Application of SNP markers in the identification of inbred rat strains and primer sequences

Abstract

The application discloses application of a SNP marker in inbred strain rat strain identification and a primer sequence. The SNP marker provided by the application comprises four SNP sites, can identify part or all of eight inbred strain rats selected from BN, F344, WKY, Lewis, DA, PVG, SHR and TSC, improves the range and efficiency of inbred strain rat strain identification, and saves identification cost.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of rat strain identification, and particularly relates to application of a SNP marker in inbred rat strain identification and a primer sequence. BACKGROUND

[0002] Experimental animals refer to animals that are artificially bred, whose microorganisms are controlled, whose genetic backgrounds are clear or sources are known, and are used for scientific research, teaching, production, verification and other scientific experiments. From the perspective of genetics, experimental animals are divided into inbred lines, closed populations and hybrid populations, and different strains of experimental animals have their unique genetic backgrounds. The genetic background information of experimental animals determines the reaction characteristics of experimental animals, and the stability of the genetic structure of experimental animals has a key influence on the accuracy and reliability of experimental results. Therefore, it is very important to use experimental animals with clear genetic backgrounds in biomedical research. Experimental rats and experimental mice are the most widely used experimental animals in biomedical research. Among them, experimental rats have the advantages of larger size, easy to study, and closer to humans in some physiological characteristics, and are irreplaceable animal models in biomedical research, and are widely used in preclinical drug safety evaluation and disease mechanism research. In the commonly used and more inbred rat strains, the genetic differences between different rat strains are large, but the differences in epigenetic traits are small, and it is almost impossible to identify inbred rat strains simply from appearance. With the development of life medicine, the requirements of scientific researchers and businesses for the quality of experimental animals are becoming higher and higher. Therefore, the identification of inbred rat strains has become a problem to be solved.

[0003] The existing inbred rat strain identification methods include biochemical markers, skin grafting, hair color genes, and immune markers. However, these methods have certain limitations and cannot meet the needs of scientific research. In order to improve the genetic detection methods of experimental rats, SNP marker technology is currently used to identify experimental rat strains. For example, patent document CN109022592A (hereinafter referred to as document 1) discloses a SNP marker for identifying four common rat strains and its application. It uses 13 SNP sites to identify four experimental rat strains, Wistar, GK, BN, and SD. Patent document CN110452969A (hereinafter referred to as document 2) discloses a SNP marker genotyping method and kit for rat genetic quality monitoring based on KASP. It uses 48 SNP sites to identify four experimental rat strains, Lewis, F344, BN, and Wistar. Patent document CN113136436A (hereinafter referred to as document 3) discloses a rat genetic background-related SNP and its use. It uses 21 SNP sites to identify five inbred rat strains, DA, PVG, Lewis, BN, and F344. Xu Yuan et al. (Establishment of a rat genetic quality detection method based on high-throughput targeted sequencing, Biochemical Engineering, 2020, 6(04), hereinafter referred to as document 4) established a rat genetic quality detection method based on high-throughput targeted sequencing. It uses 90 SNP sites to simultaneously detect the genetic quality of five rat strains: SHR, WKY, GK, F344, and SD. Wang Shujing et al. (Application of single nucleotide polymorphism analysis in inbred rat genetic detection, Chinese Journal of Experimental Animals, 2011, 19(06), hereinafter referred to as document 5) established a method for identifying seven inbred rat strains, BN, F344, WKY, LEW, SHR, MIJ, and HFJ, using nine SNP sites. However, the methods disclosed in documents 1-5 can identify multiple inbred rat strains using SNP sites. However, they require at least nine SNP sites, or even more SNP sites, to simultaneously identify up to seven rat strains, or even only four rat strains. Therefore, there are defects such as a large number of SNP sites and primers required for amplifying these SNP sites, and a small number of rat strains that can be identified simultaneously, which increases the cost of identifying inbred rat strains and reduces the efficiency of identifying inbred rat strains. SUMMARY

[0004] In view of one or more of the problems existing in the prior art, one aspect of the present application provides an application of SNP markers in inbred rat strain identification, which utilizes a combination of four SNP loci to identify part or all of eight inbred rat strains, the combination being a combination of loci named SNP1, SNP2, SNP3 and SNP4, or a combination of loci named SNP5, SNP6, SNP7 and SNP8, the inbred rat strains being BN, F344, PVG, Lewis, DA, SHR, WKY and TSC; wherein:

[0005] The Ensembl rs number of the SNP1 is rs8173311, and the alleles thereof are C or T;

[0006] The Ensembl rs number of the SNP2 is rs106506161, and the alleles thereof are T or C;

[0007] The Ensembl rs number of the SNP3 is rs65729677, and the alleles thereof are A or G;

[0008] The Ensembl rs number of the SNP4 is rs65970293, and the alleles thereof are T or C;

[0009] The Ensembl rs number of the SNP5 is rs64750237, and the alleles thereof are T or C;

[0010] The Ensembl rs number of the SNP6 is rs106888236-R, and the alleles thereof are G or T;

[0011] The Ensembl rs number of the SNP7 is rs106672258, and the alleles thereof are T or C;

[0012] The Ensembl rs number of the SNP8 is rs105466309-R, and the alleles thereof are C or T.

[0013] Another aspect of the present application provides an application of the above-mentioned SNP markers in preparing a kit or a liquid chip for identifying inbred rat strains, wherein the kit or the liquid chip is used for detecting alleles of four SNP loci of the inbred rat strains; wherein the four SNP loci are SNP1, SNP2, SNP3, SNP4, or SNP5, SNP6, SNP7, SNP8, and the inbred rat strains are selected from BN, F344, PVG, Lewis, DA, SHR, WKY and TSC.

[0014] In another aspect, the present application provides a primer combination for amplifying the SNP markers described above, which is used for identifying an inbred rat strain, comprising 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, or the primer combination comprises a fifth primer for amplifying the SNP5 site, a sixth primer for amplifying the SNP6 site, a seventh primer for amplifying the SNP7 site, an eighth primer for amplifying the SNP8 site, and the inbred rat strain is selected from the group consisting of BN, F344, PVG, Lewis, DA, SHR, WKY and TSC;

[0015] Optionally,

[0016] The first primer comprises SNP1-F and SNP1-R, wherein the nucleotide sequence of the SNP1-F is shown as SEQ ID NO: 1, and the nucleotide sequence of the SNP1-R is shown as SEQ ID NO: 2;

[0017] The second primer comprises SNP2-F and SNP2-R, wherein the nucleotide sequence of the SNP2-F is shown as SEQ ID NO: 3, and the nucleotide sequence of the SNP2-R is shown as SEQ ID NO: 4;

[0018] The third primer comprises SNP3-F and SNP3-R, wherein the nucleotide sequence of the SNP3-F is shown as SEQ ID NO: 5, and the nucleotide sequence of the SNP3-R is shown as SEQ ID NO: 6;

[0019] The fourth primer comprises SNP4-F and SNP4-R, wherein the nucleotide sequence of the SNP4-F is shown as SEQ ID NO: 7, and the nucleotide sequence of the SNP4-R is shown as SEQ ID NO: 8;

[0020] The fifth primer comprises SNP5-F and SNP5-R, wherein the nucleotide sequence of the SNP5-F is shown as SEQ ID NO: 9, and the nucleotide sequence of the SNP5-R is shown as SEQ ID NO: 10;

[0021] The sixth primer comprises SNP6-F and SNP6-R, wherein the nucleotide sequence of the SNP6-F is shown as SEQ ID NO: 11, and the nucleotide sequence of the SNP6-R is shown as SEQ ID NO: 12;

[0022] the seventh primer comprises SNP7-F and SNP7-R, wherein the nucleotide sequence of the SNP7-F is shown as SEQ ID NO: 13, and the nucleotide sequence of the SNP7-R is shown as SEQ ID NO: 14;

[0023] the eighth primer comprises SNP8-F and SNP8-R, wherein the nucleotide sequence of the SNP8-F is shown as SEQ ID NO: 15, and the nucleotide sequence of the SNP8-R is shown as SEQ ID NO: 16.

