SNP site combination and application thereof in individual identification and tracing of beijing black pigs

Abstract

The present application relates to the technical field of nucleic acid detection, and particularly relates to a SNP site combination and application thereof in individual identification and tracing of Beijing black pigs. The SNP site combination comprises SNP1-SNP20. The SNP site combination comprises nucleotide sequences as shown in SEQ ID NO. 1-20. The present application studies and screens a SNP site combination composed of 20 SNP sites. The SNP sites have a high individual identification rate, and the average identification rate can reach 98.9%. The SNP site combination has high throughput and high accuracy, can greatly reduce the labor and detection time cost, and has important application value in the quality supervision field of Beijing black pig products.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection technology, and in particular to a combination of SNP sites and its application in the identification and tracing of individual black pigs in Beijing. Background Technology

[0002] The Beijing Black Pig is a high-quality local breed of black pig, characterized by its hardiness, early maturity, high productivity, and tender, flavorful meat. It also boasts rapid growth and abundant meat production. Protecting this high-quality local breed, eliminating adulteration and counterfeiting, and maintaining the premium brand image of this valuable product necessitates conducting research on the authenticity and traceability of such products. Therefore, establishing a safe, reliable, and transparent traceability system suitable for meat product production and sales is crucial for enhancing regulatory efficiency and ensuring product quality. This system will not only protect consumers from fraud but also safeguard local specialty meat products, effectively maintaining their brand image. Furthermore, it will provide reliable technical support for protecting Beijing's local specialty meat products and their breeding and processing enterprises, and enhancing the overall brand value of Beijing's meat industry.

[0003] In recent years, extensive research has been conducted both domestically and internationally on meat product traceability. Traceability methods are broadly categorized into three types: physical methods (such as labeling traceability technology), chemical methods (such as isotope traceability technology), and biotechnology (such as DNA traceability technology). Among these, DNA traceability technology is recognized as the most promising and valuable rapid traceability technology due to its advantages such as easy typing, good repeatability, simple and rapid detection methods, and low cost. Single nucleotide polymorphisms (SNPs) are markers that identify polymorphisms caused by variations in a single nucleotide on the genome. By utilizing the SNP differences in the genomes of individual species, an SNP feature map of each individual species can be constructed, essentially creating a unique DNA identity card for each individual. DNA traceability technology not only enables full traceability of meat products from farm to table but also allows for accurate identification of individuals and breeds. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a combination of SNP sites and its application in the identification and tracing of individual black pigs in Beijing.

[0005] In a first aspect, the present invention provides a combination of SNP sites, including SNP1-SNP20; SNP1 is located at 60792655 bp on chromosome Chromosome13, and its polymorphism is A / G; SNP2 is located at 101773318 bp on chromosome 8, and the polymorphism is A / G; SNP3 is located at 29498397 bp on chromosome Chromosome3, and its polymorphism is A / G; SNP4 is located at 8479022 bp on chromosome 8 of Chromosome, and its polymorphism is A / G; SNP5 is located at 56216032 bp on chromosome Chromosome14, and its polymorphism is A / G; SNP6 is located at 3765781 bp on chromosome Chromosome7, and its polymorphism is T / C. SNP7 is located at 137712833 bp on chromosome Chromosome4, and its polymorphism is A / G; SNP8 is located at 124157393 bp on chromosome Chromosome4, and its polymorphism is C / G; SNP9 is located at 156408847 bp on chromosome Chromosome13, and its polymorphism is A / G; SNP10 is located at 88201093 bp on chromosome Chromosome2, and its polymorphism is A / G; SNP11 is located at 73144741 bp on chromosome Chromosome5, and its polymorphism is T / C. SNP12 is located at 25784942 bp on chromosome Chromosome2, and its polymorphism is A / G; SNP13 is located at 69817848 bp on chromosome Chromosome1, and its polymorphism is C / G; SNP14 is located at 95533526 bp on chromosome Chromosome2, and its polymorphism is A / C; SNP15 is located at 813652 bp on chromosome Chromosome1, and its polymorphism is T / C. SNP16 is located at 62557043 bp on chromosome Chromosome9, and its polymorphism is T / C. SNP17 is located at 82135347 bp on chromosome Chromosome16, and its polymorphism is T / C. SNP18 is located at 11109184 bp on chromosome Chromosome16, and its polymorphism is T / C. SNP19 is located at 2842626 bp on chromosome Chromosome17, and its polymorphism is T / G; SNP20 is located at 2738859 bp on chromosome 15, and its polymorphism is A / G.

