SNP (Single Nucleotide Polymorphism) site combination and application thereof in Beijing black pig individual identification and traceability

Abstract

The invention relates to the technical field of nucleic acid detection, in particular to an SNP (Single Nucleotide Polymorphism) site combination and application thereof in Beijing black pig individual identification and traceability. The SNP loci comprise SNP1-SNP20, and the SNP loci comprise SNP1-SNP20. The SNP loci comprise nucleotide sequences as shown in SEQ ID NO. 1 to 20. The invention also discloses a kit for detecting the SNP loci. The SNP site combination composed of 20 SNP sites is obtained through research and screening, the SNP sites have the high individual recognition rate, the average recognition rate can reach 98.9%, high throughput and high accuracy are achieved, manpower and detection time cost can be greatly reduced, and the SNP site combination has the important application value in the field of quality supervision of Beijing black pig products.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and in particular to a SNP site combination and its application in individual identification and tracing of Beijing black pigs. Background Art

[0002] Beijing Black Pig is a local high-quality black pig breed. It is not only tolerant to roughage, early maturing and prolific, and has tender meat, but also fast growing and meaty. It is necessary to protect local high-quality black pig breeds, eliminate adulteration and counterfeiting, maintain the high-end brand image of high-value products, and conduct authenticity identification and traceability research on such products. Therefore, it is necessary to establish a safe, reliable, and transparent traceability system suitable for the production and sales of meat products to enhance regulatory efficiency and ensure product quality. This is beneficial to protect consumers from fraud, protect local specialty meat products, and effectively maintain the brand image of local specialty products. It provides reliable technical guarantees 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, a large number of studies on the traceability of meat products have been carried out at home and abroad. Traceability methods can be roughly divided into three categories: physical methods (label traceability technology, etc.), chemical methods (isotope traceability technology, etc.) and biotechnology (DNA traceability technology, etc.). Among them, DNA traceability technology has the advantages of easy typing, good repeatability, simple and fast detection methods, and low cost. It is recognized as the most promising and application-valued rapid traceability technology. Single nucleotide polymorphism (SNP) refers to a marker method that causes polymorphism caused by the variation of a single nucleotide on the genome. Using the SNP differences in the genome of individual species to construct the SNP characteristic map of individual species is equivalent to making a unique DNA ID card for each individual species. DNA traceability technology can not only realize the traceability of meat food from breeding to dining table, but also realize the accurate identification of individuals and varieties. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a SNP site combination and its application in individual identification and traceability of Beijing black pigs.

[0005] In a first aspect, the present invention provides a SNP locus combination, including SNP1-SNP20; SNP1 is located at 60792655bp of chromosome Chromosome13, and the polymorphism is A / G; SNP2 is located at 101773318bp of chromosome Chromosome8, and the polymorphism is A / G; SNP3 is located at 29498397bp of chromosome Chromosome3, and the polymorphism is A / G; SNP4 is located at 8479022bp of chromosome Chromosome8, and the polymorphism is A / G; SNP5 is located at 56216032bp of chromosome Chromosome14, and the polymorphism is A / G; SNP6 is located at 3765781bp of chromosome Chromosome7, and the polymorphism is T / C; SNP7 is located at 137712833bp of chromosome Chromosome4, and the polymorphism is A / G; SNP8 is located at 124157393bp of chromosome Chromosome4, and the polymorphism is C / G; SNP9 is located at 156408847bp of chromosome Chromosome13, and the polymorphism is A / G; SNP10 is located at 88201093bp of chromosome Chromosome2, and the polymorphism is A / G; SNP11 is located at 73144741bp of chromosome Chromosome5, and the polymorphism is T / C; SNP12 is located at 25784942bp of chromosome Chromosome2, and the polymorphism is A / G; SNP13 is located at 69817848bp of chromosome Chromosome1, and the polymorphism is C / G; SNP14 is located at 95533526bp of chromosome Chromosome2, and the polymorphism is A / C; SNP15 is located at 813652bp of chromosome Chromosome1, and the polymorphism is T / C; SNP16 is located at 62557043bp of chromosome Chromosome9, and the polymorphism is T / C; SNP17 is located at 82135347bp of chromosome Chromosome16, and the polymorphism is T / C; SNP18 is located at 11109184bp of chromosome Chromosome16, and the polymorphism is T / C; SNP19 is located at 2842626bp of chromosome Chromosome17, and the polymorphism is T / G; SNP20 is located at 2738859bp of chromosome Chromosome15, and the polymorphism is A / G.

