CN104694629A - Kit for detecting hereditary deafness susceptibility gene mutation - Google Patents
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Abstract
The invention relates to a multiplex ligation-dependent probe amplification kit for detecting hereditary deafness susceptibility gene mutation. The multiplex ligation-dependent probe amplification kit comprises a detection probe for detecting mutation site specificity, a universal primer, a positive DNA template, a DNA ligase, a connection buffer solution, a hybridization solution and a PCR reaction reagent, wherein the detection probe is composed of a pair of short probe and long probe and corresponds to one mutation site. The method is high in sensitivity and is accurate in result, and compared with the traditional detection technology, the kit can be used for completing high-throughput screening of multiple key hereditary deafness gene mutation diagnosis at one time, and has the advantages of high economical efficiency, high efficiency and high accuracy.
Description
Technical field
The invention belongs to biological technical field, particularly relating to a kind of multiple linking probe amplification detection kit for detecting hereditary hearing impairment susceptibility gene mutation.
Background technology
Deaf disease is one of modal heredopathia clinically, is also the common disease affecting human health.According to statistics, Chinese hearing and speech impairments person has 2,780 ten thousand people, accounts for 34% of national existing Disabled persons's sum, and wherein simple hearing loss has 2,004 ten thousand people, and the existing residual rate of hearing loss is about 2.11%; Just have 1 ~ 3 routine deaf youngster in every 1000 newborn infants, within less than 7 years old, deaf youngster about has 740,000 people, and with the annual speed increment of 2 ~ 30,000.The deaf youngster of new life of about 60% is caused by inherited genetic factors.In addition in a large amount of Delayed onset auditory dysesthesia patient, many patients are caused a disease because of autogene defect, or because of genetic flaw and polymorphism to specific environmental agents susceptible the hereditary hearing impairment that causes a disease, also have 40% caused by environmental factors.Reduce and deaf focus on early stage prevention and intervention, for the deafness that environmental factors causes, can by preventing infection, strengthen pregnancy period perinatal care, avoid the measures such as application ototoxic drug effectively to prevent; But for the prevention of hereditary hearing impairment, key be clear and definite deafness disease because of.
The hereditary hearing impairment overwhelming majority is heterogeneous stronger single gene inheritance disease, and wherein the hereditary hearing impairment case of 30% belongs to syndromic deafness, the most non-NSHL of remaining hereditary hearing impairment.Up to now, 40 relevant to NSHL autosomal recessive genes, 25 autosomal dominant genes, 3 X linked genes are cloned.Extensive deaf molecule epidemic disease-ology research shows, in population of China, modal Disease-causing gene comprises gap junction protein β 2GJB2 (Gap Junction protein beta 2), gap junction protein β 3GJB3 (Gap Junction protein beta 3), SLC26A4 (PDS), Mitochondrial DNA (Mitochondrion DNA) 12S rRNA.
The Cx26 of GJB2 genes encoding belongs to inserted by connexin gene family, a complete Gap junctions is formed with the inserted by connexin of flanking cell, these passages play an important role in Information Conduction and exchange of substance, have been the important channel of iuntercellular conversion of ionogen, second messenger and meta-bolites.After hair cell is subject to external stimulus, potassium ion enters cochlear endolymph liquid through inner ear hair cells pump around circuit, and inserted by connexin passage has regulating and controlling effect in the process.Cx26 is high expression level in the cochlear hair cell of the mankind, therefore GJB2 transgenation and deafness closely related.The sudden change of major part GJB2 gene coding region causes the phase shift mutation in protein translation process, produces non-functional protein, have impact on the structure of inserted by connexin, thus affect the normal opening and closing of passage.
GJB3 transgenation can cause dominant or recessive non-syndrome deaf.Preliminary function assessment result of study discloses interaction and dual-gene dissection, physiology and the function assessment basis of the Cx31 of Cx26 and the GJB3 genes encoding of GJB2 genes encoding.Although GJB3 gene and GJB2 gene be not on same karyomit(e), all encode and connect albumen, play a role in inner ear ionic equilibrium, cause deaf pattern according to diallele, likely GJB2 gene and GJB3 gene cause the morbidity of deafness patient jointly.
