CN104404164A - Hereditary deafness gene mutation detection kit - Google Patents
Hereditary deafness gene mutation detection kit Download PDFInfo
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Abstract
The invention relates to a hereditary deafness gene mutation detection kit. The hereditary deafness gene mutation detection kit comprises sequencing primers, wherein the sequencing primers comprise sequences as follows: SEQ ID NO:1-36. The hereditary deafness gene mutation detection kit has the advantages of multiple detection site, wide coverage range, high positive rate, high clinical usage value, high sensitivity, high accuracy, high detection specificity, high repeatability and good detection stability and can be applied to premarital screening, prenatal diagnosis and hereditary deafness gene site screening and detection for fetuses during pregnancy and deaf people, so that the birth of deaf children and occurrence of tardive deafness can be greatly avoided, economic burdens on the society and families can be reduced, and the good birth and the good care can be realized.
Description
Technical field
The present invention relates to gene sequencing technology, particularly a kind of hereditary hearing impairment gene mutation detection kit.
Background technology
Deafness is one of modal sensory disturbance disease in world wide, and hereditary hearing impairment can be divided into non-syndrome deafness and syndromic deafness.According to the Second China National Sample Survey on Disability display, China's hearing loss number about 2,004 ten thousand people, accounts for 24.16% of Disabled persons's sum; The speech disabilities that deafness causes about 1,270,000 people, accounts for 1.53% of Disabled persons's sum.The sickness rate of congenital deafness in newborn infant is about 1/1000, and congenital deafness wherein over half is relevant with gene.
In non-syndrome deafness, GJB2, SLC26A4 and Mitochondrial DNA are the modal Disease-causing genes of China.GJB2 genes encoding connects albumen (connexin 26), participate in intercellular signal mediation and ion transport, the GJB2 gene of sudden change may cause connecting abnormal protein, and then affects the linkage function in intercellular substance, causes inner ear potassium ion reclaim obstacle and cause deafness.GJB2 transgenation and non-syndrome sex-controlled inheritance deafness closely related, comprise the deaf DFNA3 of autosomal dominant and autosomal recessive hereditary deafness DFNB1.In Chinese population, the prelingual infant of 26% ~ 33%, caused by GJB2 transgenation, accounts for 28% of recessive hereditary deafness, and the major way of its sudden change is that c.235delC recall rate is about 13.6% ~ 21.5%.GJB2 transgenation also can cause non-comprehensive autosomal dominant deaf.SLC26A4 transgenation and Dilated Vestibular Aqueduct Syndrome/Mondin deformity and Pendred syndrome (aqueductus vestibuli expand or accompany that Mondini is lopsided, nerve deafness and thyrocele) close relation.SLC26A4 genes encoding Pendred albumen.Pendred albumen forms primarily of hydrophobic amino acid, and its function mainly participates in the transhipment of iodine/chlorion.In inner ear, Pendred protein expression is in the neurocyte at ductus endolymphaticus and endolymphytic sac epithelial cell and Utriculus and sacculus edge.There is the abnormal transhipment that can affect intraor extracellular negatively charged ion in Pendred albumen, thus affects transmission sound and cause hearing loss.In China's Mainland, dilated vestibular aqueduct syndrome SLC26A4 transgenation recall rate is 97.9%, higher than Korea S's (78%), Japan's (92%) and Europe (40%).
