Application of SNP markers in identification of inbred mouse strains and their primer sequences

Abstract

The present invention discloses the use of a single-nucleotide polymorphism (SNP) marker for identifying inbred mouse strains and primer sequences. The SNP marker provided by the present invention includes multiple or all of eight SNP sites and can identify some or all of ten inbred mouse strains selected from DBA / 2, FVB, BALB / cJ, C57BL / 6, C57BL / 6-bg, NPI, NCPC / 2, NU / J, C3H, and T739, thereby improving the scope and efficiency of inbred mouse strain identification.

Description

Technical Field

[0001] The invention belongs to the technical field of mouse strain identification, and particularly relates to an application of a SNP marker in inbred mouse strain identification and a primer sequence. Background Art

[0002] Laboratory animals are artificially bred animals whose microorganisms are controlled, whose genetic background or provenance is clear, and which are used for scientific research, teaching, production, testing, and other scientific experiments. Common laboratory animals include laboratory rats and mice, which include thousands of strains, including closed colonies, inbred strains, recombinant congenic strains, recombinant inbred strains, and mutant strains.

[0003] Among the many common, long-established inbred mouse strains, genetic differences between strains are substantial. However, epigenetic differences are minimal, making identification of inbred mouse strains based solely on appearance nearly impossible. With the advancement of biomedicine, researchers and businesses are placing increasingly high demands on the quality of laboratory animals. Therefore, identification of inbred mouse strains has become a pressing issue.

[0004] Patent document CN114507744A (hereinafter referred to as Document 1) discloses the application of a SNP marker in the identification of inbred mouse strains and the identification primer sequences. It uses 15 SNP sites to simultaneously identify seven inbred mouse strains: C57BL / 6J, C57BL / 6N, BALB / c, FVB, DBA / 2, CBA / CaJ, and C3H. However, on the one hand, Document 1 requires the use of 15 SNP sites and 15 pairs of primer sequences for amplifying these SNP sites to identify the seven inbred mouse strains disclosed. Therefore, Document 1 uses a large number of SNP sites and primer sequences for amplifying these SNP sites, while simultaneously identifying fewer inbred mouse strains, increasing the cost of identification. On the other hand, when using the 15 pairs of primers disclosed in Document 1 to amplify the 15 SNP sites, 15 amplification reactions are required for each sample. Therefore, the operation is cumbersome, resulting in a long identification time and reducing the efficiency of inbred mouse strain identification. Summary of the Invention

[0005] In response to one or more problems existing in the prior art, one aspect of the present invention provides an application of a SNP marker in the identification of inbred mouse strains, which utilizes multiple or all of eight SNP sites to identify some or all of ten inbred mouse strains, wherein the eight SNP sites are respectively named: SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8, and the inbred mouse strains are selected from DBA / 2Nifdc, FVB / NJNjuNifdc, BALB / cJNifdc, C57BL / 6JNifdc, C57BL / 6JNifdc-bg, NPI, NCPC / 2Nifdc, NU / JNifdc, C3H / HeJNifdc, and T739 / Nifdc; wherein:

[0006] The Ensembl rs number of the SNP1 is rs3023039, and its allele is T or C;

[0007] The Ensembl rs number of SNP2 is rs3089109, and its allele is C or G;

[0008] The Ensembl rs number of SNP3 is rs3712692, and its allele is G or T;

[0009] The Ensembl rs number of the SNP4 is rs3022977, and its allele is C or T;

[0010] The Ensembl rs number of SNP5 is rs3709624, and its allele is T or C;

[0011] The Ensembl rs number of SNP6 is rs3022825, and its allele is T or C;

[0012] The Ensembl rs number of the SNP7 is rs3089984, and its allele is A or C;

[0013] The Ensembl rs number of the SNP8 is rs3023436, and its allele is T or A.

[0014] On the other hand, the present invention provides a use of a SNP marker in preparing a kit or a liquid phase chip for identifying an inbred mouse strain, wherein the kit or the liquid phase chip is used to detect the alleles of 8 SNP sites of the inbred mouse strain; wherein the 8 SNP sites are respectively named: SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8, and the inbred mouse strain is selected from DBA / 2Nifdc, FVB / NJNjuNifdc, BALB / cJNifdc, C57BL / 6JNifdc, C57BL / 6JNifdc-bg, NPI, NCPC / 2Nifdc, NU / JNifdc, C3H / HeJNifdc, and T739 / Nifdc.

[0015] On the other hand, the present invention also provides a primer combination for amplifying the above-mentioned SNP marker, wherein the primer combination is used to identify inbred mouse strains, including a first primer for amplifying the SNP1 site, a second primer for amplifying the SNP2 site, a third primer for amplifying the SNP3 site, a fourth primer for amplifying the SNP4 site, a fifth primer for amplifying the SNP5 site, a sixth primer for amplifying the SNP6 site, a seventh primer for amplifying the SNP7 site, and an eighth primer for amplifying the SNP8 site, and the inbred mouse strain is selected from DBA / 2Nifdc, FVB / NJNjuNifdc, BALB / cJNifdc, C57BL / 6JNifdc, C57BL / 6JNifdc-bg, NPI, NCPC / 2Nifdc, NU / JNifdc, C3H / HeJNifdc, and T739 / Nifdc.

[0016] In some embodiments, the first primer includes SNP1-F and SNP1-R, wherein the nucleotide sequence of SNP1-F is shown in SEQ ID NO: 1, and the nucleotide sequence of SNP1-R is shown in SEQ ID NO: 2; the second primer includes SNP2-F and SNP2-R, wherein the nucleotide sequence of SNP2-F is shown in SEQ ID NO: 3, and the nucleotide sequence of SNP2-R is shown in SEQ ID NO: 4; the third primer includes SNP3-F and SNP3-R, wherein the nucleotide sequence of SNP3-F is shown in SEQ ID NO: 5, and the nucleotide sequence of SNP3-R is shown in SEQ ID NO: 6; the fourth primer includes SNP4-F and SNP4-R, wherein the nucleotide sequence of SNP4-F is shown in SEQ ID NO: 7, and the nucleotide sequence of SNP4-R is shown in SEQ ID NO: 8; the fifth primer includes SNP5-F and SNP5-R, wherein the nucleotide sequence of SNP5-F is shown in SEQ ID NO: NO:9, the nucleotide sequence of the SNP5-R is shown in SEQ ID NO:10; the sixth primer includes SNP6-F and SNP6-R, wherein the nucleotide sequence of the SNP6-F is shown in SEQ ID NO:11, and the nucleotide sequence of the SNP6-R is shown in SEQ ID NO:12; the seventh primer includes SNP7-F and SNP7-R, wherein the nucleotide sequence of the SNP7-F is shown in SEQ ID NO:13, and the nucleotide sequence of the SNP7-R is shown in SEQ ID NO:14; the eighth primer includes SNP8-F and SNP8-R, wherein the nucleotide sequence of the SNP8-F is shown in SEQ ID NO:15, and the nucleotide sequence of the SNP8-R is shown in SEQ ID NO:16.

