CN115216533A - Biomarker for diagnosing Wilson's disease, amplification primer set, detection reagent and application - Google Patents
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Abstract
The invention belongs to the technical field of medical diagnosis, and particularly relates to a biomarker, an amplification primer set, a detection reagent and application for diagnosing Wilson's disease. The biomarker is an ATP7B mutant gene, and mutation sites comprise c.2384T > C and c.4005delG. The ATP7B mutant gene can cause the pathogenesis of Wilson's disease, enriches the pathogenic mutation spectrum of Wilson's disease, can screen or diagnose pathogenic gene mutation carriers or patients of Wilson's disease by detecting whether a subject carries the mutation so as to provide prenatal and postnatal care and therapeutic intervention guidance, provide a brand-new theoretical basis for the treatment of Wilson's disease, and provide possible drug targets for the treatment of Wilson's disease.
Description
Technical Field
The invention belongs to the technical field of medical diagnosis, and particularly relates to a biomarker, an amplification primer set, a detection reagent and application for diagnosing Wilson's disease.
Background
A gene mutation is a change in the base pair composition or arrangement of a gene in its structure. Depending on the base change, gene mutations can be generally classified into base substitution mutations, frameshift mutations, insertion mutations, deletion mutations, and the like. Wherein a base substitution refers to a mutation in a DNA molecule caused by the substitution of one base pair with a different base pair, also known as a point mutation. Point mutations are divided into two forms, transition and transversion. A mutation in a purine or pyrimidine substituted for a purine is called a transversion if one purine is substituted for another purine or one pyrimidine is substituted for another pyrimidine. Mutations can be classified into synonymous, missense, nonsense, and stop codon mutations according to their effect on amino acids of the peptide chain of a protein. In which missense mutation changes a certain codon of mRNA into a codon encoding another amino acid by base pair substitution, and the change of a single amino acid may cause structural and functional abnormalities of the protein or enzyme in the body, thereby causing diseases. A change in the structure of a gene due to a base pair deletion in a DNA molecule is called a deletion mutation, and a deletion mutation often causes a codon encoding one amino acid to be changed into a codon encoding another amino acid after the base deletion, thereby changing the amino acid type and sequence of a polypeptide chain. The compound heterozygous mutation refers to heterozygous mutation with 1 or more alleles, namely biallelic mutation, and mutation on each chromosome of a paternal source and a maternal source.
Wilson disease (Wilson disease) also called hepatolenticular degeneration (MIM 277900) is an autosomal recessive inherited copper metabolic disorder disease characterized by a brain degenerative disease mainly including liver cirrhosis and basal ganglia damage caused by copper metabolic disorder, and clinically characterized by different degrees of liver cell damage, brain degenerative changes and copper hyperpigmented rings at the corneal limbus. The worldwide incidence rate is 1/30000-1/100000, and the carrier of the pathogenic gene is about 1/90. It is better in adolescents, and more men than women will cause disability and even death if improper treatment is carried out. The disease is also one of a few of treatable neurogenetic diseases, and the key points are early discovery, early diagnosis and early treatment.
The hepatolenticular degeneration disease gene ATP7B (MIM 606882) is positioned on chromosome 13q14.3, the gene has the full length of 79.5kb, comprises 21 exons and 20 introns, codes a copper transport P-type ATPase consisting of 1466 amino acids, participates in transmembrane transport of copper, and the ATP7B protein transports copper to an inverse Golgi network and is combined with a ceruloplasmin precursor to form functional whole ceruloplasmin to enter blood; on the other hand, transport copper to bile for excretion. The ATP7B protein is mainly expressed in the liver, when ATP7B gene mutation causes dysfunction of transport of the ATP7B protein to copper, copper is excessively deposited in the liver, so that oxidative stress reaction of mitochondria of liver cells is caused, molecules such as lipid, protein, DNA and RNA are damaged, and liver cell damage and liver steatosis are caused; copper also activates hepatic stellate cells, accelerating the progression of hepatic fibrosis. When the copper exceeds the storage capacity of the liver, the copper enters the blood in the form of free copper and is excessively deposited at the brain, the kidney, the cornea, the joint, the intestinal tract and other parts, so that copper toxicity outside the liver is generated, and corresponding clinical manifestations are caused.
The human gene database has published hundreds of ATP7B gene mutation sites, most of which have clear pathogenic roles in the pathogenesis of hepatolenticular degeneration. The most common mutation in the European hepatolenticular degeneration patient population is p.His1069Gln, and the mutation frequency is 13-61%; the common mutation of Asian population is p.Arg778Leu, and the mutation frequency is 34-38%. The hepatolenticular degeneration patient in China has 3 high-frequency pathogenic mutations of p.Arg778Leu, p.Pro992Leu and p.Thr935Met, which account for 50-60% of all pathogenic mutations; also relatively common pathogenic mutations are p.Ala874Val, p.Ile1148Thr, p.Gly943Asp, p.Gln511X, p.Arg919Gly, p.Asn1270Ser, p.Arg778Gln, and the like. The gene mutation is mainly missense mutation, mainly comprises homozygous mutation and compound heterozygous mutation, and only a small number of patients find single heterozygous mutation. For patients with atypical and highly suspected clinical manifestations, hot-spot mutation detection of the ATP7B gene can be performed first, and the full-length coding region of the ATP7B gene and the flanking sequences thereof should be screened by a person without positive discoverer. Gene mutation is an important genetic basis for the development of diseases, and gene diagnosis is an important genetic standard for determining the diagnosis of Wilson's disease. Therefore, the search for gene diagnosis markers, early and definite patient diagnosis and targeted intervention are particularly important in the field of wilson disease detection at present.
Disclosure of Invention
The invention aims to provide a biomarker, an amplification primer set, a detection reagent and application for diagnosing Wilson's disease, enrich the pathogenic mutation spectrum of Wilson's disease, accurately judge Wilson's disease, be used for genetic diagnosis of Wilson's disease to guide treatment, and be used for genetic diagnosis and prenatal and postnatal care before embryo implantation.
The invention provides a biomarker for diagnosing Wilson's disease, wherein the biomarker is an ATP7B mutant gene, and the mutation sites are c.2384T > C and c.4005delG. The invention discovers compound mutation of an ATP7B gene by exome sequencing technology, wherein the 2384 base of the ATP7B gene is mutated from T to G, and the 4005 base G is deleted, which affects important functions of protein, and specifically comprises ATP7B: NM-000053.4. The invention discovers for the first time that the complex mutation of ATP7B gene c.2384T > C and c.4005delG can cause the pathogenesis of Wilson disease, and the result of detecting the mutation by uninjured members in families is negative. The ATP7B mutant gene enriches the pathogenic mutation spectrum of Wilson's disease, and on one hand, by detecting whether a subject carries the mutation, the pathogenic gene mutation carrier or patient of Wilson's disease can be screened or diagnosed to provide prenatal and postnatal care and therapeutic intervention guidance, so that a brand-new theoretical basis is provided for the treatment of Wilson's disease, and possible drug targets can be provided for the treatment of Wilson's disease.