[0006] In a second aspect, the present invention provides a combination of SNP sites, comprising the nucleotide sequences shown in SEQ ID NO.1-20; As shown in SEQ ID NO.1, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of A / G; As shown in SEQ ID NO.2, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of A / G; As shown in SEQ ID NO.3, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of A / G; As shown in SEQ ID NO.4, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of A / G; As shown in SEQ ID NO.5, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of A / G; As shown in SEQ ID NO.6, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of T / C. As shown in SEQ ID NO.7, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of A / G; As shown in SEQ ID NO.8, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of C / G; As shown in SEQ ID NO.9, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of A / G; As shown in SEQ ID NO.10, position 51 of the nucleotide sequence exhibits polymorphism, which is A / G. As shown in SEQ ID NO.11, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of T / C. As shown in SEQ ID NO.12, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of A / G; As shown in SEQ ID NO.13, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of C / G; As shown in SEQ ID NO.14, the nucleotide sequence at position 51 is polymorphic, with the polymorphism being A / C; As shown in SEQ ID NO.15, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of T / C. As shown in SEQ ID NO.16, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of T / C. As shown in SEQ ID NO.17, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of T / C. As shown in SEQ ID NO.18, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of T / C. As shown in SEQ ID NO.19, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of T / G; As shown in SEQ ID NO.20, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of A / G.

[0007] Thirdly, the present invention provides a primer combination for amplifying the aforementioned SNP site combination, comprising: The primer pair Primer1 used to amplify SNP1 consists of Primer 1F and Primer 1R, and the primer sequences are shown in SEQ ID NO:21-22; The primer pair Primer2 used to amplify SNP2 consists of Primer 2F and Primer 2R, and the primer sequences are shown in SEQ ID NO:23-24; The primer pair Primer3 used to amplify SNP3 consists of Primer 3F and Primer 3R, and the primer sequences are shown in SEQ ID NO:25-26. The primer pair Primer4 used to amplify SNP4 consists of Primer 4F and Primer 4R, and the primer sequences are shown in SEQ ID NO:27-28. The primer pair Primer5 used to amplify SNP5 consists of Primer 5F and Primer 5R, and the primer sequences are shown in SEQ ID NO:29-30. The primer pair Primer6 used to amplify SNP6 consists of Primer 6F and Primer 6R, and the primer sequences are shown in SEQ ID NO:31-32. The primer pair Primer7 used to amplify SNP7 consists of Primer 7F and Primer 7R, and the primer sequences are shown in SEQ ID NO:33-34; The primer pair Primer8 used to amplify SNP8 consists of Primer 8F and Primer 8R, and the primer sequences are shown in SEQ ID NO:35-36. The primer pair Primer9 used to amplify SNP9 consists of Primer 9F and Primer 9R, and the primer sequences are shown in SEQ ID NO:37-38; The primer pair Primer10 used to amplify SNP10 consists of Primer 10F and Primer 10R, and the primer sequences are shown in SEQ ID NO:39-40; The primer pair Primer11 used to amplify SNP11 consists of Primer 11F and Primer 11R, and the primer sequences are shown in SEQ ID NO:41-42. The primer pair Primer12 used to amplify SNP12 consists of Primer 12F and Primer 12R, and the primer sequences are shown in SEQ ID NO:43-44; The primer pair Primer13 used to amplify SNP13 consists of Primer 13F and Primer 13R, and the primer sequences are shown in SEQ ID NO:45-46; The primer pair Primer14 used to amplify SNP14 consists of Primer 14F and Primer 14R, and the primer sequences are shown in SEQ ID NO:47-48. The primer pair Primer15 used to amplify SNP15 consists of Primer 15F and Primer 15R, and the primer sequences are shown in SEQ ID NO:49-50. The primer pair Primer16 used to amplify SNP16 consists of Primer 16F and Primer 16R, and the primer sequences are shown in SEQ ID NO:51-52. The primer pair Primer17 used to amplify SNP17 consists of Primer 17F and Primer 17R, and the primer sequences are shown in SEQ ID NO:53-54. The primer pair Primer18 used to amplify SNP18 consists of Primer 18F and Primer 18R, and the primer sequences are shown in SEQ ID NO:55-56. The primer pair Primer19 used to amplify SNP19 consists of Primer 19F and Primer 19R, and the primer sequences are shown in SEQ ID NO:57-58. The primer pair Primer20 used to amplify SNP20 consists of Primer 20F and Primer 20R, and the primer sequences are shown in SEQ ID NO:59-60.