[0017] Another aspect of the present invention provides a use of the above-mentioned primer combination in preparing a kit or liquid phase chip for identifying inbred mouse strains, wherein the inbred mouse strains are selected from DBA / 2Nifdc, FVB / NJNjuNifdc, BALB / cJNifdc, C57BL / 6JNifdc, C57BL / 6JNifdc-bg, NPI, NCPC / 2Nifdc, NU / JNifdc, C3H / HeJNifdc, and T739 / Nifdc.

[0018] On the other hand, the present invention provides a kit or liquid chip for identifying inbred mouse strains, wherein the kit or liquid chip comprises: A. a primer combination, which includes a first primer for amplifying the SNP1 site, a second primer for amplifying the SNP2 site, a third primer for amplifying the SNP3 site, a fourth primer for amplifying the SNP4 site, a fifth primer for amplifying the SNP5 site, a sixth primer for amplifying the SNP6 site, a seventh primer for amplifying the SNP7 site, and an eighth primer for amplifying the SNP8 site.

[0019] In some embodiments, the kit or liquid chip further comprises:

[0020] B. Wild-type and mutant-specific ASPE primers for SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8 sites, respectively, wherein each ASPE primer consists of a tag sequence at the 5' end and a specific primer sequence for the SNP site at the 3' end; optionally, the specific primer sequences include: SEQ ID NO: 17 and SEQ ID NO: 18 for the SNP1 site, SEQ ID NO: 19 and SEQ ID NO: 20 for the SNP2 site, SEQ ID NO: 21 and SEQ ID NO: 22 for the SNP3 site, SEQ ID NO: 23 and SEQ ID NO: 24 for the SNP4 site, SEQ ID NO: 25 and SEQ ID NO: 26 for the SNP5 site, SEQ ID NO: 27 and SEQ ID NO: 28 for the SNP6 site, SEQ ID NO: 29 and SEQ ID NO: 30 for the SNP7 site, SEQ ID NO: 31 and SEQ ID NO: 32 for the SNP8 site. NO: 32 or more; further optionally, the tag sequence is a sequence selected from SEQ ID NO: 33-40; and

[0021] C. Magnetic balls respectively coated with specific anti-tag sequences, wherein the anti-tag sequences are complementary to the tag sequences in B; optionally, the anti-tag sequences are sequences selected from SEQ ID NOs: 51-58, and the magnetic balls for the same SNP site have different color numbers.

[0022] In some embodiments and methods, the kit or liquid chip includes one or more of the following 1)-8):

[0023] 1) Wild-type specific ASPE primers consisting of SEQ ID NO: 17 and SEQ ID NO: 33 and mutant-specific ASPE primers consisting of SEQ ID NO: 18 and SEQ ID NO: 34 for the SNP1 site, and the corresponding specific anti-tag sequences are SEQ ID NO: 51 and SEQ ID NO: 52;

[0024] 2) Wild-type specific ASPE primers consisting of SEQ ID NO: 19 and SEQ ID NO: 35 and mutant-specific ASPE primers consisting of SEQ ID NO: 20 and SEQ ID NO: 36 for the SNP2 site, and the corresponding specific anti-tag sequences are SEQ ID NO: 53 and SEQ ID NO: 54;

[0025] 3) Wild-type specific ASPE primers consisting of SEQ ID NO:21 and SEQ ID NO:34 and mutant-specific ASPE primers consisting of SEQ ID NO:22 and SEQ ID NO:37 for the SNP3 site, and the corresponding specific anti-tag sequences are SEQ ID NO:52 and SEQ ID NO:58;

[0026] 4) wild-type specific ASPE primers consisting of SEQ ID NO:23 and SEQ ID NO:36 and mutant-specific ASPE primers consisting of SEQ ID NO:24 and SEQ ID NO:38 for the SNP4 site, and the corresponding specific anti-tag sequences are SEQ ID NO:54 and SEQ ID NO:56;

[0027] 5) Wild-type specific ASPE primers consisting of SEQ ID NO: 25 and SEQ ID NO: 33 and mutant-specific ASPE primers consisting of SEQ ID NO: 26 and SEQ ID NO: 39 for the SNP5 site, and the corresponding specific anti-tag sequences are SEQ ID NO: 51 and SEQ ID NO: 57;

[0028] 6) Wild-type specific ASPE primers consisting of SEQ ID NO: 27 and SEQ ID NO: 40 and mutant-specific ASPE primers consisting of SEQ ID NO: 28 and SEQ ID NO: 35 for SNP6, with corresponding specific anti-tag sequences of SEQ ID NO: 55 and SEQ ID NO: 53;

[0029] 7) Wild-type specific ASPE primers consisting of SEQ ID NO: 29 and SEQ ID NO: 40 and mutant ASPE primers consisting of SEQ ID NO: 30 and SEQ ID NO: 38 targeting the SNP7 site, with corresponding anti-tag sequences of SEQ ID NO: 55 and SEQ ID NO: 56;

[0030] 8) Wild-type specific ASPE primers consisting of SEQ ID NO: 31 and SEQ ID NO: 39 and mutant-specific ASPE primers consisting of SEQ ID NO: 32 and SEQ ID NO: 37 for the SNP8 site, and the corresponding specific anti-tag sequences are SEQ ID NO: 57 and SEQ ID NO: 58.

[0031] On the other hand, the present invention also provides a method for detecting SNPs in an inbred mouse strain, which uses the above-mentioned kit or liquid phase chip, and the inbred mouse strain is selected from DBA / 2Nifdc, FVB / NJNjuNifdc, BALB / cJNifdc, C57BL / 6JNifdc, C57BL / 6JNifdc-bg, NPI, NCPC / 2Nifdc, NU / JNifdc, C3H / HeJNifdc, and T739 / Nifdc.

[0032] In some embodiments, the detection method uses the above-mentioned kit or liquid phase chip, comprising the following steps:

[0033] (1) PCR amplification of the DNA sample of the inbred mouse strain to be tested to obtain the PCR amplification product;

[0034] (2) purifying the obtained PCR amplification product to obtain a purified product;

[0035] (3) performing a primer extension reaction on the purified product obtained using the specific ASPE primer, incorporating biotin-labeled dCTP during the reaction to obtain a reaction product with multiple biotin labels;

[0036] (4) hybridizing a magnetic ball coated with a specific anti-tag sequence corresponding to the specific ASPE primer with the reaction product with multiple biotin labels to obtain a hybridization product;

[0037] (5) reacting the hybridization product with streptavidin-phycoerythrin to obtain a reaction product;

[0038] (6) Detecting the reaction product using a fluorescence detector to obtain the SNP site of the inbred mouse strain.

[0039] In some embodiments, in step (1), quadruple PCR amplification is performed on the DNA sample of the inbred mouse strain using the third primer, the fourth primer, the fifth primer and the sixth primer in the primer combination as a group, and / or quadruple PCR amplification is performed on the DNA sample of the inbred mouse strain using the first primer, the second primer, the seventh primer and the eighth primer in the primer combination as a group.

