Kit and use for high-throughput detection of variant genetic loci

Abstract

This invention belongs to the field of molecular biology technology, specifically relating to a high-throughput kit and its application for detecting variant gene loci. The kit includes: primers, including a downstream primer and an upstream primer, one of which is a specific primer. The specific primer includes a barcode recognition fragment and an upstream primer fragment, with the 3' terminal base corresponding to the variant gene locus; different first detection markers, each binding to a different barcode fragment, corresponding one-to-one with the barcode recognition fragment and being inversely complementary; and a second detection marker, used to bind to the amplification product. The first detection marker binds to the specific upstream primer for differentiation. Through the inverse complementary relationship between the barcode fragment and the barcode recognition fragment, a complex of first detection marker-amplification product-second detection marker is formed. This allows for the differentiation of variant gene loci based on the combination of the first and second detection markers, achieving simultaneous and efficient multiplex detection.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a high-throughput kit for detecting variant gene loci and its application. Background Technology

[0002] Genetic locus variations include single-base mutations in a sequence, such as substitutions, deletions, duplications, insertions, and translocations. Genotyping is a technique that identifies and analyzes genetic variations in specific regions of an individual's genome to reveal allelic differences at gene loci. It is a powerful molecular biology tool for studying genetic information. Genotyping has a wide range of applications, playing an indispensable role in everything from basic scientific research to clinical diagnosis and agricultural improvement. In agriculture, genotyping is a key technology in molecular-assisted breeding. By identifying genetic markers associated with important traits such as crop yield, disease resistance, and drought resistance, breeders can more accurately select and cultivate new varieties with ideal characteristics, accelerating the breeding process while improving the overall quality and adaptability of crops. In medicine, genotyping provides a scientific basis for the diagnosis of genetic diseases, disease risk assessment, personalized treatment, and drug response prediction. By analyzing specific variations in a patient's genome, it predicts disease risk, develops personalized treatment plans, and provides patients with more effective drug options. In addition, genotyping testing plays an important role in forensic medicine, disease mechanism research, germplasm resource protection, and biodiversity monitoring.

[0003] Genotyping technology has undergone significant development and iteration since the late 20th century, resulting in a diverse range of technologies and products. The TaqMan probe method, based on fluorescence resonance energy transfer (FRET) real-time PCR, detects sample genotypes through probe-specific hybridization, offering high accuracy and specificity. However, it requires designing specific probes for different loci and can only perform single-well, single-site detection, resulting in low throughput and high costs. Kompetitive Allele Specific PCR (KASP) also relies on single-well, single-site detection, offering lower cost per site. However, multi-site detection on a single sample requires significantly more reagents, consumables, and time and labor, making it unsuitable for medium- to high-throughput loci detection. Other methods include High Resolution Melting (HRM) and SNaPshot, but these still suffer from high dependence on instrument accuracy, high instrument or detection costs, and low throughput. Therefore, it is necessary to provide a product capable of simultaneous multi-site, multiplexed genotyping amplification and detection. Summary of the Invention

[0004] This invention provides a high-throughput kit and application for detecting variant gene loci that can simultaneously perform multi-site multiplex genotyping amplification.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A first aspect of the present invention provides a kit for detecting variant gene loci, the kit comprising:

[0007] The primers include a downstream primer and an upstream primer. The downstream primer and the upstream primer are used to amplify the target fragment containing the mutated gene site to generate an amplification product. One of the upstream primer and the downstream primer is a universal primer and the other is a specific primer. There are multiple specific primers. The specific primers include a barcode recognition fragment and a primer fragment. The 3' terminal bases of the primer fragments of the multiple specific primers correspond to different variations of the mutated gene site.

[0008] Multiple different first detection markers are combined with different barcode segments, and the barcode segments and the barcode recognition segments are respectively one-to-one correspondences and inverse complements;

[0009] A second detection marker is used to bind the amplification product.

[0010] In some embodiments of the present invention, the barcode identification fragment and the upstream primer fragment are connected by an interarm modification group.

[0011] In some embodiments of the present invention, the interarm modifying group includes at least one of an alkylene group having 3 to 20 carbon atoms and a polyethylene glycol group.

[0012] In some embodiments of the present invention, the interarm modifying group includes at least one of C3 Spacer, C6 Spacer, Spacer 9, C12 Spacer, and Spacer 18.

[0013] In some embodiments of the present invention, the length of the barcode identification segment is 10 to 30 nt.

[0014] In some embodiments of the present invention, the first detection marker includes magnetic microspheres, and different types of magnetic microspheres have different identifiable morphological features.

[0015] In some embodiments of the present invention, the morphological features include patterns on the surface of the magnetic microspheres.

[0016] In some embodiments of the present invention, the universal primer has a first binding portion, and the second detection marker has a second binding portion capable of binding with the first binding portion.

[0017] In some embodiments of the present invention, one of the first binding portion and the second binding portion is biotin, and the other is avidin or streptavidin.