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

[0007] In a third aspect, the present invention provides a primer combination for amplifying the aforementioned SNP site combination, comprising: Primer 1, a primer pair for amplifying SNP1, is composed of Primer 1F and Primer 1R, and the primer sequences are shown in SEQ ID NOs: 21-22; Primer 2 for amplifying SNP2, consisting of Primer 2F and Primer 2R, the primer sequences are shown in SEQ ID NOs: 23-24; Primer 3, a primer pair for amplifying SNP3, is composed of Primer 3F and Primer 3R, and the primer sequences are shown in SEQ ID NOs: 25-26; Primer 4, a primer pair for amplifying SNP4, is composed of Primer 4F and Primer 4R, and the primer sequences are shown in SEQ ID NOs: 27-28; Primer 5, a primer pair for amplifying SNP5, is composed of Primer 5F and Primer 5R, and the primer sequences are shown in SEQ ID NOs: 29-30; Primer 6, a primer pair for amplifying SNP6, is composed of Primer 6F and Primer 6R, and the primer sequences are shown in SEQ ID NOs: 31-32; Primer 7 for amplifying SNP7, consisting of Primer 7F and Primer 7R, the primer sequences are shown in SEQ ID NOs: 33-34; Primer 8, a primer pair for amplifying SNP 8, is composed of Primer 8F and Primer 8R, and the primer sequences are shown in SEQ ID NOs: 35-36; Primer 9 for amplifying SNP9, consisting of Primer 9F and Primer 9R, the primer sequences are shown in SEQ ID NOs: 37-38; Primer 10 for amplifying SNP 10, consisting of Primer 10F and Primer 10R, the primer sequences of which are shown in SEQ ID NOs: 39-40; Primer 11, a primer pair for amplifying SNP 11, is composed of Primer 11F and Primer 11R, and the primer sequences are shown in SEQ ID NOs: 41-42; Primer 12 for amplifying SNP12, consisting of Primer 12F and Primer 12R, the primer sequences of which are shown in SEQ ID NOs: 43-44; Primer 13, a primer pair for amplifying SNP13, is composed of Primer 13F and Primer 13R, and the primer sequences are shown in SEQ ID NOs: 45-46; Primer 14, a primer pair for amplifying SNP 14, is composed of Primer 14F and Primer 14R, and the primer sequences are shown in SEQ ID NOs: 47-48; Primer 15, a primer pair for amplifying SNP15, is composed of Primer 15F and Primer 15R, and the primer sequences are shown in SEQ ID NOs: 49-50; Primer 16 for amplifying SNP16, consisting of Primer 16F and Primer 16R, the primer sequences of which are shown in SEQ ID NOs: 51-52; Primer 17 for amplifying SNP17, consisting of Primer 17F and Primer 17R, the primer sequences of which are shown in SEQ ID NOs: 53-54; Primer 18, a primer pair for amplifying SNP18, is composed of Primer 18F and Primer 18R, and the primer sequences are shown in SEQ ID NOs: 55-56; Primer 19 for amplifying SNP19, consisting of Primer 19F and Primer 19R, the primer sequences of which are shown in SEQ ID NOs: 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 NOs: 59-60.

[0008] In a fourth aspect, the present invention provides a molecular probe combination, wherein the molecular probe combination is used to detect the aforementioned SNP site combination.

[0009] In a fifth aspect, the present invention provides a gene chip, wherein the gene chip comprises the aforementioned molecular probe combination.

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

[0011] In a seventh aspect, the present invention provides the use of the aforementioned SNP site combination as a target in the preparation of a gene chip for Beijing Black Pig breed identification or Beijing Black Pig individual tracing.

[0012] The present invention further provides the use 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) Individual traceability of 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. According to the polymorphism detection result, the variety identification or individual traceability of the sample to be tested is achieved.

[0014] Furthermore, the method of determining the variety identification of the sample to be tested or the individual traceability of the individual traceability according to the polymorphism detection result includes: The polymorphism of each SNP site in the SNP site combination of the sample to be tested is compared with the Beijing black pig positive sample library, and the specific source of the sample to be tested is determined according to the comparison results.

