CN102220434B - Hybrid membrane strip, PCR primer and kit for diagnosis of NSHI - Google Patents
Hybrid membrane strip, PCR primer and kit for diagnosis of NSHI Download PDFInfo
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Abstract
The invention discloses a hybrid membrane strip for diagnosis of NSHI (nonsyndromic hearing impairment). The membrane strip comprises a substrate and mutation detection probes, wherein each mutation detection probe respectively comprises a corresponding NSHI gene mutation locus. The gene mutation locus is at least one of the followings: a cDNA35 locus, cDNA176-191 loca, a cDNA235 locus, and cDNA299-300 loca of a GJB2 gene, a cDNA538 locus of a GJB3 gene, a cDNA1555 locus and a cDNA1494 locus of an mtDNA12srRNA gene, and a cDNA2168 locus and an IVS7-2 locus of an SLC26A4 gene; each mutation detection probe comprises sequences of 15-25 bases, and a central section of a sequence comprises a corresponding mutation base of the NSHI gene. The invention has the advantages of high accuracy and low prices, etc.
Description
Technical field
The present invention relates to a kind of for medically being used for the Hybond membrane bar of diagnosis nonsyndromic sensorineural, the invention still further relates to a kind of polymerase chain reaction be used to the sample gene fragment to be checked that increases (Polymerase Chain Reaction, initialism is PCR) primer; The invention still further relates to a kind of test kit for the diagnosis nonsyndromic sensorineural.
Background technology
Deafness is a kind of modal mankind sensory system's defective, because its cause of disease is complicated, incidence is high and treatment difficulty etc. is former thereby perplexing muchly extensive patients and surrounding population thereof, greatly affects mutual interchange and quality of life.Incidence is up to 1/800～1/1000 in the newborn infant for severe deafness, and the whole world has 70,000,000 people to suffer from dysacusis more than 55 decibels nearly.
Cause deafness that many-sided reason is arranged, inherited genetic factors is major cause.Deafness can be caused by term single gene sudden change or heterogeneic complex mutation.Also can be by environmental factors or gene and environment acting in conjunction and cause.Deafness can be divided syndrome and type and nonsyndromic, the deafness that is attended by the pathology of other histoorgans belongs to syndrome and type hearing loss (syndromic hearing impairment, SHI), deafness without other symptoms belongs to nonsyndromic hearing loss (nonyndromic hearing impairment, NSHI).And 70% hereditary hearing impairment shows as nonsyndromic sensorineural (namely except deafness without other sings and symptomses).Stop on January 1st, 2011,25 autosomal recessive genes relevant to nonsyndromic sensorineural, 36 autosomal dominant genes, 2 X linked genes are cloned, and be dispersed in numerous deaf mutational site in each gene, thereby have higher gene and site genetic heterogeneity.Recently, the extensive deaf molecule epidemic disease-ology research of carrying out at home shows, quite a few nonsyndromic sensorineural is only caused by several transgenations few in number, as GJB2, SLC26A4, mtDNA 12s rRNA and GJB3 etc., this carries out on a large scale the deaf gene examination for us and diagnosis provides theoretical foundation.
At present, tradition gene diagnosis method comprises that enzyme cuts, restriction fragment length polymorphism is analyzed (restriction fragment length polymorphism, RFLP), direct Sequencing etc., these methods or can not be qualitative, perhaps time and effort consuming, required equipment and consumptive material are expensive, the more important thing is that these methods are difficult to simultaneously heterogeneic a plurality of mutational sites be detected.Although and gene chips can be simultaneously detects the different genes site, its required equipment and consumptive material are expensive, are not suitable in clinical large-scale promotion, and application is restricted.
Summary of the invention
Technical problem to be solved by this invention is: make up above-mentioned the deficiencies in the prior art, a kind of Hybond membrane bar, a kind of PCR primer be used to the sample gene fragment to be checked that increases for the diagnosis nonsyndromic sensorineural proposed, and a kind of test kit for the diagnosis nonsyndromic sensorineural; The present invention has high-throughput, high-level efficiency, the advantage such as cheap, easy to use, is conducive to realize clinical rapid detection and large-scale crowd examination.