[0018] In some embodiments of the present invention, one of the first binding portion and the second binding portion is an antigen, and the other is an antibody.

[0019] In some embodiments of the present invention, the second detection label includes at least one of a fluorescent label, a chemiluminescent label, and a radioactive isotope.

[0020] In some embodiments of the present invention, the barcode segment and the barcode identification segment include at least one combination of BS and AS from Table 1.

[0021] In some embodiments of the present invention, the 3' end of the primer fragment of the specific primer is further modified.

[0022] In some embodiments of the invention, the modification includes modification of one or more nucleotides at the 3' end.

[0023] In some embodiments of the present invention, the modification site is located at at least one of a phosphate group, a base, and a ribose.

[0024] In some embodiments of the present invention, the modification of the phosphate group includes at least one of thio, amino, and boroalkyl substitution modifications.

[0025] In some embodiments of the present invention, the base modification includes at least one of pseudouridine, 2-thiouridine, N1-methylpseudouridine, 5-methyluridine, 5-methoxyuridine, N6-methyladenosine, and 5-methylcytidine.

[0026] In some embodiments of the present invention, ribose modification includes at least one of methoxy, methoxyethoxy, locked nucleic acid, and PMO.

[0027] A second aspect of the present invention provides a method for detecting variant gene loci using the aforementioned kit, comprising the following steps:

[0028] Take the DNA sample to be tested, add primers for amplification, and obtain the amplification product;

[0029] The amplification product is mixed with the first detection label and the second detection label and then incubated to obtain the incubation product;

[0030] The incubation product is tested, and the variation of the variant gene site is determined based on the detection results of the second detection marker corresponding to different first detection markers.

[0031] A third aspect of the present invention provides a detection system for variant gene loci, comprising any of the aforementioned reagent kits.

[0032] In a fourth aspect, the present invention provides the application of the aforementioned kit or the aforementioned detection system in the preparation of detection products for variant gene loci.

[0033] A fifth aspect of the present invention provides an application of the aforementioned reagent kit or the aforementioned detection system in any of the following A1) to A5):

[0034] A1) Assisted breeding;

[0035] A2) Preparation of products for assisted breeding;

[0036] A3) Prepare products for any one of the following: disease diagnosis, risk assessment, personalized treatment, or drug response prediction;

[0037] A4) Forensic medicine, disease mechanism research, germplasm resource protection, or biodiversity monitoring;

[0038] A5) Prepare products for any of the following purposes: forensic medicine, disease mechanism research, germplasm resource protection, or biodiversity monitoring.

[0039] In some embodiments of the present invention, the assisted breeding includes assisted breeding of plants and animals.

[0040] In some embodiments of the present invention, the plant includes at least one of crops, forest trees, fruit trees, and flowers.

[0041] In some embodiments of the present invention, the animal includes at least one of poultry, livestock, and aquatic animals.

[0042] The beneficial effects of this invention are:

[0043] This scheme utilizes a first detection marker with distinctive morphological characteristics combined with a specific upstream primer for differentiation. The first detection marker and the specific upstream primer form a complex of first detection marker-amplification product-second detection marker through the inverse complementary relationship between barcode fragments and barcode recognition fragments, realizing a one-to-one correspondence between the alleles of SNP loci and the first detection marker. Thus, the combination of the first and second detection markers can distinguish the genotype of SNP loci, ultimately achieving simultaneous and efficient multiplex detection.

[0044] Furthermore, the instruments involved in this solution are not high-precision equipment such as sequencers or mass spectrometers. They have low installation and operation thresholds, low prices, and small footprints that can be placed on a desktop. They can also be combined with automated equipment to greatly increase the testing throughput. Attached Figure Description

[0045] Figure 1 This is a schematic diagram illustrating the principle of multiplex genotyping detection in an embodiment of the present invention.

[0046] Figure 2 This is a cluster diagram of the samples from the 4 sites tested in Example 1.

[0047] Figure 3 This is a cluster diagram of the 50 sites tested in Example 2. Detailed Implementation

[0048] In the description of this invention, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first," "second," or "third" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0049] In a first aspect, the present invention provides a kit for detecting variant gene loci, the kit comprising primers, a first detection marker, and a second detection marker.

[0050] The term "primer" refers to an oligonucleotide molecule that participates in nucleic acid synthesis reactions as a starting point for elongation. In DNA double-strand amplification reactions, a pair of primers is typically required: an upstream primer (or forward primer) and a downstream primer (or reverse primer). The upstream primer binds to the sense strand of the DNA double helix, while the downstream primer binds to the sense strand. The general principle of primer design is to enhance specific amplification while suppressing non-specific amplification. There are specific requirements for primer length (usually 18–30 bases), GC content (40–60%), and annealing temperature (55–75°C). Specific primer designs can be created using primer design software, websites, etc., based on the DNA sequence.

