CN102220434A - 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 Hybond membrane bar that is used for medically being used to diagnose the non-syndrome deafness, the invention still further relates to a kind of segmental polymerase chain reaction of sample gene to be checked that is used to increase (Polymerase Chain Reaction, initialism are PCR) primer; The invention still further relates to a kind of test kit that is used to diagnose the non-syndrome deafness.
Background technology
Deafness is a kind of modal mankind sensory system's defective, and high and treatment difficulty etc. is former thereby perplexing extensive patients and surrounding population thereof muchly because of its cause of disease complexity, incidence, 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 a major cause.Deafness can be caused by term single gene sudden change or heterogeneic complex mutation.Also can be and cause by environmental factors or gene and environment acting in conjunction.Deafness can be divided syndrome and type and non-syndrome type, the deafness that is attended by the pathology of other histoorgans belongs to syndrome and type hearing loss (syndromic hearing impairment, SHI), without the deafness of other symptoms belong to non-syndrome type hearing loss (nonyndromic hearing impairment, NSHI).And 70% hereditary hearing impairment shows as non-syndrome deafness (promptly except that deafness without other sings and symptomses).End on January 1st, 2011, cloned with non-syndrome deaf relevant 25 autosomal recessive genes, 36 autosomal dominant genes, 2 X linked genes, 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 non-syndrome deafness is only caused by several transgenations few in number, as GJB2, SLC26A4, mtDNA 12s rRNA and GJB3 etc., this carries out the deaf gene examination on a large scale 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), directly check order etc., these methods or can not be qualitative, perhaps time and effort consuming, required equipment and consumptive material costliness the more important thing is that these methods are difficult to simultaneously heterogeneic a plurality of mutational sites be detected.Though and gene chips can detect the different genes site simultaneously, its required equipment and consumptive material costliness 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: remedy above-mentioned the deficiencies in the prior art, propose a kind ofly to be used to diagnose the Hybond membrane bar of non-syndrome deafness, the segmental PCR primer of a kind of sample gene to be checked that is used to increase, and a kind of test kit that is used to diagnose the non-syndrome deafness; The present invention has high-throughput, high-level efficiency, advantage such as cheap, easy to use, helps realizing clinical rapid detection and large-scale crowd examination.
A kind of following technical scheme of Hybond membrane strip adoption that is used to diagnose the non-syndrome deafness of the present invention:
The described Hybond membrane bar that is used to diagnose the non-syndrome deafness, it is characterized in that: comprise substrate, and be fixed on described suprabasil sudden change detection probes, the corresponding respectively gene mutation site that comprises a non-syndrome deafness of the described sudden change detection probes of each bar, the gene mutation site of described non-syndrome deafness is at least one in the 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; The described sudden change detection probes of each bar comprises the sequence of 15-25 base, comprises the mutating alkali yl of its corresponding non-syndrome deaf gene at the medium position of described sequence.
Preferably, described sudden change detection probes is at least one sequence among 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 the 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 among SEQ ID NO.10 ~ SEQ ID NO.18 or the DNA of its reverse complementary sequence.
Preferably, the all corresponding gene mutation site that comprises a described non-syndrome deafness of each described normal control probe, wherein, the gene mutation site of SEQ ID NO.10 correspondence is cDNA35, the gene mutation site of SEQ ID NO.11 correspondence is cDNA176-191, the gene mutation site of SEQ ID NO.12 correspondence is cDNA235, the gene mutation site of SEQ ID NO.13 correspondence is the cDNA299-300 site, the gene mutation site of SEQ ID NO.14 correspondence is cDNA538, the gene mutation site of SEQ ID NO.15 correspondence is cDNA1494, the gene mutation site of SEQ ID NO.16 correspondence is cDNA1555, the gene mutation site of SEQ ID NO.17 correspondence is cDNA2168, and the gene mutation site of SEQ ID NO.18 correspondence is IVS7-2.
Preferably, 3 ' or 5 ' of described sudden change detection probes and/or normal control probe end has amino labeled.
A kind of segmental PCR primer of sample gene to be checked that is used to increase of the present invention adopts following technical scheme:
The described described gene mutation site of the segmental PCR primer amplification of sample gene to be checked claim 1 that is used to increase, 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 following site respectively: 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 has vitamin H or fluorescent mark.
