A detection method for genotyping high-frequency mutation sites of the ATP7B gene in Wilson's disease by constant temperature multiplex amplification

Abstract

The present invention provides a primer combination, kit, detection system, detection method and application for detecting the gene typing of high-frequency mutation sites of the ATP7B gene in Wilson's disease by constant temperature multiplex amplification. The method of the present invention is based on the amplification refractory mutation system technology combined with the nicking endonuclease nucleic acid isothermal amplification detection reaction system to perform single-tube multiplex amplification on three high-frequency pathogenic ectopic sites of the ATP7B gene in Wilson's disease, and at the same time, capillary electrophoresis is combined for product analysis to achieve the purpose of gene typing detection.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and in particular relates to a detection method and a detection system for gene SNP typing based on isothermal and multiplex amplification. Background Art

[0002] Hepatolenticular degeneration, also known as Wilson's disease, is an autosomal single-gene recessive genetic disease characterized by copper metabolism disorder. The lesions mainly involve the liver, brain, kidneys, and cornea, etc., causing progressive liver cirrhosis, basal ganglia damage, kidney damage, and corneal pigment ring, etc. At present, the global prevalence of hepatolenticular degeneration is approximately 1 / 30,000. In isolated populations (such as Sardinia), the prevalence can reach 1 / 10,000, and the prevalence of Han nationality in Hong Kong, China is as high as 1 / 5400.

[0003] At present, the diagnosis of hepatolenticular degeneration mainly relies on typical clinical manifestations, laboratory tests, and gene detection. The treatments include penicillamine, zinc preparations, and liver transplantation, etc. Early diagnosis and early intervention are crucial for delaying the progression of the disease and preventing irreversible sequelae.

[0004] When the ATP7B gene mutates, the expression level and / or localization of ATP7B in cells change, its copper transport ability decreases, resulting in a decrease in the synthesis of ceruloplasmin and obstruction of copper excretion in the bile duct. Excessive copper accumulates in the body, causing cell necrosis and organ damage, seriously affecting the quality of life of patients in the later stage, and ultimately leading to death.

[0005] At present, there are many methods for detecting SNPs, such as sanger sequencing, pyrosequencing, taqman probe, arms-PCR, HRM high-resolution melting curve, etc. But almost all methods need to use PCR amplification technology, with complex operations, long detection cycles, and a process of temperature change, which leads to the need for a temperature-changing instrument module, such as a PCR instrument, with a relatively high price, restricting the popularization of this technology. Summary of the Invention

[0006] In view of these drawbacks in the prior art, the present invention provides a detection method, detection primers, and a detection system for SNP typing of high-frequency mutation sites of the ATP7B gene through isothermal multiplex detection. The present invention uses the ARMS technology in combination with the NEAR isothermal amplification system to perform single-tube multiplex amplification on 3 high-frequency pathogenic mutation sites of the ATP7B gene, and at the same time combines capillary electrophoresis for product analysis to perform gene typing detection.

[0007] The present invention has the following beneficial effects

[0008] 1) The present invention provides a new method for rapid SNP genotyping detection by ARMS-combined nicking endonuclease-mediated isothermal amplification, which can achieve rapid, efficient, and low-cost simultaneous genotyping of 3-40 SNP loci;

[0009] 2) The present invention adopts an isothermal reaction system, which can minimize the dependence on high-precision instruments. As is well known, due to the heating and cooling modules, PCR instruments have high precision requirements. Different PCR instruments are prone to unstable results or inconsistent results in different laboratories due to different heating and cooling rates. At the same time, the cost of PCR instruments is also relatively high. The isothermal system of the present invention helps to lower the threshold of molecular biology detection, remove the variable temperature module, and reduce the instrument cost;

[0010] 3) The amplification reaction is rapid and efficient: The nicking endonuclease technology has mild reaction conditions and high reaction efficiency, and the amplified fragment can be obtained within 15-30 minutes. Compared with the traditional PCR technology that requires 1.5-2 hours, the reaction time is greatly shortened, achieving rapid and efficient results, which plays a positive role in rapid diagnosis;

[0011] 4) The present invention uses a multiplex amplification system combined with a capillary to analyze the fragments of the products, making the detection results intuitive and easy to interpret. The present invention uses a multiplex amplification system combined with capillary electrophoresis analysis to achieve the simultaneous detection of the genotypes of multiple SNP loci through single-tube multiplex amplification, and the detection results are easy to analyze and interpret. Compared with other single detection methods on the market, it further reduces the detection reagent cost on the basis of improving the detection efficiency and simplifying the operation steps.

[0012] 5) The method of the present invention has high throughput, strong specificity, high sensitivity, and is simple to operate, rapid, efficient, and low-cost.

[0013] According to one aspect of the present invention, the present invention provides a primer combination for the high-frequency mutation sites of the ATP7B gene in Wilson's disease, and the primer combination can simultaneously amplify the following 3 risk sites in the same PCR system:

[0014] p.R778L site;

[0015] p.P992L site; and

[0016] p.T935M site.

[0017] According to certain embodiments of the present invention, each primer contains a recognition site for the restriction site of a nicking endonuclease, a stable region, and a nucleic acid strand of a target-binding region complementary to the target; and the SNP recognition-specific upstream primers at each detection site include a mismatch of introducing a base at the -2 or -3 position at the 3' end, and the specific base corresponding to the SNP site at the last base.

[0018] According to certain embodiments of the present invention, the primer pair for each locus includes a wild-type upstream primer, a mutant upstream primer, and a common downstream primer.

[0019] According to certain embodiments of the present invention, the primer combination sequence includes SEQ ID NO.1-9.

[0020] According to certain embodiments of the present invention, a fluorescent group recognized for later capillary electrophoresis detection is labeled, and the fluorescent group is modified on a T base of a specific primer segment. The fluorescent group can be modified with FAM, HEX, ROX, TAMER, or CY5.

[0021] According to certain embodiments of the present invention, the fluorescent group is labeled on a T base at 3-8 bases at the 3' end from the recognition site of the nicking endonuclease on the specific primer segment of the common downstream primer at each detection locus.

[0022] According to certain embodiments of the present invention, the fluorescent group is labeled on a T base at the 3rd to 8th bases at the 3' end from the recognition site of the nicking endonuclease on the specific primer segment of the common downstream primer at each detection locus, such as 3-6 bases, 3-5 bases, 5-8 bases, 5-6 bases, or 6-8 bases.

[0023] According to certain embodiments of the present invention, the fluorescent group is labeled on a T base at the 3rd, 4th, 5th, 6th, 7th, or 8th base at the 3' end from the recognition site of the nicking endonuclease on the specific primer segment of the common downstream primer at each detection locus.

[0024] According to certain embodiments of the present invention, in the primer combination, the amount of the upstream primer is 4 times that of the downstream primer.

[0025] According to one aspect of the present invention, the present invention provides a kit for genes of high-frequency mutation sites of the ATP7B gene in Wilson's disease, characterized in that the kit includes the primer combination, NERA isothermal multiplex amplification enzyme mixture, 10X reaction buffer, dNTP positive control, and internal standard required for capillary electrophoresis.