[0051] Unlike conventional primers, in this embodiment of the invention, one of the upstream and downstream primers is a universal primer, while the other is a specific primer, meaning it can only specifically amplify variant gene sites with specific variations. Specifically, the 3' terminal base of the primer fragment of the specific primer can recognize specific variations. Different specific primers recognize different specific variations at their 3' terminal bases, so primer binding and extension to the template only occur when the two primers match.

[0052] "Variant gene sites" include single-base mutations in a sequence, such as substitutions, deletions, duplications, insertions, and translocations. Substitutions include base transitions and transversions, often referred to as single nucleotide polymorphisms (SNPs). Taking variant gene sites as SNP sites as an example, in some cases, an SNP site contains two alleles, while in others, it may contain three or more alleles. Therefore, in the embodiments of this application, there may be two or more specific primers for the same SNP site (e.g., two, three, or more). The 3' terminal base of the primer fragments of multiple specific primers corresponding to different variant gene sites may be one or more 3' terminal bases. For SNP sites, the 3' terminal base of the primer fragments of multiple specific primers may be a single 3' terminal base; for other types of variant gene sites, the 3' terminal base of the primer fragments of multiple specific primers may be one or more 3' terminal bases.

[0053] In some embodiments, the 3' end of the primer fragment of the specific primer is further modified to enhance the recognition of the variant gene site by the 3' terminal base, or to avoid the situation where the 3' terminal base is accidentally cleaved during the reaction, thus preventing the completion of recognition. In some embodiments, the modification includes modification of one or more nucleotides at the 3' end. In some embodiments, the modification site may be located at at least one of a phosphate group, a base, or a ribose. In some embodiments, the modification of the phosphate group includes at least one of thio, amino, or borane substitution modifications (e.g., a sulfur atom or an amino or borane substitution for a non-bridging oxygen atom of the phosphate group). In some embodiments, the base modification includes at least one of pseudouridine, 2-thiouridine, N1-methylpseudouridine, 5-methyluridine, 5-methoxyuridine, N6-methyladenosine, and 5-methylcytidine. In some embodiments, the ribose modification includes at least one of methoxy, methoxyethoxy, locked nucleic acid, and PMO.

[0054] To distinguish each specific primer from any other specific primer in subsequent detection processes, each specific primer includes a barcode identification segment in addition to the primer fragment. Each primer fragment corresponds to a specific barcode identification segment, and different primer fragments correspond to different barcode identification segments. The barcode identification segment is used to identify the barcode segment bound to the first detection mark, specifically utilizing the inverse complementary relationship between the barcode fragment and the barcode identification segment to achieve a one-to-one correspondence.

[0055] In some implementations, the length of the barcode segment and the barcode identification segment is 10 to 30 nt, for example, it can be 10 nt, 12 nt, 14 nt, 16 nt, 18 nt, 20 nt, 22 nt, 24 nt, 26 nt, 28 nt, or 30 nt.

[0056] In some implementations, the barcode sense sequence (BS) and the barcode anti-sense sequence (AS) can be selected from any of the correspondences in Table 1 below (i.e., the correspondence between BSn and ASn, where n is 1 to 100), and one or more combinations can be selected, i.e., including at least one combination of BS and AS in Table 1 (BSn and ASn constitute a combination).

[0057] Table 1. Sequences of Barcode Fragments and Barcode Recognition Fragments

[0058]

[0059]

[0060]

[0061] In some embodiments, the barcode identification fragment and the primer fragment are linked by a spacer modification group. In some embodiments, the spacer modification group includes at least one of an alkylene group or a polyethylene glycol group having 3 to 20 carbon atoms. Specifically, the number of carbon atoms can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the alkylene group or polyethylene glycol group is a linear alkylene group or a polyethylene glycol group. In some embodiments, the spacer modification group includes at least one of C3 spacer, C6 spacer, spacer 9, C12 spacer, or spacer 18.

[0062] The high-throughput detection requirements of variant gene loci typically necessitate the use of 10, 20, 30, 40, 50, 60, 80, 100, 200, 400, 500, 1000, 2000, or even more than 4000 first detection markers. Therefore, in some embodiments, microspheres are used as carriers for the first detection markers, and different detectable features are woven onto the microspheres to form a variety of different microspheres. In some embodiments, the detectable features include, but are not limited to, the morphological characteristics of the microspheres, such as their appearance and size. In some specific embodiments, the appearance includes the microscopic morphology of the microspheres, for example, forming one or more two-dimensional or three-dimensional patterns on the surface of the microspheres through one or more methods such as etching or printing, utilizing the differences in the patterns to construct the desired variety of detection markers. In some specific embodiments, the patterns can be set at different positions on the microspheres, and each position can have multiple different patterns; for example, setting y selectable patterns at x positions on the microspheres, thereby obtaining y x Microspheres are then used. To determine the location of the microsphere to which the pattern belongs, it needs to be positioned. In some embodiments, the magnetic bead has a positioning marker. In some specific embodiments, the marker can be a notch on the magnetic bead, which disrupts the symmetry of the microsphere to achieve positioning. It is understood that the microsphere is not necessarily a perfect sphere; it can be a sphere with a notch, an ellipsoid, a near-sphere, or other shapes. In some embodiments, the pattern can also be a QR code or barcode. In some embodiments, for ease of subsequent operation and recycling, the microsphere can be a magnetic microsphere.