[0008] Fourthly, the present invention provides a molecular probe assembly for detecting the aforementioned SNP site assemblies.

[0009] Fifthly, the present invention provides a gene chip, the gene chip comprising the aforementioned molecular probe combination.

[0010] In a sixth aspect, the present invention provides a kit comprising the aforementioned SNP site combination, or the aforementioned primer pair, or the aforementioned molecular probe combination, or the aforementioned gene chip.

[0011] Seventhly, the present invention provides the application of the aforementioned SNP site combination as a target in the preparation of gene chips for the identification of Beijing Black Pig breeds or for the tracing of individual Beijing Black Pigs.

[0012] The present invention further provides the application of the aforementioned SNP site combination, or the aforementioned primer pair, or the aforementioned molecular probe combination, or the aforementioned gene chip, or the aforementioned kit in any of the following: i) Identification of Beijing Black Pig breed, ii) Traceability of individual Beijing black pigs iii) Beijing Black Pig Breeding iv) Identification of Beijing black pork products.

[0013] Furthermore, the application includes: The genome of the sample to be tested is extracted, and the polymorphism of each SNP site in the aforementioned SNP site combination is detected. Based on the polymorphism detection results, the variety identification or individual traceability of the sample to be tested is determined.

[0014] Furthermore, the step of determining the individual origination in variety identification or individual origination of the sample to be tested based on the polymorphism detection results includes: The polymorphism of each SNP site in the SNP site combination of the sample to be tested is compared with the positive sample database of Beijing black pigs, and the specific source of the sample to be tested is determined based on the comparison results.

[0015] Preferably, the Beijing Black Pig positive sample database consists of locus information from different known Beijing Black Pig individuals at the aforementioned 20 SNP loci. Specifically, during comparison, if the test result of the sample to be tested is completely consistent with a positive sample in the Beijing Black Pig positive sample database, it can be determined that the sample to be tested originated from that positive sample.

[0016] The 20 nuclear gene SNP loci provided in this invention were selected from 50,000 sequenced SNP loci through steps such as logical operations, genotyping analysis, and gene frequency calculation. This ensures a relatively balanced frequency of homozygous and heterozygous genotypes while also possessing sufficient individual identification capability (theoretically, up to 3...). 20 (This refers to over 3.4 billion individual Beijing Black pigs). By comparing the polymorphisms of 20 SNP sites in an unknown sample with those of a positive sample in the Beijing Black Pig positive sample database, it can be determined whether the unknown sample is a Beijing Black pig sample and from which individual Beijing Black pig in the positive sample database it originated.

[0017] As one specific implementation, the present invention provides a method for individual identification and traceability technology of Beijing black pigs based on multiple SNP detection, comprising: (1) Extract DNA from pork samples to obtain sample templates; (2) PCR amplification reaction; (3) Alkaline phosphatase digestion of PCR products; (4) Single base extension reaction at SNP sites; (5) Mass spectrometry detection and genotype analysis.

[0018] Furthermore, the PCR amplification reaction system in step (2) includes: sample DNA template, primers, PCR amplification enzyme, PCR buffer, MgCl2, dNTP mix and ddH2O.

[0019] Further, in step (1), an animal tissue DNA extraction kit or the CTAB method can be used to extract DNA from the sample.

[0020] Further, the PCR amplification reaction system described in step (2) comprises, in 5 μL increments, the following components: 1 μL DNA template (10-70 ng / μL), 1 μL primer mix (10-20 pmol / μL) as shown in SEQ ID NO.21-60, 0.2 μL PCR amplification enzyme HotStarTaq (5 U / μL), 0.625 μL 10×PCR Buffer, 0.325 μL MgCl2 (25 mM), 0.1 μL dNTP mix (25 mM), and the remaining volume is made up with ddH2O.

[0021] Furthermore, the PCR amplification reaction procedure described in step (2) includes: (1) pre-denaturation: 95℃, 120s; (2) denaturation: 95℃, 20s; annealing: 56℃, 30s; extension: 72℃, 60s, 45 cycles; (3) post-amplification extension: 72℃, 180s; (4) cooling to 4℃, Hold.