[0026] According to certain embodiments of the present invention, the NERA isothermal multiplex amplification enzyme mixture includes a nicking endonuclease and a DNA polymerase.

[0027] According to certain embodiments of the present invention, the nicking endonuclease can be selected from N.ALwl, Nb.BbvCI, Nt.BbvCI, Nt.BstNBI, Nb.BsmI, or other similar nicking endonucleases.

[0028] According to certain embodiments of the present invention, the nicking enzyme is preferably Nt.Bst NBI nicking enzyme.

[0029] According to certain embodiments of the present invention, the DNA polymerase is selected from Bst DNA polymerase, Klenow DNA polymerase, Vent DNA polymerase, phi29 DNA polymerase or other similar DNA polymerases with strand displacement activity.

[0030] According to certain embodiments of the present invention, the DNA polymerase is preferably Nt.BstNBI-Bst 3.0 polymerase.

[0031] According to certain embodiments of the present invention, the 10X reaction buffer is: 20 mM MgSO4, 1500 mM KCl, 100 mM (NH4)2SO4, 500 mM NaCl, 400 mM Tris-Cl (pH 8.8).

[0032] According to certain embodiments of the present invention, in the 10X buffer, the concentration of Mg+ ions can be adjusted according to the detection effect and can be adjusted between 10 mM and 40 mM to obtain the optimal amplification efficiency. According to certain embodiments of the present invention, in the 10X buffer, the concentration of Mg+ ions is 10 mM - 40 mM, 10 mM - 30 mM, 10 mM - 20 mM, 20 mM - 40 mM, 20 mM - 30 mM, 30 mM - 40 mM. According to certain embodiments of the present invention, in the 10X buffer, the concentration of Mg+ ions is 10 mM, 20 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM or 40 mM.

[0033] According to certain embodiments of the present invention, the 10X reaction buffer is: 10 mM MgSO4, 1500 mM KCl, 100 mM (NH4)2SO4, 500 mM NaCl, 400 mM Tris-Cl (pH 8.8). According to certain embodiments of the present invention, the 10X reaction buffer is: 15 mM MgSO4, 1500 mM KCl, 100 mM (NH4)2SO4, 500 mM NaCl, 400 mM Tris-Cl (pH 8.8). According to certain embodiments of the present invention, the 10X reaction buffer is: 25 mM MgSO4, 1500 mM KCl, 100 mM (NH4)2SO4, 500 mM NaCl, 400 mM Tris-Cl (pH 8.8). According to certain embodiments of the present invention, the 10X reaction buffer is: 30 mM MgSO4, 1500 mM KCl, 100 mM (NH4)2SO4, 500 mM NaCl, 400 mM Tris-Cl (pH 8.8). According to certain embodiments of the present invention, the 10X reaction buffer is: 35 mM MgSO4, 1500 mM KCl, 100 mM (NH4)2SO4, 500 mM NaCl, 400 mM Tris-Cl (pH 8.8). According to certain embodiments of the present invention, the 10X reaction buffer is: 40 mM MgSO4, 1500 mM KCl, 100 mM (NH4)2SO4, 500 mM NaCl, 400 mM Tris-Cl (pH 8.8).

[0034] According to one aspect of the present invention, the present invention provides a isothermal multiplex amplification detection system for the primer combination to detect high-frequency mutation sites of the ATP7B gene in Wilson's disease, characterized in that the isothermal multiplex amplification detection system is:

[0035] Component Amount (to be added) 10X Reaction Buffer 2.5 μL 5X Primer MIX 5 μL dNTP (10 mM) 2.5 μL DMSO 0.5 μL BSA (10 μg / μL) 0.5 μL DNA Template (10 ng / μL) 2.5 μL Make up to with double-distilled water 24 μL

[0036] Wherein, the 10X reaction buffer is: 20 mM MgSO4, 1500 mM KCl, 100 mM (NH4)2SO4, 500 mM NaCl, 400 mM Tris-Cl (pH 8.8);

[0037] The above reaction system is treated at 95 °C for 5 min, quickly placed on ice, and after cooling, nicking enzyme and polymerase are added to each tube of the reaction solution according to the amounts in the following table, as follows:

[0038] Nt.Bst NBI Nicking Enzyme (10 U / μL) 0.2 μL DNA Polymerase (8 U / μL) 0.8 μL

[0039] Among them, the nicking endonuclease can be selected from N.ALwl, Nb.BbvCI, Nt.BbvCI, Nt.BstNBI, Nb.BsmI or other similar nicking endonucleases; the DNA polymerase is selected from Bst DNA polymerase, Klenow DNA polymerase, Vent DNA polymerase, phi29 DNA polymerase or other similar DNA polymerases with strand displacement activity.

[0040] According to certain embodiments of the present invention, in the 10X buffer, the concentration of Mg+ ions can be adjusted according to the detection effect and can be adjusted between 10 mM and 40 mM to obtain the optimal amplification efficiency. According to certain embodiments of the present invention, in the 10X buffer, the concentration of Mg+ ions is 10 mM - 40 mM, 10 mM - 30 mM, 10 mM - 20 mM, 20 mM - 40 mM, 20 mM - 30 mM, 30 mM - 40 mM. According to certain embodiments of the present invention, in the 10X buffer, the concentration of Mg+ ions is 10 mM, 20 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM or 40 mM.

[0041] According to certain embodiments of the present invention, the 10X reaction buffer is: 10 mM MgSO4, 1500 mM KCl, 100 mM (NH4)2SO4, 500 mM NaCl, 400 mM Tris-Cl (pH 8.8). According to certain embodiments of the present invention, the 10X reaction buffer is: 15 mM MgSO4, 1500 mM KCl, 100 mM (NH4)2SO4, 500 mM NaCl, 400 mM Tris-Cl (pH 8.8). According to certain embodiments of the present invention, the 10X reaction buffer is: 25 mM MgSO4, 1500 mM KCl, 100 mM (NH4)2SO4, 500 mM NaCl, 400 mM Tris-Cl (pH 8.8). According to certain embodiments of the present invention, the 10X reaction buffer is: 30 mM MgSO4, 1500 mM KCl, 100 mM (NH4)2SO4, 500 mM NaCl, 400 mM Tris-Cl (pH 8.8). According to certain embodiments of the present invention, the 10X reaction buffer is: 35 mM MgSO4, 1500 mM KCl, 100 mM (NH4)2SO4, 500 mM NaCl, 400 mM Tris-Cl (pH 8.8). According to certain embodiments of the present invention, the 10X reaction buffer is: 40 mM MgSO4, 1500 mM KCl, 100 mM (NH4)2SO4, 500 mM NaCl, 400 mM Tris-Cl (pH 8.8).

[0042] According to certain embodiments of the present invention, the nicking endonuclease is preferably Nt.Bst NBI nicking endonuclease.

[0043] According to certain embodiments of the present invention, the DNA polymerase is preferably Nt.BstNBI-Bst 3.0 polymerase.