At present, the test kit technological method used carrying out detecting for deaf gene clinically has PCR-RFLP method, micro-array chip method, ARMS-PCR method, fluorescence quantitative PCR method, gel electrophoresis, fluorescence melting curve method, fluorescent capillary electrophoresis tube method.Existing China clinically mainly be micro-array chip method and fluorescence quantitative PCR method, their major technique defect is as follows:
Micro-array chip method detects need extract the step such as (30min), pcr amplification (more than 2h), products thereof analysis (more than 3h) through whole blood DNA, detection time long (more than 6h), complex operation, can not meet the clinical demand to causing a large amount of examination of deaf gene; Because operating process is too much, easily cause sample cross contamination; Secondly, lower to the discrimination power of single base, easily there is false negative and false positive, the accuracy rate of result is affected; In addition, micro-array chip high cost, need special chip scanning instrument, testing cost is high; The amplification efficiency of common ARMS-PCR product is only the 1-10% of normal amplification, therefore amplification system deficient in stability; A reaction of four primer ARMS-PCR can only examine a mutational site.The shortcoming that regular-PCR product has is: need to judge genotype by stripe size when result interpretation, lacks intuitive.Detected through gel electrophoresis result easily causes PCR crossed contamination, detection sensitivity low and length consuming time; Utilize Taqman probe to carry out SNP abrupt climatic change, often detecting a site needs 2 probes, and has higher requirements to probe specificity.Probe synthesis cost is higher, has requirement to the port number of fluorescent PCR instrument, is unfavorable for promoting; It is few that fluorescence melting curve method detects a pipe detection site, and cannot meet the detection of deaf polygene multidigit point by the restriction of fluorescence dye passage; PCR-RFLP method detection technique complex operation, needs through endonuclease reaction, long reaction time, needs detected through gel electrophoresis easily to occur sample cross contamination simultaneously.Restriction enzyme site is subject to genovariation impact; Multiple PCR primer system is more complicated, more difficult design, can form mutual interference, competitive inhibition between multipair primer.Therefore detection site is more, and required primer is more, and it is higher that PCR system optimizes difficulty, and simultaneously by the restriction of capillary electrophoresis apparatus fluorescence channel, product promotion is more difficult, and general detection site is no more than at most 20;
The shortcoming that currently available products has is that detection site is few, can only detect hereditary hearing impairment hotspot mutation, cannot detect numerous rare sites, but deaf Disease-causing gene is many, mutational site is many, and above method all cannot meet quick clinically, easy, the requirement that sensing range is wide.There is undetected possibility when clinical application, further explanation cannot be made to the cause of disease of patient, reduce the value of Clinical practice.
Summary of the invention
Technical problem to be solved by this invention is: provide the hereditary hearing impairment gene mutation detection kit that a kind of sensing range is wide, speed is fast, highly sensitive.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is:
A kind of hereditary hearing impairment gene mutation detection kit, comprise sequencing primer, described sequencing primer comprises following sequence: according to upstream primer F1 and the downstream primer R1 of GJB2 gene design, and the sequence of described F1 is if the sequence of SEQ ID NO:1, described R1 is as SEQ ID NO:2; According to upstream primer F2 and the downstream primer R2 of GJB3 gene design, the sequence of described F1 is if the sequence of SEQ ID NO:3, described R1 is as SEQ ID NO:4; According to upstream primer F3-F16 and the downstream primer R3-R16 of SLC26A4 gene design, the sequence of described F3-F16 is if the sequence of SEQ ID NO:5-18, described R3-R16 is as SEQ ID NO:19-32; According to upstream primer F17-18 and the downstream primer R17-18 of 12S rRNA gene design, the sequence of described F17-F18 is if the sequence of SEQ ID NO:33-34, described R17-R18 is as SEQ ID NO:35-36.
Beneficial effect of the present invention is:
(1) sequencing primer of hereditary hearing impairment gene mutation detection kit of the present invention designs for GJB2 gene, SLC26A4 gene, 12S rRNA gene and GJB3 gene, 37 site mutations being positioned at GJB2 gene can be detected, be positioned at 2 site mutations of GJB3 gene, be positioned at 59 site mutations of SLC26A4 gene, be positioned at 7 site mutations of 12S rRNA gene, 105 sites altogether, have that detection site is many, scope is wide, hot spot mutation and the rare mutation of the multiple gene of hereditary hearing impairment can be detected.Existing product on the market all cannot detect rare site maybe can only detect the rare site of minority, there is undetected possibility.This test kit can make up the defect of currently available products, the rare site mutation that one-time detection is numerous, thus realizes that detection site is many, wide coverage, positive rate are high, the costly object of Clinical practice;
(2) may be used for pre-marital examination, antenatal diagnosis, the pregnancy period fetus and deaf crowd the examination of hereditary hearing impairment gene locus detect, condition is created for carrying out hereditary hearing impairment gene screening comprehensively, greatly can avoid the birth of deaf youngster and the generation of Delayed onset deafness, alleviate the economical load of social family, realize prenatal and postnatal care;
(3) hereditary hearing impairment gene mutation detection kit of the present invention has highly sensitive (minimum detectability is 5ng), accuracy high (reaching more than 99%), detection specificity high (reaching more than 99%), repeatability high (more than 99%) and detects the advantage of good stability.