A kind of following technical scheme of test kit employing that is used to diagnose the non-syndrome deafness of the present invention:
The described test kit that is used to diagnose the non-syndrome deafness comprises the Hybond membrane bar that the non-syndrome deafness is diagnosed in above-mentioned being used to, and the PCR reaction solution that contains the above-mentioned segmental PCR primer of sample gene to be checked that is used to increase.
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, because the roughly mid-way that is fixed on suprabasil sudden change detection probes includes the gene mutation site of non-syndrome deafness, can directly make a definite diagnosis by PCR blooming bar hybridization technique person's to be checked genotype, normal, heterozygous mutation, sudden change homozygote etc. is easy to judge have high-throughput, high-level efficiency, advantage such as cheap, easy to use.
Description of drawings
Fig. 1 be the embodiment of the invention do not detect non-syndrome deaf gene sudden change sample result example, its genotype is: N/N;
Fig. 2 is the non-syndrome deaf gene heterozygous mutation sample result example of the embodiment of the invention, and its genotype is: 35M/N;
Fig. 3 is the non-syndrome deaf gene sudden change homozygote sample result example of the embodiment of the invention, and its genotype is: 35M/35M;
Fig. 4 is the dual heterozygous mutation sample result of the non-syndrome deaf gene example of the embodiment of the invention, and its genotype is: 35M/235M.
Embodiment
Contrast accompanying drawing and the present invention is explained in detail below in conjunction with preferred embodiment.
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 the table 2,3 ' or 5 ' end band amino labeled of sudden change detection probes and/or normal control probe, the synthetic method is the DNA synthesis method of existing conventional.
Table 1:
Table 2:
Annotate: " M " representative sudden change detection probes, " N " represents the normal control probe.
The corresponding respectively gene mutation site that comprises a non-syndrome deafness of the described sudden change detection probes of each bar, the gene mutation site of non-syndrome deafness 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 bar sudden change detection probes comprises the sequence of 15-25 base, comprises the mutating alkali yl of its corresponding non-syndrome deaf gene at the medium position of sequence.
The all corresponding gene mutation site that comprises a non-syndrome deafness of each normal control probe, wherein, the gene mutation site of SEQ ID NO.10 correspondence is cDNA35, the gene mutation site of SEQ ID NO.11 correspondence is cDNA176-191, the gene mutation site of SEQ ID NO.12 correspondence is cDNA235, the gene mutation site of SEQ ID NO.13 correspondence is the cDNA299-300 site, the gene mutation site of SEQ ID NO.14 correspondence is cDNA538, the gene mutation site of SEQ ID NO.15 correspondence is cDNA1494, the gene mutation site of SEQ ID NO.16 correspondence is cDNA1555, the gene mutation site of SEQ ID NO.17 correspondence is cDNA2168, and the gene mutation site of SEQ ID NO.18 correspondence is IVS7-2.
1.2, the preparation of Hybond membrane bar
A. use printer to go up and print site array in 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. (10mmol/L Tris is pH8.0) with 18 probe dilution to 5 μ mol/L to use probe dilution liquid respectively;
D. by the site array of printing on the nylon membrane bar, 18 probes are put respectively by probe title correspondence be added on the corresponding site of nylon membrane the carboxyl generation crosslinking reaction on amino on the probe and nylon membrane surface;
E. after treating nylon membrane bar drying, the NaOH vacuolar membrane of usefulness 0.1mol/L 5 minutes is with the unreacted carboxyl in sealing nylon membrane surface;
F. wash nylon membrane with pure water, dry back is standby.
Table 3: the probe array on the nylon membrane bar is as follows:
Annotate: " M " representative sudden change detection probes, " N " represents the normal control probe.
2, PCR primer amplification
Position relation according to this project 9 mutational sites to be checked, 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 HL1F, HL2F, HL3F, HL4F, HL5F respectively; 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 HL1R, HL2R, HL3R, HL4R, HL5R respectively; In this table, cDNA35, cDNA176-191, cDNA235, the cDNA299-300 site of primer HL1F and HL1R amplification GJB2 gene, the cDNA538 site of primer HL2F and HL2R amplification GJB3 gene, cDNA1555, the cDNA1494 site of primer HL3F and HL3R amplification mtDNA 12S rRNA gene, 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 has vitamin H or fluorescein (as Cy3, Cy5 etc.) mark.The embodiment of the invention is for to carry out biotin labeling to downstream primer (HL1R, HL2R, HL3R, HL4R, HL5R), the product behind its pcr amplification can with the probe hybridization in the embodiment of the invention, the dna sequence dna of PCR primer is as shown in table 5.