[0040] In another aspect, the present invention provides a method for identifying inbred mouse strains, which further comprises the following steps based on the above-mentioned detection method: (7) using the obtained alleles of the SNP site of the inbred mouse strain to identify the inbred mouse strain; the identification process comprises: distinguishing DBA / 2Nifdc from the other nearly 9 inbred mouse strains according to the typing results of the SNP1 site, further distinguishing FVB / NJNjuNifdc from the remaining 8 inbred mouse strains according to the typing results of the SNP2 site, and then distinguishing BALB / cJNifdc from the remaining 7 inbred mouse strains according to the typing results of the SNP3 site. Based on the typing results of SNP4, C57BL / 6JNifdc and C57BL / 6JNifdc-bg were further distinguished from the remaining five inbred mouse strains. Based on the typing results of SNP5, C57BL / 6JNifdc and C57BL / 6JNifdc-bg were distinguished. Then, the following five inbred mouse strains were distinguished using the five SNP loci: DBA / 2Nifdc, FVB / NJNjuNifdc, BALB / cJNifdc, C57BL / 6JNifdc and C57BL / 6JNifdc-bg.

[0041] In some embodiments, the identification process further includes: dividing the remaining five cross-bred mouse strains into two parts according to the typing results of the SNP6 site: NPI, NCPC / 2Nifdc and NU / JNifdc, C3H / HeJNifdc, and T739 / Nifdc, wherein NPI and NCPC / 2Nifdc are identified according to the typing results of the SNP7 site, and NU / JNifdc, C3H / HeJNifdc and T739 / Nifdc are first identified according to the typing results of the SNP7 site. Then, based on the typing results of SNP8, C3H / HeJNifdc and T739 / Nifdc were distinguished. Then, the following 10 inbred mouse strains were distinguished using 8 SNP loci: DBA / 2Nifdc, FVB / NJNjuNifdc, BALB / cJNifdc, C57BL / 6JNifdc, C57BL / 6JNifdc-bg, NPI, NCPC / 2Nifdc, NU / JNifdc, C3H / HeJNifdc, and T739 / Nifdc.

[0042] The SNP markers for identifying inbred mouse strains provided based on the above technical solution include 8 SNP sites. Multiple of these 8 SNP sites can be used to identify multiple or all of the 10 inbred mouse strains (including DBA / 2Nifdc, FVB / NJNjuNifdc, BALB / cJNifdc, C57BL / 6JNifdc, C57BL / 6JNifdc-bg, NPI, NCPC / 2Nifdc, NU / JNifdc, C3H / HeJNifdc, and T739 / Nifdc). Therefore, compared with the method disclosed in the above-mentioned document 1 for identifying 7 inbred mouse strains using 15 SNP markers, the SNP markers used in the present invention are more streamlined, and the inbred mouse strains identified are richer, which can effectively save costs. On the other hand, among the 8 pairs of primers provided by the present invention for amplifying 8 SNP sites, they can be divided into two groups, and quadruple PCR amplification can be performed on the DNA samples of inbred mouse strains respectively. Therefore, only two PCR amplification reactions are required for each sample. Compared with the method disclosed in the above-mentioned document 1 that requires 15 PCR amplification reactions for each sample, it can effectively simplify the operation, save time, and thus improve the identification efficiency of inbred mouse strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1These are gel electrophoresis images of the products of PCR amplification of genomic DNA from 10 inbred mouse strains using PCR primers targeting SNP1-16 sites; Panel A represents the gel electrophoresis image using PCR primers targeting SNP1-8 sites, and Panel B represents the gel electrophoresis image using PCR primers targeting SNP9-16 sites. DETAILED DESCRIPTION

[0044] The following describes the present invention in detail with reference to specific embodiments and accompanying drawings. The terms "first," "second," ..., and "eighth" are used to distinguish similar objects and are not intended to limit a particular order or sequence, nor to limit the number of objects.

[0045] Hereinafter, only certain exemplary embodiments are briefly described. As will be appreciated by those skilled in the art, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and description are to be considered as illustrative in nature and not restrictive.

[0046] Unless otherwise specified, the methods used in the following examples are conventional methods. For specific steps, please refer to: Molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

[0047] The methods for obtaining the various biological materials described in the examples merely provide experimental methods for achieving the disclosed objectives and should not be construed as limiting the sources of the biological materials used in the present invention. In fact, the sources of the biological materials used are diverse, and any legally and ethically accessible biological material may be substituted for and used as indicated in the examples.

[0048] Example 1: Determination of SNP markers for identifying inbred mouse strains and design of primers to amplify SNP sites

[0049] 1.1. Screening of SNP markers for identifying inbred mouse strains

[0050] PETKOV PM et al. (PETKOV PM, CASSELL MA, SARGENT EE, et al. Development of a SNP genotyping panel for genetic monitoring of the laboratory mouse [J]. Genomics, 2004, 83 (5): 902-911) have disclosed a number of SNP sites that can be used to detect the genetic quality of experimental mice. The present inventors screened out multiple groups of SNP markers from these SNP sites, as shown in Tables 1 and 2 below, which show two groups of SNP markers (respectively named the first group of SNP markers and the second group of SNP markers) to determine the SNP markers that can be used to monitor the genetic quality of 10 inbred mouse strains (including DBA / 2Nifdc (abbreviated as DBA / 2), FVB / NJNjuNif dc (abbreviated as FVB), ​​BALB / cJNifdc (abbreviated as BALB / cJ), C57BL / 6JNifdc (abbreviated as C57BL / 6), C57BL / 6JNifdc-bg (abbreviated as C57BL / 6-bg), NPI, NCPC / 2Nifdc (abbreviated as NCPC / 2), NU / JNifdc (abbreviated as NU / J), C3H / HeJNifdc (abbreviated as C3H), T739 / Nifdc (abbreviated as T739)).

[0051] Table 1: SNP sites and their positions and allele information of the first group of SNP markers

[0052] Site number Ensembl rs number Chromosome location Allele SNP1 rs3023039 Chromosome 5:25090498 T / C SNP2 rs3089109 Chromosome 10:87601155 C / G SNP3 rs3712692 Chromosome 1:31763467 G / T SNP4 rs3022977 Chromosome 4:20038613 C / T SNP5 rs3709624 Chromosome 8:15241287 T / C SNP6 rs3022825 Chromosome 1:87131859 T / C SNP7 rs3089984 Chromosome 10:8813751 A / C SNP8 rs3023436 Chromosome 16:87954922 T / A

[0053] Table 2: SNP sites and their positions and allele information of the second group of SNP markers

[0054]

[0055] 1.2 Design of PCR primers targeting SNP sites

[0056] Based on the SNP site information of the two groups of SNP markers screened in step 1.1 above (shown in Tables 1 and 2), the base sequences near each SNP site were searched in the UCSC database. PCR primers capable of amplifying the SNP site were designed using Primer 6.0 software. The fragment sizes of the amplified products should differ by approximately 50-100 bp to facilitate electrophoresis differentiation. The specific primer sequences are shown in Tables 3 and 4. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0057] Table 3: PCR primers for SNP1-8 sites

[0058]

[0059] Table 4: PCR primers for SNP9-16 sites

[0060]

[0061] The inventors used the SNP markers shown in Tables 1 and 2 above to identify 10 inbred mouse strains (including DBA / 2, FVB, BALB / cJ, C57BL / 6, C57BL / 6-bg, NPI, NCPC / 2, NU / J, C3H, and T739) according to the method in Example 2 below. The results showed that only the first group of SNP markers shown in Table 1 could identify all of the above 10 inbred mouse strains (please refer to the identification results of Example 2 below). Therefore, the present invention determined to use the first group of SNP markers shown in Table 1 to identify the above 10 inbred mouse strains.