[0024] In still another aspect, the present application provides a kit or a liquid chip for identifying a strain of inbred rats, named a first kit or a first liquid chip, comprising: A1. the above-mentioned primer combination comprising a first primer for amplifying the SNP1 site, a second primer for amplifying the SNP2 site, a third primer for amplifying the SNP3 site, and a fourth primer for amplifying the SNP4 site;

[0025] Optionally, the first kit or the first liquid chip further comprises:

[0026] B1. ASPE primers specific to wild type and mutant type of the SNP1, SNP2, SNP3 and SNP4 sites, respectively, wherein each ASPE primer is composed 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 sequence comprises: SEQ ID NO: 25 and SEQ ID NO: 26 for the SNP1 site, SEQ ID NO: 27 and SEQ ID NO: 28 for the SNP2 site, SEQ ID NO: 29 and SEQ ID NO: 30 for the SNP3 site, and SEQ ID NO: 31 and SEQ ID NO: 32 for the SNP4 site; further optionally, the tag sequence is a sequence selected from SEQ ID NO: 33-40; and

[0027] C1. magnetic balls coated with specific anti-tag sequences, which correspondingly complementarily pair with the tag sequences in B1; optionally, the anti-tag sequence is a sequence selected from SEQ ID NO: 63-64, SEQ ID NO: 67-68, SEQ ID NO: 71-74.

[0028] In some embodiments, the first kit or the first liquid chip comprises the following 1)-4):

[0029] 1) 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: 34 for SNP1 site, the corresponding specific anti-tag sequences are SEQ ID NO: 63 and SEQ ID NO: 64;

[0030] 2) wild type specific ASPE primers consisting of SEQ ID NO: 27 and SEQ ID NO: 35 and mutant specific ASPE primers consisting of SEQ ID NO: 28 and SEQ ID NO: 36 for SNP2 site, the corresponding specific anti-tag sequences are SEQ ID NO: 67 and SEQ ID NO: 68;

[0031] 3) wild type specific ASPE primers consisting of SEQ ID NO: 29 and SEQ ID NO: 37 and mutant specific ASPE primers consisting of SEQ ID NO: 30 and SEQ ID NO: 38 for SNP3 site, the corresponding specific anti-tag sequences are SEQ ID NO: 71 and SEQ ID NO: 72;

[0032] 4) 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: 40 for SNP4 site, the corresponding specific anti-tag sequences are SEQ ID NO: 73 and SEQ ID NO: 74.

[0033] Still another aspect of the present application provides a kit or liquid chip for identifying inbred rat strain, named second kit or second liquid chip, comprising: A2. the above-mentioned primer combination comprising a fifth primer for amplifying SNP5 site, a sixth primer for amplifying SNP6 site, a seventh primer for amplifying SNP7 site, and an eighth primer for amplifying SNP8 site;

[0034] Optionally, the second kit or second liquid chip further comprises:

[0035] B2. ASPE primers specific to wild type and mutant type of SNP5, SNP6, SNP7, SNP8 sites respectively, wherein each ASPE primer is composed of a tag sequence at 5' end and a specific primer sequence to SNP site at 3' end; optionally, the specific primer sequence comprises: SEQ ID NO: 41 and SEQ ID NO: 42 to SNP5 site, SEQ ID NO: 43 and SEQ ID NO: 44 to SNP6 site, SEQ ID NO: 45 and SEQ ID NO: 46 to SNP7 site, SEQ ID NO: 47 and SEQ ID NO: 48 to SNP8 site; further optionally, the tag sequence is a sequence selected from SEQ ID NO: 33-34, SEQ ID NO: 37-40, SEQ ID NO: 49-50; and

[0036] C2. magnetic balls coated with specific anti-tag sequences respectively, which correspondingly are complementary to the tag sequences in B2; optionally, the anti-tag sequence is a sequence selected from SEQ ID NO: 63-66, SEQ ID NO: 71-74.

[0037] In some embodiments, the second kit or second liquid chip comprises the following 5)-8):

[0038] 5) wild type specific ASPE primer composed of SEQ ID NO: 41 and SEQ ID NO: 33 and mutant type specific ASPE primer composed of SEQ ID NO: 42 and SEQ ID NO: 34 to SNP5 site, corresponding specific anti-tag sequences are SEQ ID NO: 63 and SEQ ID NO: 64;

[0039] 6) wild type specific ASPE primer composed of SEQ ID NO: 43 and SEQ ID NO: 49 and mutant type specific ASPE primer composed of SEQ ID NO: 44 and SEQ ID NO: 50 to SNP6 site, corresponding specific anti-tag sequences are SEQ ID NO: 65 and SEQ ID NO: 66;

[0040] 7) wild type specific ASPE primers consisting of SEQ ID NO: 45 and SEQ ID NO: 37 and mutant specific ASPE primers consisting of SEQ ID NO: 46 and SEQ ID NO: 38 for SNP7 site, and the corresponding specific anti-tag sequences are SEQ ID NO: 71 and SEQ ID NO: 72;

[0041] 8) wild type specific ASPE primers consisting of SEQ ID NO: 47 and SEQ ID NO: 39 and mutant specific ASPE primers consisting of SEQ ID NO: 48 and SEQ ID NO: 40 for SNP8 site, and the corresponding specific anti-tag sequences are SEQ ID NO: 73 and SEQ ID NO: 74.

[0042] In another aspect, the present application provides a method for detecting SNP sites of inbred rat strains, which uses the kit or liquid chip described above, and the inbred rat strains are selected from the group consisting of BN, F344, PVG, Lewis, DA, SHR, WKY and TSC.

[0043] In some embodiments, the detection method comprises the following steps:

[0044] (1) PCR amplifying the DNA sample of the inbred rat strain to be detected to obtain a PCR amplification product;

[0045] (2) purifying the obtained PCR amplification product to obtain a purified product;

[0046] (3) performing primer extension reaction on the obtained purified product with the specific ASPE primers, and incorporating biotin-labeled dCTP during the reaction to obtain a reaction product with multiple biotin labels;

[0047] (4) hybridizing the magnetic beads coated with specific anti-tag sequences corresponding to the specific ASPE primers with the reaction product with multiple biotin labels to obtain a hybridization product;

[0048] (5) reacting the hybridization product with streptavidin-phycoerythrin to obtain a reaction product;

[0049] (6) detecting the reaction product to obtain the allelic information of the SNP site of the inbred rat strain;

[0050] Optionally, in step (1), the DNA sample of the inbred rat strain is subjected to quadruple PCR amplification with the above-mentioned first, second, third and fourth primers, or the DNA sample of the inbred rat strain is subjected to quadruple PCR amplification with the above-mentioned fifth, sixth, seventh and eighth primers.

[0051] In another aspect, the present application provides a method for identifying an inbred rat strain selected from the group consisting of BN, F344, PVG, Lewis, DA, SHR, WKY and TSC, which comprises the following steps in addition to the above-mentioned detection method:

[0052] (7) identifying the inbred rat strain by using the obtained allelic information of the SNP loci of the inbred rat strain; the identification process comprises:

[0053] According to the typing results of SNP1 locus, the above-mentioned eight inbred rat strains are divided into two groups, wherein Lewis, BN, PVG and TSC are in group A, and DA, SHR, F344 and WKY are in group B; in group A, TSC is distinguished from the other three inbred rat strains according to the typing results of SNP2 locus, then PVG is distinguished from the remaining two inbred rat strains according to the typing results of SNP3 locus, and finally Lewis and BN are distinguished from each other according to the typing results of SNP4 locus; in group B, SHR is distinguished from the other three inbred rat strains according to the typing results of SNP2 locus, then WKY is distinguished from the remaining two inbred rat strains according to the typing results of SNP3 locus, and finally DA and F344 are distinguished from each other according to the typing results of SNP4 locus; or

[0054] According to the typing results of SNP5 locus, the above-mentioned eight inbred rat strains are divided into two groups, wherein Lewis, BN, TSC, PVG and DA are in group C, and F344, SHR and WKY are in group D; in group C, the five inbred rat strains are divided into two groups according to the typing results of SNP6 locus, wherein Lewis and BN are in group E, and TSC, PVG and DA are in group F, in group E, Lewis and BN are distinguished from each other according to the typing results of SNP8 locus, in group F, DA is distinguished from the remaining two inbred rat strains according to the typing results of SNP7 locus, and then TSC and PVG are distinguished from each other according to the typing results of SNP8 locus; in group D, SHR is distinguished from the other two inbred rat strains according to the typing results of SNP6 locus, and then F344 and WKY are distinguished from each other according to the typing results of SNP7 or SNP8 locus, thereby achieving the identification of the eight inbred rat strains.