Table 5:
2.1, extract template DNA: use other commercial kit from patients'blood, to extract template DNA.
2.2, synthetic 10 primers: the synthetic method is the DNA synthesis method of existing conventional.
2.3, preparation PCR reaction solution: be mixed with the PCR reaction solution of 25 μ L/ person-portions, each composition and concentration are respectively in the PCR reaction solution of everyone part: 10 primer concentrations are respectively 0.2 μ mol/L, the Taq enzyme is that 0.1U/ μ L, 1 * PCR Buffer, 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 pre-sex change 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 behind the pcr amplification comprises patient's to be measured dna fragmentation.
3, hybridization and colour developing
3.1. hybridization
With the product behind the pcr amplification and the nylon membrane bar in the step 1.2 join 12mL hybridization solution (2 * SSC, 0.1%SDS, pH7.4) in, 42 ℃ of hybridization are more than 2 ~ 4 hours in the hybridization case.
3.2. wash film
With the nylon membrane bar transfer to the washing lotion that is preheated to 42 ℃ (0.5 * SSC, 0.1%SDS, pH7.4) in, in 42 ℃ of washings 15 minutes.
C. peroxidase (Peroxidase, initialism are POD) is hatched
The film bar is put into the solution of the strepto-affinity element of freshly prepared 0.25U/mL-POD(streptin-POD), hatched 15 minutes for 37 ℃, strepto-affinity vitamin H plain and in the PCR product is combined, and POD is connected on the PCR product, the streptin-POD that flush away is unnecessary.
D. colour developing
Now join colour developing liquid (0.1mol/L Trisodium Citrate, 0.1mg/mL TMB, 0.0015% H
2O
2), the film bar is put into colour developing liquid lucifuge colour developing 15 minutes, there is the film bar correspondent probe position of hybridization product can show blue spot.
4, the result judges
Shown in Fig. 1-4, the colour developing result of film bar by naked eyes judges judge with having or not of each site color signal whether sample to be checked exists transgenation, and is sudden change homozygote or heterozygote.If the position of all normal control probe correspondences colour developing on the nylon membrane bar, not developing the color in the position of all sudden change detection probes correspondences, then can be judged as this test sample and not detect sudden change, and genotype is abbreviated as N/N(such as Fig. 1).If the position colour developing of a sudden change detection probes correspondence is arranged, and the position of all normal control probe correspondences all develops the color, and then 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 the position colour developing of a sudden change detection probes correspondence is arranged, and do not develop the color in the position of its corresponding normal control probe correspondence, then this sample can be judged position sudden change homozygote, and genotype is abbreviated as M/M, and all adds the site (as Fig. 3) of undergoing mutation before two M.If the position colour developing of two sudden change detection probes correspondences is arranged, and also all develop the color in the position of its corresponding two normal control probe correspondences, then this sample can be judged as dual heterozygous mutation, genotype is abbreviated as M/M, and adds two mutational sites (in no particular order) (as Fig. 4) before two M respectively.
4 kinds of situations of in the embodiment of the invention the above are the situation that most cases may occur, if the result exceeds above-mentioned 4 kinds of situations, are confirming under the errorless situation of experimental implementation, should each case concrete analysis result.
At modal 9 kinds of heredity non-syndrome deaf gene mutation types among the Chinese, be fixed on the film bar by the sudden change detection probes that will contain 9 kinds of heredity non-syndrome deaf gene mutational 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 heredity non-syndrome deafness 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 accuracy height, high specificity, can detect advantages such as the recessive carrier of gene.Therefore, should in pre-marital medical check-up or pregnant inspection, use this method to aim at father and mother and carry out gene test, then according to circumstances, in case of necessity fetus be carried out antenatal detection, so just can significantly reduce the birth of the deaf infant of heredity non-syndrome.
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 accuracy height, high specificity, can detect 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 is applicable to that hospital promotes the use of at home on a large scale.