The reagent and/or the kit provided by the invention can be used for quickly and effectively predicting or diagnosing Wilson's disease.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below.
FIG. 1 is a genetic map of Wilson's disease line No.1, in which,
it is indicated that the male carrier is,
denotes a female carrier ● denotes a female patient, o denotes a fetus, ↗ denotes a proband;
FIG. 2 is a graph of the results of the determination of the genotype of ATP7B: NM-000053.4; c is a family Wilson's disease patient (the arrow in the sequencing map indicates the site of mutation);
FIG. 3 is a genetic map of Wilson's disease pedigree No. 2; wherein,
it is indicated that the male carrier is,
denotes a female carrier, ■ denotes a male patient, o denotes a fetus, ↗ denotes a proband;
FIG. 4 is a graph showing the results of using the kit to detect the genotype of ATP7B: NM-000053.4 exon 9; b is wild type (arrow in sequencing map indicates mutation position);
FIG. 5 is a graph showing the results of using the kit to detect the genotype at the ATP7B: NM-000053.4 site (ex on 19); a is wild type (arrow in the sequencing diagram indicates the site where the mutation occurs).
Detailed Description
The invention provides a biomarker for diagnosing Wilson's disease, wherein the biomarker is an ATP7B mutant gene, and the mutation sites are c.2384T > C and c.4005delG.
According to the invention, exon sequencing is utilized to screen pathogenic gene mutation highly related to Wilson's disease, in order to avoid the occurrence of false positive results, sanger sequencing is used for verification, and finally, an ATP7B mutant gene for diagnosing Wilson's disease is obtained, wherein the mutation sites are c.2384T > C and c.4005delG. The ATP7B mutant gene is a compound heterozygous mutation, wherein the 2384 base of the ATP7B gene is mutated from T to G, and the 4005 base G is deleted, in particular to ATP7B: NM-000053.4 Exon 9. The mutation site c.2384T > C is from the allele on the parent 13 chromosome, and the mutation site c.4005delG is from the allele on the parent 13 chromosome. The ATP7B mutant gene provided by the invention can distinguish Wilson disease patients from normal people, is a biomarker for diagnosing Wilson disease, is beneficial to screening and diagnosing Wilson disease gene mutation, and provides new technical support for drug screening, drug effect evaluation and targeted therapy.
According to the prominent effect of the ATP7B mutant gene on Wilson's disease, the following contents are included in the protection scope of the invention: the ATP7B mutant gene is used as a detection target to prepare a reagent for diagnosing Wilson's disease; the ATP7B mutant gene is used as a detection target to prepare a kit for preventing Wilson's disease; the ATP7B mutant gene is used as a detection target to prepare a kit for detecting Wilson's disease; the ATP7B mutant gene is used as a detection target to prepare a kit for diagnosing Wilson's disease; the ATP7B mutant gene is used as a detection target to prepare a kit for adjuvant therapy of Wilson's disease; the ATP7B mutant gene is used as a detection target to prepare a pre-pregnancy and/or prenatal genetic disease screening and/or diagnosis kit.
The invention also provides a primer group for amplifying the ATP7B mutant gene in the technical scheme, which comprises a primer group 1 and a primer group 2; the primer group 1 comprises an upstream primer ATP7B-1F and a downstream primer ATP7B-1R; the primer group 2 comprises an upstream primer ATP7B-2F and a downstream primer ATP7B-2R.
The upstream primer ATP7B-1F of the invention preferably comprises any one of a 1) to a 4): a1 Single-stranded DNA described by SEQ ID NO.1 of the sequence Listing; a2 A single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or the 3' end of a 1); a3 A single-stranded DNA having an identity of 85% or more to the single-stranded DNA defined in a 1) or a 2); a4 A single-stranded DNA which hybridizes with the single-stranded DNA defined in a 1) or a 2) under stringent conditions. The downstream primer ATP7B-1R of the invention preferably comprises any one of B1) to B4): b1 Single-stranded DNA described by SEQ ID NO.2 of the sequence Listing; b2 A single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of b 1); b3 A single-stranded DNA having an identity of 85% or more to the single-stranded DNA defined in b 1) or b 2); b4 A single-stranded DNA which hybridizes under stringent conditions with the single-stranded DNA defined under b 1) or b 2). The upstream primer ATP7B-2F of the invention preferably comprises any one of the following primers c 1) to c 4): c1 Single-stranded DNA described by SEQ ID NO.31 of the sequence Listing; c2 A single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of c 1); c3 A single-stranded DNA having an identity of 85% or more to the single-stranded DNA defined in c 1) or c 2); c4 A single-stranded DNA which hybridizes with the single-stranded DNA defined in c 1) or c 2) under stringent conditions. The downstream primer ATP7B-2R of the invention preferably comprises any one of d 1) to d 4): d1 Single-stranded DNA described by SEQ ID NO.32 of the sequence Listing; d2 A single-stranded DNA obtained by adding one or several nucleotides to the 5 '-end and/or the 3' -end of d 1); d3 A single-stranded DNA having an identity of 85% or more to the single-stranded DNA defined in d 1) or d 2); d4 A single-stranded DNA which hybridizes with the single-stranded DNA defined in d 1) or d 2) under stringent conditions.
The nucleotide sequences of SEQ ID NO. 1-4 of the invention are as follows:
SEQ ID NO.1：5’-ATCCATACATCCTCCTC-3’；
SEQ ID NO.2：5’-AATCATCTACACCTCCC-3’；
SEQ ID NO.31：5’-GAATAAAGGGAAGAAAGTCG-3’；
SEQ ID NO.32：5’-ATGCCTGGTCAAAGAGCC-3’。
the invention also provides application of the primer group in the technical scheme in preparation of a kit for preventing and/or diagnosing Wilson's disease; the application in the preparation of a kit for the adjuvant therapy of Wilson's disease; the application in the preparation of a pre-pregnancy and/or prenatal genetic disease screening and/or diagnosis kit. The primer group can detect whether c.2384T > C and c.4005delG mutant sites exist on the ATP7B gene or not, and specifically, the primer group 1 specifically amplifies the ATP7B gene containing the c.2384T > C mutant sites, and the primer group 2 specifically amplifies the ATP7B gene containing the c.4005delG mutant sites. The primer group disclosed by the invention can be used for quickly and accurately diagnosing the Wilson disease.
The invention also provides a reagent for diagnosing Wilson's disease, which comprises the primer and the sequencing primer group in the technical scheme.
In the present invention, the sequencing primer set preferably includes sequencing primer sets 1 and 2; the sequencing primer group 1 preferably comprises an upstream primer ATP7B-Seq1F and a downstream primer ATP7B-Seq1R; the sequencing primer set 2 preferably comprises an upstream primer ATP7B-Seq2F and a downstream primer ATP7B-Seq2R.