[0055] The SNP markers used for identifying the inbred rat strains provided in the above technical solution include four SNP loci, and the combination of the four SNP loci can simultaneously identify multiple or all of the eight inbred rat strains (including BN, F344, WKY, Lewis, DA, PVG, SHR and TSC). Therefore, compared with the methods disclosed in documents 1-5, which use more (for example, more than 9) SNP markers to identify at most 7 or fewer inbred rat strains, the SNP markers used in the present application are more concise, the primer sequences used for amplifying the SNP loci are less, and the identified inbred rat strains are more abundant, which can effectively save costs and improve identification efficiency. On the other hand, in the four pairs of primers provided in the present application for amplifying the four SNP loci, the DNA sample of the inbred rat strain can be subjected to quadruplex PCR amplification, so that only one PCR amplification reaction is needed for each sample, thereby effectively simplifying the operation, saving time, and further improving the identification efficiency of the inbred rat strain. BRIEF DESCRIPTION OF DRAWINGS

[0056] Figure 1 Gel electrophoresis images of PCR amplification products of genomic DNA of eight inbred rat strains using PCR primers for SNP1-SNP12 loci; wherein A shows a gel electrophoresis image using PCR primers for SNP1-SNP4 loci, B shows a gel electrophoresis image using PCR primers for SNP5-SNP8 loci, and C shows a gel electrophoresis image using PCR primers for SNP9-SNP12 loci, and 1-8 in each figure represent Lewis, BN, TSC, PVG, DA, F344, SHR and WKY DNA samples, respectively. DETAILED DESCRIPTION

[0057] The content of the present application will be described in detail below in combination with specific embodiments and drawings. In this article, "first", "second", …, and "eighth" and the like are applicable to distinguish similar objects, and are not limited as a specific order or sequence, nor as a limitation of the number of objects.

[0058] In the following, only some exemplary embodiments are simply described. As those skilled in the art can recognize, the described embodiments can be modified in various different ways without departing from the spirit or scope of the present application. Therefore, the drawings and the description are considered to be exemplary in nature rather than limiting.

[0059] The methods used in the following examples are conventional methods, and the specific steps can be found in Molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor) unless otherwise specified.

[0060] The obtaining routes of various biomaterials described in the examples only provide a route for experimental obtaining to achieve the specific disclosed purposes, and should not be regarded as a limitation on the sources of the biomaterials of the present application. In fact, the sources of the biomaterials used are extensive, and any biomaterial that can be obtained without violating laws and moral ethics can be used according to the hints in the examples.

[0061] Example 1: Determination of SNP markers for identifying inbred rat strains and design of primers for amplifying SNP sites

[0062] 1.1. Screening of SNP markers for identifying inbred rat strains

[0063] The above document 4 has disclosed a plurality of SNP sites that can be used for detecting the genetic quality of experimental rats. The present inventors screened a plurality of SNP markers therefrom, as shown in Tables 1, 2 and 3 below, which show three groups of SNP markers (named as the first group of SNP markers, the second group of SNP markers and the third group of SNP markers, respectively) from which SNP markers that can be used for identifying 8 inbred rat strains (including BN / SsNslcNifdc (abbreviated as BN), F344 / NSlcNifdc (abbreviated as F344), PVG, Lewis, DA, SHR, WKY, TSC) are determined.

[0064] Table 1: SNP sites of the first group of SNP markers and their positions and allele information

[0065] Site number Ensembl rs number Chromosome location Allele SNP1 rs8173311 Chromosome 19:28152386 C / T SNP2 rs106506161 Chromosome 10:48666517 T / C SNP3 rs65729677 Chromosome 5:165382887 A / G SNP4 rs65970293 Chromosome 1:57557141 T / C

[0066] Table 2: SNP sites of the second group of SNP markers and their positions and allele information

[0067] Site number Ensembl rs number Chromosome location Allele SNP5 rs64750237 Chromosome 4:57406208 T / C SNP6 rs106888236-R Chromosome 16:25469544 G / T SNP7 rs106672258 Chromosome 12:27308022 T / C SNP8 rs105466309-R Chromosome 5:85772036 C / T

[0068] Note: rs106888236-R represents the complementary strand of rs106888236; rs105466309-R represents the complementary strand of rs105466309;

[0069] Table 3: SNP site and its position and allele information of the third group of SNP markers

[0070] Site number Ensembl rs number Chromosome location Allele SNP9 rs13453252 Chromosome 20:30513112 G / C SNP10 rs64102787 Chromosome 6:25783066 C / A SNP11 rs106647405 Chromosome 4:90154766 C / T SNP12 rs65545562 Chromosome X:92061566 T / G

[0071] 1.2, Design of PCR primers for SNP sites

[0072] According to the SNP site information of the three groups of SNP markers screened in step 1.1 above (shown in Tables 1-3), the sequences of 500 bp upstream and downstream of each SNP site were searched and downloaded in the Ensembl database, and PCR primers capable of amplifying the SNP sites were designed using primer 6.0 software, and the fragment sizes of the amplification products were made to differ by about 50 bp or more, so as to distinguish the bands of each site in the electrophoresis results. The specific primer sequences are shown in Tables 4-6 below, and the primers were synthesized by Shengong Bioengineering (Shanghai) Co., Ltd.

[0073] Table 4: PCR primers for SNP 1-4 sites

[0074]

[0075] Table 5: PCR primers for SNP 5-8 sites

[0076]

[0077] Table 6: PCR primers for SNP 9-12 sites

[0078]

[0079] The inventors identified 8 inbred rat strains (including BN, F344, PVG, Lewis, DA, SHR, WKY, TSC) using the SNP markers shown in Tables 1-3 above according to the method in Example 2 below, and the results showed that only the two groups of SNP markers shown in Tables 1 and 2 could identify all of the above 8 inbred rat strains (see the identification results in Example 2 below). Therefore, the present application determines to use the two groups of SNP markers shown in Tables 1 and 2 to identify the above 8 inbred rat strains.

[0080] Example 2: Identification of 8 inbred rat strains

[0081] This example uses the PCR primers of the SNP markers shown in Tables 1-3 designed in Example 1 above (shown in Tables 4-6) to identify 8 inbred rat strains (including BN, F344, PVG, Lewis, DA, SHR, WKY, TSC), and the identification method specifically includes the following operations.

[0082] 2.1, Extraction of DNA samples from inbred rat strains

[0083] Genomic DNA of rats was extracted using commercially available blood / cell / tissue genomic DNA extraction kit (TaKaRa) according to the kit manual, which includes the following steps:

[0084] (1) Cut 1-2 cm of rat tail and put it into a 1.5 mL EP tube. Add 180 μL of Buffer GL, 20 μL of Proteinase K and 10 μL of RNase A (10 mg / mL), and incubate in a 56°C water bath until the tissue is completely lysed (2-3 hours, and the lysis time can be appropriately extended even overnight for difficult-to-lyse materials). Note: The sample can be taken out for shaking or pipetting to accelerate lysis during incubation.

[0085] (2) Add 200 μL of Buffer GB and 200 μL of 100% ethanol to the lysate and mix well by pipetting.