Accompanying drawing explanation
Fig. 1 is the plastosome 1555A>G homozygote Sequencing chromatogram of the embodiment of the present invention;
Fig. 2 is the plastosome 1494C>T homozygote Sequencing chromatogram of the embodiment of the present invention;
Fig. 3 is the GJB2176del 16 heterozygote Sequencing chromatogram of the embodiment of the present invention;
Fig. 4 is the GJB2235del C heterozygote Sequencing chromatogram of the embodiment of the present invention;
Fig. 5 is the GJB2299del AT homozygote Sequencing chromatogram of the embodiment of the present invention;
Fig. 6 is the GJB3538C>T homozygote Sequencing chromatogram of the embodiment of the present invention;
Fig. 7 is the SLC26A4IVS7-2A>G heterozygote Sequencing chromatogram of the embodiment of the present invention;
Fig. 8 is the SLC26A42168A>G heterozygote Sequencing chromatogram of the embodiment of the present invention;
Fig. 9 is the SLC26A41174A>T homozygote Sequencing chromatogram of the embodiment of the present invention;
Figure 10 is the SLC26A41226G>A homozygote Sequencing chromatogram of the embodiment of the present invention;
Figure 11 is the SLC26A41229C>T homozygote Sequencing chromatogram of the embodiment of the present invention;
Figure 12 is the SLC26A42027T>A homozygote Sequencing chromatogram of the embodiment of the present invention;
Embodiment
By describing technology contents of the present invention in detail, realized object and effect, accompanying drawing is coordinated to be explained below in conjunction with embodiment.
The design of most critical of the present invention is: for GJB2 gene, SLC26A4 gene, 12S rRNA gene and GJB3 gene design sequencing primer, thus has that detection site is many, the advantage of wide coverage.
The present invention, according to the site of hereditary hearing impairment transgenation, designs special primer and increases, by sequencing reaction thus can carry out fast hereditary hearing impairment gene test site, high throughput testing.The present invention can detect 108 kinds of sudden changes in 105 mutational sites be positioned on hereditary hearing impairment four genes GJB2, GJB3, SLC26A4,12SrRNA, comprises hot spot mutation and rare mutation, and total primer 18 is right, corresponding special PCR primer 18 groups.
, to guarantee that increased PCR primer segment is single, special and to comprise detection site, there is not non-specific amplification in 1, sequencing primer design: sequencing primer is high to the specific requirements of primer in design simultaneously.The Tm of primer is too high, though can improve primer specificity, affects the joint efficiency of primer and template sequence, causes product amount low.Tm value is too low, though can improve joint efficiency, affects its specificity, easily occurs non-specific amplification.Balance between the specificity of primer and efficiency is the difficult point during sequencing primer of the present invention designs.Sequencing primer of the present invention, for obtain after a large amount of screening and optimizing, can be guaranteed that pcr amplification is specific and ensure its amplification efficiency simultaneously.In order to make the convenience of hereditary hearing impairment gene mutation detection kit of the present invention on using, by primer annealing temperature Uniting be about 58 DEG C (± 3-5 DEG C).The DNA sequence dna of GJB2, GJB3, SLC26A4,12SrRNA gene is obtained from ncbi database, and according to the Position Design primer in each mutational site, corresponding amplification 18 groups of PCR primer.Optimum primer after optimization and the corresponding relation of detection site as shown in table 1.Table 1 is the mapping table of sequencing primer and detection site.
Table 1
2, the determination of PCR reaction solution formula: the concentration range of primer storage liquid is at 0.1 ~ 0.5 μm of ol/L, MgCl
2final concentration selects 1.5mM-6mM, the mixed solution dNTP final concentration of isocyatic four kinds of deoxy adenosine triphosphate (dATP, dGTP, dCTP, dTTP) selects 100nM-300nM, archaeal dna polymerase final concentration selects 0.5-5IU/ reaction, utilize orthogonal test method, contrasted by great many of experiments, finally determine that optimum PCR reaction system is in table 2.Table 2 is PCR reaction solution formula table.