[0015] Preferably, the Beijing Black Pig positive sample library is composed of the site information of different known Beijing Black Pig individuals at the aforementioned 20 SNP sites. During specific 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 library, it can be determined that the sample to be tested originates from the positive sample.

[0016] The 20 nuclear gene SNP sites provided by the present invention are screened from 50,000 sequenced SNP sites through logical operations, typing analysis, and gene frequency calculation. It can ensure that the genotype frequencies of homozygous and heterozygous are relatively balanced, and have the ability to identify a sufficient number of individuals (theoretically, up to 3 can be identified). 20 By comparing the polymorphism of the 20 SNP sites of the unknown sample with a positive sample in the Beijing Black Pig positive sample library to see if they are completely consistent, it can be determined whether the unknown sample is a Beijing Black Pig sample and which Beijing Black Pig individual in the positive sample library it comes from.

[0017] As a specific embodiment, the present invention provides a method for Beijing black pig individual identification and traceability technology based on multiple SNP detection, comprising: (1) Extracting 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 site; (5) Mass spectrometry detection and genotype analysis.

[0018] Furthermore, the PCR amplification reaction system of step (2) comprises: sample DNA template, primers, PCR amplification enzyme, PCR Buffer, MgCl 2 , dNTP mix and ddH 2 O.

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

[0020] Furthermore, the PCR amplification reaction system in step (2) comprises: DNA template 1 μL (10-70 ng / μL), primer mix shown in SEQ ID NO. 21-60 1 μL (10-20 pmol / μL), PCR amplification enzyme HotStarTaq (5U / μL) 0.2 μL, 10×PCR Buffer 0.625 μL, MgCl 2 (25mM) 0.325μL, dNTP mix (25 mM) 0.1μL, and the rest of the volume was diluted with ddHO 2 O complement.

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

[0022] Furthermore, the PCR product of step (3) was digested with alkaline phosphatase, and 0.3 μL SAP Enzyme (1.7 U / μL), 0.17 μL 10× PCR Buffer and 1.53 μL ddH were added to each well of the product of the PCR amplification reaction of step (2). 2 O, digestion was performed by incubation at 37°C for 40 min, inactivation at 85°C for 5 min, and standby at 4°C.

[0023] Furthermore, for the single base extension reaction at the SNP site described in step (4), the following were added to the PCR digestion product in each well of step (3): 0.041 μL of iPLEX enzyme, 0.2 μL of iPLEX terminator, 0.2 μL of iPLEX Buffer plus, and 0.94 μL of Extend primer Mix, and the remaining volume was diluted with ddHO. 2 O complement; The single-base extension amplification program was set as follows: (1) pre-denaturation: 94°C, 30 s; (2) denaturation: 94°C, 5 s; annealing: 52°C, 5 s; extension: 80°C, 5 s, 40 large cycles, in which annealing and extension were performed for 5 small cycles in each large cycle; (3) post-amplification extension: 72°C, 180 s; (4) cooling to 4°C, hold.

[0024] Further, for the mass spectrometry detection and genotyping analysis described in step (5), the reaction product of step (4) above (9 µL in total) was diluted 3 times, desalted using a resin (6 MG), rotated on a rotary mixer for 15 minutes, and centrifuged at 3200 g (2000 rpm in a standard plate centrifuge) for 5 minutes. The desalted sample was loaded onto a sample target using a mass spectrometer, and naturally crystallized; the sample was loaded onto a mass spectrometer for mass spectrometry detection, and data was collected.

[0025] The present invention has the following beneficial effects: The present invention screened a SNP site combination consisting of 20 SNP sites. Based on the polymorphism detection results of these 20 SNP sites and the genotype data of each site in the database, the individual identification of Beijing black pigs can be achieved. The individual identification rate of each SNP site in the SNP site combination provided by the present invention reaches an average of 98.9%, which has high throughput and high accuracy, and can greatly reduce manpower and detection time costs.

[0026] The present invention provides a powerful tool for the individual traceability and identification of Beijing Black Pigs, which is conducive to the sustainable development of the brand of geographical indication products and the protection of local variety diversity. The development and research of technical methods for identifying geographical indication products can effectively curb brand adulteration and protect brand reputation, which is of great significance to enhancing the overall brand value of the local specialty livestock industry and protecting precious local specialty varieties and their breeding and processing enterprises. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 These are the test results of CNC10082042 and CNC10021852 of the Beijing Black Pig individual identification and traceability technology based on multiple SNP detection provided in Example 1 of the present invention; wherein, blue and yellow represent two homozygous genotypes, respectively, green represents heterozygous genotype, and red represents undetected.