[0086] (3) Place the Spin Column on the Collection Tube, and transfer the solution obtained in step (2) to the Spin Column, and centrifuge at 12,000 rpm for 2 min, and discard the filtrate.

[0087] (4) Add 500 μL of Buffer WA to the Spin Column, and centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0088] (5) Add 700 μL of Buffer WB to the Spin Column, and centrifuge at 12,000 rpm for 1 min, and discard the filtrate. Note: Make sure that the specified volume of 100% ethanol has been added to Buffer WB. Add Buffer WB along the wall of the Spin Column tube to help completely wash the salt adhering to the wall.

[0089] (6) Repeat step (5).

[0090] (7) Place the Spin Column on the Collection Tube, and centrifuge at 12,000 rpm for 2 min.

[0091] (8) Place the Spin Column on a new 1.5 mL centrifuge tube, and add 50-200 μL of sterile water or Elution Buffer to the center of the Spin Column membrane, and stand at room temperature for 5 min. Note: Heating the sterile water or Elution Buffer to 65°C can improve the elution efficiency.

[0092] (9) 12,000 rpm centrifugation for 2 min to elute DNA. If a larger yield is desired, the eluate can be re-applied to the center of the Spin Column membrane or 50-200 μL of sterilized water or Elution Buffer can be added, and after 5 min at room temperature, 12,000 rpm centrifugation for 2 min to elute DNA.

[0093] (10) Quantification of genomic DNA. The extracted genomic DNA can be quantified by electrophoresis or absorbance determination.

[0094] (11) The DNA solution to be extracted is stable in the refrigerator at 4°C for 1-2 days, and the DNA integrity is detected by 1.6% agarose gel electrophoresis, and the purity and concentration of the DNA are detected by a microspectrophotometer.

[0095] According to the above steps (1)-(11), genomic DNA samples of 8 inbred rat strains were extracted and obtained.

[0096] 2.2, PCR amplification of genomic DNA of inbred rat strains

[0097] In this step, the genomic DNA samples of the 8 inbred rat strains extracted and obtained in the above step 2.1 were subjected to quadruplex PCR amplification using the PCR primers of Tables 4-6 in the above Example 1, respectively, to obtain PCR amplification products of each inbred rat strain, wherein the multiplex PCR reaction system and procedure are shown in Tables 7 and 8, respectively. When the genomic DNA of the inbred rat strains is subjected to PCR amplification using the multiplex PCR primers shown in Tables 4-6, respectively, the PCR primers for SNP1-4 sites, SNP5-8 sites and SNP9-12 sites are used as a group for quadruplex PCR amplification.

[0098] Table 7: Multiplex PCR reaction system

[0099]

[0100] Table 8: Multiplex PCR reaction procedure

[0101]

[0102] 2.3, Electrophoretic detection of multiplex PCR products

[0103] Prepare 2.0% agarose gel: add 0.8 g agarose to 40 mL of 1xTAE solution, add 4 uL of Ex Red dye (note: the ratio of dye to agarose gel is 1:10000), mix well, then heat in a microwave oven for several times until the solution is transparent and bubble-free, then pour the dissolved solution into a gel plate, insert a comb, and place at room temperature for 20-30 min.

[0104] Take 4 μL multiplex PCR amplification product into 1 μL 6x DNA loading buffer, mix well, and add to the electrophoresis tank for electrophoresis detection. Use 50 bp DNA marker as the position reference of the band. Electrophorese at 120 V for 40 min, and then scan and take a picture using the ultraviolet gel imaging system. As shown in Figure 1 Figure 2, which shows the gel electrophoresis gel map, where panel A represents the results of PCR amplification of the genomic DNA of 8 inbred rat strains using the PCR primers shown in Table 4, panel B represents the results of PCR amplification of the genomic DNA of 8 inbred rat strains using the PCR primers shown in Table 5, and panel C represents the results of PCR amplification of the genomic DNA of 8 inbred rat strains using the PCR primers shown in Table 6.

[0105] 2.4, purification of multiplex PCR product

[0106] After the multiplex PCR reaction, there are remaining dNTPs, primers, single-stranded products, etc., which will affect the subsequent ASPE extension reaction. Exonuclease I can remove the remaining primers and single-stranded products, and Alkaline Phosphatase (Shrimp) enzyme can remove the remaining primers, single-stranded DNA and remaining dNTPs, especially dCTP, in the PCR product. This step uses Exonuclease I and Alkaline Phosphatase (Shrimp) reagents (both purchased from Takara Co.). Add 1 μL of Exo-SAP mixture (10 uL Exonuclease I, 62.5 uL Alkaline Phosphatase (Shrimp), 27.5 uL ddH2O to prepare Exo-SAP mixture) to every 10 μL multiplex PCR product, and then react at 37°C for 30 min and at 80°C for 15 min to inactivate the enzyme, to obtain the purified multiplex PCR product.

[0107] 2.5, site-specific primer extension reaction (ASPE)

[0108] This step uses the designed site-specific primers to perform primer extension reaction on the multiplex PCR product purified in step 2.4 above, and Biotin-14-dCTP (purchased from Invitrogen) is used in the reaction process, so that the product after the reaction is labeled with multiple biotin.

[0109] 2.5.1, design of site-specific primers (ASPE primers)

[0110] Each ASPE primer (annealing temperature should be between 51-56°C) includes two parts, the 5' end is a specific tag sequence for the anti-tag sequence on the corresponding magnetic ball, and the 3' end is a mutant or wild-type specific primer sequence. All ASPE primers were synthesized by Shanghai Generay Biotech Co., Ltd. The ASPE primers designed for SNP1-4 sites are shown in Table 9, the ASPE primers designed for SNP5-8 sites are shown in Table 10, and the ASPE primers designed for SNP9-12 sites are shown in Table 11.

[0111] Table 9: ASPE primer sequence information for SNP1-4 sites

[0112]

[0113] Table 10: ASPE primer sequence information for SNP5-8 sites

[0114]

[0115] Table 11: ASPE primer sequence information for SNP9-12 sites

[0116]

[0117] 2.5.2, ASPE reaction system and reaction procedure

[0118] The ASPE reaction system is selected as the 20 μL system recommended in the Luminex company operation guide, as shown in Table 12.

[0119] Table 12: ASPE reaction system

[0120]

[0121] After setting the PCR amplifier according to the reaction procedure in Table 13, the prepared ASPE reaction system is put into the ASPE reaction to obtain the ASPE extension reaction product.

[0122] Table 13: ASPE reaction procedure

[0123]

[0124] 2.6, hybridization reaction

[0125] 2.6.1, selection of magnetic balls

[0126] According to the ASPE primers designed in Tables 9-11, the corresponding magnetic balls (the concentration of the magnetic balls is 2.5 x 10 6(1000 individual / mL, purchased from Luminex company). Each magnetic ball has different color code, and each magnetic ball surface is connected with a specific 24bp oligonucleotide sequence (anti-tag sequence) respectively, which can specifically bind to the tag sequence at the 5' end of the corresponding ASPE primer. The color code and anti-tag sequence of the corresponding magnetic ball are shown in Table 14.