Table 2
3, the determination of PCR response procedures: the optimization of the present invention to PCR response procedures carries out on the basis of Standard PCR program.Standard PCR response procedures is as shown in table 3.Choose thermograde, time gradient, cycle number do orthogonal experiment, through great many of experiments, determine that peak optimization reaction program is as table 4.First 95 DEG C of denaturation 5min, then 95 DEG C of sex change 30s on ABI9700 instrument, 58 DEG C of annealing 30s, 72 DEG C extend 30s, and circulate 35 times, last 72 DEG C extend 10min again.Table 3 is Standard PCR response procedures table.Table 4 is preferred PCR response procedures table.
Table 3
Table 4
4, the PCR:18 group that checks order specific amplified product 2% agarose gel electrophoresis detects, and cuts PCR primer, uses the DNA gel of Axygen company to reclaim kits PCR primer.Get 1 μ L purified after each group of PCR primer use corresponding upstream and downstream primer (sequencing primer) to carry out order-checking PCR respectively on ABI9700 as template.Order-checking PCR system and response procedures are as shown in table 5, table 6.Table 5 is order-checking PCR system table.Table 6 is order-checking PCR response procedures table.
Table 5
Table 6
5, check order: adopt sodium-acetate/Ethanol Method to carry out purifying to sequencing reaction product, be loaded to 3500DX/3500xL DX sequenator, check order.Compare in NCBI nucleic acid database sequence after the splicing of sequencing sequence bioinformatics software, whether suddenly change to analyze each detection site.
The main operational steps of the detection method of hereditary hearing impairment gene mutation detection kit of the present invention is as follows: (1) pcr amplification: the people's heredity group DNA profiling adding 50-200ng respectively in every pipe PCR reaction solution carries out pcr amplification, response procedures is 95 DEG C of 5min first, then 95 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 30s, circulate 35 times, last 72 DEG C of 10min; (2) PCR primer electrophoresis detection and purifying: the PCR primer sepharose of 2% concentration carries out electrophoresis detection, and reclaim test kit recovery purifying with the DNA gel of Axygen company; (3) check order PCR: after adding purifying by table 6 in order-checking PCR reaction solution, PCR primer 1 μ L, upstream primer/downstream primer 1 μ L carries out pcr amplification, and response procedures is 96 DEG C of 1min first, then 96 DEG C of 10s, 50 DEG C of 5s, 60 DEG C of 4min, circulate 25 times, last 4 DEG C of insulations; (4) check order product purification: adopt sodium-acetate/Ethanol Method purifying order-checking PCR primer; (5) Genetic Analyser order-checking: carry out loading sequencing procedures by 3500DX/3500xL DX sequenator operation instructions; (6) data collection process and analysis: measured sequence compared in NCBI nucleic acid database, whether analyzing and testing site exists sudden change.
6, effect analysis
6.1 hereditary hearing impairment gene mutation detection kit of the present invention are used for the situation of clinical detection: the sample choosing 10 routine congenital deafness patients, the sample of 10 routine Dilated Vestibular Aqueduct Syndrome patients, the sample of 10 routine drug induced deafness patients and the sample of 10 routine Subjects With Normal Hearing groups, detect with hereditary hearing impairment gene mutation detection kit (this test kit) of the present invention.Detected result is as shown in table 7.Table 7 is the information slip that this test kit detects clinical sample.Table 8 is this test kit detected result and patient's Audiometry Comparative result table.
Table 7
Table 8
By table 7, table 8 is known, this test kit is used to detect clinical 40 routine samples, 10 examples are plastosome 1555A>G homozygous mutation as a result, 4 examples are SLC26A42168A>G homozygous mutation, 5 examples are SLC26A4IVS7-2 A>G homozygous mutation, 1 routine SLC26A41229C>T homozygous mutation, 5 examples are GJB2235del C homozygous mutation, 1 routine GJB2235del G and the sudden change of 299del AT double heterozygous, 4 routine GJB2299del AT homozygous mutations totally 30 routine positive sample, 10 routine negative sample.Compared with clinical audition detected result, its positive coincidence rate and negative match-rate are 100%.Repeated test experience is carried out to above-mentioned positive sample, choose 10 routine samples, different lot number product, different people (2 people) operates, within one day, do 2 times, do 2 days altogether, each sample of each test repeats 3 times and detects, evaluate repeatability, the detected result of each repeated test experience is all as shown in table 10.Table 10 is the repetition stability experiment table of this test kit.