[0044] According to one aspect of the present invention, the present invention provides the use of the primer combination or isothermal multiplex amplification detection system in the preparation of a reagent for genotyping detection of high-frequency mutation sites of the ATP7B gene.

[0045] According to one aspect of the present invention, the present invention provides a method for isothermal multiplex amplification detection of genotyping of high-frequency mutation sites of the ATP7B gene in Wilson's disease, characterized in that the method includes:

[0046] a) Configure the isothermal amplification system and perform isothermal amplification at 58 °C for 25 minutes;

[0047] b) Capillary electrophoresis detection. According to whether specific amplification peak spectra appear in the wild-type and mutant peak maps at each detection site, the genotype of the sample at this site is interpreted.

[0048] According to certain embodiments of the present invention, the method includes

[0049] a) Configure the isothermal amplification system and perform isothermal amplification at 58°C for 25 minutes;

[0050] b) Prepare a loading mixture mixed with a molecular weight internal standard and formamide: (0.5 μL molecular weight internal standard + 8.5 μL formamide) × the number of test samples, vortex for 1 - 15 seconds, use a pipette to dispense 9 μL of the formamide and internal standard mixture into each detection well, take 1 μL of the amplification product and add it to the loading mixture of the molecular weight internal standard and formamide, and perform electrophoresis detection according to the steps in the user manual of the genetic analyzer; Data analysis: Import relevant files into the GeneMapper software, import the original data detected by the genetic analyzer, and analyze the data; Judgment of detection: According to whether specific amplification peak spectra (the peak height threshold is set to Rf100) appear in the wild-type and mutant peak maps at each detection site, the genotype of the sample at this site is interpreted.

[0051] According to one aspect of the present invention, the present invention provides a method for detecting the genotyping of high-frequency mutation sites of the ATP7B gene in Wilson's disease by isothermal multiplex amplification. The method uses a rapid and efficient SNP genotyping technology, namely the ARMS technology combined with NEAR isothermal amplification, and combines capillary electrophoresis analysis for SNP genotyping.

[0052] According to one aspect of the present invention, the present invention provides a method for detecting the genotyping of high-frequency mutation sites of the ATP7B gene in Wilson's disease by isothermal multiplex amplification. The detection method includes a primer combination for detecting 3 high-frequency pathogenic variant SNP sites of the ATP7B gene.

[0053] According to certain embodiments of the present invention, the primer combination comprises primer sequences for an ARMS-NEAR isothermal amplification system for detecting three sites, namely p.R778L, p.P992L, and p.T935M, of the ATP7B gene. Each primer contains a recognition site for a restriction site (5'-GAGTCNNNN-3', such as Nt.BstNBI), a stable region, and a nucleic acid strand of a target-binding region complementary to the target. Moreover, the upstream primers for amplification at each site are further designed according to the allele-specific principle. Each specific primer can only bind to the DNA template of the corresponding genotype and perform specific amplification. In order to improve the resolution ability of the specific primers for SNP sites, according to the design principle of ARMS primers, in addition to the last base at the 3' end of each SNP recognition-specific upstream primer corresponding to the specific base of the SNP site, a base mismatch is introduced at the -2 or -3 position at the 3' end. The primer sequences are as shown in SEQ1-9.

[0054] According to certain embodiments of the present invention, the downstream primer shared by each detection site is labeled with a fluorescent group recognized for later capillary electrophoresis detection. The fluorescent group is modified on a T base at 3-8 bases from the 3' end of the specific primer segment away from the recognition site of the nicking endonuclease. The fluorescent group can be modified with FAM, HEX, ROX, TAMER, or CY5.

[0055] According to certain embodiments of the present invention, in the primer composition, the amount of the upstream primer is 4 times that of the downstream primer.

[0056] According to certain embodiments of the present invention, the 10X reaction buffer is: 20 mM MgSO4, 1500 mM KCl, 100 mM (NH4)2SO4, 500 mM NaCl, 400 mM Tris-Cl (pH 8.8).

[0057] According to certain embodiments of the present invention, the NERA isothermal complex amplification enzyme mixture includes a nicking endonuclease and a DNA polymerase. The nicking endonuclease can be selected from N.ALwl, Nb.BbvCI, Nt.BbvCI, Nt.BstNBI, Nb.BsmI, or other similar nicking endonucleases; the DNA polymerase can be selected from Bst DNA polymerase, Klenow DNA polymerase, VentDNA polymerase, phi29 DNA polymerase, or other similar DNA polymerases with strand displacement activity.

[0058] According to certain embodiments of the present invention, the preparation method of the primer combination MIX and the preparation method of the isothermal amplification system.

[0059] According to one aspect of the present invention, the present invention provides a detection method for genotyping high-frequency mutation sites of the ATP7B gene for hepatolenticular degeneration by isothermal multiplex amplification and a specific application method for performing isothermal multiplex amplification detection, including the following steps: isothermal amplification, detection of amplification products by a genetic analyzer, data analysis, and a specific usage method for judgment of detection results.

[0060] Definition

[0061] Hepatolenticular degeneration (Wilson's disease)

[0062] Hepatolenticular degeneration (HLD), also known as Wilson's disease (WD), is an autosomal recessive inherited copper metabolism disorder disease. First reported and described by Wilson, it is a liver cirrhosis caused by hereditary copper metabolism disorder and a degenerative disease of the brain mainly in the basal ganglia. Clinically, it is manifested as progressively aggravated extrapyramidal symptoms, liver cirrhosis, mental symptoms, renal function damage, and corneal pigment ring. The specific manifestations of copper metabolism disorder in this disease are: the total serum copper and ceruloplasmin are reduced while the free copper is increased, the amount of copper excreted by the liver into the bile is reduced, the urinary copper excretion is increased, and excessive copper is deposited in many organs and tissues, especially significantly in the liver, brain, cornea, kidney, etc. The excessive deposited copper can damage the tissue structure and function of these organs and cause diseases.

[0063] ATP7B gene

[0064] Hepatolenticular degeneration is caused by mutations in the ATP7B gene, which encodes copper-transporting P-type ATPase (ATP7B). ATP7B gene mutations are characterized by high-frequency mutations accompanied by widespread rare mutations, with obvious geographical and ethnic distribution characteristics. ATP7B is a membrane protein expressed in multiple organs, and its main function is to promote the excretion of copper with blood and bile. When the ATP7B gene mutates, the expression level and / or localization of ATP7B in cells change, its copper-transporting ability decreases, resulting in reduced synthesis of serum ceruloplasmin and blocked copper excretion in bile ducts. Excessive copper accumulates in the body, causing cell necrosis and organ damage, seriously affecting the quality of life of patients in the later stage, and ultimately leading to death.