[0062] Example 2: Identification of 10 inbred mouse strains

[0063] This example uses the SNP markers shown in Table 1 determined in Example 1 above and the PCR primers for SNP1-8 sites shown in Table 3 to identify 10 inbred mouse strains (including DBA / 2, FVB, BALB / cJ, C57BL / 6, C57BL / 6-bg, NPI, NCPC / 2, NU / J, C3H, and T739). The identification method specifically includes the following operations.

[0064] 2.1. Extraction of DNA Samples from Inbred Mouse Strains

[0065] In this step, a commercially available blood / cell / tissue genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used to extract mouse genomic DNA. The kit instructions specifically included the following steps:

[0066] (1) Cut a 1-2 cm section of the mouse tail and place it in a 1.5 mL EP tube. First, add 50 μL of proteinase K, then add 500 μL of extraction buffer to the EP tube. Seal the tube with sealing film, mix well, and place it on a float. Place it in a 53.5°C water bath overnight, mixing it several times. Make sure to put enough purified water in the water bath and cover it tightly.

[0067] (2) On the next day, remove the float from the water bath, remove the EP tube from the float, add 500 μL of Tris-phenol solution, mix well, and centrifuge at 12,000 rpm / min for 2 minutes. Take the supernatant and put it into a new EP tube. When aspirating the supernatant, be careful to keep the tip of the pipette close to the wall of the EP tube to avoid aspirating the oily substance at the bottom.

[0068] (3) Add an equal volume of chloroform:isoamyl alcohol (24:1) to the new EP tube from step (2), ensuring ventilation. Mix thoroughly by inversion and centrifuge at 12,000 rpm / min for 2 minutes. Remove the supernatant and place it in a new EP tube.

[0069] (4) Add 25 μL of ammonium acetate and 750 μL of isopropanol to the new EP tube from step (3). Mix gently by shaking up and down until a clear flocculent precipitate is visible. Centrifuge at 12,000 rpm for 5 min.

[0070] (5) Pour out the liquid in the EP tube at once, add 500 μL of 70% ethanol, shake it up and down gently to mix, and then centrifuge it at 12000 rpm for 5 minutes, and discard the supernatant.

[0071] (6) Place the EP tube after discarding the supernatant on filter paper and place it at room temperature for about 30 minutes. After the ethanol is completely removed, add 200 μL of the prepared TE solution to dissolve it. Store at -20°C until ready for use.

[0072] (7) After the extracted DNA solution is stabilized in a 4°C refrigerator for 1-2 days, the DNA integrity is detected by 1.6% agarose gel electrophoresis and the DNA purity and concentration are detected by a micro-spectrophotometer.

[0073] According to the above steps (1)-(7), genomic DNA samples of 10 inbred mouse strains were extracted and obtained.

[0074] 2.2 PCR Amplification of Genomic DNA from Inbred Mouse Strains

[0075] In this step, the PCR primers in Table 3 and Table 4 of Example 1 were used to perform PCR amplification on the genomic DNA samples of the 10 inbred mouse strains extracted in step 2.1 (artificially synthesized plasmids were used as positive controls, and sterile water was used as negative controls) to obtain PCR amplification products for each inbred mouse strain, wherein the PCR reaction system and procedure are shown in Tables 5 and 6 below, respectively. When the PCR primers shown in Table 3 were used to perform PCR amplification on the genomic DNA of the inbred mouse strains, quadruple PCR amplification was performed using PCR primers targeting SNP3-6 sites as a group, and quadruple PCR amplification was performed using PCR primers targeting SNP1, 2, 7, and 8 sites as a group; when the PCR primers shown in Table 4 were used to perform PCR amplification on the genomic DNA of the inbred mouse strains, quadruple PCR amplification was performed using PCR primers targeting SNP9, 11, 14, and 15 sites as a group, and quadruple PCR amplification was performed using PCR primers targeting SNP10, 12, 13, and 16 sites as a group.

[0076] Table 5: Multiplex PCR reaction system

[0077]

[0078] Table 6: Multiplex PCR reaction program

[0079]

[0080] 2.3 Electrophoresis detection of multiplex PCR products

[0081] Prepare 2.5% agarose gel: add 1.0 g agarose to 40 mL of 1×TAE solution, add Ex Red dye (note: the ratio of dye to agarose gel is 1:10000), mix well and put into the microwave, heat several times until the solution is transparent and free of bubbles. During the process, pay attention to whether the agarose gel emerges from the conical flask. If it emerges, stop heating immediately and shorten the heating time. Then pour the dissolved solution into the gel plate, insert the comb, and leave it at room temperature for 20-30 minutes.

[0082] Take 5 μL of PCR amplification product and add 1 μL of 6× DNA loading buffer, mix well and add to the electrophoresis tank for electrophoresis detection. Use a 50 bp DNA marker as a reference for the position of the band. Run the electrophoresis at 130V for 30 minutes, then scan and photograph using a UV gel imaging system. Figure 1 As shown, a gel electrophoresis gel image is shown, wherein panel A shows the results of PCR amplification of genomic DNA of 10 inbred mouse strains using the PCR primers shown in Table 3, and panel B shows the results of PCR amplification of genomic DNA of 10 inbred mouse strains using the PCR primers shown in Table 4.

[0083] 2.4. Purification of Multiplex PCR Products

[0084] After the multiplex PCR reaction is completed, there will be residual dNTPs, primers, single-stranded products, etc., which will affect the subsequent ASPE extension reaction. ExoⅠ can be used to remove the residual primers and single-stranded products. SAP enzyme removes the residual primers, single-stranded DNA and residual dNTPs, especially dCTP, in the PCR products. This step uses the ExoSAP-IT kit (purchased from USB Company, USA) to add 1μL of Exo-SAP mixture to every 10μL of multiplex PCR product. Then, the reaction is carried out at 37℃ for 30min and the enzyme is inactivated at 80℃ for 15min to obtain purified multiplex PCR products.

[0085] 2.5 Site-Specific Primer Extension (ASPE)

[0086] In this step, the designed site-specific primers were used to perform primer extension reaction on the multiplex PCR products purified in step 2.4 above. Biotin-labeled dCTP (purchased from Shanghai Sangon Biotechnology Service Co., Ltd.) was incorporated during the reaction, so that the reaction products were labeled with multiple biotins.

[0087] 2.5.1. Design of site-specific primers (ASPE primers)

[0088] Each ASPE primer (annealing temperature should be between 51-56°C) consists of two parts: a tag sequence specific for the anti-tag sequence on the corresponding magnetic sphere at the 5' end and a mutant or wild-type specific primer sequence at the 3' end. All ASPE primers were synthesized by Shanghai Sangon Biotechnology Service Co., Ltd. The ASPE primers designed in this step for SNPs 1-8 are shown in Table 7 below, and the ASPE primers designed for SNPs 9-16 are shown in Table 8 below.