[0029] Figure 2 These are the test results of CNC10010024 and CNC10170056 of the Beijing Black Pig individual identification and traceability technology based on multiple SNP detection provided in Example 1 of the present invention; wherein, blue and yellow represent two homozygous genotypes, respectively, green represents heterozygous genotype, and red represents undetected.

[0030] Figure 3 These are the test results of CNC10091242 and CNC10042330 of the Beijing Black Pig individual identification and traceability technology based on multiple SNP detection provided in Example 1 of the present invention; wherein, blue and yellow represent two homozygous genotypes, respectively, green represents heterozygous genotype, and red represents undetected.

[0031] Figure 4 These are the detection results of CNC10011430 and CNCB10001966 of the Beijing Black Pig individual identification and traceability technology based on multiple SNP detection provided in Example 1 of the present invention; wherein, blue and yellow represent two homozygous genotypes, respectively, green represents heterozygous genotype, and red represents undetected.

[0032] Figure 5 These are the test results of CNC10160241 and CNC10070071 of the Beijing Black Pig individual identification and traceability technology based on multiple SNP detection provided in Example 1 of the present invention; wherein, blue and yellow represent two homozygous genotypes, respectively, green represents heterozygous genotype, and red represents undetected.

[0033] Figure 6 These are the test results of CNC10020552 and CNCB10002454 of the Beijing Black Pig individual identification and traceability technology based on multiple SNP detection provided in Example 1 of the present invention; wherein, blue and yellow represent two homozygous genotypes, respectively, green represents heterozygous genotype, and red represents undetected.

[0034] Figure 7 These are the test results of CNC10150052 and CNC10042609 of the Beijing Black Pig individual identification and traceability technology based on multiple SNP detection provided in Example 1 of the present invention; wherein, blue and yellow represent two homozygous genotypes, respectively, green represents heterozygous genotype, and red represents undetected.

[0035] Figure 8 These are the test results of CNC10133084 and CNCB10004066 of the Beijing Black Pig individual identification and traceability technology based on multiple SNP detection provided in Example 1 of the present invention; wherein, blue and yellow represent two homozygous genotypes, respectively, green represents heterozygous genotype, and red represents undetected.

[0036] Fig. 9 These are the test results of CNC10141074 and CNC10080151 of the Beijing Black Pig individual identification and traceability technology based on multiple SNP detection provided in Example 1 of the present invention; wherein, blue and yellow represent two homozygous genotypes, respectively, green represents heterozygous genotype, and red represents undetected.

[0037] Fig.10 These are the test results of CNC10161593 and CNCB10008679 of the Beijing Black Pig individual identification and traceability technology based on multiple SNP detection provided in Example 1 of the present invention; wherein, blue and yellow represent two homozygous genotypes, respectively, green represents heterozygous genotype, and red represents undetected.

[0038] Fig.11 This is the practical application of the Beijing black pig individual identification and traceability technology based on multiple SNP detection provided in Example 2 of the present invention; wherein, the QR code on the left is different downstream products of black pigs, and the right is the scanning result, which includes the genotypes of 20 SNP sites of the product for individual identification and traceability.

[0039] Fig.12 This is the result of RT-PCR high-resolution melting curve analysis of the Beijing Black Pig individual identification and traceability technology based on multiple SNP detection provided in Example 3 of the present invention; wherein, the red box in the table above shows the genotypes of different samples in the SNP site, corresponding to the curve typing of different samples in the high-resolution melting curve below.

[0040] Fig.13 This is the SNP site screening and optimization result (CNC10141079 and CNC10072418) of the Beijing Black Pig individual identification and traceability technology based on multiple SNP detection provided in Comparative Example 1 of the present invention; wherein, blue and yellow represent two homozygous genotypes, green represents heterozygous genotype, and red represents undetected.

[0041] Fig.14 This is the SNP site screening and optimization result of the Beijing black pig individual identification and traceability technology based on multiple SNP detection provided in Comparative Example 1 of the present invention (CNC10150932 and CNC100082598); wherein, blue and yellow represent two homozygous genotypes, green represents heterozygous genotype, and red represents undetected.