Of the present invention a kind of for the technical scheme below the Hybond membrane strip adoption of diagnosis nonsyndromic sensorineural:
described Hybond membrane bar for the diagnosis nonsyndromic sensorineural, it is characterized in that: comprise substrate, and be fixed on described suprabasil sudden change detection probes, each described sudden change detection probes is the corresponding gene mutation site that comprises a nonsyndromic sensorineural respectively, the gene mutation site of described nonsyndromic sensorineural is at least one in following site: the cDNA35 of GJB2 gene, cDNA176-191, cDNA235, the cDNA299-300 site, the cDNA538 site of GJB3 gene, the cDNA1555 of mtDNA 12s rRNA gene, the cDNA1494 site, the cDNA2168 of SLC26A4 gene, the IVS7-2 site, each described sudden change detection probes comprises the sequence of 15-25 base, comprises the mutating alkali yl of its corresponding nonsyndromic sensorineural gene at the medium position of described sequence.
Preferably, described sudden change detection probes is at least one sequence in SEQ ID NO.1～SEQ ID NO.9 or the DNA of its reverse complementary sequence.
Preferably, also be fixed with the normal control probe in described substrate, itself and described sudden change detection probes are fixed on the different positions of described substrate.
Preferably, described normal control probe is at least one sequence in SEQ ID NO.10～SEQ ID NO.18 or the DNA of its reverse complementary sequence.
preferably, the corresponding gene mutation site that comprises a described nonsyndromic sensorineural of each described normal control probe, wherein, the gene mutation site that SEQ ID NO.10 is corresponding is cDNA35, the gene mutation site that SEQ ID NO.11 is corresponding is cDNA176-191, the gene mutation site that SEQ ID NO.12 is corresponding is cDNA235, the gene mutation site that SEQ ID NO.13 is corresponding is the cDNA299-300 site, the gene mutation site that SEQ ID NO.14 is corresponding is cDNA538, the gene mutation site that SEQ ID NO.15 is corresponding is cDNA1494, the gene mutation site that SEQ ID NO.16 is corresponding is cDNA1555, the gene mutation site that SEQ ID NO.17 is corresponding is cDNA2168, the gene mutation site that SEQ ID NO.18 is corresponding is IVS7-2.
Preferably, 3 ' or 5 ' of described sudden change detection probes and/or normal control probe end is with amino labeled.
A kind of PCR primer be used to the sample gene fragment to be checked that increases of the present invention adopts following technical scheme:
Described PCR primer amplification gene mutation site claimed in claim 1 be used to the sample gene fragment to be checked that increases, described PCR primer is at least one pair of of following 5 centerings: SEQ ID NO.19 and SEQ ID NO.20; SEQ ID NO.21 and SEQ ID NO.22; SEQ ID NO.23 and SEQ ID NO.24; SEQ ID NO.25 and SEQ ID NO.26; SEQ ID NO.27 and SEQ ID NO.28; Wherein SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27 are upstream primers; SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28 are downstream primers.
preferably, described amplified production comprises respectively following site: the cDNA35 of primer SEQ ID NO.19 and SEQ ID NO.20 amplification GJB2 gene, cDNA176-191, cDNA235, the cDNA299-300 site, the cDNA538 site of primer SEQ ID NO.21 and SEQ ID NO.22 amplification GJB3 gene, the cDNA1555 of primer SEQ ID NO.23 and SEQ ID NO.24 amplification mtDNA 12S rRNA gene, the cDNA1494 site, the cDNA2168 site of primer SEQ ID NO.25 and SEQ ID NO.26 amplification SLC26A4 gene, the IVS7-2 site of primer SEQ ID NO.27 and SEQ ID NO.28 amplification SLC26A4 gene.
Preferably, 5 ' of described upstream primer and/or downstream primer end is with vitamin H or fluorescent mark.