[0063] After the first detection marker completely binds to the amplification product containing the specific allele, the system also contains some free primers that have not bound to the amplification product due to excess. Therefore, a complex of the first detection marker, amplification product, and second detection marker is formed using a second detection marker to distinguish between them and complete the detection. Unlike the first detection marker, the second detection marker is usually only used to determine "yes" or "no" and does not need to distinguish between different second detection markers. Therefore, the type of the second detection marker can be any type of marker known in the art that can be detected by binding through one or more physical, chemical, or biological methods. In some embodiments, the second detection marker includes, but is not limited to, fluorescent markers, chemiluminescent markers, and radioactive isotopes. In some specific embodiments, fluorescent labeling includes fluorescent dyes such as anthocyanins (e.g., Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Cy7.5, etc.), rhodamine derivatives (e.g., Rhodamine 101, Rhodamine 6G, Tetramethylrhodamine, Rhodamine B, X-rhodamine, Texas Red, etc.), fluorescein derivatives (e.g., fluorescein, fluorescein isothiocyanate, 5(6)-carboxyhexachlorofluorescein, 5(6)-carboxynaphthylfluorescein, Oregon Green, Pennsylvania Green, etc.), fluorescent proteins (e.g., green fluorescent protein, phycoerythrin, phycocyanin, allophycocyanin), quantum dots, nanocrystals, etc. In some specific embodiments, chemiluminescent labeling includes luminol, isoluminol, acridine esters, acridine salts, imidazole, oxalates, etc. In some specific embodiments, radioactive isotopes include tritium, carbon-14, phosphorus-32, sulfur-35, iodine-125, etc.

[0064] In some embodiments, the universal primer has a first binding portion, and the second detection label has a second binding portion capable of binding to the first binding portion, thereby binding the second detection label to the amplification product. In some embodiments, the first and second binding portions bind via covalent bonds (e.g., amide bonds, gold-thiol bonds, coordination bonds, etc.), non-covalent interactions (e.g., the biotin-avidin system), or antigen-antibody interactions. In some specific embodiments, one of the first and second binding portions is biotin, and the other is avidin or streptavidin. In some specific embodiments, one of the first and second binding portions is an antigen, and the other is an antibody, such as digoxigenin-anti-digoxigenin antibody.

[0065] In some implementations, the kit also includes other components necessary for expansion, such as dNTPs, buffers, polymerases, etc., which will not be described in detail here.

[0066] A second aspect of the present invention provides a method for detecting variant gene sites using the aforementioned kit, comprising the following steps:

[0067] Take the DNA sample to be tested, add primers for amplification, and obtain the amplification product;

[0068] The amplification product is mixed with the first detection label and the second detection label and then incubated to obtain the incubation product;

[0069] The incubation products are tested, and the variation of the variant gene loci is determined based on the detection results of the second detection marker corresponding to different first detection markers.

[0070] In some implementations, the amount of DNA sample added to the amplification reaction system is 10ng-300ng / 10μL, for example, it can be 10ng / 10μL, 20ng / 10μL, 40ng / 10μL, 50ng / 10μL, 60ng / 10μL, 80ng / 10μL, 100ng / 10μL, 200ng / 10μL, or 300ng / 10μL.

[0071] In some embodiments, the concentration of specific primers in the amplification reaction system is 0.05–2 μM, for example, 0.05 μM, 0.06 μM, 0.08 μM, 0.1 μM, 0.12 μM, 0.14 μM, 0.15 μM, 0.16 μM, 0.18 μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, 1 μM, 1.2 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.8 μM, or 2 μM.

[0072] In some embodiments, the concentration of universal primers in the amplification reaction system is 0.05–2 μM, for example, 0.05 μM, 0.06 μM, 0.08 μM, 0.1 μM, 0.12 μM, 0.14 μM, 0.15 μM, 0.16 μM, 0.18 μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, 1 μM, 1.2 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.8 μM, or 2 μM.

[0073] In some embodiments, the amplification reaction system is 10 to 50 μL, for example, 10 μL, 20 μL, 30 μL, 40 μL, or 50 μL.

[0074] In some implementations, the amplification program is as follows: 95°C for 2–15 min, 5–20 cycles (95°C for 10–20 s, 67–61°C for 30–60 s), 10–20 cycles (95°C for 10–20 s, 61°C for 15–25 s), 61°C for 15–25 s, and hold at 4–12°C.