[0127] Table 14: Color code and anti-tag sequence of magnetic ball

[0128]

[0129] 2.6.2, Hybridization reaction of ASPE extension PCR product and magnetic ball coated with anti-tag sequence

[0130] The hybridization method recommended in Luminex user guide is divided into two kinds, the method of washing magnetic ball and the method of not washing magnetic ball, this embodiment adopts the method of not washing magnetic ball:

[0131] (1) Before use, the magnetic ball is oscillated by vortex oscillator for 30-60s to prevent the magnetic ball from being unable to be absorbed to the precipitate at the bottom of the bottle, 10 μL of each coded magnetic ball is taken to prepare the magnetic ball stock solution, and then the stock solution is diluted into working solution, the working solution concentration is about 100 individual / μL, 22.5 μL of magnetic ball working solution is taken into the PCR eight continuous row reaction tube;

[0132] (2) 2.5 μL of the ASPE extension reaction product to be tested is added into the reaction tube, so that the total reaction volume is 25 μL;

[0133] (3) 2.5 μL of ddH2O is added into the negative control, and 2.5 μL of the ASPE extension reaction product of the synthesized plasmid is added into the positive control;

[0134] (4) After labeling, the PCR eight continuous row tube is mixed on the vortex oscillator and then centrifuged in the micro centrifuge for about 10s, and then placed in the PCR amplifier, the reaction condition is: 96℃ denaturation for 90s, and then 37℃ reaction for 30min;

[0135] (5) 100 μL of 1x hybridization buffer containing 8 μg / mL streptavidin phycoerythrin and 0.01% BSA is added into the PCR eight continuous row reaction tube, and the precipitated magnetic ball is resuspended by slowly blowing with a pipette, and it is noted that vortex oscillator is not used for mixing;

[0136] (6) Again put the PCR eight connection row reaction tube into the PCR amplification instrument to carry out streptavidin phycoerythrin hybridization. Adjust the temperature to 37℃, react at this temperature for 20 min, and then use Luminex200 instrument to detect, and the system output value is median fluorescence intensity (MFI);

[0137] (7) Before using the Luminex instrument to detect the sample, the machine needs to be preheated for 30 min, and then the instrument is verified, and only after the verification is completed can the sample detection be carried out;

[0138] (8) After the sample detection is completed, the detection probe needs to be cleaned, and deionized water and 10% chlorine-containing disinfectant are added to the corresponding position of the calibration plate for probe cleaning. After the cleaning is completed, the software can be closed, and attention should be paid to regularly clean the waste liquid in the waste liquid barrel.

[0139] 2.7, Data analysis

[0140] After the hybridization reaction of the above step 2.6.2, the Luminex200 reading system is used to excite red laser and green laser respectively to detect the magnetic ball system, and the output value is median fluorescence intensity (MFI), which is used to calculate the allele MFI ratio, allele MFI ratio = MFI 目标碱基 / (MIF 野生型 +MFI 突变型 ). The typing principle is: allele MFI ratio > 0.75 or < 0.25 is homozygous wild type or homozygous mutant, 0.25 < allele MFI ratio < 0.75 is heterozygous mutant, and Excel 2019 software is used for statistical analysis.

[0141] The Luminex detection results and the calculated allele MFI ratio of the first group of SNP markers shown in Table 1 are shown in Tables 15 and 16 respectively, the Luminex detection results and the calculated allele MFI ratio of the second group of SNP markers shown in Table 2 are shown in Tables 17 and 18 respectively. The Luminex detection results and the calculated allele MFI ratio of the third group of SNP markers shown in Table 3 are shown in Tables 19 and 20 respectively.

[0142] Table 15: Luminex detection results of the first group of SNP markers

[0143]

[0144] Table 16: Allele MFI ratios calculated from Luminex assay results for the first set of SNP markers

[0145] Lewis 0.963029025 C 0.93466731 T 0.920697598 A 0.044715447 C BN 0.951440768 C 0.940932461 T 0.9264 A 0.935545531 T TSC 0.963388101 C 0.037673461 C 0.914943037 A 0.036711479 C PVG 0.968964719 C 0.926226636 T 0.060058049 G 0.051119561 C DA 0.090619469 T 0.932112492 T 0.870540722 A 0.908675799 T F344 0.10341986 T 0.933372195 T 0.910523354 A 0.033348472 C SHR 0.08534432 T 0.046879129 C 0.050799136 G 0.949073709 T WKY 0.085220398 T 0.930535636 T 0.072390348 G 0.933770562 T

[0146] Table 17: Luminex assay results for the second set of SNP markers

[0147]

[0148] Table 18: Allele MFI ratios calculated from Luminex assay results for the second set of SNP markers

[0149] Lewis 0.925047239 T 0.930255403 G 0.927131589 T 0.051014425 T BN 0.950470219 T 0.948484206 G 0.923911703 T 0.95627907 C TSC 0.957576731 T 0.0390484 T 0.059095232 C 0.030813435 T PVG 0.953744493 T 0.053433288 T 0.040763968 C 0.961662892 C DA 0.954829172 T 0.044799719 T 0.945769487 T 0.97602208 C F344 0.066875879 C 0.947075828 G 0.942233278 T 0.95717417 C SHR 0.054855669 C 0.040091265 T 0.086214506 C 0.060847107 T WKY 0.06661251 C 0.943705036 G 0.054180776 C 0.050996252 T

[0150] Table 19: Luminex assay results for the third set of SNP markers

[0151]

[0152] Table 20: Allele MFI ratios calculated from Luminex assay results for the third set of SNP markers

[0153] Lewis 0.964047846 G 0.908195812 C 0.929244014 C 0.879558948 T BN 0.961769115 G 0.945225916 C 0.935450502 C 0.906564969 T TSC 0.947396107 G 0.946189376 C 0.930878699 C 0.061576581 G PVG 0.955301884 G 0.930268538 C 0.942684452 C 0.87491065 T F344 0.962590299 G 0.934489843 C 0.94851184 C 0.910794183 T SHR 0.960195268 G 0.05258467 A 0.070179759 T 0.049885728 G DA 0.053041018 C 0.941086458 C 0.962971698 C 0.060447605 G WKY 0.048606746 C 0.043122465 A 0.058162078 T 0.056956876 G

[0154] According to the typing results calculated from the allele MFI ratios shown in Table 16, the typing information of Lewis at SNP1-SNP4 loci is C, T, A, C; the typing information of BN at SNP1-SNP4 loci is C, T, A, T; the typing information of TSC at SNP1-SNP4 loci is C, C, A, C; the typing information of PVG at SNP1-SNP4 loci is C, T, G, C; the typing information of DA at SNP1-SNP4 loci is T, T, A, T; the typing information of F344 at SNP1-SNP4 loci is T, T, A, C; the typing information of SHR at SNP1-SNP4 loci is T, C, G, T; and the typing information of WKY at SNP1-SNP4 loci is T, T, G, T. Therefore, according to the typing combination information at SNP1-SNP4 loci, the 8 rat strains of BN, F344, PVG, Lewis, DA, SHR, WKY and TSC can be identified. The specific identification process is as follows: according to the typing results at rs8173311 locus, the 8 inbred rat strains can be divided into two groups (Lewis, BN, PVG and TSC belong to group A, and DA, SHR, F344 and WKY belong to group B); in group A, TSC can be distinguished from the other 3 strains according to the typing results at rs106506161 locus, then PVG can be distinguished from the remaining 2 strains according to the typing results at rs65729677 locus, and finally Lewis and BN can be distinguished according to the typing results at rs65970293 locus; in group B, SHR can be distinguished from the other 3 strains according to the typing results at rs106506161 locus, then WKY can be distinguished from the remaining 2 strains according to the typing results at rs65729677 locus, and finally DA and F344 can be distinguished according to the typing results at rs65970293 locus. Therefore, it can be seen that the first group of SNP markers provided by the present application, which comprises the 4 SNP loci of SNP1-SNP4, can distinguish the 8 inbred rat strains (including BN, F344, PVG, Lewis, DA, SHR, WKY and TSC), and thus the identification of the 8 inbred rat strains can be realized.