A kind of test kit for the diagnosis nonsyndromic sensorineural of the present invention adopts following technical scheme:
Described test kit for the diagnosis nonsyndromic sensorineural comprises the above-mentioned Hybond membrane bar that is used for the diagnosis nonsyndromic sensorineural, and the PCR reaction solution that contains above-mentioned PCR primer be used to the sample gene fragment to be checked that increases.
The beneficial effect that the present invention is compared with the prior art is: the present invention is based on the gene tester of PCR-film bar hybridization technique, the gene mutation site that includes nonsyndromic sensorineural due to the roughly mid-way that is fixed on suprabasil sudden change detection probes, can directly make a definite diagnosis person's to be checked genotype by PCR blooming bar hybridization technique, normal, heterozygous mutation, no mutant homozygote was etc. are easy to judgement, have high-throughput, high-level efficiency, the advantage such as cheap, easy to use.
Description of drawings
Fig. 1 be the embodiment of the present invention do not detect nonsyndromic sensorineural transgenation sample result example, its genotype is: N/N;
Fig. 2 is the nonsyndromic sensorineural transgenation heterozygote sample result example of the embodiment of the present invention, and its genotype is: 35M/N;
Fig. 3 is the nonsyndromic sensorineural transgenation homozygote sample result example of the embodiment of the present invention, and its genotype is: 35M/35M;
Fig. 4 is the dual heterozygous mutation sample result of the nonsyndromic sensorineural gene example of the embodiment of the present invention, and its genotype is: 35M/235M.
Embodiment
The below contrasts accompanying drawing and in conjunction with preferred embodiment, the present invention is explained in detail.
1, the preparation of Hybond membrane bar
1.1, the preparation of 9 sudden changes detection probes and 9 normal control probes
According to synthetic 18 probes of the sequence in table 1 and table 2,3 ' or 5 ' end band amino labeled of sudden change detection probes and/or normal control probe, synthetic method is the DNA synthesis method of existing routine.
Table 1:
Table 2:
Annotate: " M " representative sudden change detection probes, " N " represents the normal control probe.
each described sudden change detection probes is the corresponding gene mutation site that comprises a nonsyndromic sensorineural respectively, the gene mutation site of nonsyndromic sensorineural is following site: the cDNA35 of GJB2 gene, cDNA176-191, cDNA235, the cDNA299-300 site, the cDNA538 site of GJB3 gene, the cDNA1555 of mtDNA 12s rRNA gene, the cDNA1494 site, the cDNA2168 of SLC26A4 gene, the IVS7-2 site, each sudden change detection probes comprises the sequence of 15-25 base, the mutating alkali yl that comprises its corresponding nonsyndromic sensorineural gene at the medium position of sequence.
the corresponding gene mutation site that comprises a nonsyndromic sensorineural of each normal control probe, wherein, the gene mutation site that SEQ ID NO.10 is corresponding is cDNA35, the gene mutation site that SEQ ID NO.11 is corresponding is cDNA176-191, the gene mutation site that SEQ ID NO.12 is corresponding is cDNA235, the gene mutation site that SEQ ID NO.13 is corresponding is the cDNA299-300 site, the gene mutation site that SEQ ID NO.14 is corresponding is cDNA538, the gene mutation site that SEQ ID NO.15 is corresponding is cDNA1494, the gene mutation site that SEQ ID NO.16 is corresponding is cDNA1555, the gene mutation site that SEQ ID NO.17 is corresponding is cDNA2168, the gene mutation site that SEQ ID NO.18 is corresponding is IVS7-2.