[0089] Table 7: ASPE primer sequence information for SNP1-8 sites

[0090]

[0091] Table 8: ASPE primer sequence information for SNP9-16 sites

[0092]

[0093] 2.5.2 ASPE reaction system and reaction procedure

[0094] The ASPE reaction system selected was the 20 μL system recommended in the Luminex operating guide, as shown in Table 9 below.

[0095] Table 9: ASPE reaction system

[0096]

[0097] After setting up the PCR amplification instrument according to the reaction program in Table 10 below, the prepared ASPE reaction system was placed in the instrument to perform the ASPE reaction and obtain the ASPE extension reaction product.

[0098] Table 10: ASPE reaction procedure

[0099]

[0100] 2.6 Hybridization reaction

[0101] 2.6.1. Selection of magnetic balls and anti-tag sequence coating

[0102] In this step, the ASPE primers designed in Table 7 and Table 8 were selected according to the corresponding magnetic balls (the concentration of the magnetic balls was 2.5×10 5 Each magnetic ball is color-coded and carries a 24-bp specific oligonucleotide sequence (anti-tag sequence, synthesized by Haisheng Bioengineering Technology Service Co., Ltd.; a 5-10 T spacer sequence is attached between the anti-tag sequence and the magnetic ball, i.e., a 5-10 T spacer sequence is added before each anti-tag sequence). These anti-tag sequences specifically bind to the tag sequence at the 5' end of the corresponding ASPE primer. The color codes of the corresponding magnetic balls and the anti-tag sequences they carry are shown in Table 11 below.

[0103] Table 11: Color coding of magnetic balls and anti-tag sequences they carry

[0104]

[0105] The process of coating the magnetic sphere with the anti-tag sequence is as follows:

[0106] Take 5×10 6 Each of the above-numbered carboxylated magnetic spheres (purchased from Luminex) was suspended in 50 μL of 0.1 mol / L MES solution (pH 4.5), and 10 μL of a synthetic anti-tag molecule (100 nmol / ml) was added. A 10 ng / ml working solution of EDC (N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide) (purchased from Pierce Chemical) was prepared. 2.5 μL of the EDC working solution was added to the magnetic sphere suspension, and the mixture was incubated at a constant temperature for 30 minutes. Another 2.5 μL of the EDC working solution was added, and the mixture was incubated at a constant temperature for another 30 minutes. After the reaction, the mixture was washed once with 0.02% Tween-20 and once with 0.1% SDS. The washed magnetic beads coated with the anti-tag sequence were resuspended in 100 μL of Tris-EDTA solution [10 mmol / L Tris (pH 8.0), 1 mmol / L EDTA] and stored at 2-8° C. in the dark.

[0107] 2.6.2. Hybridization reaction between ASPE-extended PCR products and magnetic beads coated with anti-tag sequences

[0108] The Luminex user guide recommends two hybridization methods: one with magnetic beads washed and one without. This example uses the method without washing the magnetic beads:

[0109] (1) Before use, vortex the magnetic balls for 30-60 seconds to prevent the magnetic balls that have settled to the bottom of the bottle from being sucked up. Pipette 10 μL of each coded magnetic ball to prepare a magnetic ball stock solution. Then dilute the stock solution to a working solution with a concentration of approximately 50 balls / μL. Pipette 22.5 μL of the magnetic ball working solution into eight consecutive PCR reaction tubes.

[0110] (2) Add 2.5 μL of the ASPE extension reaction product to be tested to the reaction tube, making the total reaction volume 25 μL;

[0111] (3) For negative control, 2.5 μL ddH2O was added, and for positive control, 2.5 μL of the ASPE extension reaction product of the synthesized plasmid was added;

[0112] (4) After labeling, place the eight-tube strip on a vortex shaker to mix thoroughly, then centrifuge in a microcentrifuge for about 10 seconds. Then place it in a PCR amplification instrument. The reaction conditions are: denaturation at 96°C for 90 seconds, followed by reaction at 37°C for 30 minutes.

[0113] (5) Take 100 μL of 1× hybridization buffer containing 8 μg / mL streptavidin phycoerythrin and 0.01% BSA and add it to eight PCR reaction tubes in a row. Use a pipette to slowly pipette and remember not to use a vortex shaker to mix and resuspend the precipitated magnetic balls.

[0114] (6) Place the eight PCR reaction tubes in the PCR amplifier again for streptavidin-phycoerythrin hybridization. Adjust the temperature to 37°C and allow the reaction to proceed for 20 minutes. After the reaction is complete, the Luminex 200 instrument can be used for detection. The system output value is the median fluorescence intensity (MFI).

[0115] (7) Before using the Luminex instrument to test samples, turn on the machine and preheat it for 30 minutes, then verify the instrument. Only after the verification is completed can the sample be tested. Be careful to adjust the height of the probe at any time;

[0116] (8) After the sample test is completed, the detection probe needs to be cleaned. Deionized water and 84 disinfectant are added to the corresponding position of the calibration plate to clean the probe. After the cleaning is completed, the software can be closed. Note that the waste liquid in the waste liquid bucket should be cleaned regularly.

[0117] 2.7 Data Analysis

[0118] After the hybridization reaction in step 2.6.2 above, the magnetic ball system was detected using the Luminex200 reading system to excite the red laser and the green laser respectively. The output value was the median fluorescence intensity (MFI), which was used to calculate the allele MFI ratio. The allele MFI ratio = MFI 目标碱基 / (MIF 野生型 +MFI 突变型 The typing principle is as follows: an allele MFI ratio > 0.75 or < 0.25 indicates a homozygous wild type or homozygous mutant type, and an allele MFI ratio < 0.25 indicates a heterozygous mutant type. Statistical analysis, such as CV, was performed using Excel 2019 software.

[0119] The Luminex detection results and calculated allele MFI ratios using the first set of SNP markers shown in Table 1 are shown in Tables 12 and 13 below, respectively. The Luminex detection results and calculated allele MFI ratios using the second set of SNP markers shown in Table 2 are shown in Tables 14 and 15 below, respectively.

[0120]

[0121]

[0122] From the calculation results of the allele MFI ratios shown in Table 13 above, it can be seen that the DBA / 2 strain can be distinguished from other inbred mouse strains based on the typing results of the rs3023039 site, the FVB strain can be further distinguished from the remaining 8 strains of mice based on the typing results of the rs3089109 site, and then the BALB / cJ strain can be distinguished from the remaining 7 strains based on the typing results of the rs3712692 site, and the C57BL / 6 and C57BL / 6-bg strains can be further distinguished from the remaining 5 strains based on the typing results of the rs3022977 site. The typing results of locus 709624 distinguished C57BL / 6 from C57BL / 6-bg. The remaining five strains could be divided into NPI, NCPC / 2 and NU / J, C3H, and T739 according to the typing results of locus rs3022825. The first two strains (NPI and NCPC / 2) could be distinguished according to the typing results of locus rs3089984. The last three strains (NU / J, C3H, and T739) could be distinguished by first distinguishing Nu / J according to the typing results of locus rs3089984, and then distinguishing C3H and T739 according to the typing results of locus rs3023436. Therefore, it can be seen that the first group of SNP markers provided by the present invention, including the 8 SNP sites SNP1-8, can distinguish 10 inbred mouse strains (including DBA / 2, FVB, BALB / cJ, C57BL / 6, C57BL / 6-bg, NPI, NCPC / 2, NU / J, C3H, T739), thereby realizing the identification of these 10 inbred mouse strains.