[0155] According to the typing results calculated from the allele MFI ratios shown in Table 18, the typing information of Lewis at the SNP5-SNP8 loci is T, G, T, T; the typing information of BN at the SNP5-SNP8 loci is T, G, T, C; the typing information of TSC at the SNP5-SNP8 loci is T, T, C, T; the typing information of PVG at the SNP5-SNP8 loci is T, T, C, C; the typing information of DA at the SNP5-SNP8 loci is T, T, T, C; the typing information of F344 at the SNP5-SNP8 loci is C, G, T, C; the typing information of SHR at the SNP5-SNP8 loci is C, T, C, T; and the typing information of WKY at the SNP5-SNP8 loci is C, G, C, T. Therefore, according to the typing combination information at the SNP5-SNP8 loci, the eight rat strains of BN, F344, PVG, Lewis, DA, SHR, WKY and TSC can be identified. The specific identification process is as follows: according to the typing results at the rs64750237 locus, the eight inbred rat strains can be divided into two groups (Lewis, BN, TSC, PVG and DA belong to group C, and F344, SHR and WKY belong to group D), in group C, according to the typing results at the rs106888236-R locus, the five strains can be divided into two groups (Lewis and BN belong to group E, and TSC, PVG and DA belong to group F), in group E, Lewis and BN can be distinguished according to the typing results at the rs105466309-R locus, in group F, DA can be distinguished from the remaining two strains according to the typing results at the rs106672258 locus, and TSC and PVG can be distinguished according to the typing results at the rs105466309-R locus. In group D, SHR can be distinguished from the other two strains according to the typing results at the rs106888236-R locus, and F344 and WKY can be distinguished according to the typing results at the rs106672258 or rs105466309-R locus. Therefore, it can be seen that the second group of SNP markers provided by the present application, which comprises the four SNP loci of SNP5-SNP8, can distinguish the eight inbred rat strains (including BN, F344, PVG, Lewis, DA, SHR, WKY and TSC), and further realize the identification of the eight inbred rat strains.

[0156] From the typing results calculated from the allele MFI ratios shown in Table 20, the typing information of PVG at SNP9-SNP12 loci is G, C, C, T; the typing information of Lewis at SNP9-SNP12 loci is G, C, C, T; the typing information of F344 at SNP9-SNP12 loci is G, C, C, T; the typing information of BN at SNP9-SNP12 loci is G, C, C, T; the typing information of TSC at SNP9-SNP12 loci is G, C, C, G; the typing information of SHR at SNP9-SNP12 loci is G, A, T, G; the typing information of DA at SNP9-SNP12 loci is C, C, C, G; the typing information of WKY at SNP9-SNP12 loci is C, A, T, G. Since the typing results of PVG, Lewis, BN and F344 at SNP9-SNP12 loci are consistent, this group of loci cannot be used to distinguish these four rat strains, thus this group of loci information cannot be used to identify the eight rat strains of BN, F344, PVG, Lewis, DA, SHR, WKY and TSC at the same time, but can only be used to identify the four rat strains of TSC, SHR, DA and WKY. The specific identification process is as follows: according to the typing results of rs13453252 locus, the eight inbred rat strains can be divided into two groups (PVG, Lewis, BN, SHR, TSC and F344 belong to group G, and DA and WKY belong to group H), in group G, according to the typing results of rs64102787 locus, SHR can be distinguished from the other five strains, and according to the typing results of rs106647405 locus, TSC can be distinguished from the other four strains (PVG, Lewis, BN and F344), but the remaining four strains (PVG, Lewis, BN and F344) cannot be further distinguished. In group H, DA and WKY can be distinguished according to the typing results of rs64102787 or rs106647405 locus. Therefore, it can be seen that the third group of SNP markers including SNP9-SNP12 cannot distinguish the eight inbred rat strains, and at most can only identify four inbred rat strains (including SHR, TSC, DA and WKY).

[0157] Example 3: Kit or liquid chip for identifying inbred rat strains

[0158] This example provides two kinds of kits or liquid chips for identifying eight inbred rat strains, which are respectively named as the first kit or the first liquid chip and the second kit or the second liquid chip, wherein the eight inbred rat strains include BN, F344, PVG, Lewis, DA, SHR, WKY and TSC.

[0159] The first kit or the first liquid chip provided includes the following components:

[0160] A1. The PCR primers shown in Table 4 in Example 1 for respectively amplifying SNP1-SNP4 sites (SEQ ID NO: 1-SEQ ID NO: 8, which can be abbreviated as SEQ ID NO: 1-8, same below) ;

[0161] B1. One or more of the wild type and mutant specific ASPE primers respectively for SNP1, SNP2, SNP3, SNP4 sites, wherein each ASPE primer is composed of a tag sequence at 5' end and a specific primer sequence for SNP site at 3' end; optionally, the specific primer sequence comprises one or more of SEQ ID NO: 25 and SEQ ID NO: 26 for SNP1 site, SEQ ID NO: 27 and SEQ ID NO: 28 for SNP2 site, SEQ ID NO: 29 and SEQ ID NO: 30 for SNP3 site, SEQ ID NO: 31 and SEQ ID NO: 32 for SNP4 site shown in Table 9 in Example 2; further optionally, the tag sequence is a sequence selected from SEQ ID NO: 33-40 in Table 9 in Example 2; and

[0162] C1. Magnetic balls respectively coated with specific anti-tag sequences, which can be complementarily paired with the tag sequences in B1 respectively; optionally, the anti-tag sequence is a sequence selected from SEQ ID NO: 63-64, SEQ ID NO: 67-68, SEQ ID NO: 71-74 in Table 14, and the magnetic balls for the same SNP site have different color numbers.

[0163] Specifically, the first kit or liquid chip provided in this embodiment comprises the following 1)-4):

[0164] 1) Wild type specific ASPE primer for SNP1 site composed of SEQ ID NO: 25 and SEQ ID NO: 33, and mutant specific ASPE primer composed of SEQ ID NO: 26 and SEQ ID NO: 34, and the corresponding specific anti-tag sequences are SEQ ID NO: 63 and SEQ ID NO: 64;

[0165] 2) wild type specific ASPE primers consisting of SEQ ID NO: 27 and SEQ ID NO: 35 and mutant specific ASPE primers consisting of SEQ ID NO: 28 and SEQ ID NO: 36 for SNP2 site, the corresponding specific anti-tag sequences are SEQ ID NO: 67 and SEQ ID NO: 68;

[0166] 3) wild type specific ASPE primers consisting of SEQ ID NO: 29 and SEQ ID NO: 37 and mutant specific ASPE primers consisting of SEQ ID NO: 30 and SEQ ID NO: 38 for SNP3 site, the corresponding specific anti-tag sequences are SEQ ID NO: 71 and SEQ ID NO: 72;

[0167] 4) 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: 40 for SNP4 site, the corresponding specific anti-tag sequences are SEQ ID NO: 73 and SEQ ID NO: 74.

[0168] The second kit or second liquid chip provided comprises the following components:

[0169] A2. PCR primers (SEQ ID NO: 9-16) shown in Table 5 in Example 1 for amplifying SNP5-SNP8 sites, respectively;

[0170] B2. wild type and mutant specific ASPE primers for SNP5, SNP6, SNP7, SNP8 sites, respectively, wherein each ASPE primer consists of a tag sequence at 5' end and a specific primer sequence for SNP site at 3' end; optionally, the specific primer sequence comprises: SEQ ID NO: 41 and SEQ ID NO: 42 for SNP5 site, SEQ ID NO: 43 and SEQ ID NO: 44 for SNP6 site, SEQ ID NO: 45 and SEQ ID NO: 46 for SNP7 site, SEQ ID NO: 47 and SEQ ID NO: 48 for SNP8 site shown in Table 10 in Example 2; further optionally, the tag sequence is a sequence selected from SEQ ID NO: 33-34, SEQ ID NO: 37-40, SEQ ID NO: 49-50 in Table 10 in Example 2; and

[0171] C2. magnetic balls coated with specific anti-tag sequences respectively, which can be respectively paired with the tag sequences in B2; optionally, the anti-tag sequences are sequences selected from SEQ ID NO: 63-66, SEQ ID NO: 71-74 in Table 14, and the magnetic balls for the same SNP site have different color numbers.