1.2, the preparation of Hybond membrane bar
A. use printer in the upper printing site array of substrate (being nylon membrane in the present embodiment), and indicate the probe title in corresponding site, the normal control probe is fixed on the different positions of substrate with the sudden change detection probes;
B. used 5% EDAC solution vacuolar membrane 30 minutes, with the carboxyl on activation nylon membrane surface;
C. use respectively probe dilution liquid (10mmol/L Tris, pH8.0) with 18 probe dilution to 5 μ mol/L;
D. by the site array of printing on the nylon membrane bar, 18 probes are put respectively by probe title correspondence be added on nylon membrane corresponding site, the carboxyl generation crosslinking reaction on the amino on probe and nylon membrane surface;
E. after nylon membrane bar drying, the NaOH vacuolar membrane of use 0.1mol/L 5 minutes is with the unreacted carboxyl in sealing nylon membrane surface;
F. wash nylon membrane with pure water, dry rear standby.
Table 3: the probe array on the nylon membrane bar is as follows:
| 35M | 176-191M | 235M | 299- |
538M | 1494M | |
| 35N | 176- |
235N | 299- | 538N | 1494N | |
| 1555N | 2168N | IVS7- |
1555M | 2168M | IVS7-2M |
Annotate: " M " representative sudden change detection probes, " N " represents the normal control probe.
2, PCR primer amplification
The position relationship in 9 mutational sites to be checked according to this project, having designed 5 pairs of primers increases respectively, as shown in table 4, wherein SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27 are upstream primers, are abbreviated as respectively HL1F, HL2F, HL3F, HL4F, HL5F; SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28 are downstream primers, are abbreviated as respectively HL1R, HL2R, HL3R, HL4R, HL5R; In this table, cDNA35, the cDNA176-191 of primer HL1F and HL1R amplification GJB2 gene, cDNA235, cDNA299-300 site, the cDNA538 site of primer HL2F and HL2R amplification GJB3 gene, the cDNA1555 of primer HL3F and HL3R amplification mtDNA 12S rRNA gene, cDNA1494 site, the cDNA2168 site of primer HL4F and HL4R amplification SLC26A4 gene, the IVS7-2 site of primer HL5F and HL5R amplification SLC26A4 gene, as shown in table 4:
Table 4:
5 ' end of upstream primer and/or downstream primer is with vitamin H or fluorescein (as Cy3, Cy5 etc.) mark.The embodiment of the present invention is for to carry out biotin labeling to downstream primer (HL1R, HL2R, HL3R, HL4R, HL5R), the product after its pcr amplification can with the embodiment of the present invention in probe hybridization, the DNA sequence dna of PCR primer is as shown in table 5.
Table 5:
2.1, extract template DNA: use other commercial kit to extract template DNA from patient's blood.
2.2, synthetic 10 primers: synthetic method is the DNA synthesis method of existing routine.
2.3, preparation PCR reaction solution: be mixed with the PCR reaction solution of 25 μ L/ person-portions, in the PCR reaction solution of every person-portion, each composition and concentration are respectively: 10 primer concentrations are respectively 0.2 μ mol/L, the Taq enzyme is that 0.1U/ μ L, 1 * PCRBuffer, MgCl2 are that 1.5mmol/L, dNTP are 0.2mmol/L, template DNA 1～10ng/ μ L.
2.4, pcr amplification: the response procedures of pcr amplification is: 95 ℃ of denaturations 5 minutes; 95 ℃ of sex change 30 seconds, 55 ℃ of renaturation 30 seconds, 72 ℃ were extended 45 seconds, and circulated 35 times; 72 ℃ were extended 5 minutes again.Product after pcr amplification comprises patient's to be measured DNA fragmentation.
3, hybridization and colour developing
3.1. hybridization
Product after pcr amplification and the nylon membrane bar in step 1.2 are joined in the hybridization solution (2 * SSC, 0.1%SDS, pH7.4) of 12mL, and 42 ℃ of hybridization are more than 2～4 hours in the hybridization case.
3.2. wash film
The nylon membrane bar is transferred in the washing lotion (0.5 * SSC, 0.1%SDS, pH7.4) that is preheated to 42 ℃, in 42 ℃ of washings 15 minutes.
3.3. peroxidase (Peroxidase, initialism are POD) is hatched
The film bar is put into the solution of the streptavidin of freshly prepared 0.25U/mL-POD(streptin-POD), hatched 15 minutes for 37 ℃, the vitamin H of streptavidin in the PCR product is combined, and POD is connected on the PCR product, washes away unnecessary streptin-POD.