[0065] To date, more than 1,000 pathogenic variants of the ATP7B gene have been reported, distributed throughout the entire gene length. Therefore, the genotypes of patients with hepatolenticular degeneration are very complex. Most patients have compound heterozygous mutations (i.e., each of the pair of chromosomal genes carries two different mutations). Different hotspot mutations also show population and regional differences. Among them, the most common mutation site in the Asian population is p.R778L (c.2333G>T), and the carrier rate in patients from mainland China and Taiwan, China can reach 30%. There are mainly three high-frequency pathogenic variants in Chinese patients with Wilson disease, namely p.R778L, p.P992L, and p.T935M, accounting for 60%-70% of all pathogenic variants. Therefore, patients highly suspected of having Wilson disease clinically can first be screened for these pathogenic variants. Those who are not detected can continue to be screened for the entire coding region of the ATP7B gene and its flanking sequences. The three high-frequency pathogenic variants p.R778L, p.P992L, and p.T935M are all single-base variants, that is, single-base SNP variants. However, there is a lack of products and technologies for accurate and rapid mutation screening of the high mutation sites of the ATP7B gene in Wilson disease among the existing products and technologies currently.

[0066] Nicking enzyme-assisted reaction

[0067] Nicking enzyme-assisted reaction (NEAR) is a new nucleic acid isothermal amplification technology that depends on nicking enzymes and is developed on the basis of strand displacement isothermal amplification technology (SDA). Compared with ordinary PCR reactions, NEAR has the advantages of a constant reaction temperature, simple operation, short time consumption, high specificity, and high sensitivity. The reaction system mainly consists of primers, DNA polymerase with strand displacement activity, nicking enzymes, and dNTPs as the main reaction components. The main principle is that nicking enzymes can recognize specific DNA sequences and cut one strand of the double-stranded DNA molecule at specific sites. The DNA polymerase with strand displacement activity uses the 3' end of the nicking enzyme incision as the starting point, uses the uncut single strand as a template, synthesizes a new strand, and strips the old strand; the strand that restores the nicking enzyme recognition site can repeat the above amplification reaction in a cycle, ultimately achieving exponential amplification of the target sequence. The entire reaction can be carried out at a constant temperature of 50-60°C, and the entire process can reach the detection level in 15-30 minutes. The detection methods for NEAR products include gel electrophoresis, real-time fluorescence detection, chemiluminescence immunoassay, etc.

[0068] Amplification refractory mutation system

[0069] Amplification Refractory Mutation System (ARMS), also known as Allele-Specific Detection System, is based on the reaction of DNA polymerase for allele-specific extension. Only when the base at the 3'-end of a certain allele-specific primer is complementary to the base at the mutation site can the extension reaction occur. Primers with bases at the 3'-end that do not fully match the template cannot bind and extend normally, and no specific extension products can be obtained. It is a reliable genotyping method.

[0070] Method for multiplex detection of SNP genotyping of high mutation sites of ATP7B gene based on ARMS technology combined with NEAR isothermal amplification system

[0071] The present invention provides a method for multiplex detection of SNP genotyping of high mutation sites of ATP7B gene based on ARMS technology combined with NEAR isothermal amplification system, as well as detection primers and system. The present invention is achieved through the following scheme:

[0072] A primer combination for detecting 3 high-frequency pathogenic variant SNP sites of ATP7B gene based on ARMS combined with NEAR isothermal amplification method. The primer combination includes primers for ARMS-NEAR isothermal amplification system for detecting 3 sites of p.R778L, p.P992L and p.T935M of ATP7B gene. The primer composition can simultaneously perform multiplex amplification of these 3 sites in the same NEAR system.

[0073] The primer composition includes upstream and downstream primer sets for detecting p.R778L site, upstream and downstream primer sets for detecting p.P992L site, and upstream and downstream primers for detecting p.T935M site; the primers are different from ordinary primers and are specially designed for nicking endonucleases. Usually, each primer contains a recognition site for a restriction site (5'-GAGTCNNNN-3', such as Nt.BstNBI), a stable region, and a nucleic acid strand of a target-binding region complementary to the target. According to the principle of allele specificity, the upstream primer for each site can only bind to the DNA template of the corresponding genotype and perform amplification. The primer design for each site is as follows (SEQ ID NO.1-9), where the bold letters represent the bases mutated or changed according to the principle of ARMS:

[0074] Among them, the upstream and downstream primer sequences for detecting p.R778L site are: (146-148bp)

[0075] Wild-type upstream inner primer (SEQ ID NO.1):

[0076] 5’-AACCTGAAGC GAGTCAACT CTTTGCCAAGTGTTCCAGCCTCC-3’

[0077] Mutant upstream internal primer (SEQ ID NO.2):

[0078] 5’-AACCTGAAGC GAGTCAACT ACCTTTGCCAAGTGTTCCAGCCAGA-3’

[0079] Common downstream internal primer (SEQ ID NO.3):

[0080] 5’-GTGCTCATCG GAGTCAACT CAAGCA(FAM-dT)TGCTTATGTTTATTCT-3’

[0081] The upstream and downstream primer sequences for detecting the p.P992L site are: (87 - 89bp)

[0082] Wild-type upstream internal primer (SEQ ID NO.4):

[0083] 5’-TTCTGCGCGG GAGTCAACT TGCCCACCATGACAGCCGAGG-3’

[0084] Mutant upstream internal primer (SEQ ID NO.5):

[0085] 5’-TTCTGCGCGG GAGTCAACT GGTGCCCACCATGACAGCCGTTA-3’

[0086] Common downstream internal primer (SEQ ID NO.6):

[0087] 5’-CAGACAGAGG GAGTCAACT GG(FAM-dT)TTGCTTTCCAGACGTCCATC-3’

[0088] The upstream and downstream primer sequences for detecting the p.T935M site are: (153 - 155bp)

[0089] Wild-type upstream primer (SEQ ID NO.7):

[0090] 5’-ACCAAAATCG GAGTCAACT ATTACAATCCATACCACCACCG-3’

[0091] Mutant upstream primer (SEQ ID NO.8):

[0092] 5’-ACCAAAATCG GAGTCAACT CGATTACAATCCATACCACCAAAG-3’

[0093] Common downstream primer (SEQ ID NO.9):

[0094] 5’-TTAGAAATAGG GAGTCAACT GTG(FAM-dT)TTTATTTCTTCATAGGT-3’

[0095] Preferably, among more than 40 bases of the upstream and downstream primers at each detection site, about 20 - 23 bp at the 3' end are specific ARMS primers complementary to the template, and there is also a recognition site for nicking endonuclease (indicated by "") at the 5' end, and there are about 10 - 14 bp of protective bases at the 5' end of the recognition site to ensure that the nicking endonuclease can perform enzymatic digestion quickly and efficiently.

[0096] In order to solve the problem that the products after NEAR multiplex isothermal amplification can be detected and recognized by the capillary electrophoresis detection platform, the present invention labels the common downstream primer at each detection site with a fluorescent group recognized for later capillary electrophoresis detection. This fluorescent group is different from conventional fluorescent labels that can be modified at the 5' end of the primer. Because there is a recognition site for nicking endonuclease at the 5' end of the primer of the present invention, and there are about 10 - 14 bp of protective bases at the 5' end of the recognition site, this part of the sequence fragment will be recognized by the nicking endonuclease and spliced off during the isothermal reaction stage. Therefore, the fluorescent group needs to be modified at the 3' end on the specific ARMS primer segment complementary to the template. Therefore, the present invention modifies the fluorescent group on a T base of the specific primer segment (as shown above as the modified sequence information).