[0022] Further, the PCR product described in step (3) is digested with alkaline phosphatase. 0.3 μL SAP Enzyme (1.7 U / μL), 0.17 μL 10×PCR Buffer and 1.53 μL ddH2O are added to each well of the product from the PCR amplification reaction in step (2). The digestion is carried out by incubation at 37°C for 40 min, followed by inactivation at 85°C for 5 min, and then stored at 4°C for later use.

[0023] Furthermore, in step (4), the single base extension reaction of the SNP site is performed by adding the following to each well of the PCR digestion product in step (3): iPLEX enzyme 0.041 μL, iPLEX terminator 0.2 μL, iPLEX Buffer plus 0.2 μL, Extend primer Mix 0.94 μL, and the remaining volume is made up with ddH2O. The single-base extension amplification program was set as follows: (1) Pre-denaturation: 94℃, 30s; (2) Denaturation: 94℃, 5s; Annealing: 52℃, 5s; Extension: 80℃, 5s, 40 large cycles, of which annealing and extension were performed in 5 small cycles in each large cycle; (3) Extension after amplification: 72℃, 180s; (4) Cool down to 4℃, Hold.

[0024] Further, in step (5), the mass spectrometry detection and genotype analysis described above involve diluting the reaction product (total 9 µL) from step (4) by 3 times, desalting it using resin (6 mg), rotating it in a rotary mixer for 15 minutes, and centrifuging it at 3200 g (2000 rpm on a standard plate centrifuge) for 5 minutes. The desalted sample is then loaded onto a sample target using a mass spectrometer and allowed to crystallize naturally. Mass spectrometry detection is then performed, and data are collected.

[0025] The present invention has the following beneficial effects: This invention screened and obtained a combination of 20 SNP loci. Based on the polymorphism detection results of these 20 SNP loci, and compared with the genotype data of each locus in the database, individual identification of Beijing Black pigs can be achieved. The average individual identification rate of each SNP locus in the combination of SNP loci provided by this invention reaches 98.9%, which has high throughput and high accuracy, and can significantly reduce manpower and detection time costs.

[0026] This invention provides a powerful tool for the individual traceability and identification of Beijing Black Pigs, contributing to the sustainable development of geographical indication products and the protection of local breed diversity. Developing and researching technical methods for identifying geographical indication products can effectively curb brand adulteration, protect brand reputation, and is of great significance for enhancing the overall brand value of local specialty livestock industries and protecting valuable local specialty breeds and their breeding and processing enterprises. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 The results are the detection results of CNC10082042 and CNC10021852 of the Beijing Black Pig Individual Identification and Tracing Technology based on Multiple SNP Detection provided in Embodiment 1 of the present invention; wherein, blue and yellow represent two homozygous genotypes respectively, green represents heterozygous genotype, and red represents not detected.

[0029] Figure 2 The results are the CNC10010024 and CNC10170056 detection results of the Beijing Black Pig Individual Identification and Tracing Technology based on Multiple SNP Detection provided in Embodiment 1 of the present invention; wherein, blue and yellow represent two homozygous genotypes respectively, green represents heterozygous genotype, and red indicates no detection.

[0030] Figure 3 The results are the CNC10091242 and CNC10042330 detection results of the Beijing Black Pig Individual Identification and Tracing Technology based on Multiple SNP Detection provided in Embodiment 1 of the present invention; wherein, blue and yellow represent two homozygous genotypes respectively, green represents heterozygous genotype, and red represents not detected.

[0031] Figure 4 The results are the CNC10011430 and CNCB10001966 detection results of the Beijing Black Pig Individual Identification and Tracing Technology based on Multiple SNP Detection provided in Embodiment 1 of the present invention; wherein, blue and yellow represent two homozygous genotypes respectively, green represents heterozygous genotype, and red represents not detected.

[0032] Figure 5 The results are the detection results of CNC10160241 and CNC10070071 of the Beijing Black Pig Individual Identification and Tracing Technology based on Multiple SNP Detection provided in Embodiment 1 of the present invention; wherein, blue and yellow represent two homozygous genotypes respectively, green represents heterozygous genotype, and red represents not detected.

[0033] Figure 6 The results are the CNC10020552 and CNCB10002454 detection results of the Beijing Black Pig Individual Identification and Tracing Technology based on Multiple SNP Detection provided in Embodiment 1 of the present invention; wherein, blue and yellow represent two homozygous genotypes respectively, green represents heterozygous genotype, and red represents not detected.