3.4. colour developing
Now join nitrite ion (0.1mol/L Trisodium Citrate, 0.1mg/mL TMB, 0.0015%H
2O
2), the film bar was put into nitrite ion lucifuge colour developing 15 minutes, there is the film bar correspondent probe position of hybridization product can show blue spot.
4, result judgement
As shown in Fig. 1-4, the colour developing result of film bar judges by naked eyes, with each site color signal have or not to judge whether sample to be checked exists transgenation, and be no mutant homozygote was or heterozygote.If the position that on the nylon membrane bar, all normal control probes are corresponding colour developing, position corresponding to all sudden change detection probes do not develop the color, can be judged as this test sample and not detect sudden change, genotype is abbreviated as N/N(such as Fig. 1).If a sudden change detection probes corresponding position colour developing is arranged, and position corresponding to all normal control probes all develop the color, and this sample can be judged as heterozygous mutation, and genotype is abbreviated as M/N, and adds the site (as Fig. 2) of undergoing mutation before M.If a position colour developing that the sudden change detection probes is corresponding is arranged, and do not develop the color in position corresponding to its corresponding normal control probe, this sample can judge a no mutant homozygote was, and genotype is abbreviated as M/M, and all adds the site (as Fig. 3) of undergoing mutation before two M.If two position colour developings that the sudden change detection probes is corresponding are arranged, and also all develop the color in its corresponding two positions corresponding to normal control probe, this sample can be judged as dual heterozygous mutation, genotype is abbreviated as M/M, and adds respectively two mutational sites (in no particular order) (as Fig. 4) before two M.
4 kinds of situations of the above in the embodiment of the present invention are the situation that most cases may occur, if result exceeds above-mentioned 4 kinds of situations, in the situation that confirm that experimental implementation is errorless, should each case concrete analysis result.
For modal 9 kinds of heredity nonsyndromic sensorineural gene mutation types in Chinese, be fixed on the film bar by the sudden change detection probes that will contain 9 kinds of heredity nonsyndromic sensorineural gene mutation sites, PCR product hybridization with person's corresponding DNA fragments to be checked can detect 35delG, the 176-191del16,235delC, 299-300delAT, the 538C that cause the heredity nonsyndromic sensorineural simultaneously〉T, 1555A〉G, 1494C〉T, 2168A〉G, IVS7-2A〉these 9 kinds of mutation types of G.The present invention is based on the gene tester of PCR-film bar hybridization technique, can directly make a definite diagnosis person's to be checked genotype, has that accuracy is high, high specificity, can detect the advantages such as the recessive carrier of gene.Therefore, should use the method to aim at father and mother in pre-marital medical check-up or pregnant inspection and carry out gene test, then according to circumstances, in case of necessity fetus be carried out antenatal detection, so just can greatly reduce the birth of heredity nonsyndromic sensorineural infant.
The present invention is based on the gene tester of PCR-reversal point hybridization technique, can directly make a definite diagnosis person's to be checked genotype, has that accuracy is high, high specificity, can detect the advantages such as the recessive carrier of gene.Detecting with present method does not need valuable instrumentation, only needs the equipment of PCR Lab routine to get final product, and being applicable at home, hospital promotes the use of on a large scale.
Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that concrete enforcement of the present invention is confined to these explanations.For the general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, make some being equal to substitute or obvious modification, and performance or purposes identical, all should be considered as belonging to protection scope of the present invention.