[0172] Specifically, the second kit or liquid chip provided in this embodiment comprises the following 5)-8):

[0173] 5) wild type specific ASPE primers consisting of SEQ ID NO: 41 and SEQ ID NO: 33 and mutant specific ASPE primers consisting of SEQ ID NO: 42 and SEQ ID NO: 34 for SNP5 site, and the corresponding specific anti-tag sequences are SEQ ID NO: 63 and SEQ ID NO: 64;

[0174] 6) wild type specific ASPE primers consisting of SEQ ID NO: 43 and SEQ ID NO: 49 and mutant specific ASPE primers consisting of SEQ ID NO: 44 and SEQ ID NO: 50 for SNP6 site, and the corresponding specific anti-tag sequences are SEQ ID NO: 65 and SEQ ID NO: 66;

[0175] 7) wild type specific ASPE primers consisting of SEQ ID NO: 45 and SEQ ID NO: 37 and mutant specific ASPE primers consisting of SEQ ID NO: 46 and SEQ ID NO: 38 for SNP7 site, and the corresponding specific anti-tag sequences are SEQ ID NO: 71 and SEQ ID NO: 72;

[0176] 8) wild type specific ASPE primers consisting of SEQ ID NO: 47 and SEQ ID NO: 39 and mutant specific ASPE primers consisting of SEQ ID NO: 48 and SEQ ID NO: 40 for SNP8 site, and the corresponding specific anti-tag sequences are SEQ ID NO: 73 and SEQ ID NO: 74.

[0177] In the above kit or liquid chip product, the PCR amplification primers for each SNP site can be mixed and packaged or packaged separately, and when packaged separately, mixed when used; the specific ASPE primers for each SNP site can be packaged separately, and the magnetic balls coated with specific anti-tag sequences can be packaged separately.

[0178] In addition, the kit or liquid chip provided by the present application (the first and second) also includes a method for detecting the SNP site of an inbred rat strain using the kit or liquid chip or a method for identifying an inbred rat strain, wherein:

[0179] The method for detecting the SNP site of an inbred rat strain includes the following steps:

[0180] (1) using the PCR primers provided in the kit or liquid chip to amplify the DNA sample obtained from the inbred rat strain to be tested (multi-PCR amplification can be used, such as four-PCR amplification by combining the first to fourth primers, or four-PCR amplification by combining the fifth to eighth primers) to obtain PCR amplification products;

[0181] (2) purifying the obtained PCR amplification products to obtain purified products;

[0182] (3) performing primer extension reaction on the obtained purified products using the specific ASPE primers provided in the kit or liquid chip, and incorporating biotin-labeled dCTP during the reaction to obtain reaction products with multiple biotin labels;

[0183] (4) hybridizing the magnetic beads coated with specific anti-tag sequences corresponding to the specific ASPE primers with the reaction products with multiple biotin labels to obtain hybridization products;

[0184] (5) reacting the hybridization products with streptavidin-phycoerythrin to obtain reaction products;

[0185] (6) detecting (for example, by a fluorescence detector) the reaction products to obtain the allele information of the SNP site of the inbred rat strain.

[0186] The method for identifying (or distinguishing) an inbred rat strain further includes the following steps based on the above-mentioned method for detecting the SNP site of an inbred rat strain:

[0187] (7) using the obtained allele information of the SNP site of the inbred rat strain to identify the inbred rat strain; the identification process includes:

[0188] According to the typing result of SNP1 site, the above 8 inbred rat strains are divided into two groups, wherein Lewis, BN, PVG and TSC are group A, and DA, SHR, F344 and WKY are group B; in group A, TSC is distinguished from the other 3 inbred rat strains according to the typing result of SNP2 site, then PVG is distinguished from the remaining 2 inbred rat strains according to the typing result of SNP3 site, and finally Lewis and BN are distinguished according to the typing result of SNP4 site; in group B, SHR is distinguished from the other 3 inbred rat strains according to the typing result of SNP2 site, then WKY is distinguished from the remaining 2 inbred rat strains according to the typing result of SNP3 site, and finally DA and F344 are distinguished according to the typing result of SNP4 site; or

[0189] According to the typing result of SNP5 site, the above 8 inbred rat strains are divided into two groups, wherein Lewis, BN, TSC, PVG and DA are group C, and F344, SHR and WKY are group D; in group C, the 5 inbred rat strains are divided into two groups according to the typing result of SNP6 site, wherein Lewis and BN are group E, and TSC, PVG and DA are group F, in group E, Lewis and BN are distinguished according to the typing result of SNP8 site, in group F, DA is distinguished from the remaining 2 inbred rat strains according to the typing result of SNP7 site, and then TSC and PVG are distinguished according to the typing result of SNP8 site; in group D, SHR is distinguished from the other 2 inbred rat strains according to the typing result of SNP6 site, and then F344 and WKY are distinguished according to the typing result of SNP7 or SNP8 site;

[0190] Further, the purpose of identifying and distinguishing the 8 inbred rat strains by using 4 SNP sites is achieved.

[0191] Finally, it should be noted that: the above only for the preferred embodiments of the present application, and not for limiting the present application, although the present application has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it still can be modified to the technical solutions recorded in the foregoing embodiments, or equivalent replacement of part of the technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be within the scope of the present application.

Claims

1. The application of a reagent for detecting SNP marker alleles in the identification of inbred rat strains, characterized in that, The SNP markers are combinations of four SNP loci, and these combinations of four SNP loci are used to identify eight inbred rat strains. The combinations are loci named SNP1, SNP2, SNP3 and SNP4, respectively. The inbred rat strains are BN / SsNSlcNifdc, F344 / NSlcNifdc, PVG, Lewis, DA, SHR, WKY and TSC. The SNP1 has an Ensembl rs number of rs8173311 and its allele is C or T. The SNP2 has an Ensembl rs number of rs106506161 and its alleles are T or C. The SNP3 has an Ensembl rs number of rs65729677 and its allele is A or G. The SNP4 has an Ensembl rs number of rs65970293, and its alleles are T or C.

2. The use of the reagent for detecting the SNP marker allele mentioned in claim 1 in the preparation of a first kit or a first liquid-phase chip for identifying inbred rat strains, characterized in that, The first kit or the first liquid phase chip is used to detect alleles of the SNP1, SNP2, SNP3 and SNP4 loci combinations as described in claim 1 of the inbred rat strain; the inbred rat strain is BN / SsNSlcNifdc, F344 / NSlcNifdc, PVG, Lewis, DA, SHR, WKY and TSC.

3. The application of the reagent for detecting the SNP marker allele mentioned in claim 1 as described in claim 2 in the preparation of a first kit or a first liquid-phase chip for identifying inbred rat strains, characterized in that... The first kit includes: A1. a primer combination for amplifying the SNP marker mentioned in claim 1, comprising a first primer for amplifying SNP1, a second primer for amplifying SNP2, a third primer for amplifying SNP3, and a fourth primer for amplifying SNP4. 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 first reagent kit also includes: B1. Wild-type and mutant-specific ASPE primers for SNP1, SNP2, SNP3, and SNP4, respectively, wherein each ASPE primer consists of a 5' tag sequence and a 3' specific primer sequence for the SNP site; and C1. Magnetic spheres each coated with a specific anti-tag sequence, wherein the anti-tag sequence is complementary to the tag sequence in B1; in: The wild-type specific ASPE primer for the SNP1 site consists of SEQ ID NO:25 at the 3' end and SEQ ID NO:33 at the 5' end, while the mutant specific ASPE primer for the SNP1 site consists of SEQ ID NO:26 at the 3' end and SEQ ID NO:34 at the 5' end. The corresponding specific anti-tag sequences are SEQ ID NO:63 and SEQ ID NO:

64. The wild-type specific ASPE primer for the SNP2 site consists of SEQ ID NO:27 at the 3' end and SEQ ID NO:35 at the 5' end, while the mutant specific ASPE primer for the SNP2 site consists of SEQ ID NO:28 at the 3' end and SEQ ID NO:36 at the 5' end. The corresponding specific anti-tag sequences are SEQ ID NO:67 and SEQ ID NO:

68. The wild-type specific ASPE primer for SNP3 consists of SEQ ID NO:29 at the 3' end and SEQ ID NO:37 at the 5' end, while the mutant specific ASPE primer for SNP3 consists of SEQ ID NO:30 at the 3' end and SEQ ID NO:38 at the 5' end. The corresponding specific anti-tag sequences are SEQ ID NO:71 and SEQ ID NO:

72. The wild-type specific ASPE primer for SNP4 consists of SEQ ID NO:31 at the 3' end and SEQ ID NO:39 at the 5' end, while the mutant specific ASPE primer for SNP4 consists of SEQ ID NO:32 at the 3' end and SEQ ID NO:40 at the 5' end. The corresponding specific anti-tag sequences are SEQ ID NO:73 and SEQ ID NO:

74.