Above content be in conjunction with concrete preferred implementation to further describing that the present invention did, 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 are 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 to diagnose Hybond membrane bar, PCR primer and the test kit of non-syndrome deafness
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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
＜213〉artificial sequence
<400> 24
tggtttggct?aaggttgtct?ggta 24
<210> 25
<211> 21
<212> DNA
＜213〉artificial sequence
<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
＜213〉artificial sequence
<400> 28
cccttgggat?ggattta 17
Claims (10)
1. Hybond membrane bar that is used to diagnose the non-syndrome deafness, it is characterized in that: comprise substrate, and be fixed on described suprabasil sudden change detection probes, the corresponding respectively gene mutation site that comprises a non-syndrome deafness of the described sudden change detection probes of each bar, the gene mutation site of described non-syndrome deafness is at least one in the 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; The described sudden change detection probes of each bar comprises the sequence of 15-25 base, comprises the mutating alkali yl of its corresponding non-syndrome deaf gene at the medium position of described sequence.
2. Hybond membrane bar according to claim 1 is characterized in that: described sudden change detection probes is at least one sequence among SEQ ID NO.1 ~ SEQ ID NO.9 or the DNA of its reverse complementary sequence.
3. Hybond membrane bar according to claim 1 and 2 is characterized in that: also be fixed with the normal control probe in the described substrate, itself and described sudden change detection probes are fixed on the different positions of described substrate.
4. Hybond membrane bar according to claim 3 is characterized in that: described normal control probe is at least one sequence among SEQ ID NO.10 ~ SEQ ID NO.18 or the DNA of its reverse complementary sequence.
5. Hybond membrane bar according to claim 4, it is characterized in that: all corresponding gene mutation site that comprises a described non-syndrome deafness of each described normal control probe, wherein, the gene mutation site of SEQ ID NO.10 correspondence is cDNA35, the gene mutation site of SEQ ID NO.11 correspondence is cDNA176-191, the gene mutation site of SEQ ID NO.12 correspondence is cDNA235, the gene mutation site of SEQ ID NO.13 correspondence is the cDNA299-300 site, the gene mutation site of SEQ ID NO.14 correspondence is cDNA538, the gene mutation site of SEQ ID NO.15 correspondence is cDNA1494, the gene mutation site of SEQ ID NO.16 correspondence is cDNA1555, the gene mutation site of SEQ ID NO.17 correspondence is cDNA2168, and the gene mutation site of SEQ ID NO.18 correspondence is IVS7-2.
6. Hybond membrane bar according to claim 5 is characterized in that: 3 ' or 5 ' end of described sudden change detection probes and/or normal control probe has amino labeled.
7. segmental PCR primer of the sample gene to be checked that is used to increase is characterized in that: the described gene mutation site of described PCR primer amplification claim 1, and 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.
8. PCR primer according to claim 7, it is characterized in that: described amplified production comprises following site respectively: 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.
9. according to claim 7 or 8 described PCR primers, it is characterized in that: 5 ' end of described upstream primer and/or downstream primer has vitamin H or fluorescent mark.
10. test kit that is used to diagnose the non-syndrome deafness, it is characterized in that: comprise the described Hybond membrane bar that is used to diagnose the non-syndrome deafness of claim 1, and the PCR reaction solution that contains the described segmental PCR primer of sample gene to be checked that is used to increase of claim 7.
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| CN105200040A (en) * | 2015-10-22 | 2015-12-30 | 安诺优达基因科技(北京)有限公司 | Reagent kit for simultaneously detecting multiple deafness genes on single cell level |
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| CN105695582A (en) * | 2016-03-11 | 2016-06-22 | 东莞市儿科研究所 | Non-syndromic deafness gene detection membrane strip and PCR (polymerase chain reaction) 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 |
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| 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 |
| US10982267B2 (en) | 2013-12-05 | 2021-04-20 | Capitalbio Technology Corporation | Luminophore-labeled molecules coupled with particles for microarray-based assays |
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| CN104498609A (en) * | 2014-12-18 | 2015-04-08 | 亚能生物技术（深圳）有限公司 | Nucleic acid membrane strip and kit for detecting gene of hereditary hearing loss |
| CN105200040A (en) * | 2015-10-22 | 2015-12-30 | 安诺优达基因科技(北京)有限公司 | Reagent kit for simultaneously detecting multiple deafness genes on single cell level |
| 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 |
| CN105695582A (en) * | 2016-03-11 | 2016-06-22 | 东莞市儿科研究所 | Non-syndromic deafness gene detection membrane strip and PCR (polymerase chain reaction) primer |
| CN105648089A (en) * | 2016-03-11 | 2016-06-08 | 东莞市第八人民医院 | Hepatitis B surface antigen immune escape mutant gene detection membrane strip and PCR primer |
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| 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 |
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