Sequence table
＜110〉the danger plum is beautiful
＜120〉be used for Hybond membrane bar, PCR primer and the test kit of diagnosis nonsyndromic sensorineural
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<160> 28
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<212> DNA
＜213〉artificial sequence
<400> 1
cgatcctggg ggtgtga 17
<210> 2
<211> 21
<212> DNA
＜213〉artificial sequence
<400> 2
Cctgcagcca gctacgatca c 21
<210> 3
<211> 17
<212> DNA
＜213〉artificial sequence
<400> 3
Ctatgggcct gcagctg 17
<210> 4
<211> 21
<212> DNA
＜213〉artificial sequence
<400> 4
ctaccggaga cgagaagaag a 21
<210> 5
<211> 20
<212> DNA
＜213〉artificial sequence
<400> 5
ctacattgcc tgacctaccg 20
<210> 6
<211> 22
<212> DNA
＜213〉artificial sequence
<400> 6
ccgtcaccct tctcaagtat ac 22
<210> 7
<211> 21
<212> DNA
＜213〉artificial sequence
<400> 7
tagaggaggc aagtcgtaac a 21
<210> 8
<211> 18
<212> DNA
＜213〉artificial sequence
<400> 8
tgacggtccg tgatgcta 18
<210> 9
<211> 21
<212> DNA
＜213〉artificial sequence
<400> 9
ttatttcgga cgataattgc t 21
<210> 10
<211> 17
<212> DNA
＜213〉artificial sequence
<400> 10
gatcctgggg ggtgtga 17
<210> 11
<211> 23
<212> DNA
＜213〉artificial sequence
<400> 11
caggctgcaa gaacgtgtgc tac 23
<210> 12
<211> 17
<212> DNA
＜213〉artificial sequence
<400> 12
ctatgggccc tgcagct 17
<210> 13
<211> 22
<212> DNA
＜213〉artificial sequence
<400> 13
ctaccggaga catgagaaga ag 22
<210> 14
<211> 19
<212> DNA
＜213〉artificial sequence
<400> 14
ctacattgcc cgacctacc 19
<210> 15
<211> 20
<212> DNA
＜213〉artificial sequence
<400> 15
ccgtcaccct cctcaagtat 20
<210> 16
<211> 22
<212> DNA
＜213〉artificial sequence
<400> 16
gaggagacaa gtcgtaacat gg 22
<210> 17
<211> 21
<212> DNA
＜213〉artificial sequence
<400> 17
tgacggtcca tgatgctata c 21
<210> 18
<211> 24
<212> DNA
＜213〉artificial sequence
<400> 18
gttttatttc agacgataat tgct 24
<210> 19
<211> 23
<212> DNA
＜213〉artificial sequence
<400> 19
agagcaaacc gcccagagta gaa 23
<210> 20
<211> 24
<212> DNA
＜213〉artificial sequence
<400> 20
gaagatgacc cggaagaaga tgct 24
<210> 21
<211> 21
<212> DNA
＜213〉artificial sequence
<400> 21
gccccctgcc ccaacatcgt g 21
<210> 22
<211> 21
<212> DNA
＜213〉artificial sequence
<400> 22
gtggcagcgg caggtggaag c 21
<210> 23
<211> 23
<212> DNA
＜213〉artificial sequence
<400> 23
ttaagggtcg aaggtggatt tag 23
<210> 24
<211> 24
<212> DNA
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<400> 24
tggtttggct aaggttgtct ggta 24
<210> 25
<211> 21
<212> DNA
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<400> 25
aatgcgggtt ctttgacgac a 21
<210> 26
<211> 23
<212> DNA
＜213〉artificial sequence
<400> 26
aaatggaacc ttgaccctct tga 23
<210> 27
<211> 19
<212> DNA
＜213〉artificial sequence
<400> 27
cagcattatt tggttgaca 19
<210> 28
<211> 17
<212> DNA
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<400> 28
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Claims (9)