[0097] Preferably, the fluorescent group can be modified with FAM, HEX, ROX, TAMER or CY5.

[0098] The present invention also provides an isothermal multiplex detection system for detecting 3 high-frequency pathogenic variant SNP sites of the ATP7B gene. The isothermal multiplex detection system includes the above primer composition. This isothermal multiplex amplification system is a system combining multiplex fluorescent ARMS isothermal amplification and capillary electrophoresis detection. Specifically, in this system, the detection is performed by capillary electrophoresis detection, and the amplification is performed by nicking endonuclease-mediated multiplex allele-specific isothermal amplification.

[0099] Specifically, in the isothermal multiplex amplification system, three primers are set for each detection site. For the two SNP genotyping, one specific primer with different lengths (differing by 2-3 bp) is set respectively, and one fluorescently labeled downstream primer. Each specific primer can only bind to the DNA template of the corresponding genotype and amplify. After completing the multiplex isothermal amplification, capillary electrophoresis detection is carried out. Through specific fluorescent labeling, the presence or absence of amplification products of specific lengths can be used to determine whether a specific genotype exists at a specific site in the sample. In the setting of the present invention, the target fragment amplified by the wild-type primer is 2-3 bp smaller than the fragment amplified by the mutant primer.

[0100] Preferably, in order to improve the resolution ability of the specific primer for the SNP site, according to the design principle of ARMS primers, in addition to the last base at the 3' end of each SNP recognition specific upstream primer corresponding to the specific base of the SNP site, a base mismatch is introduced at the -2 or -3 position at the 3' end. The mismatched base at the 3' end of the primer will slow down the primer extension speed. When the mismatch reaches a certain degree, the primer extension will terminate and a PCR product of specific length cannot be obtained, indicating that the template DNA does not have a base complementary to the 3' end of the primer.

[0101] Preferably, in the isothermal reaction system, the amount of the upstream primer is usually 4 times that of the downstream primer.

[0102] The isothermal amplification system also includes nicking endonuclease, DNA polymerase, dNTP, bovine serum albumin (BSA), DMSO and Mg+, buffer solution, etc.

[0103] Preferably, in the NEAR reaction system, the nicking endonuclease can be selected from N.ALwl, Nb.BbvCI, Nt.BbvCI, Nt.BstNBI, Nb.BsmI or other similar nicking endonucleases; the DNA polymerase can be selected from Bst DNA polymerase, Klenow DNA polymerase, Vent DNA polymerase, phi29 DNA polymerase or other similar DNA polymerases with strand displacement activity.

[0104] Preferably, the nicking endonuclease used in the present invention is Nt.BstNBI, and the DNA polymerase used is Bst DNA polymerase, both of which can be purchased from NEB Company, USA.

[0105] Preferably, the 10X reaction buffer is: 20 mM MgSO4, 1500 mM KCl, 100 mM (NH4)2SO4, 500 mM NaCl, 400 mM Tris-Cl (pH 8.8).

[0106] Preferably, DMSO is further added to the system of the present invention to disrupt the secondary structure of DNA and improve the efficiency of the isothermal reaction.

[0107] Preferably, BSA is further added to the system of the present invention to stabilize the reaction system.

[0108] The present invention also provides the use of the above primer combination or multiplex amplification system in a reagent for detecting the genotyping of 3 high-frequency pathogenic variants of the ATP7B gene.

[0109] The present invention also provides a method for genotyping 3 high-frequency pathogenic SNP sites of the ATP7B gene, including the step of amplifying a sample with the above primer composition or multiplex amplification system.

[0110] The present invention also provides a method for using the above detection system or kit, mainly including the following steps: configuring an isothermal system, isothermal amplification, detecting amplification products with a genetic analyzer, data analysis, and judging the detection results.

[0111] Preferably, the sample that can be detected by the detection system of the present invention is human whole blood or peripheral blood sample.

[0112] According to one aspect of the present invention, the present invention provides a detection method for genotyping 3 high-frequency pathogenic SNPs of the ATP7B gene based on ARMS combined with NEAR isothermal amplification method, and the steps are as follows:

[0113] (1) Extract the DNA of the sample to be tested as a template, and adjust the concentration to 10 ng / μL for standby.

[0114] (2) Configure a 5X primer MIX for detecting gene mutations at 3 loci of p.R778L, p.P992L and p.T935M of the ATP7B gene, as shown in the following table:

[0115]

[0116]

[0117] (3) Configure the NEAR pre-reaction solution (24 μL); wherein the 10X reaction buffer is: 20 mM MgSO4, 1500 mM KCl, 100 mM (NH4)2SO4, 500 mM NaCl, 400 mM Tris-Cl (pH 8.8)

[0118] Component Amount (to be added) 10X Reaction Buffer 2.5 μL 5X Primer MIX 5 μL dNTP (10 mM) 2.5 μL DMSO 0.5 μL BSA (10 μg / μL) 0.5 μL DNA Template (10 ng / μL) 2.5 μL Make up to with double-distilled water 24 μL

[0119] (4) Treat the reaction solution prepared in step (3) at 95 °C for 5 min, quickly place it on ice, and add nicking enzyme and polymerase according to the following amounts after cooling

[0120] Nt.Bst NBI Nicking Enzyme (10 U / μL) 0.2 μL Nt.BstNBI-Bst 3.0 Polymerase (8 U / μL) 0.8 μL

[0121] (5) After the above reaction configuration is completed, place it in a metal bath with the temperature controlled at 58°C and perform isothermal amplification for 25 min;

[0122] (6) Detect the amplification products by capillary electrophoresis: Prepare a loading mixture mixed with a molecular weight internal standard and formamide: (0.5 μL molecular weight internal standard + 8.5 μL formamide) × the number of test samples, vortex for 1 - 15 seconds, use a pipette to dispense 9 μL of the formamide and internal standard mixture into each detection well, take 1 μL of the amplification product and add it to the loading mixture of the molecular weight internal standard and formamide, and perform electrophoresis detection according to the steps in the user manual of the genetic analyzer;

[0123] (7) Data analysis: Import relevant files into the GeneMapper software, import the original data detected by the genetic analyzer, and analyze the data;

[0124] (8) Judgment of detection: Based on whether specific amplification peak spectra appear in the wild-type and mutant peak maps at each detection site, judge the genotype of the sample at this site:

[0125] 1) The wild-type peak at the p.R778L site is 146 bp, and the mutant peak is 148 bp. When only a specific amplification peak appears at the position of 146 bp in the detection graph and there is no specific amplification peak at 148 bp, it can be judged that the site of the test sample is homozygous wild-type; when specific amplification peaks appear at both 146 bp and 148 bp in the detection graph, it can be judged that the site of the test sample is heterozygous mutant; when only a specific amplification peak appears at the position of 148 bp in the detection graph and there is no detection of a specific amplification peak at 146 bp, it can be judged that the site of the test sample is homozygous mutant;

[0126] 2) The wild-type peak at the p.P992L site is 87 bp, and the mutant peak is 89 bp. When only a specific amplification peak appears at the position of 87 bp in the detection graph and there is no specific amplification peak at 89 bp, it can be judged that the site of the test sample is homozygous wild-type; when specific amplification peaks appear at both 87 bp and 898 bp in the detection graph, it can be judged that the site of the test sample is heterozygous mutant; when only a specific amplification peak appears at the position of 89 bp in the detection graph and there is no detection of a specific amplification peak at 87 bp, it can be judged that the site of the test sample is homozygous mutant;

[0127] 3) The wild-type peak at the p.T935M locus is 153 bp, and the mutant peak is 155 bp. When only a specific amplification peak appears at the 153 bp position in the detection graph and no specific amplification peak appears at 155 bp, it can be determined that this locus of the detected sample is homozygous wild-type; when specific amplification peaks appear at both 153 bp and 155 bp in the detection graph, it can be determined that this locus of the detected sample is heterozygous mutant; when only a specific amplification peak appears at the 155 bp position in the detection graph and no specific amplification peak is detected at 153 bp, it can be determined that this locus of the detected sample is homozygous mutant.