SLC26A4 gene, or claim PDS gene, manufactured protein is pendrin, is the transport protein of a chlorine and iodide ion.Generally think that it mainly plays the part of in inner ear and regulate ion equilibrium and the effect of endolymph fluid, if undergo mutation, inner ear developmental malformation will be caused and listen barrier.SLC26A4 gene belongs to recessive inheritance, the hereditary hearing impairment that the sudden change of diallele pathologic can cause.This transgenation and Dilated Vestibular Aqueduct Syndrome and Cochlear malformation have very close relationship.
Mitochondrial DNA Mutation easily causes drug induced deafness, and in pathogenic process, some patients has susceptibility to aminoglycosides antibiotics, and the medicine namely applying normal dose or trace just can cause patient's hearing loss, this susceptibility normally matrilinear inheritance.Hair cell injury of mitochondria causes its production capacity afunction, is the Cytological Basis of hearing impairment.MtDNA is the closed-circular DNA in human body cell matter, and total length is 16569bp, 13 key enzyme subunits in coding respiratory chain, 2 rRNA, 22 tRNA.Because mtDNA protects without histone, be exposed, so it suddenlys change comparatively nuclear gene height 10-20 times.MtDNA is not containing intron in addition, and subregion also has gene overlap phenomenon.Therefore any sudden change of mtDNA all can involve the some critical function districts in genome.
Above-mentioned Disease-causing gene comprises multiple mutational site, GJB2:35delG, 299delAT; GJB3:250G>A, 538C>T, 547G>A; SLC26A4 (PDS): 707T>C, 1174A>T, 1226G>A, 1229C>T, 1975G>C, 2027T>A, 2168A>G, IVS7-2A>G, IVS15+5G>A; MtDNA 12S rRNA:1494C>T, 1555A>G.Above 16 gene mutation sites belong to Chinese population mutantional hotspot, detect it, and in conjunction with clinical interrogation with have a medical check-up, can diagnose the hereditary hearing impairment of China more than 80%.
Present clinical comprises restricted length polymorphism analysis (RFLP), dot hybridization (ASO), genescan, dhplc analysis (DHPLC), direct Sequencing, method for gene chip etc. to the main method of hereditary hearing impairment Disease-causing gene diagnosis.But these technology or time and effort consuming, or cost is higher, or be difficult to the different mutational sites simultaneously detecting multiple gene.For solving the problem of clinical rapid detection and large-scale crowd examination, need a kind of efficient, high-throughput, fast, easily method detection transgenation.Multiplex ligation-dependent probe amplification has efficiently, can the feature of Parallel testing, fully meet this demand.Compared with chip technology, multiplex ligation-dependent probe amplification has that detection sensitivity is high, good stability, economical and efficient, short and flux advantages of higher consuming time, its superiority being applied to clinical deaf gene examination is obvious, as intervened for prevention before pregnant, pregnancy period and examination in postpartum etc.
In sum, the report adopting multiplex ligation-dependent probe amplification diagnostic techniques simultaneously to select some polymorphism mutation sites formation composite amplification detection system on GJB2, GJB3, SLC26A4 and chondriogen is had no in currently available technology, need badly a kind of easy and simple to handle and practical in practice, and fast, efficiently, economy, intuitively detection kit.
Summary of the invention
Technical problem to be solved
Technical problem to be solved by this invention is to provide a kind of based on multiplex ligation-dependent probe amplification, detect the detection kit of hereditary hearing impairment susceptibility gene mutation situation, only carry out detecting and the defect of complicated operation poor practicability for single or minority fractional mutations site to overcome available reagent box.