The upstream primer ATP7B-Seq1F of the invention preferably comprises any one of e 1) to e 4): e1 Single-stranded DNA described by SEQ ID NO.5 of the sequence Listing; e2 A single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of e 1); e3 A single-stranded DNA having an identity of 85% or more to the single-stranded DNA defined in e 1) or e 2); e4 A single-stranded DNA which hybridizes with the single-stranded DNA defined in e 1) or e 2) under stringent conditions. The downstream primer ATP7B-Seq1R of the invention preferably comprises any one of f 1) to f 4): f1 Single-stranded DNA described by SEQ ID NO.6 of the sequence Listing; f2 A single-stranded DNA obtained by adding one or several nucleotides to the 5 '-end and/or 3' -end of f 1); f3 A single-stranded DNA having an identity of 85% or more to the single-stranded DNA defined in f 1) or f 2); f4 A single-stranded DNA which hybridizes under stringent conditions with the single-stranded DNA defined under f 1) or f 2). The upstream primer ATP7B-Seq2F of the invention preferably comprises any one of g 1) to g 4): g1 Single-stranded DNA described by SEQ ID NO.7 of the sequence Listing; g2 A single-stranded DNA obtained by adding one or several nucleotides to the 5 '-end and/or 3' -end of g 1); g3 A single-stranded DNA having an identity of 85% or more to the single-stranded DNA defined in g 1) or g 2); g4 A single-stranded DNA which hybridizes with the single-stranded DNA defined in g 1) or g 2) under stringent conditions. The downstream primer ATP7B-Seq2R of the invention preferably comprises any one of the following components h 1) -h 4): h1 Single-stranded DNA described by SEQ ID NO.8 of the sequence Listing; h2 A single-stranded DNA obtained by adding one or more nucleotides to the 5 'end and/or the 3' end of h 1); h3 A single-stranded DNA having an identity of 85% or more to the single-stranded DNA defined in h 1) or h 2); h4 Single-stranded DNA which hybridizes under stringent conditions with the single-stranded DNA defined in h 1) or h 2).
The nucleotide sequence of SEQ ID NO. 61-64 is specifically as follows:
SEQ ID NO.61：5’-AACACCACGCTTGTGACTC-3’；
SEQ ID NO.62：5’-GTCAATACAACATGGGCATC-3’；
SEQ ID NO.63：5’-GCTGTGGGTGCTGGGAGG-3’；
SEQ ID NO.64：5’-GGAACCTGGGAGACAGAAGC-3’。
in the present invention, the reagent preferably further comprises other reagents in the PCR amplification reaction, including but not limited to dNTPs, PCR buffer, magnesium ions and Tap polymerase. In the specific implementation process of the invention, other reagents can be selected conventionally according to actual needs. The PCR buffer solution of the present invention preferably comprises 50mmol/L KCl, 10mmol/L Tris-HCl pH8.3, and 1.5mmol/L MgCl 2 。
The invention also provides the application of the reagent in the technical scheme in the preparation of a kit for preventing and/or diagnosing Wilson's disease; the application in the preparation of a kit for the adjuvant therapy of Wilson's disease; the application in the preparation of a pre-pregnancy and/or prenatal genetic disease screening and/or diagnosis kit. The sequencing primer in the reagent can sequence an amplification product of a primer group for amplifying the ATP7B mutant gene, so that whether c.2384T > C and c.4005delG mutant sites exist on the ATP7B gene or not is judged, and the Wilson disease is rapidly and accurately diagnosed.
The invention also provides a kit for diagnosing Wilson's disease, which comprises the reagent in the technical scheme. The kit diagnoses whether an individual has Wilson's disease by detecting the genotype of the mutation site in a sample, and particularly judges that a compound heterozygous mutation exists in a 7B gene and the individual is a patient when the genotype of the ATP7B: NM-000053.4; if the genotype of the two sites is 'C.2384T > C heterozygous mutation' or 'c.4005delG heterozygous mutation', judging that the ATP7B gene has single heterozygous mutation and the individual is a carrier; if the genotype of the locus is wild-type, i.e., there is no mutation, the individual is a normal human. The test sample of the present invention preferably comprises blood or amniotic fluid.
In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the molecular genetics, nucleic acid chemistry and molecular biology-related terms and laboratory procedures used herein are all terms and routine procedures used extensively in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.
The term "diagnosis" as used herein includes prediction of risk of disease, diagnosis of the presence or absence of disease, and assessment of prognosis of disease.
The term "mutation" as used herein refers to an alteration in the wild-type polynucleotide sequence, which refers to the addition, deletion and/or substitution of one or several (e.g., several) bases in the gene sequence or DNA sequence, into a variant, which may be naturally occurring or non-naturally occurring. The term "mutation" when used to describe a product or protein encoded by a gene refers to the addition, deletion and/or substitution of one or several (e.g., several) amino acid residues in the protein or encoded product.
In the present invention, the term "heterozygous mutation" means that the mutation is present in only one gene of a pair of alleles.
In the present invention, the term "compound heterozygous mutation" means a heterozygous mutation wherein 1 or more alleles are present, that is, a biallelic mutation, and each chromosome is mutated.
The term "prenatal diagnosis" refers to the definite diagnosis of high-risk fetus based on genetic counseling, mainly through genetic detection and imaging examination, and the purpose of fetus selection is achieved through the selective abortion of affected fetus, so that the birth defect rate is reduced, and the prenatal quality and the population quality are improved.
In the present invention, "primer" refers to a polynucleotide fragment, typically an oligonucleotide, for amplifying a target nucleic acid in a PCR reaction, e.g., a polynucleotide fragment containing at least 5 bases, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more bases. The primer does not have to be completely complementary to the gene of interest to be amplified or its complementary strand, as long as it can specifically amplify the gene of interest. As used herein in the context of the present invention,
the term "specifically amplify" means that the primers are capable of amplifying the gene of interest by PCR reaction without amplifying other genes. For example, specifically amplifying the ATP7B gene means that only the ATP7B gene is amplified by primers in a PCR reaction, and other genes are not amplified.
For further explanation of the present invention, the following detailed descriptions of the biomarker, amplification primer set, detection reagent and application for diagnosing wilson's disease provided by the present invention are provided with reference to the drawings and examples, but they should not be construed as limiting the scope of the present invention.
Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: conditions described in the laboratory Manual (New York: cold Spring Harbor laboratory Press, 1989), or according to the manufacturer's recommendations.
Example 1
1. Diagnostic criteria:
the diagnosis standard of Wilson's disease can be determined by referring to the "guide for diagnosis and treatment of hepatolenticular degeneration" 2022 edition, in which Leipzig scoring system is applied to diagnosis of hepatolenticular degeneration, the diagnosis can be confirmed when the total score is not less than 4, the diagnosis can be excluded when the total score is 3, and the diagnosis is not more than 2 (Table 1).
TABLE 1 diagnostic criteria of Laibtin 8th International conference on Wilson's disease in 2001 (Leipzig scoring System)
Note: a represents that liver copper can not be quantified in time; ULN is the upper limit of normal.