[0128] In this application, when "about" is used to modify a numerical value, it means that the numerical value can fluctuate within the range of ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% or ±1%.

[0129] Unless otherwise specified in this application or in obvious contradiction to the context, the terms "a", "an", "the", "said", "this" and "at least one" and similar references used in the context of describing this application (including the context of the claims) are construed to cover both the singular and the plural. Unless otherwise specified in this application or in obvious contradiction to the context, the terms "comprising", "having", "including" and "containing" used in this application are construed as open-ended terms (i.e., "including but not limited to"). Unless otherwise specified in this application or in obvious contradiction to the context, all the methods described in this application can be carried out in any suitable order according to the understanding of those skilled in the art.

[0130] All patents, patent applications and references cited in this application are incorporated herein by reference in their entirety, to the same extent as if each document were individually cited as a reference. If there is a conflict between this application and the documents provided herein, the content of this application shall prevail. Brief Description of the Drawings

[0131] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for description in the embodiments. Obviously, the drawings described below are only some embodiments of this application. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0132] Figure 1 It is a detection graph of a homozygous control plasmid, showing the detection results that the three loci of p.R778L, p.P992L, and p.T935M are all homozygous wild-type according to certain embodiments of the present invention;

[0133] Figure 2It is a detection graph of the heterozygous control plasmid, showing the detection results of the heterozygous mutant type at the three loci of p.R778L, p.P992L, and p.T935M according to certain embodiments of the present invention;

[0134] Figure 3 It is a detection graph of the homozygous mutant plasmid, showing the detection results of the homozygous mutant type at the three loci of p.R778L, p.P992L, and p.T935M according to certain embodiments of the present invention;

[0135] Figure 4 It shows the detection result graph of clinical sample 1 (a sample with wild type at all three loci) according to certain embodiments of the present invention;

[0136] Figure 5 It shows the detection result graph of clinical sample 2 (heterozygous mutation at p.R778L and p.P992L, wild type at p.T935M) according to certain embodiments of the present invention;

[0137] Figure 6 It shows the detection result graph of clinical sample 3 (homozygous mutation at p.R778L, wild type at p.P992L and p.T935M) according to certain embodiments of the present invention;

[0138] Figure 7 It shows the detection result graph of clinical sample 4 (wild type at p.R778 and p.P992L, heterozygous mutant type at p.T935M) according to certain embodiments of the present invention; and

[0139] Figure 8 It shows the detection result graph of clinical sample 5 (wild type at p.R778, heterozygous mutant type at p.P992L, wild type mutation at p.T935M) according to certain embodiments of the present invention. Specific embodiments

[0140] The present invention will be further clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all embodiments, and the protection scope of the present invention is not limited thereto.

[0141] Example 1

[0142] Five samples (sample sources are clinical test samples from the Second Affiliated Hospital of Zhejiang University) with the three loci of p.R778L, p.P992L, and p.T935M of the ATP7B gene typed by the gold standard method (Sanger sequencing method) and three plasmid controls (wild, heterozygous, homozygous mutant) were detected using the present invention.

[0143] Among them, the typing results of the three loci of the five samples are as follows:

[0144] p.R778L p.P992L p.T935M Sample 1 Wild type Wild type Wild type Sample 2 Heterozygous mutant Heterozygous mutant ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

[0145] 1. Primer design

[0146] Specific primers were designed based on the mutant sequences of the three sites p.R778L, p.P992L, and p.T935M of the ATP7B gene or their complementary sequences. According to the ARMS primer design principle, a base mismatch was introduced at the -1 or -2 position at the 3' end of the specific primers to ensure the genotyping effect. After the design of the specific primer ends was completed, a recognition site for a nicking endonuclease was introduced at the 5' end, and about 10 - 14 bp of protective bases were added at the 5' end of the recognition site to ensure that the nicking endonuclease could perform enzymatic digestion quickly and efficiently.

[0147] 2. Sample DNA extraction:

[0148] The EDTA-anticoagulated whole blood of 5 samples with known genotyping results was provided by the Second Affiliated Hospital of Zhejiang University School of Medicine, and 2 ml of EDTA-anticoagulated blood was taken from each sample. The genomic DNA in the blood was extracted using the Qiagen DNeasy Blood Tissue Kit (Qiagen, Germany, catalog number 69506). After measuring the concentration with Nanodrop, it was uniformly diluted to 10 ng / μL for later use.

[0149] 3. Prepare 5X primer MIX for the detection of gene mutations at the three sites p.R778L, p.P992L, and p.T935M of the ATP7B gene, as shown in the following table:

[0150] ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

[0151] 4. Prepare 5 tubes of NEAR reaction pre-liquid; among them, the 10X reaction buffer is: 20 mM MgSO4, 1500 mM KCl, 100 mM (NH4)2SO4, 500 mM NaCl, 400 mM Tris-Cl (pH 8.8)

[0152] ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

[0153] 5. Treat the reaction solution prepared in step (3) at 95°C for 5 min, quickly place it on ice, and after cooling, add the nicking enzyme and polymerase according to the amounts in the following table

[0154] ​ ​ ​ ​

[0155] 6. After the above reaction is configured, place it in a metal bath with a temperature controlled at 58°C and perform constant temperature amplification for 25 minutes

[0156] 7. Detection of the amplification product by capillary electrophoresis: Prepare a loading mixture containing a molecular weight internal standard and formamide: (0.5 μL of molecular weight internal standard + 8.5 μL of formamide) × the number of test samples, vortex for 1 - 15 seconds, and use a pipette to dispense 9 μL of the formamide and internal standard mixture into each test well. Take 1 μL of the amplification product and add it to the loading mixture of the molecular weight internal standard and formamide, and perform electrophoresis detection according to the steps in the user manual of the genetic analyzer.

[0157] 8. Data analysis: Import relevant files into the GeneMapper software, import the original data detected by the genetic analyzer, and analyze the data.