1. Hybond membrane bar that is used for the diagnosis nonsyndromic sensorineural, it is characterized in that: comprise substrate, and be fixed on described suprabasil sudden change detection probes, each described sudden change detection probes is the corresponding gene mutation site that comprises a nonsyndromic sensorineural respectively, the gene mutation site of described nonsyndromic sensorineural comprises following site: the cDNA35 of GJB2 gene, cDNA176-191, cDNA235 and cDNA299-300 site, the cDNA538 site of GJB3 gene, the cDNA1555 of mtDNA 12s rRNA gene and cDNA1494 site, the cDNA2168 of SLC26A4 gene and IVS7-2 site, each described sudden change detection probes comprises the sequence of 15-25 base, comprises the mutating alkali yl of its corresponding nonsyndromic sensorineural gene at the medium position of described sequence, described sudden change detection probes is the DNA of SEQ ID NO.1 as follows～SEQ ID NO.9 sequence or its reverse complementary sequence:
SEQ ID NO.1：5’-CGATCCTGGGGGTGTGA-3’
SEQ ID NO.2：5’-CCTGCAGCCAGCTACGATCAC-3’
SEQ ID NO.3：5’-CTATGGGCCTGCAGCTG-3’
SEQ ID NO.4：5’-CTACCGGAGACGAGAAGAAGA-3’
SEQ ID NO.5：5’-CTACATTGCCTGACCTACCG-3’
SEQ ID NO.6：5’-CCGTCACCCTTCTCAAGTATAC-3’
SEQ ID NO.7：5’-TAGAGGAGGCAAGTCGTAACA-3’
SEQ ID NO.8：5’-TGACGGTCCGTGATGCTA-3’
SEQ ID NO.9：5’-TTATTTCGGACGATAATTGCT-3’。
2. Hybond membrane bar according to claim 1, it is characterized in that: also be fixed with the normal control probe in described substrate, itself and described sudden change detection probes are fixed on the different positions of described substrate.
3. Hybond membrane bar according to claim 2 is characterized in that: described normal control probe is at least one sequence in SEQ ID NO.10 as follows～SEQ ID NO.18 or the DNA of its reverse complementary sequence:
SEQ ID NO.10：5’-GATCCTGGGGGGTGTGA-3’
SEQ ID NO.11：5’-CAGGCTGCAAGAACGTGTGCTAC-3’
SEQ ID NO.12：5’-CTATGGGCCCTGCAGCT-3’
SEQ ID NO.13：5’-CTACCGGAGACATGAGAAGAAG-3’
SEQ ID NO.14：5’-CTACATTGCCCGACCTACC-3’
SEQ ID NO.15：5’-CCGTCACCCTCCTCAAGTAT-3’
SEQ ID NO.16：5’-GAGGAGACAAGTCGTAACATGG-3’
SEQ ID NO.17：5’-TGACGGTCCATGATGCTATAC-3’
SEQ ID NO.18：5’-GTTTTATTTCAGACGATAATTGCT-3’。
4. Hybond membrane bar according to claim 3, it is characterized in that: the corresponding gene mutation site that comprises a described nonsyndromic sensorineural of each described normal control probe, wherein, the gene mutation site that SEQ ID NO.10 is corresponding is cDNA35, the gene mutation site that SEQ ID NO.11 is corresponding is cDNA176-191, the gene mutation site that SEQ ID NO.12 is corresponding is cDNA235, the gene mutation site that SEQ ID NO.13 is corresponding is the cDNA299-300 site, the gene mutation site that SEQ ID NO.14 is corresponding is cDNA538, the gene mutation site that SEQ ID NO.15 is corresponding is cDNA1494, the gene mutation site that SEQ ID NO.16 is corresponding is cDNA1555, the gene mutation site that SEQ ID NO.17 is corresponding is cDNA2168, the gene mutation site that SEQ ID NO.18 is corresponding is IVS7-2.
5. Hybond membrane bar according to claim 4 is characterized in that: 3 ' or 5 ' end of described sudden change detection probes and/or normal control probe is with amino labeled.