[0034] Figure 7 The results are the detection results of CNC10150052 and CNC10042609 of the Beijing Black Pig Individual Identification and Tracing Technology based on Multiple SNP Detection provided in Embodiment 1 of the present invention; wherein, blue and yellow represent two homozygous genotypes respectively, green represents heterozygous genotype, and red represents not detected.

[0035] Figure 8 The results are the CNC10133084 and CNCB10004066 detection results of the Beijing Black Pig Individual Identification and Tracing Technology based on Multiple SNP Detection provided in Embodiment 1 of the present invention; wherein, blue and yellow represent two homozygous genotypes respectively, green represents heterozygous genotype, and red indicates no detection.

[0036] Figure 9 The results are the detection results of CNC10141074 and CNC10080151 of the Beijing Black Pig Individual Identification and Tracing Technology based on Multiple SNP Detection provided in Embodiment 1 of the present invention; wherein, blue and yellow represent two homozygous genotypes respectively, green represents heterozygous genotype, and red represents not detected.

[0037] Figure 10 The results are CNC10161593 and CNCB10008679 detections of the Beijing Black Pig individual identification and traceability technology based on multiple SNP detection provided in Embodiment 1 of the present invention; wherein, blue and yellow represent two homozygous genotypes respectively, green represents heterozygous genotype, and red represents not detected.

[0038] Figure 11 This is a practical application of the Beijing Black Pig individual identification and traceability technology based on multiple SNP detection provided in Embodiment 2 of the present invention; wherein, the QR code on the left represents different downstream products of the black pig, and the right side is the scanning result, which contains the genotypes of 20 SNP loci of the product, for individual identification and traceability.

[0039] Figure 12 The results of the RT-PCR high-resolution melting curve analysis of the Beijing Black Pig individual identification and traceability technology based on multiple SNP detection provided in Embodiment 3 of the present invention are shown. In the table above, the red box shows the genotypes of different samples at the SNP site, which correspond to the curve typing of different samples in the high-resolution melting curve below.

[0040] Figure 13 The results of SNP site screening and optimization (CNC10141079 and CNC10072418) for individual identification and tracing technology of Beijing black pigs based on multiple SNP detection provided in Comparative Example 1 of this invention are shown. In this example, blue and yellow represent two homozygous genotypes, green represents heterozygous genotypes, and red represents undetected genotypes.

[0041] Figure 14 The results of SNP site screening and optimization (CNC10150932 and CNC100082598) for individual identification and tracing technology of Beijing black pigs based on multiple SNP detection provided in Comparative Example 1 of this invention are shown. In this example, blue and yellow represent two homozygous genotypes, green represents heterozygous genotypes, and red represents undetected genotypes.

[0042] Figure 15 The results of SNP site screening and optimization (CNC10170458 and CNC10082724) for individual identification and tracing technology of Beijing black pigs based on multiple SNP detection provided in Comparative Example 1 of this invention are shown. In this example, blue and yellow represent two homozygous genotypes, green represents heterozygous genotypes, and red represents undetected genotypes.

[0043] Figure 16 The results of SNP site screening and optimization for the Beijing Black Pig individual identification and traceability technology based on multiple SNP detection provided in Comparative Example 1 of this invention are (CNCB10003393 and CNC10093030). Among them, blue and yellow represent two homozygous genotypes, green represents heterozygous genotype, and red represents undetected.

[0044] Figure 17 The results of SNP site screening and optimization of the Beijing Black Pig individual identification and traceability technology based on multiple SNP detection provided in Comparative Example 1 of this invention are (CNC10133433 and CNCB10008209); where blue and yellow represent two homozygous genotypes respectively, green represents heterozygous genotype, and red represents undetected. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0046] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0047] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0048] Main instruments and equipment: 384-well PCR instrument (Applied Biosystems Veriti384), Massarray mass spectrometer (Agena), Massarray mass spectrometer spotter (Agena RS1000), high-speed benchtop centrifuge (Eppendorf 5417R), fluorescence microplate reader (Bio tek Synergy H4), NanoDrop (Thermo 2000), vortex mixer (Qilinbell QL-901), micropipettes (2.5μL, 10μL, 100μL, 1000μL), etc.

[0049] Main reagents: Animal tissue genome extraction kit (QIAGEN), PCR Enzyme custom (Roche), PCR Accessory Set (Sequenom), IPLEX Gold Reagent Kit (Sequenom), Spectro CHIPResin Kit (Sequenom), etc.