2. Detecting an object
A Wilson disease family (called as family 1 for short) is taken as a subject to be detected, the clinical information of partial members of the family 1 is shown in Table 2, the family map is shown in figure 1, wherein,
it is indicated that the male carrier is,
representing a female carrier, ● representing a female patient, o, representing a fetus, ↗ representing a proband.
TABLE 2 clinical information of family Member of Wilson's disease No.1
Note: i and II sequentially represent the first generation and the second generation, the peripheral blood DNA of family personnel No. 1I 1, I2 and II 1 is used for sequencing, and the amniotic fluid DNA II 2 is used for sequencing; the detection result of the patient with prochiral disease of family No.1 is 1379.52 mug/24 h (the reference value is 10-60 mug/24 h), the copper-orchid protein is 0.0774g/L (the reference value is 0.31-0.55 g/L), the glutamic-pyruvic transaminase is 103 mug/L (the reference value is 0-40 mug/L), the glutamic-oxaloacetic transaminase is 46 mug/L (the reference value is 5-34 mug/L), the liver echo enhancement of the B-ultrasound is coarse and super, and the detection result accords with the change of diffuse liver injury.
Example 2
Exon sequencing
1. The instrument equipment comprises: high throughput sequencer NextSeq500 (Illumina); a quatitfluorometer nucleic acid quantifier (Invitrogen); PCR instrument (Bio-RAD); centrifuge 5810R (Eppendorf); centrifuge 5424 (Eppendorf); 5418 small high speed centrifuge (Eppendorf); biological safety cabinets (sujing); super clean bench (sujing); an ice maker (Grant); UPS power supply (Santa); milliQ ultra pure water instruments (Millipore); high performance computers (including servers, cabinets, switches, storage, etc.) (DELLs); -25 degree refrigerator (meiling); ultra-low temperature refrigerators (Eppendorf); microwave oven (american).
2. Reagent consumable
Human whole exon sequencing kit (Agilent), DNA 1000 kit (Agilent), 96-well plate (Axygen), different model tips (Axygen), 200 μ L centrifuge tube (Eppendorf), 1.5mL centrifuge tube (Eppendorf), capillary electrophoresis buffer (Thermo), sequencing standards (Thermo), absolute ethanol (Thermo), bigDye Terminator V3.1 (Thermo), peripheral blood gDNA extraction kit (TIANGEN), agarose (TIANGEN), and EB stain (amereco).
3. Reagent formulation
1) 5 × TBE electrophoretic solution stock solution: 5.4g Tris, 750mg boric acid, 2mL EDTA (pH 8.0,0.5 mol/L), plus ddH 2 Make up to 100mL
2) Working solution of 0.5 XTBE electrophoresis solution, ddH 2 Diluting the 5 × TBE electrophoresis solution stock solution by 10 times with O.
3) 10 × erythrocyte lysate: 82.9g NH 4 Cl、10g KHCO 3 0.37g of EDTA, plus dH 2 Supplementing O to 1000mL, autoclaving, and storing at 4 deg.C
4) 1 × formulation of cell nucleus lysate: 0.5mL of 2M Tris-HCl, pH8.2, 10mL of 4M NaCl, and 0.4mL of 2mM EDTA.
4. Experimental procedure
After signing an informed consent, 3-5 mL of peripheral blood of the members I1, I2 and II 1 and 5-10 mL of amniotic fluid of the members III 2 in the family are collected as research samples.
4.1 sample DNA extraction
1) If the sample is a heparin anticoagulation peripheral blood sample, 3-5 mL of peripheral blood is put into a 15mL centrifuge tube, 1 Xerythrocyte lysate with 2-3 times volume is added, the mixture is uniformly mixed, and the mixture is kept stand on ice for 30 minutes until the solution becomes transparent; and if the amniotic fluid is adopted, directly performing the step 2).
2) Centrifuge at 3000rpm for 10 minutes at 4 ℃ and carefully remove the supernatant. The pellet was mixed with 1mL of 1 Xcell nucleus lysate, followed by addition of 2mL of 1 Xcell nucleus lysate and 150. Mu.L of 20% SDS, and the mixture was shaken until it became viscous and transparent. Add 10. Mu.L of 20mg/mL proteinase K and shake well. Digestion was carried out at 37 ℃ for more than 6 hours or overnight.
3) Add equal volume of saturated phenol, shake gently and mix well, centrifuge at 3000rpm for 10 minutes at room temperature.
4) The supernatant was carefully transferred to another centrifuge tube, mixed with an equal volume of phenol/chloroform (1:1) and centrifuged at 3000rpm for 10 minutes at room temperature.
5) The supernatant was carefully removed and, if it was not clear, extracted once more with an equal volume of chloroform.
6) The supernatant was transferred to another centrifuge tube, and two times the volume of absolute ethanol was added thereto, followed by shaking to obtain white flocculent DNA. The DNA was hooked out using a flame-sterilized glass hook needle, washed twice with 70% ethanol, dried at room temperature for 5 minutes, and then dissolved in 200. Mu.L of 1 XTE and drum-dissolved overnight. Measuring the OD value by ultraviolet.
7) TE-solubilized DNA can be stored at 4 ℃ for one year, and if long-term storage is required, 2 times the volume of absolute ethanol is added and the DNA is stored at-70 ℃.
4.2 exon sequencing
1) Taking 2 mu g of DNA, mechanically breaking the DNA to ensure that the size of the fragment is about 200bp, cutting the gel and recovering 150-250 bp fragments;
2) Carrying out end repair on the DNA fragment and adding A at the 3' end;
3) Connecting a sequencing joint, purifying a connecting product, performing PCR amplification, and purifying an amplification product;
4) Adding the purified amplification product into an Agilent kit probe for hybridization capture, eluting and recovering the hybridization product, performing PCR amplification, recovering the final product, and performing agarose gel electrophoresis on a small sample for quality control analysis;
5) NextSeq500 sequencer and data analysis.
4.3 results
Obtaining 2 gene complex hybrid mutations ATP7B of pathogenic significance NM — 000053.4; wherein the mutation of exon9: c.2384T > C may result in the change of amino acid residue 469 of the encoded protein from leucine to proline, and the mutation of exon19: c.4005delG may result in a frameshift mutation after amino acid residue 1335 of the encoded protein and a termination after amino acid residue 56. The genotype at ATP7B: NM — 000053.4.
Example 3
Sanger sequencing validation
The sequencing results of family exome No.1 were further verified using Sanger sequencing for ATP7B: NM — 000053.4. ATP7B: NM — 000053.4 for 4 family members in example 1 and 100 family members for external normal persons were subjected to gene detection at site nos.
The method comprises the following specific steps:
DNA extraction
Genomic DNA was extracted according to the method of example 2.
2. Candidate primer design, validation and optimization
2.1 candidate primer design reference human genome sequence database hg 19/built 36.3.