[0158] 9. Results: Determine the genotype of the sample at each detection site based on whether specific amplification peak spectra appear in the wild-type and mutant peak patterns. As ​ shown, the detection result diagram of the pure wild-type plasmid; ​ shown, the detection result of the heterozygous plasmid control. Specific peaks appeared at the positions of the wild-type peak and the mutant peak for the p.R778L locus, p.P992L, and p.T935M. ​ shown, the detection result of the homozygous mutant plasmid control: Peaks appeared only at the mutant peak position for p.R778L, p.P992L, and p.T935M. ​ shown, the detection result diagram of sample 1. Peaks appeared only at the wild-type peak position for p.R778L, p.P992L, and p.T935M, indicating that all three loci of this sample are wild-type, which is consistent with the sequencing results. ​ shown, the detection result diagram of sample 2. Specific peaks appeared at the positions of the wild-type peak and the mutant peak for the p.R778L and p.P992L loci, and a specific peak appeared only at the wild-type peak position for p.T935M. ​ shown, the detection result diagram of sample 4. A specific peak appeared only at the mutant peak position for the p.R778L locus, and specific peaks appeared only at the wild-type peak position for p.P992L and p.T935M. ​ shown, the detection result diagram of sample 4. Specific peaks appeared only at the wild-type peak position for the p.R778L and p.P992L loci, and specific peaks appeared at the positions of the wild-type peak and the mutant peak for the p.T935M locus. ​ shown, the detection result diagram of sample 5. Specific peaks appeared only at the wild-type peak position for the p.R778L and p.T935M loci, and specific peaks appeared at the positions of the wild-type peak and the mutant peak for the p.P992L locus.

[0159] The innovation of the present invention lies in the constant-temperature multiplex rapid detection, which reduces the demand for amplification instruments, shortens the detection time, and saves the detection cost. It has great significance in clinical gene typing detection, especially in scenarios such as single-gene genetic diseases, pharmacogenomics, and microbial drug resistance, and has important value.

[0160] Example 2

[0161] In order to verify the universality of the method of the present invention, the inventor conducted the following verification experiments:

[0162] Twenty patient and control samples (already verified by Sanger sequencing) from the Second Affiliated Hospital of Zhejiang University School of Medicine were used as the samples to be tested, and the experimental methods and steps, as well as the analysis and determination conditions, were the same as those in Example 1 for detection. The results are as follows:

[0163] The results obtained by the method of the present invention are as follows: 8 samples are wild-type at all 3 detected loci; 5 samples are heterozygous mutations at the p.R778L locus, and the p.P992L and p.T935M loci are wild-type; 3 samples are double-site heterozygous mutations at the R778Lp. and T935M loci, and the p.T935M is wild-type; 2 samples are heterozygous mutations at the p.P992L locus, and the p.R778L and p.T935M loci are wild-type, 1 sample is double-site heterozygous mutations at the p.R778L and p.T935M loci, and the p.P992L is wild-type, and there is also 1 sample that is a homozygous mutant at the p.R778L locus, and the p.P992L and p.T935M loci are wild-type.

[0164] The results obtained in this experiment are completely consistent with the results obtained by detecting according to the gold standard method. Therefore, the results of this example further illustrate the accuracy and stability of the detection results obtained by the method of the present invention.

[0165] Comparative Example 1

[0166] According to the NEAR isothermal amplification primer design principle, upstream and downstream primer sets for the p.R778L locus, upstream and downstream primer sets for the p.P992L locus, and upstream and downstream primer sets for the p.T935M locus were designed. Each primer also contains an identification site for a restriction site (5'-GAGTCNNNN-3', such as Nt.BstNBI), a stable region, and a nucleic acid strand of a target-binding region complementary to the target. However, the upstream primer for amplification at each locus is simply exactly the same as the target detection locus, and the primer design for each locus is as follows:

[0167] Among them, the upstream and downstream primer sequences for detecting the p.R778L locus are: (146-148bp)

[0168] Wild-type upstream internal primer (SEQ ID NO.10):

[0169] 5’-AACCTGAAGC ​ CTTTGCCAAGTGTTCCAGCCACC-3’

[0170] Mutant upstream internal primer (SEQ ID NO.11):

[0171] 5’-AACCTGAAGC ​ ACCTTTGCCAAGTGTTCCAGCCACA-3’

[0172] Downstream common internal primer (SEQ ID NO.12):

[0173] 5’-GTGCTCATCG ​ CAAGCA(FAM-dT)TGCTTATGTTTATTCT-3

[0174] The upstream and downstream primer sequences for detecting the p.P992L site are: (87-89bp)

[0175] Wild-type upstream internal primer (SEQ ID NO.13):

[0176] 5’-TTCTGCGCGG ​ TGCCCACCATGACAGCCGTGG-3’

[0177] Mutant upstream internal primer (SEQ ID NO.14):

[0178] 5’-TTCTGCGCGG ​ GGTGCCCACCATGACAGCCGTGA-3’

[0179] Common downstream primer (SEQ ID NO.15):

[0180] 5’-CAGACAGAGG ​ GG(FAM-dT)TTGCTTTCCAGACGTCCATC-3’

[0181] The upstream and downstream primer sequences for detecting the p.T935M site are: (153-155bp)

[0182] Wild-type upstream primer (SEQ ID NO.16):

[0183] 5’-ACCAAAATCG ​ ATTACAATCCATACCACCAACG-3’

[0184] Mutant upstream primer (SEQ ID NO.17):

[0185] 5’-ACCAAAATCG ​ CGATTACAATCCATACCACCAACG-3’

[0186] Common downstream primer (SEQ ID NO.18):

[0187] 5’-TTAGAAATAGG ​ GTG(FAM-dT)TTTATTTCTTCATAGG-3’

[0188] That is, other modifications and primer sequences are the same as those in Example 1, except that a base mismatch is no longer introduced at the -2 or -3 position at the 3' end of the specificity, and experiments are carried out.

[0189] The above primers fully meet the requirements for the design of NEAR isothermal amplification primers, that is, each primer contains a recognition site for a restriction site (5'-GAGTCNNNN-3', such as Nt.BstNBI), a stable region, and a nucleic acid strand of a target-binding region complementary to the target. Using this primer set, 5 known samples were detected according to the method described in Example 1, and the results are shown in the following table:

[0190] ​ ​ ​ Sample 1 Heterozygous mutant Heterozygous mutant Heterozygous mutant Sample 2 Heterozygous mutant Heterozygous mutant Heterozygous mutant Sample 3 Homozygous mutant Heterozygous mutant Heterozygous mutant Sample 4 Heterozygous mutant Heterozygous mutant Heterozygous mutant Sample 5 Heterozygous mutant Heterozygous mutant Heterozygous mutant

[0191] When detecting with the above primers, there was a situation where the detection results were inconsistent with the gold standard method. Each site of each sample was detected as a heterozygous mutant type, indicating that this primer does not have the ability of SNP genotyping. Therefore, if only the NEAR isothermal amplification method is simply used to detect samples, the genotyping of 3 high-frequency mutation sites of the ATP7B gene cannot be stably achieved.