[0050] Example 1 This invention provides a method for individual identification and tracing, specifically including the following process: 1. Sample DNA extraction and concentration adjustment Sample DNA extraction: A small amount of muscle tissue was accurately weighed according to the ratio of meat to water 1:4 (m / m) and homogenized. The homogenizer was rotated at 12,000 rpm for 10 min. Genomic DNA was extracted according to the instructions of the Qiagen DNA Extraction Kit. Finally, the DNA was dissolved in 200 μL of TE buffer, and the absorbance at wavelengths of 280 nm and 260 nm was measured using a NanoDrop One ultra-micro UV-Vis spectrophotometer. The DNA purity and concentration were calculated, and the DNA was diluted with TE buffer to 10-70 ng / μL for later use.

[0051] 2. Primer sequence The nucleotide sequences of the 20 pairs of specific primers of this invention are SEQ ID NO.21-60. Primers Primer 1 to Primer 20 are synthesized (see table below).

[0052] Table 1 SNP primer sequences

[0053] 3. PCR reaction system and amplification procedure Prepare the working solution mix according to the PCR reaction system (Table 2). After mixing all components thoroughly, aliquot the mixture into 384-well plates (4µL / well). Load the DNA sample (1µL) into the 384-well plate. Seal the plate with a special sealing film and centrifuge it briefly at 1200rpm at 4℃. Ensure that there are no air bubbles in the reaction solution at the bottom of the well before proceeding with the PCR program (Table 3).

[0054] Table 2 PCR reaction system (4µL)

[0055] Table 3 PCR amplification procedure (three-step method)

[0056] 4. Alkaline phosphatase digestion of PCR products The PCR products were digested with alkaline phosphatase to convert the 5'-P end to a 5'-OH end. In each well of the 384-well plate, 0.3 μL of SAP Enzyme (1.7 U / μL), 0.17 μL of 10×PCR Buffer, and 1.53 μL of ddH2O were added. Digestion was performed by incubation at 37°C for 40 min, followed by inactivation at 85°C for 5 min, and then storage at 4°C.

[0057] 5. Single base extension reaction Using specific primers to pair complementary bases with SNP sites in the PCR product, DNA polymerase, buffer, reverse transcriptase, and other reaction components are added to extend a single base at the target nucleotide position. The single-base extension reaction solution is prepared according to the PCR reaction system (Table 4) and added to 384-well plates. After sealing the plates with a special sealing film, the 384-well plates are briefly centrifuged at 1200 rpm at 4°C to ensure no air bubbles remain at the bottom of the wells before proceeding with the PCR program (Table 5).

[0058] Table 4 Single-base extension reaction system

[0059] Table 5. Single-base extension amplification procedure (three-step method)

[0060] 6. Mass spectrometry detection The reaction product (total 9 µL) was diluted 3-fold and desalted using resin (6 mg). The mixture was rotated for 15 minutes and centrifuged at 3200 g (2000 rpm for a standard plate centrifuge) for 5 minutes. The desalted sample was loaded onto a sample target using a mass spectrometer and allowed to crystallize naturally. Mass spectrometry was then performed, and data were collected. Genotypes of 20 nuclear SNP loci in the sample were analyzed. Figures 1-10 The individual identification and traceability of the sample can be completed by comparing the test results with the Beijing Black Pig Positive Sample Database.

[0061] The Beijing Black Pig Positive Sample Database consists of locus information from different known Beijing Black Pig individuals at the aforementioned 20 SNP sites. During comparison, if the test result of the sample to be tested is completely identical to that of a positive sample in the Beijing Black Pig Positive Sample Database, it can be determined that the sample to be tested originated from that positive sample.

[0062] Example 2 Based on the method provided in Example 1 above, this invention performed mass spectrometry detection on 306 Beijing black pig samples and statistically analyzed the sample identification rate (Table 6). The average individual sample identification rate reached 98.9%, indicating that these SNP loci have high throughput and high accuracy, and can significantly reduce manpower and detection time costs.

[0063] Table 6 Individual Identification Rate of SNP Detection in Beijing Black Pigs

[0064] In practical applications, black pork products can be labeled with traceability codes to identify their biometric identity, thereby verifying the product's authenticity and tracing its production source. Figure 11 ).