[0123] From the calculation results of the allele MFI ratios shown in Table 15 above, it can be seen that the DBA / 2 strain can be distinguished from other inbred mouse strains based on the typing results of the rs3023039 site, and the C57BL / 6 strain can be distinguished from the remaining 8 strains of mice based on the typing results of the rs3023450 site. Subsequently, the C57BL / 6-bg strain can be distinguished from the remaining 7 strains based on the typing results of the rs3022977 site, and the BALB / cJ strain can be further distinguished from the remaining 6 strains based on the typing results of the rs3023442 site. The typing results of the rs3022883 locus can further distinguish the T739 strain from the remaining five strains, and the typing results of the rs3023436 locus can further distinguish the Nu / J strain from the remaining four strains. Subsequently, the typing results of the rs3022825 locus can further distinguish the FVB and C3H strains from the remaining NPI and NCPC / 2 strains. However, the FVB and C3H strains cannot be further distinguished, that is, the typing results of the rs3023226 locus cannot further distinguish the NPI and NCPC / 2 strains. Therefore, it can be seen that the second group of SNP markers including the 8 SNP sites SNP9-16 cannot distinguish the 10 inbred mouse strains (including DBA / 2, FVB, BALB / cJ, C57BL / 6, C57BL / 6-bg, NPI, NCPC / 2, NU / J, C3H, T739), and can only identify 6 of the inbred mouse strains (including DBA / 2, BALB / cJ, C57BL / 6, C57BL / 6-bg, NU / J, T739).

[0124] Example 3: Kit or liquid phase chip for identifying inbred mouse strains

[0125] This embodiment provides a kit or liquid phase chip for identifying 10 inbred mouse strains, wherein the 10 inbred mouse strains include DBA / 2, FVB, BALB / cJ, C57BL / 6, C57BL / 6-bg, NPI, NCPC / 2, NU / J, C3H, and T739. The provided kit or liquid phase chip includes the following components:

[0126] A. one or more pairs of primers among the PCR primers shown in Table 3 of Example 1 for amplifying SNP1-8 sites respectively;

[0127] B. One or more of the wild-type and mutant-specific ASPE primers for SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8 sites, respectively, wherein each ASPE primer consists of a tag sequence at the 5' end and a specific primer sequence for the SNP site at the 3' end; optionally, the specific primer sequences include: SEQ ID NO: 17 and SEQ ID NO: 18 for the SNP1 site, SEQ ID NO: 19 and SEQ ID NO: 20 for the SNP2 site, SEQ ID NO: 21 and SEQ ID NO: 22 for the SNP3 site, SEQ ID NO: 23 and SEQ ID NO: 24 for the SNP4 site, SEQ ID NO: 25 and SEQ ID NO: 26 for the SNP5 site, SEQ ID NO: 27 and SEQ ID NO: 28 for the SNP6 site, SEQ ID NO: 29 and SEQ ID NO: 30 for the SNP7 site, SEQ ID NO: 31 for the SNP8 site shown in Table 7 of Example 2. ID NO: 31 and SEQ ID NO: 32 or more; further optionally, the tag sequence is a sequence selected from SEQ ID NO: 33-40 in Table 7 in Example 2; and

[0128] C. Magnetic spheres each coated with a specific anti-tag sequence, wherein the anti-tag sequence can complementarily pair with the tag sequence in B; optionally, the anti-tag sequence is a sequence selected from SEQ ID NO: 51-58 in Table 11, and the magnetic spheres for the same SNP site have different color numbers.

[0129] Specifically, the kit or liquid phase chip provided in this embodiment includes one or more of the following 1)-8):

[0130] 1) Wild-type specific ASPE primers consisting of SEQ ID NO: 17 and SEQ ID NO: 33 and mutant-specific ASPE primers consisting of SEQ ID NO: 18 and SEQ ID NO: 34 for the SNP1 site, and the corresponding specific anti-tag sequences are SEQ ID NO: 51 and SEQ ID NO: 52;

[0131] 2) Wild-type specific ASPE primers consisting of SEQ ID NO: 19 and SEQ ID NO: 35 and mutant-specific ASPE primers consisting of SEQ ID NO: 20 and SEQ ID NO: 36 for the SNP2 site, and the corresponding specific anti-tag sequences are SEQ ID NO: 53 and SEQ ID NO: 54;

[0132] 3) Wild-type specific ASPE primers consisting of SEQ ID NO:21 and SEQ ID NO:34 and mutant-specific ASPE primers consisting of SEQ ID NO:22 and SEQ ID NO:37 for the SNP3 site, and the corresponding specific anti-tag sequences are SEQ ID NO:52 and SEQ ID NO:58;

[0133] 4) wild-type specific ASPE primers consisting of SEQ ID NO:23 and SEQ ID NO:36 and mutant-specific ASPE primers consisting of SEQ ID NO:24 and SEQ ID NO:38 for the SNP4 site, and the corresponding specific anti-tag sequences are SEQ ID NO:54 and SEQ ID NO:56;

[0134] 5) Wild-type specific ASPE primers consisting of SEQ ID NO: 25 and SEQ ID NO: 33 and mutant-specific ASPE primers consisting of SEQ ID NO: 26 and SEQ ID NO: 39 for the SNP5 site, and the corresponding specific anti-tag sequences are SEQ ID NO: 51 and SEQ ID NO: 57;

[0135] 6) Wild-type specific ASPE primers consisting of SEQ ID NO: 27 and SEQ ID NO: 40 and mutant-specific ASPE primers consisting of SEQ ID NO: 28 and SEQ ID NO: 35 for SNP6, with corresponding specific anti-tag sequences of SEQ ID NO: 55 and SEQ ID NO: 53;

[0136] 7) Wild-type specific ASPE primers consisting of SEQ ID NO: 29 and SEQ ID NO: 40 and mutant ASPE primers consisting of SEQ ID NO: 30 and SEQ ID NO: 38 targeting the SNP7 site, with corresponding anti-tag sequences of SEQ ID NO: 55 and SEQ ID NO: 56;

[0137] 8) Wild-type specific ASPE primers consisting of SEQ ID NO: 31 and SEQ ID NO: 39 and mutant-specific ASPE primers consisting of SEQ ID NO: 32 and SEQ ID NO: 37 for the SNP8 site, and the corresponding specific anti-tag sequences are SEQ ID NO: 57 and SEQ ID NO: 58.