2.2 design 15 pairs of candidate primers for c.2384T > C and c.4005delG sites respectively (see tables 3-8), and verify and evaluate the quality of each pair of candidate primers by PCR experiment
Table 3 shows the basic conditions of candidate primers for c.2384T > C site and the results of verification experiment
Table 4 basic conditions of candidate primers for c.4005delG site and results of verification experiment
Note: only one specific band exists after electrophoresis of a normal PCR amplification result, and if a primer dimer band and a non-specific product band appear, the primer dimer band and the non-specific product band are both the results of primer abnormal reaction; the target primer avoids the primer as much as possible, and simultaneously avoids the conditions of hairpin, dimer, abnormal G + C content and the like as much as possible.
2.3 candidate primer PCR validation reactions
Performing PCR according to the reaction system in Table 5 and keeping the reaction system on ice; 8 reaction test tubes (Nos. 1 to 8 in Table 5) were provided for each pair of primers.
TABLE 5 primer detection PCR reaction System
Note: 10 XPCR buffer from 500mmol/L KCl, 100mmol/L Tris-HCl (pH8.3) and 15mmol/L MgCl 2 And (4) forming.
Reaction conditions are as follows: placing the test reaction tube into a PCR instrument, and executing the following reaction procedures:
the first step is as follows: pre-denaturation at 95 ℃ for 5min; the second step is that: 30 cycles (denaturation at 95 ℃ 30sec → Tm annealing 30sec → extension at 72 ℃ 60 sec); setting PCR amplification parameters according to Tm values of the primers in Table 5); the third step: extending for 7min at 72 ℃; the fourth step: 4 ℃ until sampling.
2.4 agarose gel electrophoresis detection of the candidate primer PCR results to assess the effectiveness, specificity of the primer reaction:
1) The two ends of the washed and dried gel sample former are sealed by an adhesive tape, the gel sample former is placed on a horizontal table, and a comb is placed at a position of about 1cm of one end of the sample former.
2) Weighing 2g agar powder into a conical flask, adding 100mL 0.5 XTBE electrophoresis buffer, shaking, heating in microwave oven or electric furnace (adding asbestos gauze), boiling, shaking, heating until the gel is completely melted, and cooling at room temperature.
3) And when the gel is cooled to about 50 ℃, pouring the gel into a sealed gel sample injector to ensure that the thickness is about 5 mm.
4) The gel is solidified, the adhesive tape is removed, and the gel and the sample injector are placed into an electrophoresis tank.
5) Adding electrophoresis buffer solution to make the liquid surface 1-2 mm higher than the glue surface, and pulling out the comb upwards; and (3) respectively and uniformly mixing the sample and the DNA size standard substance with the sample carrying liquid by using a micropipette, and adding the mixture into each sample adding hole, wherein the DNA sinks into the bottom of the hole due to the large specific gravity of the sucrose in the sample carrying liquid.
6) Covering the electrophoresis tank, switching on the power supply, adjusting to proper voltage, and starting electrophoresis. And judging the approximate position of the sample according to the indication of bromophenol blue in the sample carrier liquid, and determining whether to terminate electrophoresis.
7) The power was turned off, the gel was taken out and stained in 0.5g/ml EB aqueous solution for 10 to 15 minutes.
8) The gel was placed under a transmission ultraviolet irradiator for observation at a wavelength of 254nm, and the results of electrophoresis were photographed with a camera with a red filter or recorded with a gel scanning system.
2.5 evaluation of results:
1) If the No.7 tube only has a bright band and no band, the pair of primers and the reaction system are judged to have good effectiveness and strong specificity;
2) If no target band appears in the No.7 tube, judging that the pair of primers and the reaction system are invalid;
3) If the primer-primer dimer band outside the target band appears in the No.7 tube and the primer dimer band also appears in the No.2, 3, 4, 5 and 6 tubes, judging that the effectiveness of the pair of primers and the reaction system is poor;
4) If the non-specific band outside the target band appears in the No.7 tube and also appears in the No.5 and No.6 tubes, judging that the specificity of the pair of primers and the reaction system is poor;
5) If the primer dimer and the non-specific band appear outside the target band in the No.7 tube, and the primer dimer and the non-specific band also appear in the No.2, 3, 4, 5, and 6 tubes, the effectiveness and the specificity of the pair of primers and the reaction system are judged to be poor.
2.6 according to table 5 to verify the results of the statistics after the test, SEQ ID No.1 and SEQ ID No.2 in table 3 were selected as amplification primers for ATP7B: NM — 000053.4exon 9 c.2384t > c; SEQ ID nos. 31 and 32 in table 4 were selected as amplification primers for ATP7B: NM — 000053.4exon19 c.4005delg.
3. PCR amplification of mutation sites of family No.1 and 100 family members
And (3) PCR reaction system: 2.0. Mu.L of 10 XPCR buffer, 0.4. Mu.L of 10mmol/L dNTPs, 0.5. Mu.L of 100 ng/. Mu.L APC-F, 0.5. Mu.L of 100 ng/. Mu.L APC-R, 1.0. Mu.L of 100 ng/. Mu.L peripheral blood extracted DNA, 0.2. Mu.L of 5 u/. Mu.L Taq enzyme, 15.4. Mu.L ddH 2 And O. PCR was performed as described above and the reaction system was kept on ice.
The reaction conditions are as follows: the reaction system was placed in a PCR instrument and the following reaction sequence was performed:
the first step is as follows: pre-denaturation at 95 ℃ for 5min; the second step: 30 cycles (denaturation at 95 ℃ 30sec → annealing at 40 ℃ 30sec → extension at 72 ℃ 60 sec); the third step: extending for 7min at 72 ℃; the fourth step: 4 ℃ until sampling.
4. Agarose gel electrophoresis detection
Refer to step 2.4 above.
5. And (3) carrying out enzymolysis purification on the PCR product: mu.L of each of exonuclease I (Exo I) and alkaline phosphatase (AIP) was added to 5. Mu.L of the PCR product, digested at 37 ℃ for 15min, and inactivated at 85 ℃ for 15min.
6. BigDye reaction
BigDye reaction system: : 2.0. Mu.L of purified DNA from PCR product, 1.0. Mu.L of 3.2 pmol/. Mu.L sequencing primer, 0.5. Mu.L of BigDye, 2.0. Mu.L of 5 XBigDye sequencing buffer, and 4.5. Mu.L of ddH 2 O。
Sequencing PCR cycling conditions:
the first step is as follows: pre-denaturation at 96 ℃ for 1min; the second step is that: 33 cycles (denaturation at 96 ℃ 30sec → annealing at 55 ℃ 15sec → extension at 60 ℃ for 4 min); the third step: 4 ℃ until sampling.