Technical scheme
One of technical scheme of the present invention is to provide a kind of test kit utilizing multiple linking probe augmentation detection hereditary hearing impairment susceptibility gene mutation site, comprises detection probes and universal primer; Described detection probes is each short probe and a corresponding mutational site of long probe; Described catastrophe point is:
GJB2：299-300delAT、35delG；
GJB3：250G>A、538C>T、547G>A；
12SrRNA：1494C>T、1555A>G；
SLC26A4：707T>C、IVS7-2A>G、1174A>T、1226G>A、1229C>T、IVS15+5G>A、1975G>C、2027T>A、2168A>G。
One of optimal technical scheme of mentioned reagent box is, the short probe corresponding to described mutational site respectively is:
GJB2：SEQ ID NO:1、SEQ ID NO:2；
GJB3：SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5；
12SrRNA：SEQ ID NO:6、SEQ ID NO:7；
SLC26A4：SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16。
Two of the optimal technical scheme of mentioned reagent box is that the probe sequence that the long probe 5 ' corresponding to described mutational site is held respectively is:
GJB2：SEQ ID NO:17、SEQ ID NO:18；
GJB3：SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21；
12SrRNA：SEQ ID NO:22、SEQ ID NO:23；
SLC26A4：SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32。
Three of the optimal technical scheme of mentioned reagent box is that described long probe length range is 30-600bp, and with in once multiple linking probe amplified reaction, the length difference of each bar long probe is within the scope of 3-100bp.
Four of the optimal technical scheme of mentioned reagent box is, described short probe is made up of general upstream primer sequence and probe sequence two portions from 5 ' end successively to 3 ' end; Described long probe is followed successively by probe sequence, non-human genome sequence and general downstream primer reverse complementary sequence from 5 ' end to 3 ' end; Described universal primer is non-human genome sequence SEQ ID NO:33 and SEQ ID NO:34.
Five of the optimal technical scheme of mentioned reagent box is that described short probe is by synthetic.
Six of the optimal technical scheme of mentioned reagent box is, described long probe is prepared by synthetic or PCR method.
Seven of the optimal technical scheme of mentioned reagent box is, described test kit also comprise in positive DNA profiling, DNA hybridization liquid, DNA ligase, connection buffered soln and PCR reaction reagent one or more.
Eight of the optimal technical scheme of mentioned reagent box is that described DNA hybridization liquid and detection probes coordinate the solution carrying out hybridization together with the genomic dna extracted or positive DNA profiling; Described connection buffered soln coordinates the solution carrying out ligation; PCR reaction reagent be with described ligation deactivation ligase enzyme after product carry out the agent combination of multiplexed PCR amplification reaction for template.
Nine of the optimal technical scheme of mentioned reagent box is, described PCR reaction reagent comprises archaeal dna polymerase, PCR reacts in buffered soln, dNTPs and magnesium ion one or more.
Two of technical scheme of the present invention is to provide the application of above-mentioned multiple linking probe amplification detection kit in non-diagnostic non-treatment field.
In described detection probes, the probe sequence that short probe sequence and long probe 5 ' are held is as following table:
Table 1 probe sequence table
Described universal primer sequence is as following table:
Table 2 universal primer sequence table
| SEQ ID NO | Sequence (5 '-3 ') |
| SEQ ID NO:33 | GCCGTTCCTTGTCTATGTTC |
| SEQ ID NO:34 | TTGTTCGCTATCGGTCTCTC |
Described ligase enzyme refers to the 5 '-P end had DNA chain 3 '-OH end and another DNA chain, makes the two generate phosphodiester bond, thus two sections of adjacent DNA chains are linked to be the enzyme of the effect of complete chain.Preferably, described ligase enzyme is T4 ligase enzyme, T7 ligase enzyme, heat-staple DNA ligase or Taq enzyme or its combination.
Described hybridization solution refers to have shock absorption to pH value of solution and the damping fluid promoting DNA hybridization.Preferably, described damping fluid is PBS damping fluid.
Described PCR reaction reagent can preferably include archaeal dna polymerase, PCR reacts buffered soln, dNTPs and MgCl
2.
Described archaeal dna polymerase refers to the enzyme that detecting target base sequence can be made to carry out pcr amplification reaction.Preferably, described archaeal dna polymerase is Tub enzyme, Tth enzyme, Taq enzyme, Pfu enzyme, Pfx enzyme, Tfl enzyme, Tsp enzyme, Tli enzyme or its combination.
The multiple linking probe amplification detection kit of hereditary hearing impairment Disease-causing gene of the present invention, can preferably use following method steps to realize testing goal:
(1) from biomaterial, genomic dna is extracted, or the positive DNA profiling of synthesis, as detection sample;
(2) add detection probes and hybridization solution in the sample to which, carry out hybridization;
(3) add in the solution after hybridization and connect damping fluid and ligase enzyme, carry out ligation, reaction terminates rear deactivation ligase enzyme, preferably, at 65 DEG C of deactivation 10min;
(4) with the product after ligation deactivation for template carries out multiplexed PCR amplification reaction;
(5) agarose gel electrophoresis or SDS-PAGE gel electrophoresis or capillary electrophoresis analysis are carried out to reacted product.