Table 9
The performance index of 6.2 test kits: it is 5ng that this test kit can stablize the concentration minimum detectability detecting human blood genomic dna; The accuracy that this test kit detects hereditary hearing impairment gene reaches more than 99% in (in carry out 40 routine samples, 30 routine deafness patient sample standard deviation test positive results, the normal sample standard deviations of 10 example are detected as negative findings); This test kit detects hereditary hearing impairment gene mutation site specificity and reaches more than 99% in (in 40 routine samples, the sample of 10 routine hearing normal patient, detected result is feminine gender); The repeatability of this test kit is more than 99% (choose 10 routine samples, different lot number product, different people (2 people) operates, and does 2 times in one day, does 2 days altogether, and each sample of each test repeats 3 times and detects, and result is consistent); This product uses before the deadline and can meet above each index completely.
Embodiments of the invention one are: the hereditary hearing impairment gene mutation detection kit of the present embodiment by sequencing to 105 mutational sites on people's clinical gene group DNA sample four focus genes relevant to hereditary hearing impairment totally 108 kinds of sudden changes carry out qualitative detection, wherein GJB235, all there are two kinds of sudden changes in 176,187 3 sites.Can the details of detection site in table 1.This test kit, for deaf four focus genes, respectively at conserved regions design special primer, adopts PCR method in conjunction with sequencing technologies, detects the catastrophe of each gene.The moiety of the test kit of the present embodiment is in table 11.Table 11 is the moiety table of the test kit of the present embodiment.
Table 10
In less than-18 DEG C storages.Avoid multigelation.Test kit uncork be placed on 2 ~ 8 DEG C or less than-18 DEG C storages.Validity period is 6 months.Qualitative PCR instrument uses ABI Veriti DX Thermal Cycler, Applied Biosystems, Inc.; Unexpected rival 9600; Genetic Analyser uses ABI 3500DX/3500xL Dx Genetic Analyzer.
3, the method for inspection
3.1DNA obtains: " the trace sample genome DNA extracting reagent kit (centrifugal column type) " of human gene group DNA's recommendation Yaneng Biotechnology (Shenzhen) Co., Ltd. that this test kit is used extracts, also Available experimental room ordinary method (phenol-chloroform extraction process) or other test kit extract, and the whole blood DNA of test kit recommendation QIAGEN company extracts test kit.Extract gained DNA, ultraviolet spectrophotometer should be adopted to carry out DNA concentration and purity testing, and OD260/OD280 should reach 1.7 ~ 2.1, carries out if desired concentrating or diluting, and just can carry out test experience after DNA concentration being adjusted to 1 ~ 100ng/ μ L.
3.2PCR amplification
(1) reaction solution preparation: take out reaction solution from test kit, primer melts and mixing of vibrating in room temperature, the low-speed centrifugal several seconds.Get N number of (N=sample to be tested number of cases × 18+HL quality control product × 18+ blank tube contrast × 1) reaction tubes, prepare single part PCR reaction solution according to system shown in table 12, table 12 is single part PCR reaction solution component list.Note: forward and reverse primer amplification of H1 ~ H18 used respectively by every routine sample, expands 18 pipes altogether.
Table 11
(2) application of sample: get each 4 μ L of DNA sample to be measured, be added to respectively in corresponding PCR reaction solution pipe, cover tightly pipe lid, carry out mark, the low-speed centrifugal several seconds to be placed in PCR instrument and to record sample puts order.Blank pipe and Quality Control QC synchronously carry out.
(3) amplification condition: pcr amplification parameter the following is first 95 DEG C of 5min, then 95 DEG C of 15s, and 58 DEG C of 30s, 72 DEG C of 30s, circulate 35 times, last 72 DEG C of 5min.
PCR primer electrophoresis detection and purifying: get 3 ~ 5 μ L and increase, with 1.5% sepharose (inside adding suitable quantities of nucleic acid dyestuff), under 5V/cm voltage, electrophoresis is about 30min, observe and whether have object band (each stripe size sees the following form), as observed obvious single purpose band, then purifying is carried out to PCR primer.The PCR primer obtained after purifying is carried out electrophoresis or is surveyed OD concentration definite value (concentration range is 10 ~ 50ng), can check order.Table 13 is the mapping table of each sequencing primer and object fragment, and table 14 is each am-plified fragments and detection site mapping table.
Table 12
Table 13
3.3 order-checking PCR: each sequencing primer first follows water with the dilution proportion of 1:2 respectively, re-uses.PCR primer after definite value, prepares order-checking PCR system by table 5.Increase by table 6 parameter with qualitative PCR instrument.