[0075] In some embodiments, the incubation time for the amplified products is 10 to 40 minutes, for example, 10 minutes, 12 minutes, 14 minutes, 15 minutes, 16 minutes, 18 minutes, or 20 minutes.

[0076] In some embodiments, the incubation temperature of the amplified product is 40–60°C, for example, 40°C, 45°C, 50°C, 55°C, or 60°C.

[0077] In some implementations, after incubation, the process includes cleaning with a cleaning solution, which may be performed 1 to 3 times, for example, 2 times.

[0078] In some embodiments, the incubation product further includes a demagnetization step. In some embodiments, the incubation product further includes a replenishment step.

[0079] In some implementations, the variation of the variant gene site is determined based on the detection results of the second detection marker corresponding to different first detection markers. This includes determining the detection amount of the second detection marker corresponding to different variation of the variant gene site based on the combination results of the first detection marker and the second detection marker of the complex formed by the first detection marker-amplification product-second detection marker corresponding to different variation of the variant gene site, and thus obtaining the variation of the variant gene site.

[0080] In some implementations, the variant gene locus includes SNP loci, and determining the variation of the variant gene locus based on the detection results of the second detection marker corresponding to different first detection markers includes determining the genotyping of the SNP locus based on the detection results of the second detection marker corresponding to different first detection markers.

[0081] In some implementations, the genotyping of SNP sites is determined based on the detection results of the second detection markers corresponding to different first detection markers. This includes determining the detection amount of the second detection marker for each allele in the SNP site based on the combination result of the first detection marker and the second detection marker of the complex formed by the first detection marker, amplification product, and second detection marker corresponding to multiple alleles of the SNP site, and thus obtaining the genotyping of the SNP site.

[0082] In some embodiments, the second detection marker includes a fluorescent marker, and the genotyping of the SNP locus is determined based on the difference in fluorescence intensity of different alleles (e.g., a ratio). In some embodiments, the difference in fluorescence intensity can be determined by methods such as clustering.

[0083] In some implementations, the number of SNP sites to be detected is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or more than 50.

[0084] In some implementations, the detection of incubated products is performed using a liquid biochip reader to read the results.

[0085] In some implementations, detecting mutated gene loci does not directly involve the diagnosis or treatment of disease, and therefore this method falls under the category of non-disease diagnosis or treatment methods.

[0086] Compared to existing TaqMan probe methods and KASP technologies, the genotyping technology provided by this invention can detect dozens of sites in a single reaction, basically meeting the testing needs of common single-sample multi-site testing, and greatly saving the reagent, material, and manpower costs associated with single-sample multi-site testing. Furthermore, this hybridization method can be used for testing different sites without the need to re-prepare microbeads for different panels; only sequence modifications during primer design need to be adjusted. Simultaneously, this invention does not involve highly sophisticated instrument-dependent testing reactions such as sequencing, mass spectrometry, or Vera Code, and avoids complex and time-consuming detection procedures. It only involves multiplex amplification and hybridization reactions, resulting in relatively low reagent and material costs, shorter testing time and cycles, and the ability to be integrated with automated equipment to significantly increase testing throughput and reduce manpower consumption.

[0087] A third aspect of the present invention provides a detection system for variant gene loci, comprising any of the aforementioned reagent kits.

[0088] In some implementations, the primers, first detection marker, second detection marker, etc. in the kit can be independently distributed in different positions in the detection system. During detection, the primers are mixed and reacted according to the corresponding steps by manual or automated means, and the reaction results are detected.

[0089] In some implementations, the detection system for variant gene sites includes a detection system for SNP sites.

[0090] A fourth aspect of the present invention provides the application of the aforementioned kit or the aforementioned detection system in the preparation of detection products for variant gene loci.

[0091] In some implementations, the detection products for variant gene loci include genotyping detection products.

[0092] A fifth aspect of the present invention provides an application of the aforementioned reagent kit or the aforementioned detection system in any of the following A1) to A5):

[0093] A1) Assisted breeding;

[0094] A2) Preparation of products for assisted breeding;

[0095] A3) Prepare products for any one of the following: disease diagnosis, risk assessment, personalized treatment, or drug response prediction;

[0096] A4) Forensic medicine, disease mechanism research, germplasm resource protection, or biodiversity monitoring;

[0097] A5) Prepare products for any of the following purposes: forensic medicine, disease mechanism research, germplasm resource protection, or biodiversity monitoring.

[0098] In some embodiments, the assisted breeding includes assisted breeding of plants and animals.

[0099] In some embodiments, the plant includes at least one of crops, trees, fruit trees, and flowers.

[0100] In some embodiments, the animal includes at least one of poultry, livestock, and aquatic animals.

[0101] In some implementations, the disease includes a genetic disease.

[0102] In some implementations, the disease includes at least one of the following: Duchenne muscular dystrophy, hemophilia A, hemophilia B, hereditary deafness, thalassemia, spinal muscular atrophy, congenital adrenocortical hyperplasia, phenylketonuria, albinism, galactosemia, Wilson's disease, polycystic kidney disease, glycogen storage disease type II, citrullinemia, Gaucher disease, mucopolysaccharide type I, and mucopolysaccharide type II.