In aforesaid method step (1), described biomaterial is, such as, containing the blood of genomic dna, saliva, hair, tooth, cast-off cells, seminal stain or bone.
Described positive DNA profiling is the DNA containing mutational site, can be used for positive verification experiment; Source can be prepared by synthetic, also can be prepared by sequence clone.
Test kit principle of work of the present invention is as follows: with DNA sample to be checked for template, in Hybridization Buffer system, adds detection probes and hybridization solution.When in DNA profiling containing corresponding with detection probes 1 or several catastrophe point time, this catastrophe point combines with corresponding probe, and acquired results is the positive; When DNA profiling be normal gene namely containing catastrophe point time, the detection site of probe not in DNA profiling is combined, acquired results be feminine gender.After hybridization terminates, add the combining length probe of ligase enzyme and connect, then carry out multiplex polymerase chain re-action, with agarose gel electrophoresis or capillary detection amplified production to connect product for template.Because long probe length is different, the object band length that positive findings corresponding to the often pair of detection probes amplifies is different, can determine whether that amplified production length is in table 3 containing corresponding catastrophe point according to the presence or absence of object band in electrophoresis detection result and clip size.
Table 3
| Numbering | Target product length (bp) | Numbering | Target product length (bp) |
[0060]
| L1 | 173 | L9 | 394 |
| L2 | 185 | L10 | 404 |
| L3 | 256 | L11 | 430 |
| L4 | 278 | L12 | 460 |
| L5 | 311 | L13 | 466 |
| L6 | 338 | L14 | 486 |
| L7 | 354 | L15 | 528 |
| L8 | 381 | L16 | 547 |
Beneficial effect
This test kit is based on multiplex ligation-dependent probe amplification, for detecting hereditary hearing impairment susceptibility gene mutation situation, highly sensitive, result is accurate, disposablely can complete the high-throughput examination of multiple crucial hereditary hearing impairment transgenation diagnosis, there is advantage that is quick, economic, efficient and high-accuracy.
Accompanying drawing explanation
Fig. 1 is multiple linking probe amplification experimental principle figure, and wherein, 1 is that probe and target-gene sequence are hybridized, and 2 is hybridization probe connection, 3 products differed for pcr amplification linking probe acquisition length.
Fig. 2 is that negative sample detects gel electrophoresis figure.Left side swimming lane is DNA molecular amount mark, and be 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp from top to bottom, right lanes is negative sample.
Fig. 3-12 is 1-10 group blind check gel electrophoresis figure.The left side swimming lane of Fig. 3,5,6,8-12 is DNA molecular amount mark, and be 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp from top to bottom, right lanes is positive findings; Otherwise about Fig. 4 and Fig. 7.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
Embodiment 1: negative sample detects
The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, as: molecular cloning protocols handbook, or according to the condition that manufacturer advises.All inorganic chemical reagents and organic solvent are purchased from traditional Chinese medicines reagent company limited, blood DNA extracts test kit purchased from Beijing Quanshijin Biotechnology Co., Ltd, T4DNALigase-E is purchased from Niu Yinglun Bioisystech Co., Ltd, TransStart TopTaq warm start enzyme purchased from Beijing Quanshijin Biotechnology Co., Ltd, and primer and probe synthesize by Shanghai JaRa company.Eppendorf gradient type PCR amplification instrument is purchased from German Eppendorf company.(1) DNA extraction: to agree to or its guardian knows the inside story under condition at me, gather its peripheral blood.Adopt commercially available full formula gold blood DNA to extract test kit to extract, OD230/260 is greater than 2.0, OD280/260 and is greater than 1.8, and to demarcate its concentration be 100ng/ μ l.This sample clinical diagnosis confirmation is normal people's negative sample.