7. Purification of BigDye reaction product:
1) Add 1. Mu.L 125mM EDTA (pH8.0) to each tube, add to the bottom of the tube, add 1. Mu.L 3mol/L NaAc (pH5.2);
2) Adding 70 μ L70% ethanol, shaking and mixing for 4 times, standing at room temperature for 15min;
3) 3000g, centrifuging at 4 deg.C for 30min; immediately inverting 96-well plate, centrifuging at 185g for 1min;
4) Standing at room temperature for 5min, allowing residual ethanol to evaporate at room temperature, adding 10 μ LHi-Di formamide to dissolve DNA, denaturing at 96 deg.C for 4min, rapidly placing on ice for 4min, and performing sequencing on machine.
8. Sequencing
DNA sequencing is carried out on the purified BigDye reaction product, a sequencing primer is designed on the basis of the SEQ ID NO.1 and the SEQ ID NO.2 and the SEQ ID NO.31 and the SEQ ID NO.32 as a nested primer (a second group of primers is designed in the range of the product sequence obtained by amplifying the first group of primers), and the primer sequence is shown as follows: .
Sequencing primer sequences for ATP7B: NM — 000053.4exon 9 c.2384t > c.l 795p site are as follows:
5’-AACACCACGCTTGTGACTC-3’(SEQ ID NO.61)
5’-GTCAATACAACATGGGCATC-3’(SEQ ID NO.62)
the sequencing primer sequences for ATP7B: NM — 000053.4:
5’-GCTGTGGGTGCTGGGAGG-3’(SEQ ID NO.63)
5’-GGAACCTGGGAGACAGAAGC-3’(SEQ ID NO.64)。
9. analysis of results
Sequencing results figure 2, from figure 2 it can be seen that the ATP7B: NM — 000053.4; the genotype of 3 carriers in family No.1 at this site was "c.2384t > C heterozygous mutation" or "c.4005delg heterozygous mutation", respectively, and the genotype of 100 normal controls without relationship at ATP7B: NM — 000053.4. The position indicated by the arrow in the sequencing diagram of FIG. 2: layers a and D show that the carrier individual ATP7B: NM — 000053.4 in family No. 1; layer B shows that the carrier individuals in family No.1 ATP7B: NM — 000053.4.
Example 4
Wilson's disease diagnostic kit and application
1. The kit comprises the following components:
1) An amplification primer: as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.31 and SEQ ID NO.32 of example 3; 2) 10 XPCR buffer (from 500mmol/L KCl, 100mmol/L Tris-HCl pH8.3, 15mmol/L MgCl 2 ddH of sum remainder 2 O composition); 3) Taq enzyme; 4) dNTPs; 5) c.2384T ATP7B>C and c.4005delG positive mutation reference substance DNA, wherein the reference substance is a section of double-stranded DNA, c.2384T>The specific sequence of the C mutation site positive reference substance is shown as SEQ ID NO.65, and specifically: 5' -ATCCATACATCCTCCTCAAGGTCATGCCATTTTTAACACCCTTCATTGTCCCCAACACTGAAATGTATCTTACAAGAGAATGGAACTCAGGCCCTAAAAGAAATTAGATTGAACCTAAAAGAAAATGGTCAGTGGGAAGACTGATGTTTGTTTCCAGTCTGAAATGTAATTCCCGGACTTTAGTGGCACAGGCACTGTGCATCACTGTGGAAGTGACATGTGGCCATGTGTGGTGGATAGCAAGTAACGCCCACCTGCAGAGCCTTTTATCGTGCCGTGCGGCTGTTTCTCTCGCACCAGCTGTCTCTAACACCACGCTTGTGACTCTCAGGCTGGGTTTGGACAGGTCTGCTTTCGATAGCTCTCATTTCACATTCTGGTTATTTCCTAGAGCAAAACCTCAGAAGCCCTGGCTAAACCCATGTCTCTCCAAGCCACAGAAGCCACCGTTGTGACCCTTGGTGAGGACAATTTAATCATCAGGTGAGTTATGGTTATCAAATGTCTTTGTGGTTGGTATCTATCAATCTGTGTGAGCTGCATCAGATGCCCATGTTGTATTGACATTGCAATAGACCTTGTGAGTGTGGGCAGAGACACAGTAAGATCACCACTCTCAATCCAGCTACGAAAGCAAGGCATTGAACTATAAAACTAGCAAGATTTGTAGGTGGTGTGTGTGTGCGAAATGGCACATGGGAGGTGTAGATGATT-3'; the specific sequence of the positive reference substance of the c.4005delG mutant site is shown as SEQ ID NO.66, and specifically: 5'-GAATAAAGGGAAGAAAGTCGCCATGGTGGGGGATGGGGTCAATGACTCCCCGGCCTTGGCCCAGGCAGACATGGGTGTGGCCATTGGCACCGGCACGGATGTGGCCATCGAGGCAGCCGACGTCGTCCTTATCAGAGTGAGCGTGGCTGCAGCCAGGCTGTGGGTGCTGGGAGGGCAATGGGCAGACCCCTTCCTCACTGTGTGCTCCTCTCCATCAGAATGATTTGCTGGATGTGGTGGCTAGCATTCACCTTTCCAAGAGGACTGTCCGAAGGATACGCATCAACCTGGTCCTGGCACTGATTTATAACCTGGTTGGATACCCATTGCAGCAGGTAGGCAGCTCTTACCCACTGTGCTCCAGCTGCGCCCAGAAAGGCTTCTGTCTCCCAGGTTCCTGCTGGGGTTAGTGAGTGGCTCACTCACTGGCTGGCTAGAGGCGTTTTAGAAAGGCTGTTTTTTTTTTTTTTTCATGTCCCCTGCTTTATATGTCTTTAAATGAGAAACTGTGGAGAGGACCTGAAACCTTCATTCACTCTGGTCCTTTCTAAAAAGCTAGTGGAGATGCTTGGCTGTATTTTCTGATCCCTATTTTATTGTGGCCTTATTTTCTGAGCCATTTGTTAAGTCATATAGTCTGAGAAATAATCATTAGTAAGAATTATGGGGCATCTGCTACATGCTAGGCACAATTATTGGTGTTTCAGAACCTTATCCTGAACATTAATCCTGCCCAAGTTTCACTCTGAGTTACACAAATGATCATGGAAAGAAATGGCTCTTTGACCAGGCAT-3'; 6) Sequencing primer: as shown in step 8 of example 3.
2. The using method comprises the following steps:
the family map is shown in FIG. 3, wherein FIG. 3 is shown in FIG. 3, the clinical information is shown in Table 6, and the family map is used in family 2
It represents a male carrier of the human,
representing a female carrier, ■ a male patient, o representing a fetus, ↗ representing a proband.
TABLE 6 clinical information of Wilson's disease family Member
Note: i, II and III represent the first, second and third generations in this order.
The method comprises the following steps that No.2 family personnel I1 and I2 peripheral blood DNA are used for sequencing, and II 2 amniotic fluid DNA is used for kit detection:
1) Extracting genome DNA: sample genomic DNA was extracted according to the procedure of example 2.
