 1 to 3.

5. The reagent according to claim 4, characterized in that The reagent is a solution; In the reagent, the final concentration of the amplification primers of the first group of STR sites is 0.2 μmol / L; the final concentration of the amplification primers of the second group of STR sites is 0.15 μmol / L; the final concentration of the amplification primers of the third group of STR sites is 0.25 μmol / L; and the final concentration of the amplification primers of the fourth group of STR sites is 0.18 μmol / L.

6. A kit for detecting pig relatedness and / or individual identification, characterized in that: The kit comprises the primer composition according to any one of claims 1 to 3 or the reagent according to claim 4 or 5.

7. The kit according to claim 6, characterized in that The kit also includes a PCR amplification reaction system; Preferably, the PCR amplification reaction system is 10 μL, including 4 μL 2.5×Master Mix, 1 μL of each primer mixture, 4 μL nuclease-free pure water and 1 μL DNA template.

8. A method for detecting pig relatedness and / or individual identification, characterized in that: The steps include: 1) Extract DNA from the sample to be tested; 2) using the DNA as a template, performing PCR amplification using the primer composition according to any one of claims 1 to 3, the reagent according to claim 4 or 5, or the kit according to claim 6 or 7 to obtain an amplified product; 3) Detection by capillary electrophoresis; 4) Data analysis.

9. The method according to claim 8, characterized in that The PCR reaction procedure includes: Step 1: pre-denaturation at 95°C for 5 minutes; Step 2: denaturation at 95°C for 10 seconds, annealing and extension at 58°C for 2 minutes, and cycle step 2 30 times; Step 3: 60℃ extension for 45 minutes; Step 4: Store at 4°C; Preferably, step 4) is to obtain 21 STR typing pattern information through Genemapper6.0 software analysis.

10. Use of the primer composition according to any one of claims 1 to 3, the reagent according to claim 4 or 5, the kit according to claim 6 or 7, or the method according to claim 8 or 9 in any of the following: (a) pig individual identification or products for preparing pig individual identification; (b) pig parentage identification or products for preparing pig parentage identification; (c) Pig population genetics analysis or the preparation of products for pig population genetics analysis; (d) pig DNA related case tests or products for preparing pig DNA related case tests; (e) Construction of a pig DNA database or products for the construction of a pig DNA database.

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