[0042] Fig.15 This is the SNP site screening and optimization result (CNC10170458 and CNC10082724) of the Beijing black pig individual identification and traceability technology based on multiple SNP detection provided in Comparative Example 1 of the present invention; wherein, blue and yellow represent two homozygous genotypes, green represents heterozygous genotype, and red represents undetected.

[0043] Fig.16 This is the SNP site screening and optimization result of the Beijing black pig individual identification and traceability technology based on multiple SNP detection provided in Comparative Example 1 of the present invention (CNCB10003393 and CNC10093030); wherein, blue and yellow represent two homozygous genotypes, green represents heterozygous genotype, and red represents undetected.

[0044] Fig.17 This is the SNP site screening and optimization result of the Beijing black pig individual identification and traceability technology based on multiple SNP detection provided in Comparative Example 1 of the present invention (CNC10133433 and CNCB10008209); wherein, blue and yellow represent two homozygous genotypes, green represents heterozygous genotype, and red represents undetected. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not 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.

[0046] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art, for example, reference may be made to experimental manuals in the art, or the conditions recommended in the manufacturer's instructions.

[0047] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources.

[0048] Main instruments and equipment: 384-well PCR instrument (Applied Biosystems Veriti384), Massarray mass spectrometer (Agena), Massarray mass spectrometer (Agena RS1000), high-speed desktop centrifuge (Eppendorf 5417R), fluorescence microplate reader (Bio tek Synergy H4), NanoDrop (Thermo 2000), vortex mixer (Qilin Bell QL-901), micropipette (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 The present invention provides a method for individual identification and tracing, which specifically includes the following process: 1. Sample DNA extraction and concentration adjustment Sample DNA extraction: A small amount of muscle tissue was accurately weighed and homogenized at a ratio of 1:4 (m / m) between meat and water. The homogenizer speed was 12,000 rpm and the homogenization was performed 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 TE buffer and the absorbance at wavelengths of 280 nm and 260 nm was measured using a NanoDrop One ultra-micro UV-visible spectrophotometer to calculate the DNA purity and concentration. The DNA was diluted to 10-70 ng / μL with TE buffer for later use.

[0051] 2. Primer sequence The nucleotide sequences of 20 pairs of specific primers of the present invention are SEQ ID NOs. 21-60, synthetic primers Primer 1 to Primer 20 (see the table below).

[0052] Table 1 SNP primer sequences

[0053] 3. PCR reaction system and amplification procedure Refer to the PCR reaction system (Table 2) to prepare the working solution Mix. After mixing all components evenly, dispense them into a 384-well plate (4µL / well), and load the DNA sample (1µL) into the 384-well plate. After sealing the well plate with a special sealing film, place the 384-well plate in a 4°C centrifuge at 1200rpm for instant centrifugation. After ensuring that there are no bubbles in the reaction solution at the bottom of the well, perform 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 product was digested with alkaline phosphatase to convert the 5'-P end of the product into a 5'-OH end. In the above 384-well plate, 0.3μL SAP Enzyme (1.7 U / μL), 0.17μL 10×PCR Buffer and 1.53μL ddH2O were added to each well, and the digestion was incubated at 37℃ for 40min, inactivated at 85℃ for 5min, and stored at 4℃ for later use.

[0057] 5. Single base extension reaction Use specific primers to complement the bases at the SNP site in the PCR product, add DNA polymerase, buffer, reverse transcriptase and other reaction components to extend a base at the target nucleotide position. Prepare the single base extension reaction solution according to the PCR reaction system (Table 4) and add it to the 384-well plate. After sealing the well plate with a special sealing film, place the 384-well plate in a 4℃ centrifuge and centrifuge it at 1200rpm for an instant. After ensuring that there are no bubbles in the reaction solution at the bottom of the well, perform 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 The reaction product (9 µL in total) was diluted 3 times, desalted using resin (6 mg), rotated on a rotary mixer for 15 minutes, and centrifuged at 3200 g (2000 rpm for a standard plate centrifuge) for 5 minutes. The desalted sample was loaded onto the sample target using a mass spectrometer and allowed to crystallize naturally. The sample was loaded onto a mass spectrometer for mass spectrometry detection and data collection. The genotypes of 20 nuclear gene SNP sites of the sample were detected ( Figure 1-Figure 10 ), and compare the test results with the Beijing black pig positive sample library to complete the individual identification and traceability of the samples.