2) Firstly, carrying out PCR amplification reaction by adopting PCR amplification primers, taq enzyme, 10 XPCR buffer solution, dNTPs, sample genome DNA and the like in the kit;
3) Purifying PCR amplification products;
4) Carrying out BigDye reaction on the purified PCR product by using a sequencing primer in the kit;
5) Purifying the BiyDye reaction product;
6) The BiyDye reaction products were sequenced and the sequence compared to the normal sequence.
The results are shown in fig. 4 and 5, wherein fig. 4 is a graph of the results of using the kit to detect the genotype of ATP7B: NM — 000053.4; b is the genotype of wild type (the arrow in the sequencing diagram indicates the site where the mutation occurs). FIG. 5 is a graph showing the results of using the kit to detect the genotype at the ATP7B: NM-000053.4 site (ex on 19); a is the genotype, wild type (the arrow in the sequence diagram indicates the site where the mutation occurs). Combining the detection results of the kit in fig. 4 and 5, the fetus II 2 is a complex heterozygous mutation of c.2384T > C and c.4005delG, namely a Wilson patient.
According to the embodiments, the ATP7B mutant gene can be used as a biomarker for diagnosing Wilson's disease and provides a possible drug target for treating Wilson's disease. .
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and all of the embodiments are included in the scope of the present invention.
Sequence listing
<110> Hunan Jiahui Biotechnology Ltd
<120> biomarker, amplification primer set, detection reagent and application for diagnosing Wilson disease
<160> 66
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atccatacat cctcctc 17
<210> 2
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
aatcatctac acctccc 17
<210> 3
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
acatccatac atcctcctc 19
<210> 4
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tgaagtttcc cttgacc 17
<210> 5
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ccatacatcc tcctcaa 17
<210> 6
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
aatcatctac acctccc 17
<210> 7
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
acatccatac atcctcctc 19
<210> 8
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
caccacctac aaatcttgc 19
<210> 9
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gggaagactg atgtttg 17
<210> 10
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ctttcgtagc tggattg 17
<210> 11
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
agacatccat acatcctcct c 21
<210> 12
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tcaatgcctt gctttcg 17
<210> 13
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
acatccatac atcctcctc 19
<210> 14
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gtttcccttg accaaca 17
<210> 15
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
agacatccat acatcctcct c 21
<210> 16
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
caccacctac aaatcttgc 19
<210> 17
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
aagtaacgcc cacctgc 17
<210> 18
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
caccacctac aaatcttgc 19
<210> 19
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
aatggtcagt gggaaga 17
<210> 20
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
caccacctac aaatcttgc 19
<210> 21
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gactttagtg gcacagg 17
<210> 22
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
atgccttgct ttcgtagc 18
<210> 23
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
agacatccat acatcctcct c 21
<210> 24
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
aatactaatc atctacacct ccc 23
<210> 25
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
gtgggaagac tgatgtttgt t 21
<210> 26
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
cttgctttcg tagctggatt g 21
<210> 27
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gtaattcccg gactttagtg g 21
<210> 28
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
tagttcaatg ccttgctttc g 21
<210> 29
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
atcctcctca aggtcatgcc attt 24
<210> 30
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
aatactaatc atctacacct ccc 23
<210> 31
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
gaataaaggg aagaaagtcg 20
<210> 32
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
atgcctggtc aaagagcc 18
<210> 33
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
ataaagggaa gaaagtcgc 19
<210> 34
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
atgcctggtc aaagagcc 18
<210> 35
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
tccagaataa agggaagaa 19
<210> 36
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
tgtgcctagc atgtagca 18
<210> 37
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
ataaagggaa gaaagtcgc 19
<210> 38
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
ccatgcctgg tcaaagag 18
<210> 39
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
tccagaataa agggaagaa 19
<210> 40
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
aaaggaccag agtgaatga 19
<210> 41
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
gctccagaat aaagggaa 18
<210> 42
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
aaacaccaat aattgtgcc 19
<210> 43
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
gctccagaat aaagggaa 18
<210> 44
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
aaatacagcc aagcatctc 19
<210> 45
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
tccagaataa agggaagaa 19
<210> 46
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
aaggaccaga gtgaatgaag 20
<210> 47
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
gaggtttctg ctgctatct 19
<210> 48
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
cagccaagca tctccact 18
<210> 49
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
ccaggcagac atgggtgt 18
<210> 50
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
agtgaaactt gggcagga 18
<210> 51
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
tgctgctatc tgataccttt 20
<210> 52
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
aaatacagcc aagcatctc 19
<210> 53
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
gtcgtcctta tcagagtgag 20
<210> 54
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
taattgtgcc tagcatgtag 20
<210> 55
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
gcccaggcag acatgggtgt 20
<210> 56
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
tgacttaaca aatggctca 19
<210> 57
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
taggttggca tcaacaaagt c 21
<210> 58
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
aaggaccaga gtgaatgaag gtttc 25
<210> 59
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
gtccttatca gagtgagcgt ggct 24
<210> 60
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
attgtgccta gcatgtagca g 21
<210> 61
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
aacaccacgc ttgtgactc 19
<210> 62
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
gtcaatacaa catgggcatc 20
<210> 63
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
gctgtgggtg ctgggagg 18
<210> 64
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
ggaacctggg agacagaagc 20
<210> 65
<211> 714
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
atccatacat cctcctcaag gtcatgccat ttttaacacc cttcattgtc cccaacactg 60
aaatgtatct tacaagagaa tggaactcag gccctaaaag aaattagatt gaacctaaaa 120
gaaaatggtc agtgggaaga ctgatgtttg tttccagtct gaaatgtaat tcccggactt 180
tagtggcaca ggcactgtgc atcactgtgg aagtgacatg tggccatgtg tggtggatag 240
caagtaacgc ccacctgcag agccttttat cgtgccgtgc ggctgtttct ctcgcaccag 300
ctgtctctaa caccacgctt gtgactctca ggctgggttt ggacaggtct gctttcgata 360
gctctcattt cacattctgg ttatttccta gagcaaaacc tcagaagccc tggctaaacc 420
catgtctctc caagccacag aagccaccgt tgtgaccctt ggtgaggaca atttaatcat 480
caggtgagtt atggttatca aatgtctttg tggttggtat ctatcaatct gtgtgagctg 540
catcagatgc ccatgttgta ttgacattgc aatagacctt gtgagtgtgg gcagagacac 600
agtaagatca ccactctcaa tccagctacg aaagcaaggc attgaactat aaaactagca 660
agatttgtag gtggtgtgtg tgtgcgaaat ggcacatggg aggtgtagat gatt 714
<210> 66
<211> 794
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
gaataaaggg aagaaagtcg ccatggtggg ggatggggtc aatgactccc cggccttggc 60
ccaggcagac atgggtgtgg ccattggcac cggcacggat gtggccatcg aggcagccga 120
cgtcgtcctt atcagagtga gcgtggctgc agccaggctg tgggtgctgg gagggcaatg 180
ggcagacccc ttcctcactg tgtgctcctc tccatcagaa tgatttgctg gatgtggtgg 240
ctagcattca cctttccaag aggactgtcc gaaggatacg catcaacctg gtcctggcac 300
tgatttataa cctggttgga tacccattgc agcaggtagg cagctcttac ccactgtgct 360
ccagctgcgc ccagaaaggc ttctgtctcc caggttcctg ctggggttag tgagtggctc 420
actcactggc tggctagagg cgttttagaa aggctgtttt tttttttttt tcatgtcccc 480
tgctttatat gtctttaaat gagaaactgt ggagaggacc tgaaaccttc attcactctg 540
gtcctttcta aaaagctagt ggagatgctt ggctgtattt tctgatccct attttattgt 600
ggccttattt tctgagccat ttgttaagtc atatagtctg agaaataatc attagtaaga 660
attatggggc atctgctaca tgctaggcac aattattggt gtttcagaac cttatcctga 720
acattaatcc tgcccaagtt tcactctgag ttacacaaat gatcatggaa agaaatggct 780
ctttgaccag gcat 794
Claims (10)
1. A biomarker for diagnosing Wilson's disease, wherein the biomarker is an ATP7B mutant gene, and the mutation sites comprise c.2384T > C and c.4005delG.