[0192] Comparative Example 2

[0193] Using the currently relatively mature multiplex SNP genotyping method, that is, the method of combining ARMS-PCR and capillary electrophoresis detection, the samples in Example 1 were detected. The specific experimental steps are as follows:

[0194] (1) Design ARMS-PCR amplification primers. According to the ARMS-PCR primer design principle, we designed upstream and downstream primer sets for the p.R778L site, upstream and downstream primer sets for the p.P992L site, and upstream and downstream primer sets for the p.T935M site. The primer design for each site is as follows:

[0195] Among them, the upstream and downstream primer sequences for detecting the p.R778L site are: (146 - 148bp)

[0196] Wild-type upstream internal primer (SEQ ID NO.19): 5’-CTTTGCCAAGTGTTCCAGCCTCC-3’

[0197] Mutant upstream internal primer (SEQ ID NO.20): 5’-ACCTTTGCCAAGTGTTCCAGCCAGA-3’

[0198] Downstream common internal primer (SEQ ID NO.21): 5’-FAM-CAAGCATGCTTATGTTTATTCT-3’

[0199] The upstream and downstream primer sequences for detecting the p.P992L site are: (87-89bp)

[0200] Wild-type upstream internal primer (SEQ ID NO.22): 5’-TGCCCACCATGACAGCCGAGG-3’

[0201] Mutant upstream internal primer (SEQ ID NO.23): 5’-GGTGCCCACCATGACAGCCGTTA-3’

[0202] Common downstream primer (SEQ ID NO.24): 5’-FAM-GGTTGCTTTCCAGACGTCCATC-3’

[0203] The upstream and downstream primer sequences for detecting the p.T935M site are: (153-155bp)

[0204] Wild-type upstream primer (SEQ ID NO.25): 5’-ATTACAATCCATACCACCACCG-3’

[0205] Mutant upstream primer (SEQ ID NO.26): 5’-CGATTACAATCCATACCACCAAAG-3’

[0206] Common downstream primer (SEQ ID NO.27): 5’-FAM-GTG(TTTATTTCTTCATAGGT-3’

[0207] That is, the primer no longer introduces the NEAR isothermal digestion site sequence, and only the specific detection primers for amplification of each site are designed.

[0208] (2) Configure the PCR amplification system

[0209] Component Added amount 2×PCR amplification enzyme mix 5 μL p.R778L wild-type upstream primer (10 μM) 0.2 μL p.R778L mutant upstream primer (10 μM) 0.2 μL p.R778L common downstream primer (10 μM) 0.4 μL p.P992L wild-type upstream primer (10 μM) 0.2 μL p.P992L mutant upstream primer (10 μM) 0.2 μL p.P992L common downstream primer (10 μM) 0.5 μL p.T935M wild-type upstream primer (10 μM) 0.2 μL p.T935M mutant upstream primer (10 μM) 0.2 μL p.T935M common downstream primer (10 μM) 0.4 μL Deionized water 1.6 μL DNA (template) 1 μL

[0210] (3) PCR amplification

[0211] Using a PCR instrument, perform the following amplification procedure:

[0212]

[0213] (4) Detect the amplified product by capillary electrophoresis: Perform the electrophoresis detection steps as in Example 1 and perform data analysis. Using this primer set and amplification method to detect the 5 known samples in Example 1, the obtained results are shown in the following table:

[0214]

[0215]

[0216] That is, it is completely consistent with the detection results obtained in Example 1, and is also completely consistent with the detection results by the Sanger sequencing gold standard method.

[0217] It shows that the detection method in Example 1 is consistent with the detection method in Comparative Example 2 in terms of accuracy; however, comparing the entire experimental processes of Comparative Example 1 and Comparative Example 2, in Comparative Example 2, because there is a step of cycling temperature change, a relatively expensive PCR instrument is required, and the PCR amplification process takes 90 minutes, while Example 1 only takes 25 minutes to complete the isothermal amplification process.

[0218] Through this comparative experiment, it further demonstrates that the method of the present invention can achieve isothermal multiplex rapid detection, reduce the requirement for amplification instruments, shorten the detection time, and save the detection cost.

[0219] The above are only the preferred embodiments of the present application and are not used to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A primer combination for genes at high-frequency mutation sites of the ATP7B gene in Wilson's disease, characterized in that, The primer combination can simultaneously amplify the following 3 mutation sites in the same PCR system: p.R778L site; p.P992L site; and p.T935M site; Among them, the primer combination includes a wild-type upstream primer, a mutant upstream primer, and a common downstream primer for each site; the primer combination sequence includes SEQ ID NO. 1-9; The common downstream primer is labeled with a fluorescent group recognized for later capillary electrophoresis detection, and the fluorescent group is labeled on a T base at 3-8 bases at the 3' end of the specific primer segment of the common downstream primer for each detection site, away from the recognition site of the nicking endonuclease.

2. The primer combination according to claim 1, wherein The fluorescent group can be modified with FAM, HEX, ROX, TAMER, or CY5.

3. The primer combination according to claim 2, wherein In the primer combination, the amount of the upstream primer is 4 times that of the downstream primer.

4. A kit for genes of high-frequency mutation sites of the ATP7B gene in Wilson's disease, characterized in that, The kit includes the primer combination according to any one of claims 1-3, the NERA isothermal multiplex amplification enzyme mixture, 10X reaction buffer, 10 mM dNTP, positive control, and internal standard required for capillary electrophoresis.

5. The kit according to claim 4, characterized in that, The NERA isothermal multiplex amplification enzyme mixture includes a nicking endonuclease and a DNA polymerase, where the nicking endonuclease is Nt.BstNBI; the DNA polymerase is selected from Bst DNA polymerase, Klenow DNA polymerase, Vent DNA polymerase, or phi29 DNA polymerase.

6. The kit according to claim 5, wherein The 10X reaction buffer is: 20 mM MgSO4, 1500 mM KCl, 100 mM (NH4)2SO4, 500 mM NaCl, 400 mM pH 8.8 Tris-HCl.

7. Use of the primer combination according to any one of claims 1-3 in the preparation of a constant temperature multiplex amplification detection system for genes at high-frequency mutation sites of the ATP7B gene in Wilson's disease, characterized in that, The isothermal multiplex amplification detection system is: Among them, The 10X reaction buffer is: A mixture of 20 mM MgSO4, 1500 mM KCl, 100 mM (NH4)2SO4, 500 mM NaCl, 400 mM pH 8.8 Tris-HCl; The above detection system is treated at 95 °C for 5 min, quickly placed on ice, and after cooling, the nicking enzyme and polymerase are added to each tube of the reaction solution according to the amounts in the following table, as follows: Among them, the nicking endonuclease is Nt.BstNBI; The DNA polymerase is selected from Bst DNA polymerase, Klenow DNA polymerase, Vent DNA polymerase, or phi29 DNA polymerase.

8. The constant temperature multiplex amplification detection system according to claim 7, wherein The DNA polymerase is selected from Bst 3.0 polymerase. Use of the primer combination according to any one of claims 1-3 or the isothermal multiplex amplification detection system according to claim 7 or 8 in the preparation of a reagent for genotyping of high-frequency mutation sites of the ATP7B gene.

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