(2) hybridization: add following component in 200 μ l PCR pipe after, mixing, 65 DEG C of reactions 16 hours;
| Component | Volume |
| Sample (100ng/ μ l) | 2μl |
| Every bar probe (10 μm of ol/L, totally 16 to) | 0.8μl |
| 1 × hybridization solution | 7.5μl |
| ddH 2O | Mend to 40 μ l |
(3) ligation: add following reagent in the solution after hybridization, after mixing, at room temperature places 30min and carries out ligation, at 65 DEG C of deactivation 10min after reaction terminates;
| Component | Volume |
| 10 × connect damping fluid | 5μl |
| T 4Ligase enzyme | 1μl |
| ddH 2O | Mend to 50 μ l |
(4) PCR reaction: add following reagent in a new PCR pipe;
| Component | Volume |
| Ligation product | 3μl |
| 10 × PCR damping fluid | 5μl |
| dNTPs(2.5mmol/L) | 5μl |
| General upstream primer (10 μm of ol/L) | 1.5μl |
| General downstream primer (10 μm of ol/L) | 1.5μl |
[0075]
| Top Taq enzyme | 2μl |
| ddH 2O | Mend to 50 μ l |
PCR reaction conditions is:
(5) electrophoresis detection: adopt 3% agarose gel electrophoresis to detect PCR reaction product, electrophoresis result gel imaging instrument is taken pictures, and detected result as shown in Figure 1.When sample is negative, electrophoresis detection result has no amplified band, conforms to expected results.
Embodiment 2: carry out blind check with positive template
(1) DNA extraction: with the positive DNA of synthetic containing detection transgenation point for detecting sample.
(2) transgenation piont mark is 1-16 by hybridization: ascending according to amplified band, adopts accompanying software to extract 10 sets of numbers and carries out blind check.
Data randomly drawed by table 3
(3) according to extraction number, in hybridization system, the positive DNA sample containing single catastrophe point corresponding with extracting number is added.Add following component in 200 μ l PCR pipe after, mixing, at 65 DEG C of reaction 16h;
| Component | Volume |
| DNA profiling (10 μm of ol/L) | 2μl |
| Every bar probe (10 μm of ol/L) | 0.8μl |
| 1 × hybridization solution | 7.5μl |
| ddH 2O | Mend to 40 μ l |
(4) ligation: add following reagent in the solution after hybridization, after mixing, at room temperature places 30min and carries out ligation, at 65 DEG C of deactivation 10min after reaction terminates;
| Component | Volume |
| 10 × connect damping fluid | 5μl |
| T4 ligase enzyme | 1μl |
| ddH 2O | Mend to 50 μ l |
(5) PCR reaction: add following reagent in a new PCR pipe;
| Component | Volume |
| Ligation product | 3μl |
| 10 × PCR damping fluid | 5μl |
| dNTPs(2.5mmol/L) | 5μl |
| General upstream primer (10 μm of ol/L) | 1.5μl |
| General downstream primer (10 μm of ol/L) | 1.5μl |
| Top Taq enzyme | 2μl |
| ddH 2O | Mend to 50 μ l |
PCR reaction conditions is:
(6) electrophoresis detection: adopt 3% agarose gel electrophoresis to detect PCR reaction product, electrophoresis result gel imaging instrument is taken pictures, and detected result is as shown in Fig. 2 to Figure 11.Fig. 2 is the 1st group of blind check result, the amplification target stripe of visible 466,547bp; Fig. 3 is the 2nd group of blind check result, visible 173,311, the amplification target stripe of 466bp; Fig. 4 is the 3rd group of blind check result, visible 173,311, the amplification target stripe of 394bp; Fig. 5 is the 4th group of blind check result, the amplification target stripe of visible 381,430bp; Fig. 6 is 5 groups of blind check results, visible 185,256,381, the amplification target stripe of 430bp; Fig. 7 is 6 groups of blind check results, the amplification target stripe of visible 311,486bp; Fig. 8 is 7 groups of blind check results, visible 185,311,466, the amplification target stripe of 547bp; Fig. 9 is 8 groups of blind check results, visible 173,394, the amplification target stripe of 430bp; Figure 10 is 9 groups of blind check results, visible 173,278,381, the amplification target stripe of 460bp; Figure 11 is 10 groups of blind check results, visible 311,381,384,404,430, the amplification target stripe of 466bp.
(7) conclusion: above electrophoresis detection result, target stripe is significantly clear, and size, number all conform to expected results.