2. A primer set for amplifying the ATP7B mutant gene of claim 1, comprising a primer set 1 and a primer set 2;
the primer group 1 comprises an upstream primer ATP7B-1F and a downstream primer ATP7B-1R;
the primer group 2 comprises an upstream primer ATP7B-2F and a downstream primer ATP7B-2R;
the upstream primer ATP7B-1F comprises any one of a 1) to a 4):
a1 Single-stranded DNA described by SEQ ID NO.1 of the sequence Listing;
a2 A single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or the 3' end of a 1);
a3 A single-stranded DNA having an identity of 85% or more to the single-stranded DNA defined in a 1) or a 2);
a4 A single-stranded DNA which hybridizes with the single-stranded DNA defined in a 1) or a 2) under stringent conditions;
the downstream primer ATP7B-1R comprises any one of B1) to B4):
b1 Single-stranded DNA described by SEQ ID NO.2 of the sequence Listing;
b2 A single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of b 1);
b3 A single-stranded DNA having an identity of 85% or more to the single-stranded DNA defined in b 1) or b 2);
b4 A single-stranded DNA which hybridizes with the single-stranded DNA defined in b 1) or b 2) under stringent conditions;
the upstream primer ATP7B-2F comprises any one of c 1) -c 4):
c1 Single-stranded DNA described by SEQ ID NO.31 of the sequence Listing;
c2 A single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of c 1);
c3 A single-stranded DNA having an identity of 85% or more to the single-stranded DNA defined in c 1) or c 2);
c4 A single-stranded DNA which hybridizes with the single-stranded DNA defined in c 1) or c 2) under stringent conditions;
the downstream primer ATP7B-2R comprises any one of d 1) to d 4):
d1 Single-stranded DNA described by SEQ ID NO.32 of the sequence Listing;
d2 A single-stranded DNA obtained by adding one or several nucleotides to the 5 '-end and/or the 3' -end of d 1);
d3 A single-stranded DNA having an identity of 85% or more to the single-stranded DNA defined in d 1) or d 2);
d4 A single-stranded DNA which hybridizes under stringent conditions with the single-stranded DNA defined under d 1) or d 2).
3. Use of the primer set according to claim 2 for the preparation of a reagent for diagnosing wilson's disease.
4. A reagent for diagnosing wilson's disease, comprising the primer set according to claim 2 and a sequencing primer set.
5. The reagent of claim 4, wherein the sequencing primer set comprises sequencing primer sets 1 and 2;
the sequencing primer group 1 comprises an upstream primer ATP7B-Seq1F and a downstream primer ATP7B-Seq1R;
the sequencing primer group 2 comprises an upstream primer ATP7B-Seq2F and a downstream primer ATP7B-Seq2R;
the upstream primer ATP7B-Seq1F comprises any one of e 1) to e 4):
e1 Single-stranded DNA described by SEQ ID NO.61 of the sequence Listing;
e2 A single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of e 1);
e3 A single-stranded DNA having an identity of 85% or more to the single-stranded DNA defined in e 1) or e 2);
e4 A single-stranded DNA which hybridizes with the single-stranded DNA defined in e 1) or e 2) under stringent conditions;
the downstream primer ATP7B-Seq1R comprises any one of f 1) to f 4):
f1 Single-stranded DNA described by SEQ ID NO.62 of the sequence Listing;
f2 A single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of f 1);
f3 A single-stranded DNA having an identity of 85% or more to the single-stranded DNA defined in f 1) or f 2);
f4 A single-stranded DNA which hybridizes with the single-stranded DNA defined in f 1) or f 2) under stringent conditions;
the upstream primer ATP7B-Seq2F comprises any one of g 1) to g 4):
g1 Single-stranded DNA described by SEQ ID NO.63 of the sequence Listing;
g2 A single-stranded DNA obtained by adding one or several nucleotides to the 5 'end and/or 3' end of g 1);
g3 A single-stranded DNA having an identity of 85% or more to the single-stranded DNA defined in g 1) or g 2);
g4 A single-stranded DNA which hybridizes with the single-stranded DNA defined in g 1) or g 2) under stringent conditions;
the downstream primer ATP7B-Seq2R comprises any one of h 1) to h 4):
h1 Single-stranded DNA described by SEQ ID NO.64 of the sequence Listing;
h2 A single-stranded DNA obtained by adding one or more nucleotides to the 5 'end and/or the 3' end of h 1);
h3 A single-stranded DNA having an identity of 85% or more to the single-stranded DNA defined in h 1) or h 2);
h4 Single-stranded DNA which hybridizes under stringent conditions with the single-stranded DNA defined in h 1) or h 2).
6. The reagent of claim 4 or 5, wherein the reagent further comprises one or more of dNTPs, PCR buffer, magnesium ions and Tap polymerase.
7. Use of the primer set according to claim 2 or the reagent according to any one of claims 4 to 6 for producing a kit for preventing and/or diagnosing Wilson's disease.
8. Use of the primer set according to claim 2 or the reagent according to any one of claims 4 to 6 for the preparation of a kit for the adjuvant treatment of Wilson's disease.
9. Use of the primer set of claim 2 or the reagent of any one of claims 4 to 6 for the preparation of a pre-pregnancy and/or prenatal genetic disorder screening and/or diagnostic kit.
10. A kit for diagnosing Wilson's disease, comprising the reagent according to any one of claims 4 to 6.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116656801A (en) * | 2022-12-02 | 2023-08-29 | 湖南家辉生物技术有限公司 | Application of Cowcdock syndrome pathogenic gene AIFM1 mutation site, detection reagent and application thereof |
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| CN116656801A (en) * | 2022-12-02 | 2023-08-29 | 湖南家辉生物技术有限公司 | Application of Cowcdock syndrome pathogenic gene AIFM1 mutation site, detection reagent and application thereof |
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