[0065] Example 3 This invention is based on the method provided in Example 1, using the same sample template and SNP primer combination, and employs high-resolution RT-PCR melting curve analysis for comparative verification. The implementation steps are as follows: 1. Sample DNA extraction and concentration adjustment Sample DNA extraction: A small amount of muscle tissue was accurately weighed according to the ratio of meat to water 1:4 (m / m) and homogenized. The homogenizer speed was 12,000 rpm and the homogenization time was 10 min. Genomic DNA was extracted according to the instructions of the Qiagen DNA Extraction Kit. Finally, the DNA was dissolved in 200 μL of TE buffer and the absorbance at wavelengths of 280 nm and 260 nm was measured using a NanoDrop One ultra-micro UV-Vis spectrophotometer. The DNA purity and concentration were calculated and diluted with TE buffer to 10 ng / μL for later use.

[0066] 2. RT-PCR reaction system and amplification procedure The working solution was prepared according to the RT-PCR reaction system (Table 7). After thorough mixing of all components, the mixture was dispensed into 8-tube strips (8 µL / well). 2 µL of DNA sample was loaded into each 8-tube strip, and the PCR program was executed. The program was as follows: pre-denaturation at 95°C for 300 s; denaturation at 95°C for 15 s, annealing and extension at 55°C for 45 s, for 40 cycles; then the melting curve program was executed: denaturation at 95°C for 60 s, extension at 70°C for 60 s, with the temperature continuously increased to 95°C at a rate of 0.02°C / s; finally, cooling at 40°C for 60 s to end the program.

[0067] Table 7 RT-PCR reaction system (8µL)

[0068] 3. High-resolution melting curve analysis Because the amplification products of different SNP genotypes exhibit subtle differences in Tm values ​​during melting, high-resolution melting curves can be used to distinguish between different genotypes of the amplification products. The melting curve morphology of the samples corresponds to three different genotypes ( Figure 12 The genotype of a sample can be determined by comparing the results with those from the SNP chip.

[0069] Comparative Example 1 This comparative example serves as a comparison of the individual identification and traceability technology for Beijing black pigs based on multiple SNP detection, demonstrating the process of screening and optimizing SNP locus combinations. The same sample template, experimental methods, and procedures were used for comparative verification.

[0070] The 20 SNP sites finally obtained in this invention were obtained by screening and optimization of the initial 30 SNP sites. The 10 pairs of specific primers (Primer 21 to Primer 30) corresponding to the 10 SNP sites (SNP21 to SNP30) that were screened out are shown in Table 8.

[0071] Table 8 SNP primer sequences (screened out)

[0072] Using the same method as in Example 1, mass spectrometry was performed on 306 samples, and the sample identification rate was calculated (Table 9). SNP21 to SNP27 were all successfully genotyped by mass spectrometry, but the detection rate was relatively low. Figures 13-17 The mass spectrometry results for SNP28, SNP29, and SNP30 were abnormal and could not be classified. Analysis suggests this may be due to the low amplification efficiency of the specific primers for these SNP sites on the template, resulting in an abnormal amount of PCR product with the tag (ACGTTGGATG) during final mass spectrometry detection. Therefore, after screening, the final combination of SNP1~SNP20 sites and their corresponding specific primers determined in this invention were obtained.

[0073] Table 9. Screening of SNP loci identification rates in Beijing black pigs

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A combination of SNP feature sequences, characterized in that, The nucleotide sequence is shown in SEQ ID NO.1-20; As shown in SEQ ID NO.1, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of A / G; As shown in SEQ ID NO.2, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of A / G; As shown in SEQ ID NO.3, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of A / G; As shown in SEQ ID NO.4, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of A / G; As shown in SEQ ID NO.5, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of A / G; As shown in SEQ ID NO.6, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of T / C. As shown in SEQ ID NO.7, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of A / G; As shown in SEQ ID NO.8, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of C / G; As shown in SEQ ID NO.9, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of A / G; As shown in SEQ ID NO.10, position 51 of the nucleotide sequence exhibits polymorphism, which is A / G. As shown in SEQ ID NO.11, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of T / C. As shown in SEQ ID NO.12, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of A / G; As shown in SEQ ID NO.13, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of C / G; As shown in SEQ ID NO.14, the nucleotide sequence at position 51 is polymorphic, with the polymorphism being A / C; As shown in SEQ ID NO.15, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of T / C. As shown in SEQ ID NO.16, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of T / C. As shown in SEQ ID NO.17, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of T / C. As shown in SEQ ID NO.18, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of T / C. As shown in SEQ ID NO.19, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of T / G; As shown in SEQ ID NO.20, the nucleotide sequence at position 51 is polymorphic, with a polymorphism of A / G.