[0061] The Beijing Black Pig positive sample library is composed of the locus information of different known Beijing Black Pig individuals at the aforementioned 20 SNP loci. During specific 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 library, it can be determined that the sample to be tested is derived from the positive sample.

[0062] Example 2 Based on the method provided in Example 1 above, the present invention performed mass spectrometry detection on 306 Beijing black pig samples and calculated the sample recognition rate (Table 6). The average individual recognition rate of the samples reached 98.9%, indicating that these SNP sites have high throughput and high accuracy, which can greatly reduce the manpower and detection time costs.

[0063] Table 6 Individual identification rate of Beijing black pig SNP detection

[0064] In practical applications, the product can be biometrically identified by adding a traceability code to the black pork product label to verify the authenticity of the product and trace its production source ( Fig.11 ).

[0065] Example 3 The present invention is based on the method provided in Example 1, using the same sample template and SNP primer combination, and using RT-PCR high-resolution 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 and homogenized at a ratio of 1:4 (m / m) between meat and water. The homogenizer speed was 12,000 rpm and the homogenization was performed 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 TE buffer and the absorbance at wavelengths of 280 nm and 260 nm was measured using a NanoDrop One ultra-micro UV-visible spectrophotometer to calculate the DNA purity and concentration. The DNA was diluted to 10 ng / μL with TE buffer for later use.

[0066] 2. RT-PCR reaction system and amplification procedure Refer to the RT-PCR reaction system (Table 7) to configure the working solution Mix. After the components are evenly mixed, they are dispensed into 8-tube strips (8µL / well). DNA samples (2µL) are loaded into the 8-tube strips and the PCR program is performed. The program is: pre-denaturation at 95℃ for 300s; denaturation at 95℃ for 15s, annealing and extension at 55℃ for 45s, and 40 cycles; then enter the melting curve program, denaturation at 95℃ for 60s, extension at 70℃ for 60s, and continue to heat to 95℃ at a rate of 0.02℃ / s; finally cool at 40℃ for 60s to end the program.

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

[0068] 3. High-resolution melting curve analysis Since the amplified products of different SNP genotypes have slight differences in Tm value changes during melting, high-resolution melting curves can be used to distinguish the different genotypes of amplified products. The melting curve morphology of the sample corresponds to 3 different genotypes ( Fig.12 ), the sample genotype can be determined by comparing the SNP chip detection results.

[0069] Comparative Example 1 This comparative example is a comparative example of Beijing Black Pig individual identification and traceability technology based on multiple SNP detection, reflecting the process of screening and optimizing SNP site combinations. The same sample template, experimental method and steps were used for comparison and verification.

[0070] The 20 SNP sites finally obtained in the present invention are obtained by screening and optimizing 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] The same method as in Example 1 was used to perform mass spectrometry on 306 samples and the sample identification rates were calculated (Table 9). Among them, SNP21 to SNP27 were successfully typed after mass spectrometry, but the detection rate was relatively low ( Figure 13-Figure 17 ); The mass spectrometry results of SNP28, SNP29, and SNP30 were abnormal and could not be typed. The reason for this may be that the specific primers of the SNP sites had low amplification efficiency for the template, resulting in abnormal amounts of PCR products with tag labels (ACGTTGGATG) in the final mass spectrometry detection. Therefore, after screening, the site combination of SNP1 to SNP20 and its corresponding specific primers were finally determined by the present invention.