[0103] In some implementations, the disease includes at least one of cardiovascular disease, autoimmune disease, neurodegenerative disease, and tumor.

[0104] In some implementations, cardiovascular diseases include cardiomyopathy, cardiac ion channelopathies, monogenic hereditary hypertension, hereditary aortic disease, pulmonary hypertension, hereditary thrombophilia, familial hypercholesterolemia, etc.

[0105] In some implementations, autoimmune diseases include alopecia areata (ALO), ankylosing spondylitis (AS), celiac disease (CED), Crohn's disease (CRD), eosinophilic esophagitis (EE), Graves' disease (GRD), juvenile idiopathic arthritis (JIA), multiple sclerosis (MS), primary biliary cirrhosis (PBC), psoriatic arthritis (PA), psoriasis (PSO), rheumatoid arthritis (RA), Sjögren's syndrome (SJS), systemic lupus erythematosus (SLE), systemic scleroderma / sclerosis (SSC), type 1 diabetes mellitus (T1D), ulcerative colitis (ULC), and vitiligo (VIT), etc.

[0106] In some implementations, neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, spinocerebellar ataxia, Pick's disease dementia, etc.

[0107] In some embodiments, the tumor includes at least one of solid tumors and non-solid tumors. Solid tumors include, but are not limited to, at least one of brain cancer, head and neck cancer, lung cancer, liver cancer, breast cancer, stomach cancer, colon cancer, rectal cancer, cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, bladder cancer, thyroid cancer, lymphoma, and skin cancer. Non-solid tumors include leukemia. Brain cancer includes primary and secondary brain cancer. Primary brain cancer includes gliomas and non-gliomas (such as meningiomas, medulloblastomas, medullary pituitary adenomas, primary CNS lymphomas, and central nervous system germ cell tumors). Secondary brain cancer includes malignant tumors originating elsewhere but that have spread (metastasized) to the brain, such as breast and lung cancer that have metastasized to the brain. Head and neck cancers include oral / oropharyngeal cancer, nasopharyngeal cancer, nasal / sinus cancer, laryngeal / laryngopharyngeal cancer, and salivary gland cancer. Lung cancers include small cell lung cancer and non-small cell lung cancer. Liver cancers include hepatocellular carcinoma, cholangiocarcinoma, and hepatoblastoma. Breast cancers include carcinoma in situ (such as lobular carcinoma in situ and ductal carcinoma in situ) and invasive breast cancer (invasive lobular carcinoma and invasive ductal carcinoma). Stomach cancers include gastric adenocarcinoma. Gastrointestinal tumors, including leiomyosarcomas, carcinoid tumors, and lymphomas; cervical cancer, including squamous cell carcinoma and adenocarcinoma; ovarian cancer, including epithelial tumors, germ cell tumors, and stromal tumors; pancreatic cancer, including pancreatic cancer, cystic tumors, acinar cell carcinoma, sarcoma, and ampullary carcinoma; bladder cancer, including urothelial carcinoma, squamous cell carcinoma, adenocarcinoma, clear cell carcinoma, small cell carcinoma, and carcinoid tumors; thyroid cancer, including papillary thyroid carcinoma, follicular thyroid carcinoma, and Herxheimer's cell carcinoma; lymphoma, including Hodgkin's lymphoma and non-Hodgkin's lymphoma; skin cancer, including non-melanoma and melanoma; and leukemia, including acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, and chronic myeloid leukemia.

[0108] The present invention will be further described in detail below through specific embodiments.

[0109] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0110] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0111] Example 1: 4-fold SNP genotyping detection

[0112] The principle reference of this embodiment Figure 1 This method utilizes the hybridization of microbeads with specific barcode fragments and barcode recognition fragments to detect amplification products formed by specific upstream and downstream primers. Based on the correlation between the displayed specific coding and fluorescence intensity, the amount of different alleles is determined, thereby deriving the actual genotyping of the SNP locus. The specific steps are as follows:

[0113] SNP genotyping site primer design: Primers were designed using four rice SNP markers. Two upstream specific primers and one downstream primer were designed for each SNP site, following the design principle:

[0114] Upstream typing primer 1: AS sequence 1 + C6 spacer + upstream specific primer sequence 1;

[0115] Upstream typing primer 2: AS sequence 2 + C6 spacer + upstream specific primer sequence 2;

[0116] Downstream primer: The downstream primer for this SNP site + biotin modification.

[0117] The upstream primer is named AS number-site number_SNP allele, and the downstream primer is named site number_R.

[0118] The primer sequences are shown in Table 2, where C6 is the C6-spacer, and the 5' end of the downstream primer is modified with biotin. The upstream specific primer sequence and the downstream primer are derived from the industry standard "SNP Marker Method for Identification of Authentic Rice Varieties" NY / T 2745-2021.