4. A method for identifying inbred rat strains, characterized in that, The inbred rat strains are BN / SsNSlcNifdc, F344 / NSlcNifdc, PVG, Lewis, DA, SHR, WKY, and TSC. The method includes detecting the DNA of the inbred rat strains using the first kit of claim 3, comprising the following steps: (1) PCR amplification of DNA samples from inbred rat strains to obtain PCR amplification products; (2) The obtained PCR amplification products were purified to obtain purified products; (3) The purified product was extended using the specific ASPE primers, and biotin-labeled dCTP was incorporated into the reaction to obtain the post-reaction product with multiple biotin labels. (4) Hybridize the magnetic beads coated with specific anti-tag sequences corresponding to the specific ASPE primers with the reaction products labeled with multiple biotin to obtain hybridization products; (5) The hybridization product is reacted with streptavidin-phycoerythrin to obtain the reaction product; (6) Detect the reaction products to obtain the allele information of the SNP locus combination of the inbred rat strain; (7) Identify the inbred rat strains using the allele information of the SNP locus combinations obtained from the obtained inbred rat strains; the identification process includes: Based on the SNP1 genotyping results, the above eight inbred rat strains were divided into two groups: Lewis, BN / SsNSlcNifdc, PVG, and TSC were group A, and DA, SHR, F344 / NSlcNifdc, and WKY were group B. In group A, TSC was distinguished from the other three inbred rat strains based on the SNP2 genotyping results, PVG was distinguished from the remaining two inbred rat strains based on the SNP3 genotyping results, and Lewis and BN / SsNSlcNifdc were distinguished based on the SNP4 genotyping results. In group B, SHR was distinguished from the other three inbred rat strains based on the SNP2 genotyping results, WKY was distinguished from the remaining two inbred rat strains based on the SNP3 genotyping results, and DA and F344 / NSlcNifdc were distinguished based on the SNP4 genotyping results.

5. The application of a reagent for detecting SNP marker alleles in the identification of inbred rat strains, characterized in that, The SNP markers are combinations of four SNP loci, and these combinations are used to identify eight inbred rat strains. The combinations are named SNP5, SNP6, SNP7 and SNP8, respectively. The inbred rat strains are BN / SsNSlcNifdc, F344 / NSlcNifdc, PVG, Lewis, DA, SHR, WKY and TSC. The SNP5 has an Ensembl rs number of rs64750237 and its alleles are T or C. The SNP6 Ensembl rs number is rs106888236-R, and its allele is G or T. The SNP7 Ensembl rs number is rs106672258, and its allele is T or C; The SNP8 Ensembl rs number is rs105466309-R, and its allele is C or T.

6. The use of the reagent for detecting the SNP marker alleles mentioned in claim 5 in the preparation of a second kit or a second liquid-phase chip for identifying inbred rat strains, characterized in that, The second kit or the second liquid phase chip is used to detect alleles of the SNP5, SNP6, SNP7 and SNP8 loci combinations as described in claim 5 of the inbred rat strain; the inbred rat strain is BN / SsNSlcNifdc, F344 / NSlcNifdc, PVG, Lewis, DA, SHR, WKY and TSC.

7. The use of the reagent as described in claim 6 for detecting the SNP marker allele mentioned in claim 5 in the preparation of a second kit or a second liquid-phase chip for identifying inbred rat strains, characterized in that, The second kit includes: A2. a primer combination for amplifying the SNP marker mentioned in claim 5, comprising a fifth primer for amplifying SNP5, a sixth primer for amplifying SNP6, a seventh primer for amplifying SNP7, and an eighth primer for amplifying SNP8. 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. The second kit also includes: B2. Wild-type and mutant-specific ASPE primers for SNP5, SNP6, SNP7, and SNP8, respectively, wherein each ASPE primer consists of a 5' tag sequence and a 3' specific primer sequence for the SNP site; and C2. Magnetic spheres each coated with a specific anti-tag sequence, wherein the anti-tag sequence is complementary to the tag sequence in B2; in, The wild-type specific ASPE primer for SNP5 consists of SEQ ID NO:41 at the 3' end and SEQ ID NO:33 at the 5' end, while the mutant specific ASPE primer for SNP5 consists of SEQ ID NO:42 at the 3' end and SEQ ID NO:34 at the 5' end. The corresponding specific anti-tag sequences are SEQ ID NO:63 and SEQ ID NO:

64. The wild-type specific ASPE primer for SNP6 consists of SEQ ID NO:43 at the 3' end and SEQ ID NO:49 at the 5' end, while the mutant specific ASPE primer for SNP6 consists of SEQ ID NO:44 at the 3' end and SEQ ID NO:50 at the 5' end. The corresponding specific anti-tag sequences are SEQ ID NO:65 and SEQ ID NO:

66. The wild-type specific ASPE primer for SNP7 consists of SEQ ID NO:45 at the 3' end and SEQ ID NO:37 at the 5' end, while the mutant specific ASPE primer for SNP7 consists of SEQ ID NO:46 at the 3' end and SEQ ID NO:38 at the 5' end. The corresponding specific anti-tag sequences are SEQ ID NO:71 and SEQ ID NO:

72. The wild-type specific ASPE primer for SNP8 consists of SEQ ID NO:47 at the 3' end and SEQ ID NO:39 at the 5' end, while the mutant specific ASPE primer for SNP8 consists of SEQ ID NO:48 at the 3' end and SEQ ID NO:40 at the 5' end. The corresponding specific anti-tag sequences are SEQ ID NO:73 and SEQ ID NO:

74.

8. A method for identifying inbred rat strains, wherein the inbred rat strains are BN / SsNSlcNifdc, F344 / NSlcNifdc, PVG, Lewis, DA, SHR, WKY, and TSC, characterized in that, The method includes detecting DNA from inbred rat strains using the second kit of claim 7, comprising the following steps: (1) PCR amplification of DNA samples from inbred rat strains to obtain PCR amplification products; (2) The obtained PCR amplification products were purified to obtain purified products; (3) The purified product was extended using the specific ASPE primers, and biotin-labeled dCTP was incorporated into the reaction to obtain the post-reaction product with multiple biotin labels. (4) Hybridize the magnetic beads coated with specific anti-tag sequences corresponding to the specific ASPE primers with the reaction products labeled with multiple biotin to obtain hybridization products; (5) The hybridization product is reacted with streptavidin-phycoerythrin to obtain the reaction product; (6) Detect the reaction products to obtain the allele information of the SNP locus combination of the inbred rat strain; (7) Identify the inbred rat strains using the allele information of the SNP locus combinations obtained from the obtained inbred rat strains; the identification process includes: Based on the SNP5 genotyping results, the above eight inbred rat strains were divided into two groups: Lewis, BN / SsNSlcNifdc, TSC, PVG, and DA were group C, and F344 / NSlcNifdc, SHR, and WKY were group D. Within group C, based on the SNP6 genotyping results, five inbred rat strains were further divided into two groups: Lewis and BN / SsNSlcNifdc were group E, and TSC, PVG, and DA were group F. Within group E, based on the SNP8 genotyping results, Lewis... In group F, DA was distinguished from the remaining two inbred rat strains based on the SNP7 genotype, and TSC and PVG were further distinguished based on the SNP8 genotype. In group D, SHR was distinguished from the other two inbred rat strains based on the SNP6 genotype, and F344 / NSlcNifdc and WKY were further distinguished based on the SNP7 or SNP8 genotype, thus achieving the identification of these eight inbred rat strains.

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