[0073] Table 9 Beijing Black Pig SNP site recognition rate screening

[0074] 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 make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A SNP site combination, characterized in that: Including SNP1-SNP20; SNP1 is located at 60792655bp of chromosome Chromosome13, and the polymorphism is A / G; SNP2 is located at 101773318bp of chromosome Chromosome8, and the polymorphism is A / G; SNP3 is located at 29498397bp of chromosome Chromosome3, and the polymorphism is A / G; SNP4 is located at 8479022bp of chromosome Chromosome8, and the polymorphism is A / G; SNP5 is located at 56216032bp of chromosome Chromosome14, and the polymorphism is A / G; SNP6 is located at 3765781bp of chromosome Chromosome7, and the polymorphism is T / C; SNP7 is located at 137712833bp of chromosome Chromosome4, and the polymorphism is A / G; SNP8 is located at 124157393bp of chromosome Chromosome4, and the polymorphism is C / G; SNP9 is located at 156408847bp of chromosome Chromosome13, and the polymorphism is A / G; SNP10 is located at 88201093bp of chromosome Chromosome2, and the polymorphism is A / G; SNP11 is located at 73144741bp of chromosome Chromosome5, and the polymorphism is T / C; SNP12 is located at 25784942bp of chromosome Chromosome2, and the polymorphism is A / G; SNP13 is located at 69817848bp of chromosome Chromosome1, and the polymorphism is C / G; SNP14 is located at 95533526bp of chromosome Chromosome2, and the polymorphism is A / C; SNP15 is located at 813652bp of chromosome Chromosome1, and the polymorphism is T / C; SNP16 is located at 62557043bp of chromosome Chromosome9, and the polymorphism is T / C; SNP17 is located at 82135347bp of chromosome Chromosome16, and the polymorphism is T / C; SNP18 is located at 11109184bp of chromosome Chromosome16, and the polymorphism is T / C; SNP19 is located at 2842626bp of chromosome Chromosome17, and the polymorphism is T / G; SNP20 is located at 2738859bp of chromosome Chromosome15, and the polymorphism is A / G.

2. A SNP site combination, characterized in that: comprising the nucleotide sequence shown in SEQ ID NO.1-20; As shown in SEQ ID NO.1, the nucleotide sequence has a polymorphism at position 51, and the polymorphism is A / G; As shown in SEQ ID NO.2, the nucleotide sequence has a polymorphism at position 51, and the polymorphism is A / G; As shown in SEQ ID NO.3, the nucleotide sequence at position 51 has a polymorphism, and the polymorphism is A / G; As shown in SEQ ID NO.4, the nucleotide sequence has a polymorphism at position 51, and the polymorphism is A / G; The nucleotide sequence shown in SEQ ID NO.5 has a polymorphism at position 51, and the polymorphism is A / G; The nucleotide sequence shown in SEQ ID NO.6 has a polymorphism at position 51, and the polymorphism is T / C; As shown in SEQ ID NO.7, the nucleotide sequence has a polymorphism at position 51, and the polymorphism is A / G; As shown in SEQ ID NO.8, the nucleotide sequence has a polymorphism at position 51, and the polymorphism is C / G; As shown in SEQ ID NO.9, the nucleotide sequence has a polymorphism at position 51, and the polymorphism is A / G; As shown in SEQ ID NO.10, the nucleotide sequence has a polymorphism at position 51, and the polymorphism is A / G; As shown in SEQ ID NO.11, the nucleotide sequence at position 51 has a polymorphism, and the polymorphism is T / C; As shown in SEQ ID NO. 12, the nucleotide sequence at position 51 has a polymorphism, and the polymorphism is A / G; As shown in SEQ ID NO.13, the nucleotide sequence at position 51 has a polymorphism, and the polymorphism is C / G; As shown in SEQ ID NO. 14, the nucleotide sequence at position 51 has a polymorphism, and the polymorphism is A / C; As shown in SEQ ID NO.15, the nucleotide sequence has a polymorphism at position 51, and the polymorphism is T / C; As shown in SEQ ID NO. 16, the nucleotide sequence at position 51 has a polymorphism, and the polymorphism is T / C; As shown in SEQ ID NO.17, the nucleotide sequence at position 51 has a polymorphism, and the polymorphism is T / C; As shown in SEQ ID NO.18, the nucleotide sequence at position 51 has a polymorphism, and the polymorphism is T / C; As shown in SEQ ID NO. 19, the nucleotide sequence at position 51 has a polymorphism, and the polymorphism is T / G; As shown in SEQ ID NO.20, the nucleotide sequence has a polymorphism at position 51, and the polymorphism is A / G.