2. A primer combination for amplifying polymorphic sites in the characteristic sequence combination of claim 1, characterized in that, for: The primer pair Primer1 used to amplify SNP1 consists of Primer 1F and Primer 1R, and the primer sequences are shown in SEQ ID NO:21-22; The primer pair Primer2 used to amplify SNP2 consists of Primer 2F and Primer 2R, and the primer sequences are shown in SEQ ID NO:23-24; The primer pair Primer3 used to amplify SNP3 consists of Primer 3F and Primer 3R, and the primer sequences are shown in SEQ ID NO:25-26; The primer pair Primer4 used to amplify SNP4 consists of Primer 4F and Primer 4R, and the primer sequences are shown in SEQ ID NO:27-28; The primer pair Primer5 used to amplify SNP5 consists of Primer 5F and Primer 5R, and the primer sequences are shown in SEQ ID NO:29-30; The primer pair Primer6 used to amplify SNP6 consists of Primer 6F and Primer 6R, and the primer sequences are shown in SEQ ID NO:31-32; The primer pair Primer7 used to amplify SNP7 consists of Primer 7F and Primer 7R, and the primer sequences are shown in SEQ ID NO:33-34; The primer pair Primer8 used to amplify SNP8 consists of Primer 8F and Primer 8R, and the primer sequences are shown in SEQ ID NO:35-36; The primer pair Primer9 used to amplify SNP9 consists of Primer 9F and Primer 9R, and the primer sequences are shown in SEQ ID NO:37-38; The primer pair Primer10 used to amplify SNP10 consists of Primer 10F and Primer 10R, and the primer sequences are shown in SEQ ID NO:39-40. The primer pair Primer11 used to amplify SNP11 consists of Primer 11F and Primer 11R, and the primer sequences are shown in SEQ ID NO:41-42. The primer pair Primer12 used to amplify SNP12 consists of Primer 12F and Primer 12R, and the primer sequences are shown in SEQ ID NO:43-44. The primer pair Primer13 used to amplify SNP13 consists of Primer 13F and Primer 13R, and the primer sequences are shown in SEQ ID NO:45-46. The primer pair Primer14 used to amplify SNP14 consists of Primer 14F and Primer 14R, and the primer sequences are shown in SEQ ID NO:47-48. The primer pair Primer15 used to amplify SNP15 consists of Primer 15F and Primer 15R, and the primer sequences are shown in SEQ ID NO:49-50. The primer pair Primer16 used to amplify SNP16 consists of Primer 16F and Primer 16R, and the primer sequences are shown in SEQ ID NO:51-52. The primer pair Primer17 used to amplify SNP17 consists of Primer 17F and Primer 17R, and the primer sequences are shown in SEQ ID NO:53-54. The primer pair Primer18 used to amplify SNP18 consists of Primer 18F and Primer 18R, and the primer sequences are shown in SEQ ID NO:55-56. The primer pair Primer19 used to amplify SNP19 consists of Primer 19F and Primer 19R, and the primer sequences are shown in SEQ ID NO:57-58. The primer pair Primer20 used to amplify SNP20 consists of Primer 20F and Primer 20R, and the primer sequences are shown in SEQ ID NO:59-60.

3. A reagent kit, characterized in that, The kit includes the primer pair as described in claim 2.

4. The use of the primer pair of claim 2, or the kit of claim 3, in any of the following: i) Identification of Beijing Black Pig breed, ii) Traceability of individual Beijing black pigs iii) Breeding of Beijing Black Pigs related to breed, iv) Identification of Beijing black pork products.

5. The application according to claim 4, characterized in that, The applications include: The genome of the sample to be tested is extracted, and the polymorphism of polymorphic sites in the characteristic sequence combination described in claim 1 is detected. Based on the polymorphism detection results, the variety identification or individual traceability of the sample to be tested is achieved.

6. The application according to claim 5, characterized in that, The process of determining the variety identification or individual tracing of the sample based on the polymorphism detection results includes: The polymorphism of each SNP site in the SNP site combination of the sample to be tested is compared with the positive sample database of Beijing black pigs, and the specific source of the sample to be tested is determined based on the comparison results.

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