[0138] In addition, the kit or liquid phase chip provided by the present invention also includes a method for detecting SNP sites of inbred mouse strains or a method for identifying inbred mouse strains using the kit or liquid phase chip, wherein:

[0139] The method for detecting SNP sites in an inbred mouse strain comprises the following steps:

[0140] (1) using the PCR primers provided in the kit or liquid phase chip to amplify (either by multiplex PCR amplification or singleplex PCR amplification) the DNA sample obtained from the inbred mouse strain to be tested to obtain a PCR amplification product;

[0141] (2) purifying the obtained PCR amplification product to obtain a purified product;

[0142] (3) performing a primer extension reaction on the purified product using the specific ASPE primers provided in the kit or liquid phase chip, incorporating biotin-labeled dCTP during the reaction to obtain a reaction product with multiple biotin labels;

[0143] (4) hybridizing a magnetic ball coated with a specific anti-tag sequence corresponding to the specific ASPE primer with the reaction product with multiple biotin labels to obtain a hybridization product;

[0144] (5) reacting the hybridization product with streptavidin-phycoerythrin to obtain a reaction product;

[0145] (6) Detecting the reaction product using a fluorescence detector to obtain the SNP site of the inbred mouse strain.

[0146] The method for identifying an inbred mouse strain further includes the following steps based on the above-mentioned method for detecting SNP sites in an inbred mouse strain:

[0147] (7) Identifying inbred mouse strains using the alleles of the SNP sites of the inbred mouse strains obtained. The identification process includes: distinguishing DBA / 2 from the other 9 inbred mouse strains based on the typing results of the rs3023039 site (SNP1), further distinguishing FVB from the remaining 8 inbred mouse strains based on the typing results of the rs3089109 site (SNP2), then distinguishing BALB / cJ from the remaining 7 inbred mouse strains based on the typing results of the rs3712692 site (SNP3), further distinguishing C57BL / 6 and C57BL / 6-bg from the remaining 5 inbred mouse strains based on the typing results of the rs3022977 site (SNP4), and then distinguishing C57BL / 6 from C57BL / 6-bg based on the typing results of the rs3709624 site (SNP5), so as to achieve the identification of inbred mouse strains using the SNP site alleles of the inbred mouse strains obtained. Five SNP sites are used to identify and distinguish five inbred mouse strains; further, the identification process also includes dividing the remaining five inbred mouse strains into two parts, NPI, NCPC / 2 and NU / J, C3H, and T739, according to the typing results of the rs3022825 site (SNP6). The first two strains (NPI and NCPC / 2) are distinguished according to the typing results of the rs3089984 site (SNP7). The last three strains (NU / J, C3H, and T739) first distinguish Nu / J according to the typing results of the rs3089984 site (SNP7), and then distinguish C3H and T739 according to the typing results of the rs3023436 site (SNP8), thereby realizing the identification and distinction of 10 inbred mouse strains using eight SNP sites.

[0148] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Application of a SNP marker in identifying inbred mouse strains, characterized in that: The SNP marker is a combination of 8 SNP sites, and these 8 SNP sites are used to identify 10 inbred mouse strains, the 8 SNP sites are respectively named: SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 and SNP8, and the inbred mouse strains are selected from DBA / 2Nifdc, FVB / NJNjuNifdc, BALB / cJNifdc, C57BL / 6JNifdc, C57BL / 6JNifdc-bg, NPI, NCPC / 2Nifdc, NU / JNifdc, C3H / HeJNifdc and T739 / Nifdc; wherein: The Ensembl rs number of the SNP1 is rs3023039, and its allele is T or C; The Ensembl rs number of SNP2 is rs3089109, and its allele is C or G; The Ensembl rs number of SNP3 is rs3712692, and its allele is G or T; The Ensembl rs number of the SNP4 is rs3022977, and its allele is C or T; The Ensembl rs number of SNP5 is rs3709624, and its allele is T or C; The Ensembl rs number of SNP6 is rs3022825, and its allele is T or C; The Ensembl rs number of SNP7 is rs3089984, and its allele is A or C; The Ensembl rs number of the SNP8 is rs3023436, and its allele is T or A.

2. Use of the SNP marker mentioned in claim 1 in preparing a kit or liquid phase chip for identifying inbred mouse strains, characterized in that: The kit or liquid phase chip is used to detect the alleles of 8 SNP sites of the inbred mouse strain; wherein the 8 SNP sites are: SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 and SNP8, and the inbred mouse strain is selected from DBA / 2Nifdc, FVB / NJNjuNifdc, BALB / cJNifdc, C57BL / 6JNifdc, C57BL / 6JNifdc-bg, NPI, NCPC / 2Nifdc, NU / JNifdc, C3H / HeJNifdc and T739 / Nifdc.

3. Use of the primer combination for amplifying the SNP marker mentioned in claim 1 in preparing a kit or liquid phase chip for identifying inbred mouse strains, characterized in that: The inbred mouse strain is selected from DBA / 2Nifdc, FVB / NJNjuNifdc, BALB / cJNifdc, C57BL / 6JNifdc, C57BL / 6JNifdc-bg, NPI, NCPC / 2Nifdc, NU / JNifdc, C3H / HeJNifdc and T739 / Nifdc; The primer combination includes a first primer for amplifying the SNP1 site, a second primer for amplifying the SNP2 site, a third primer for amplifying the SNP3 site, a fourth primer for amplifying the SNP4 site, a fifth primer for amplifying the SNP5 site, a sixth primer for amplifying the SNP6 site, a seventh primer for amplifying the SNP7 site, and an eighth primer for amplifying the SNP8 site.

4. The use according to claim 3, characterized in that The first primer includes SNP1-F and SNP1-R, wherein the nucleotide sequence of SNP1-F is shown in SEQ ID NO: 1, and the nucleotide sequence of SNP1-R is shown in SEQ ID NO: 2; The second primer includes SNP2-F and SNP2-R, wherein the nucleotide sequence of SNP2-F is shown in SEQ ID NO: 3, and the nucleotide sequence of SNP2-R is shown in SEQ ID NO: 4; The third primer includes SNP3-F and SNP3-R, wherein the nucleotide sequence of SNP3-F is shown in SEQ ID NO: 5, and the nucleotide sequence of SNP3-R is shown in SEQ ID NO: 6; The fourth primer includes SNP4-F and SNP4-R, wherein the nucleotide sequence of SNP4-F is shown in SEQ ID NO: 7, and the nucleotide sequence of SNP4-R is shown in SEQ ID NO: 8; The fifth primer includes SNP5-F and SNP5-R, wherein the nucleotide sequence of SNP5-F is shown in SEQ ID NO: 9, and the nucleotide sequence of SNP5-R is shown in SEQ ID NO: 10; The sixth primer includes SNP6-F and SNP6-R, wherein the nucleotide sequence of SNP6-F is shown in SEQ ID NO: 11, and the nucleotide sequence of SNP6-R is shown in SEQ ID NO: 12; The seventh primer includes SNP7-F and SNP7-R, wherein the nucleotide sequence of SNP7-F is shown in SEQ ID NO: 13, and the nucleotide sequence of SNP7-R is shown in SEQ ID NO: 14; The eighth primer includes SNP8-F and SNP8-R, wherein the nucleotide sequence of SNP8-F is shown in SEQ ID NO: 15, and the nucleotide sequence of SNP8-R is shown in SEQ ID NO:

16.