3. A primer combination for amplifying the SNP site combination according to claim 1 or 2, characterized in that: include: Primer 1, a primer pair for amplifying SNP1, is composed of Primer 1F and Primer 1R, and the primer sequences are shown in SEQ ID NOs: 21-22; Primer 2 for amplifying SNP2, consisting of Primer 2F and Primer 2R, the primer sequences are shown in SEQ ID NOs: 23-24; Primer 3, a primer pair for amplifying SNP3, is composed of Primer 3F and Primer 3R, and the primer sequences are shown in SEQ ID NOs: 25-26; Primer 4, a primer pair for amplifying SNP4, is composed of Primer 4F and Primer 4R, and the primer sequences are shown in SEQ ID NOs: 27-28; Primer 5, a primer pair for amplifying SNP5, is composed of Primer 5F and Primer 5R, and the primer sequences are shown in SEQ ID NOs: 29-30; Primer 6, a primer pair for amplifying SNP6, is composed of Primer 6F and Primer 6R, and the primer sequences are shown in SEQ ID NOs: 31-32; Primer 7 for amplifying SNP7, consisting of Primer 7F and Primer 7R, the primer sequences of which are shown in SEQ ID NOs: 33-34; Primer 8 for amplifying SNP8, consisting of Primer 8F and Primer 8R, the primer sequences are shown in SEQ ID NOs: 35-36; Primer 9 for amplifying SNP9, consisting of Primer 9F and Primer 9R, the primer sequences are shown in SEQ ID NOs: 37-38; Primer 10 for amplifying SNP10, consisting of Primer 10F and Primer 10R, the primer sequences are shown in SEQ ID NOs: 39-40; Primer 11, a primer pair for amplifying SNP 11, is composed of Primer 11F and Primer 11R, and the primer sequences are shown in SEQ ID NOs: 41-42; Primer 12 for amplifying SNP12, consisting of Primer 12F and Primer 12R, the primer sequences are shown in SEQ ID NOs: 43-44; Primer 13, a primer pair for amplifying SNP 13, is composed of Primer 13F and Primer 13R, and the primer sequences are shown in SEQ ID NOs: 45-46; Primer 14 for amplifying SNP 14, consisting of Primer 14F and Primer 14R, the primer sequences are shown in SEQ ID NOs: 47-48; Primer 15, a primer pair for amplifying SNP 15, is composed of Primer 15F and Primer 15R, and the primer sequences are shown in SEQ ID NOs: 49-50; Primer 16 for amplifying SNP16, consisting of Primer 16F and Primer 16R, the primer sequences of which are shown in SEQ ID NOs: 51-52; Primer 17 for amplifying SNP17, consisting of Primer 17F and Primer 17R, the primer sequences of which are shown in SEQ ID NOs: 53-54; Primer 18, a primer pair for amplifying SNP18, is composed of Primer 18F and Primer 18R, and the primer sequences are shown in SEQ ID NOs: 55-56; Primer 19 for amplifying SNP19, consisting of Primer 19F and Primer 19R, the primer sequences are shown in SEQ ID NOs: 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 NOs: 59-60.

4. A molecular probe combination, characterized in that: The molecular probe combination is used to detect the SNP site combination described in claim 1 or 2.

5. A gene chip, characterized in that: The gene chip comprises the molecular probe combination according to claim 4.

6. A kit, characterized in that: The kit comprises the SNP site combination according to claim 1 or 2, or the primer pair according to claim 3, or the molecular probe combination according to claim 4, or the gene chip according to claim 5.

7. Use of the SNP site combination according to claim 1 or 2 as a target in the preparation of a gene chip for Beijing black pig breed identification, Beijing black pork product identification or Beijing black pig individual tracing.

8. Use of the SNP site combination according to claim 1 or 2, or the primer pair according to claim 3, or the probe combination according to claim 4, or the gene chip according to claim 5, or the kit according to claim 6 in any of the following: i) Identification of Beijing Black Pig Breed, ii) Individual traceability of Beijing Black Pigs, iii) Beijing Black Pig Breeding, iv) Identification of Beijing black pork products.

9. The use according to claim 8, characterized in that: The applications include: Extract the genome of the sample to be tested, detect the polymorphism of each SNP site in the SNP site combination as described in claim 1 or 2, and determine the variety identification or individual traceability of the sample to be tested based on the polymorphism detection result.

10. The use according to claim 9, characterized in that: The method of determining the variety identification of the sample to be tested or the individual traceability of the individual traceability according to the polymorphism detection result includes: The polymorphism of each SNP site in the SNP site combination of the sample to be tested is compared with the Beijing black pig positive sample library, and the specific source of the sample to be tested is determined according to the comparison results.

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