Claims (11)
1. utilize the test kit in multiple linking probe augmentation detection hereditary hearing impairment susceptibility gene mutation site, comprise detection probes and universal primer; Described detection probes is each short probe and a corresponding mutational site of long probe; Described catastrophe point is:
GJB2：299-300delAT、35delG；
GJB3：250G>A、538C>T、547G>A；
12SrRNA：1494C>T、1555A>G；
SLC26A4：707T>C、IVS7-2A>G、1174A>T、1226G>A、1229C>T、IVS15+5G>A、1975G>C、2027T>A、2168A>G。
2. test kit according to claim 1, is characterized in that, the short probe corresponding to described mutational site respectively is:
GJB2：SEQ ID NO:1、SEQ ID NO:2；
GJB3：SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5；
12SrRNA：SEQ ID NO:6、SEQ ID NO:7；
SLC26A4：SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16。
3. test kit according to claim 1, is characterized in that, the probe sequence that the long probe 5 ' corresponding to described mutational site is held respectively is:
GJB2：SEQ ID NO:17、SEQ ID NO:18；
GJB3：SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21；
12SrRNA：SEQ ID NO:22、SEQ ID NO:23；
SLC26A4：SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32。
4. test kit according to claim 1, is characterized in that, described long probe length range is 30-600bp; For in same once multiple linking probe amplified reaction, there is the length difference be separated on running gel between each bar long probe, preferably within the scope of 3-100bp, more preferably within the scope of 6-71bp.
5. test kit according to claim 1, is characterized in that, described short probe is made up of general upstream primer sequence and probe sequence two portions from 5 ' end successively to 3 ' end; Described long probe is followed successively by probe sequence, non-human genome sequence and general downstream primer reverse complementary sequence from 5 ' end to 3 ' end; Described universal primer is non-human genome sequence SEQ ID NO:33 and SEQ ID NO:34.
6. test kit according to claim 1, is characterized in that, described short probe is prepared by synthetic or PCR method.
7. test kit according to claim 1, is characterized in that, described long probe is prepared by synthetic or PCR method, and PCR preparation method is shown in another patent supporting with this patent.
8. test kit according to claim 1, is characterized in that, described test kit also comprise in positive DNA profiling, DNA hybridization liquid, DNA ligase, connection buffered soln and PCR reaction reagent one or more.
9. test kit according to claim 8, is characterized in that, described DNA hybridization liquid and detection probes coordinate the solution carrying out hybridization together with the genomic dna extracted or positive DNA profiling; Described connection buffered soln coordinates the solution carrying out ligation; PCR reaction reagent be with described ligation deactivation ligase enzyme after product carry out the agent combination of multiplexed PCR amplification reaction for template.
10. test kit according to claim 8 or claim 9, is characterized in that, described PCR reaction reagent comprises archaeal dna polymerase, PCR reacts in buffered soln, dNTPs and magnesium ion one or more.
The application of multiple linking probe amplification detection kit in non-diagnostic non-treatment field described in 11. claims 1.
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| CN106811533A (en) * | 2017-03-06 | 2017-06-09 | 亚能生物技术（深圳）有限公司 | A kind of hereditary hearing impairment gene detecting kit |
| CN106811533B (en) * | 2017-03-06 | 2020-10-16 | 亚能生物技术（深圳）有限公司 | Genetic deafness gene detection kit |
| CN107058588A (en) * | 2017-06-09 | 2017-08-18 | 北京博奥医学检验所有限公司 | A kind of hereditary hearing impairment genetic test product |
| CN108676869A (en) * | 2018-06-14 | 2018-10-19 | 中山大学达安基因股份有限公司 | A kind of method and kit of detection hereditary hearing impairment gene mutation site |
| CN108676869B (en) * | 2018-06-14 | 2021-11-05 | 广州达安基因股份有限公司 | Method and kit for detecting genetic deafness gene mutation site |
| CN116656804A (en) * | 2023-05-24 | 2023-08-29 | 北京阅微基因技术股份有限公司 | Genotyping kit for hereditary hearing loss |
| CN116656804B (en) * | 2023-05-24 | 2023-12-22 | 北京阅微基因技术股份有限公司 | Genotyping kit for hereditary hearing loss |
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| CN104694629B (en) | 2018-05-18 |
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