[0119] Table 2. Sequences of each primer in Example 1

[0120]

[0121] Sample preparation: 12 samples each of Nipponbare and 93-11 rice varieties, 12 samples of hybrid rice of Nipponbare and 93-11, and 2 samples of template-free negative control (NTC) were prepared, for a total of 38 samples for testing.

[0122] The testing process is as follows:

[0123] (1) Whole genome DNA extraction: Extraction was performed using the instructions of the plant DNA extraction kit. The concentration of the extracted DNA sample was determined using an enzyme-linked immunosorbent assay (ELISA) reader. The extracted DNA sample was required to meet the following requirements: concentration >10 ng / μL, OD260 / OD280 >1.6, OD260 / OD230 >0.8.

[0124] (4) Multiplex amplification reaction: PCR reaction was carried out according to Table 3 and Table 4 to obtain multiplex amplification products.

[0125] The reaction system for the amplification process is shown in Table 3:

[0126] Table 3. Amplification Reaction System

[0127]

[0128]

[0129] Table 2 shows the amount of each component added when amplifying one sample. The volume of each component should be added according to the number of test samples. Note that each sample should be tested in one reaction well.

[0130] The amplification procedure is shown in Table 4:

[0131] Table 4. PCR Amplification Procedure

[0132]

[0133] (5) Microbead hybridization detection reaction:

[0134] The microbeads are produced by BGI Group. The microbead models involved in Example 1 are CZ-0003, CZ-0056, CZ-0066, CZ-0160, CZ-0320, CZ-1024, CZ-1161, and CZ-1169.

[0135] The correspondence between microbeads and primers is shown in Table 5:

[0136] Table 5. Correspondence between microbeads and primers

[0137]

[0138] Construct the hybridization reaction system according to a) and perform hybridization according to the procedure in b).

[0139] a) The hybridization detection reaction system is shown in Table 6:

[0140] Table 6. Hybridization Detection Reaction System

[0141]

[0142]

[0143] The table shows the amount of each component added when amplifying one sample. The volume of each component should be added according to the number of test samples. Note that each sample should be tested in one well.

[0144] b) The hybridization reaction procedure is shown in Table 7:

[0145] Table 7. Hybridization Reaction Procedure

[0146]

[0147] The reaction steps shown in the table are standard test reactions; for optical imaging, please refer to the equipment manual.

[0148] (6) Obtain sample genotyping information

[0149] The results of the optical imaging were processed and analyzed to obtain hybridization fluorescence value data for 38 samples at four loci in one go. The genotypes of the samples were then determined based on the fluorescence values. The fluorescence value data and the genotype results for each of the four SNP loci are shown in Table 8. Simultaneously, a scatter plot of the samples can be drawn based on the data from each test locus, resulting in an intuitive sample genotyping cluster diagram. The genotyping cluster diagrams for the four samples tested in this embodiment are shown in Table 8. Figure 2 .

[0150] Table 8. Hybridization fluorescence values ​​and sample genotype interpretation information

[0151]

[0152]

[0153] In this embodiment, one sample of each of the three positive samples (Nipponbare, 93-11, and heterozygous rice samples of Nipponbare and 93-11) was extracted for Sanger sequencing of the above four SNP loci. The results are shown in Table 9. The results of multiplex genotyping using this invention are consistent with the results of the Sanger sequencing report.

[0154] Table 9. Sanger sequencing results

[0155] CNR034 CNR116 CNR389 CNR244 Nippon Haru C / C C / C A / A A / A 93-11 A / A A / A G / G C / C heterozygous samples A / C A / C A / G A / C

[0156] Example 2: 50-fold SNP genotyping detection

[0157] SNP genotyping site primer design: 50 rice SNP markers were selected for primer design. The primer design principles and naming were the same as in Example 1, and the primer sequences are shown in Table 10, which are also derived from the industry standard "SNP Marker Method for Identifying Authenticity of Rice Varieties" NY / T 2745-2021.

[0158] Table 10. Primer sequences in Example 2

[0159]

[0160]

[0161]

[0162]

[0163]

[0164] Sample preparation and whole-genome DNA extraction were performed according to Example 1.

[0165] Multiplex amplification reaction: Perform PCR reaction according to Table 11 to obtain multiplex amplification products.

[0166] The reaction system for the amplification process is shown in the table below.

[0167] Table 11.50 SNP site genotyping and amplification reaction system

[0168]

[0169] The table shows the amount of each component to be added for amplification of one sample. Add the appropriate volume of each component based on the number of samples to be tested, ensuring one reaction well per sample. Additionally, the two upstream specific primers and the downstream universal primers of the 50 SNP primers must be added.

[0170] The amplification procedure is the same as in Example 1.