6. the PCR primer of the sample gene fragment to be checked that is used for increasing, it is characterized in that: described PCR primer amplification gene mutation site claimed in claim 1, described PCR primer are following 5 pairs: SEQ ID NO.19 and SEQ ID NO.20; SEQ ID NO.21 and SEQ ID NO.22; SEQ ID NO.23 and SEQ ID NO.24; SEQ ID NO.25 and SEQ ID NO.26; SEQ ID NO.27 and SEQ ID NO.28; Wherein SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.27 are upstream primers; SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.26, SEQ ID NO.28 are downstream primers; The DNA sequence dna of described PCR primer is as follows:
SEQ ID NO.19：5’-AGAGCAAACCGCCCAGAGTAGAA-3’
SEQ ID NO.20：5’-GAAGATGACCCGGAAGAAGATGCT-3’
SEQ ID NO.21：5’-GCCCCCTGCCCCAACATCGTG-3’
SEQ ID NO.22：5’-GTGGCAGCGGCAGGTGGAAGC-3’
SEQ ID NO.23：5’-TTAAGGGTCGAAGGTGGATTTAG-3’
SEQ ID NO.24：5’-TGGTTTGGCTAAGGTTGTCTGGTA-3’
SEQ ID NO.25：5’-AATGCGGGTTCTTTGACGACA-3’
SEQ ID NO.26：5’-AAATGGAACCTTGACCCTCTTGA-3’
SEQ ID NO.27：5’-CAGCATTATTTGGTTGACA-3’
SEQ ID NO.28：5’-CCCTTGGGATGGATTTA-3’。
7. PCR primer according to claim 6, it is characterized in that: described amplified production comprises respectively following site: the cDNA35 of primer SEQ ID NO.19 and SEQ ID NO.20 amplification GJB2 gene, cDNA176-191, cDNA235, the cDNA299-300 site, the cDNA538 site of primer SEQ ID NO.21 and SEQ ID NO.22 amplification GJB3 gene, the cDNA1555 of primer SEQ ID NO.23 and SEQ ID NO.24 amplification mtDNA 12S rRNA gene, the cDNA1494 site, the cDNA2168 site of primer SEQ ID NO.25 and SEQ ID NO.26 amplification SLC26A4 gene, the IVS7-2 site of primer SEQ ID NO.27 and SEQ ID NO.28 amplification SLC26A4 gene.
8. according to claim 6 or 7 described PCR primers is characterized in that: 5 ' end of described upstream primer and/or downstream primer is with vitamin H or fluorescent mark.
9. test kit that is used for the diagnosis nonsyndromic sensorineural, it is characterized in that: comprise the Hybond membrane bar for the diagnosis nonsyndromic sensorineural claimed in claim 1, and the PCR reaction solution that contains the PCR primer be used to the sample gene fragment to be checked that increases claimed in claim 6.
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| WO2012016357A1 (en) | 2010-08-06 | 2012-02-09 | Capitalbio Corporation | Microarray-based assay integrated with particles for analyzing molecular interactions |
| CN102559913A (en) * | 2012-02-17 | 2012-07-11 | 中国人民解放军总医院 | Probe and kit for detecting common mutations of large vestibular aqueduct-related deafness gene |
| CN103760355B (en) | 2013-12-05 | 2015-09-16 | 博奥生物集团有限公司 | Micro-array chip detects the particle marker method of nucleotide sequence |
| CN104498609B (en) * | 2014-12-18 | 2016-08-31 | 亚能生物技术（深圳）有限公司 | The nucleic acid film bar of a kind of hereditary hearing impairment gene test and test kit |
| CN105200040B (en) * | 2015-10-22 | 2020-05-15 | 浙江安诺优达生物科技有限公司 | Kit for simultaneously detecting multiple deafness genes on single cell level |
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| CN105695582B (en) * | 2016-03-11 | 2023-12-22 | 东莞市儿科研究所 | Non-syndrome deafness gene detection membrane strip and PCR primer |
| CN106636438A (en) * | 2017-02-17 | 2017-05-10 | 深圳市亿立方生物技术有限公司 | Blotting membrane strip, PCR primer and kit for diagnosis of PAH gene mutation |
| CN114958571B (en) * | 2022-05-23 | 2023-11-21 | 博迈德生物科技（固安）有限公司 | Automatic change magnetic bead application of sample device |
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Denomination of invention: Hybridized membrane strips, PCR primers and kits for diagnosing non syndromic deafness Effective date of registration: 20220928 Granted publication date: 20130612 Pledgee: Bank of Communications Limited Shenzhen Branch Pledgor: SHENZHEN YILIFANG BIOTECHNOLOGY Co.,Ltd. Registration number: Y2022980016869 |