5. The use according to claim 3 or 4, characterized in that The kit or liquid phase chip further comprises: B. Wild-type and mutant-specific ASPE primers for SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, and SNP8, respectively, wherein each ASPE primer consists of a tag sequence at the 5' end and a specific primer sequence for the SNP site at the 3' end; the specific primer sequences include: SEQ ID NO: 17 and SEQ ID NO: 18 for SNP1, SEQ ID NO: 19 and SEQ ID NO: 20 for SNP2, SEQ ID NO: 21 and SEQ ID NO: 22 for SNP3, SEQ ID NO: 23 and SEQ ID NO: 24 for SNP4, SEQ ID NO: 25 and SEQ ID NO: 26 for SNP5, SEQ ID NO: 27 and SEQ ID NO: 28 for SNP6, SEQ ID NO: 29 and SEQ ID NO: 30 for SNP7, and SEQ ID NO: 31 and SEQ ID NO: 32 for SNP8. NO:32; the tag sequence is a sequence selected from SEQ ID NO:33-40; and C. Magnetic spheres coated with specific anti-tag sequences, respectively. The anti-tag sequences are complementary to the tag sequences in B.

6. The use according to claim 5, characterized in that The anti-tag sequence is a sequence selected from SEQ ID NO: 51-58, and the magnetic balls targeting the same SNP site have different color numbers.

7. The use according to claim 6, characterized in that The kit or liquid phase chip includes the following 1)-8): 1) Wild-type specific ASPE primers consisting of SEQ ID NO: 17 and SEQ ID NO: 33 and mutant-specific ASPE primers consisting of SEQ ID NO: 18 and SEQ ID NO: 34 for the SNP1 site, and the corresponding specific anti-tag sequences are SEQ ID NO: 51 and SEQ ID NO: 52; 2) Wild-type specific ASPE primers consisting of SEQ ID NO: 19 and SEQ ID NO: 35 and mutant-specific ASPE primers consisting of SEQ ID NO: 20 and SEQ ID NO: 36 for the SNP2 site, and the corresponding specific anti-tag sequences are SEQ ID NO: 53 and SEQ ID NO: 54; 3) Wild-type specific ASPE primers consisting of SEQ ID NO:21 and SEQ ID NO:34 and mutant-specific ASPE primers consisting of SEQ ID NO:22 and SEQ ID NO:37 for the SNP3 site, and the corresponding specific anti-tag sequences are SEQ ID NO:52 and SEQ ID NO:58; 4) wild-type specific ASPE primers consisting of SEQ ID NO:23 and SEQ ID NO:36 and mutant-specific ASPE primers consisting of SEQ ID NO:24 and SEQ ID NO:38 for the SNP4 site, and the corresponding specific anti-tag sequences are SEQ ID NO:54 and SEQ ID NO:56; 5) Wild-type specific ASPE primers consisting of SEQ ID NO: 25 and SEQ ID NO: 33 and mutant-specific ASPE primers consisting of SEQ ID NO: 26 and SEQ ID NO: 39 for the SNP5 site, and the corresponding specific anti-tag sequences are SEQ ID NO: 51 and SEQ ID NO: 57; 6) Wild-type specific ASPE primers consisting of SEQ ID NO: 27 and SEQ ID NO: 40 and mutant-specific ASPE primers consisting of SEQ ID NO: 28 and SEQ ID NO: 35 for SNP6, with corresponding specific anti-tag sequences of SEQ ID NO: 55 and SEQ ID NO: 53; 7) wild-type specific ASPE primers consisting of SEQ ID NO: 29 and SEQ ID NO: 40 and mutant ASPE primers consisting of SEQ ID NO: 30 and SEQ ID NO: 38 for the SNP7 site, and the corresponding anti-tag sequences are SEQ ID NO: 55 and SEQ ID NO: 56; and 8) Wild-type specific ASPE primers consisting of SEQ ID NO: 31 and SEQ ID NO: 39 and mutant-specific ASPE primers consisting of SEQ ID NO: 32 and SEQ ID NO: 37 for the SNP8 site, and the corresponding specific anti-tag sequences are SEQ ID NO: 57 and SEQ ID NO:

58.

8. A method for identifying an inbred mouse strain, characterized in that: The method comprises performing the following steps using the kit or liquid phase chip mentioned in any one of claims 3 to 7: (1) PCR amplification of the DNA sample of the inbred mouse strain to be tested to obtain the PCR amplification product; (2) purifying the obtained PCR amplification product to obtain a purified product; (3) performing a primer extension reaction on the purified product obtained using the specific ASPE primer, incorporating biotin-labeled dCTP during the reaction to obtain a reaction product with multiple biotin labels; (4) hybridizing a magnetic ball coated with a specific anti-tag sequence corresponding to the specific ASPE primer with the reaction product with multiple biotin labels to obtain a hybridization product; (5) reacting the hybridization product with streptavidin-phycoerythrin to obtain a reaction product; (6) detecting the reaction product by a fluorescence detector to obtain the SNP site of the inbred mouse strain; and (7) using the obtained alleles of the SNP sites of the inbred mouse strain to identify the inbred mouse strain; the identification process includes: distinguishing DBA / 2Nifdc from the other nearly 9 inbred mouse strains according to the typing results of the SNP1 site, further distinguishing FVB / NJNjuNifdc from the remaining 8 inbred mouse strains according to the typing results of the SNP2 site, and then distinguishing BALB / cJNifdc from the remaining 7 inbred mouse strains according to the typing results of the SNP3 site, and further distinguishing FVB / NJNjuNifdc from the remaining 8 inbred mouse strains according to the typing results of the SNP4 site. C57BL / 6JNifdc and C57BL / 6JNifdc-bg were distinguished from the remaining five inbred mouse strains. C57BL / 6JNifdc and C57BL / 6JNifdc-bg were then distinguished based on the typing results of the SNP5 locus. The following five inbred mouse strains were further distinguished using the five SNP loci: DBA / 2Nifdc, FVB / NJNjuNifdc, BALB / cJNifdc, C57BL / 6JNifdc, and C57BL / 6JNifdc-bg. The remaining five crossbred mouse strains were divided into two parts according to the typing results of SNP6 locus: NPI and NCPC / 2Nifdc and NU / JNifdc, C3H / HeJNifdc and T739 / Nifdc. Among them, NPI and NCPC / 2Nifdc were identified according to the typing results of SNP7 locus, and NU / JNifdc, C3H / HeJNifdc and T739 / Nifdc were first identified according to the typing results of SNP7 locus, and then the NU / JNifdc was identified according to the typing results of SNP8 locus. The locus typing results distinguished C3H / HeJNifdc and T739 / Nifdc, and then used 8 SNP loci to distinguish the following 10 inbred mouse strains: DBA / 2Nifdc, FVB / NJNjuNifdc, BALB / cJNifdc, C57BL / 6JNifdc, C57BL / 6JNifdc-bg, NPI, NCPC / 2Nifdc, NU / JNifdc, C3H / HeJNifdc and T739 / Nifdc.

9. The method according to claim 8, characterized in that In step (1), A quadruple PCR amplification of the DNA sample of the inbred mouse strain is performed using the third primer, the fourth primer, the fifth primer and the sixth primer mentioned in claim 3 as a group, and A quadruple PCR amplification is performed on the DNA sample of the inbred mouse strain using the first primer, the second primer, the seventh primer and the eighth primer mentioned in claim 3 as a group.

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