[0171] The correspondence between microbeads and primers is shown in Table 12:

[0172] Table 12.50 Primer-Microbead Correspondence Table

[0173]

[0174]

[0175] Hybridization detection reaction system (Table 13):

[0176] Table 13. Hybridization Detection Reaction System

[0177]

[0178] The table shows the amount of each component to be added for a single sample amplification. Add the appropriate volume of each component based on the number of samples to be tested, ensuring one reaction well per sample. Additionally, all 100 barcode-coded microbeads corresponding to the upstream primers of the 50 SNP primers should be added.

[0179] The hybridization reaction procedure is the same as in Example 1.

[0180] The results of the optical imaging were processed and analyzed to obtain hybridization fluorescence values ​​for 38 samples at 50 loci in one go. A scatter plot was plotted based on the data for each test locus, and the genotype of the samples was determined based on the fluorescence data and clustering. The scatter plot of the 38 samples at the 50 SNP loci is shown below. Figure 3 .

[0181] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A kit for detecting a variant genetic locus, characterized in that, include: The primers include a downstream primer and an upstream primer. The downstream primer and the upstream primer are used to amplify the target fragment containing the mutated gene site to generate an amplification product. One of the upstream primer and the downstream primer is a universal primer and the other is a specific primer. There are multiple specific primers. The specific primers include a barcode recognition fragment and a primer fragment. The 3' terminal bases of the primer fragments of the multiple specific primers correspond to different variations of the mutated gene site. Multiple different first detection markers are combined with different barcode segments, and the barcode segments and the barcode recognition segments are respectively one-to-one correspondences and inverse complements; A second detection marker is used to bind the amplification product.

2. The reagent kit according to claim 1, characterized in that: The barcode identification fragment and the primer fragment are connected by an interarm modification group; Preferably, the inter-arm modifying group includes at least one of an alkylene group having 3 to 20 carbon atoms and a polyethylene glycol group; preferably, the inter-arm modifying group includes at least one of C3 Spacer, C6 Spacer, Spacer 9, C12 Spacer, and Spacer 18. Preferably, the length of the barcode identification segment is 10 to 30 nt.

3. The reagent kit according to claim 1, characterized in that: The first detection marker includes magnetic microspheres, and different types of magnetic microspheres have different identifiable morphological features; Preferably, the morphological features include the pattern on the surface of the magnetic microspheres.

4. The kit according to claim 1, characterized in that: The universal primer has a first binding portion, and the second detection marker has a second binding portion capable of binding to the first binding portion; Preferably, one of the first binding portion and the second binding portion is biotin, and the other is avidin or streptavidin; Preferably, one of the first binding portion and the second binding portion is an antigen, and the other is an antibody.

5. The reagent kit according to claim 1, characterized in that: The second detection label includes at least one of fluorescent label, chemiluminescent label, and radioactive isotope.

6. The reagent kit according to claim 1, characterized in that: The barcode segment and the barcode identification segment include at least one combination of BS and AS from Table 1.

7. The kit according to claim 1, characterized in that: The 3' end of the primer fragment of the specific primer is also modified; Preferably, the modification includes modification of one or more nucleotides at the 3' end; Preferably, the modification site is located at at least one of a phosphate group, a base, and a ribose; Preferably, the modification of the phosphate group includes at least one of thio, amino, and boronyl substitution modifications; Preferably, the base modification includes at least one of pseudouridine, 2-thiouridine, N1-methylpseudouridine, 5-methyluridine, 5-methoxyuridine, N6-methyladenosine, and 5-methylcytidine. Preferably, the ribose modification includes at least one of methoxy, methoxyethoxy, locked nucleic acid, and PMO.

8. A method of detecting a variant gene site using the kit according to any one of claims 1 to 7, characterized in that, Includes the following steps: Take the DNA sample to be tested, add primers for amplification, and obtain the amplification product; The amplification product is mixed with the first detection label and the second detection label and then incubated to obtain the incubation product; The incubation product is tested, and the variation of the variant gene site is determined based on the detection results of the second detection marker corresponding to different first detection markers.

9. A detection system for variant gene loci, characterized in that: The kit includes the kit described in any one of claims 1 to 7.

10. The kit according to any one of claims 1 to 7, or the detection system according to claim 9, in the preparation of detection products for variant gene loci.

11. The application of the kit according to any one of claims 1 to 7, or the detection system according to claim 9, in any one of the following A1) to A5): A1) Assisted breeding; A2) Preparation of products for assisted breeding; A3) Prepare products for any one of the following: disease diagnosis, risk assessment, personalized treatment, or drug response prediction; A4) Forensic medicine, disease mechanism research, germplasm resource protection, or biodiversity monitoring; A5) Prepare products for any of the following purposes: forensic medicine, disease mechanism research, germplasm resource protection, or biodiversity monitoring; Preferably, the assisted breeding includes assisted breeding of plants and animals; Preferably, the plant includes at least one of crops, trees, fruit trees, and flowers; Preferably, the animal includes at least one of poultry, livestock, and aquatic animals.

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