3.4 order-checking product purifications: get the PCR pipe that order-checking PCR reaction terminates, each PCR reaction tubes adds 2 μ L 125mmol/L EDTA and 2 μ L 3mol/L sodium-acetate (pH5.2).Add 50 μ L 100% dehydrated alcohols again, cover tightly pipe lid, mixing, room temperature lucifuge places 15min.In 4 DEG C of 12,000g centrifugal 30min, carefully remove supernatant (if can not immediate operation, then centrifugal 3min again before removing supernatant) immediately.Often pipe adds 150 μ L precooling 70% ethanol, 4 DEG C of 12,000g centrifugal 10min, carefully go supernatant (to blot only by raffinate immediately as far as possible, if can not immediate operation, then centrifugal 3min again before removing supernatant), this step can repetitive operation once (optional).Room temperature lucifuge places 15 ~ 30min (allowing 70% ethanol volatilization totally).Add 10 μ L Hi-Di Formamide, vibration dissolving DNA, low-speed centrifugal several seconds.Sample is placed on qualitative PCR instrument, 95 DEG C of sex change 5min, puts rapidly after cooling 4min in ice, prepares loading electrophoresis.According to kits, recommend BigDyeR Terminator v3.1Cycle Sequencing Kit.
3.5 Genetic Analyser order-checkings: the order-checking product after sex change adds 96 orifice plates supporting with Genetic Analyser, builds, editing sample list.Seq_std_BDTV3.1_ASSYXL_P0P7 or Seq_std_BDTV3.1_ASSY_P0P7 with IVD mark is selected to check order according to sequenator model.When using 3500Dx, application Data CollectionR and Sequencing Analysis software carry out data collection and analysis.Sequencing result is kept at predeterminated position automatically, opens sequencing result analysis, obtain the file of .abl .phd.1 form after reaction terminates.
3.6 quality control standards: table 15 is quality control standard table.Everyly standard must be reached, otherwise all experiments should re-start.
Table 14
3.7 results judge: all sample standard deviations there will be each fragment of H1 ~ H18, and as wherein a pipe is without amplified band, then the experiment of this pipe is invalid; When there is heterozygous mutant gene type, some bases occur bimodal or there will be cover peak from some bases; According to genotypic difference, each peak figure is variant, and partial genotype sequencing result figure is shown in accompanying drawing 1-12 in detail.
Fig. 1 is plastosome 1555A>G homozygote Sequencing chromatogram; The collection of illustrative plates that base pair in Fig. 1 in circle is answered is shown as G, and this base is gene mutation site, then as shown in Figure 1, transgenation appears in plastosome 1555A>G homozygote; Fig. 2 is plastosome 1494C>T homozygote Sequencing chromatogram; The collection of illustrative plates that base pair in Fig. 2 in circle is answered is shown as T, and this base is gene mutation site, then as shown in Figure 2, transgenation appears in plastosome 1494C>T homozygote; Fig. 3 is GJB2176del 16 heterozygote Sequencing chromatogram; There is cover peak situation in the collection of illustrative plates that the base pair in Fig. 3 in circle is answered, this base is gene mutation site, then transgenation appears in GJB2176del 16 heterozygote as shown in Figure 3; Fig. 4 is GJB2235del C heterozygote Sequencing chromatogram; There is cover peak situation in the collection of illustrative plates that the base pair in Fig. 4 in circle is answered, this base is gene mutation site, then transgenation appears in GJB2235del C heterozygote as shown in Figure 4; Fig. 5 is GJB2299del AT homozygote Sequencing chromatogram; The collection of illustrative plates that base pair in Fig. 5 in circle is answered is base C, and this base is after gene mutation site AT one, then transgenation appears in GJB2299del AT homozygote as shown in Figure 5; Fig. 6 is GJB3538C>T homozygote Sequencing chromatogram; The collection of illustrative plates that base pair in Fig. 6 in circle is answered is base T, and this base is gene mutation site, then transgenation appears in GJB3538C>T homozygote as shown in Figure 6; Fig. 7 is SLC26A4IVS7-2A>G heterozygote Sequencing chromatogram; The collection of illustrative plates that base pair in Fig. 7 in circle is answered occurs bimodal, and this base is gene mutation site, then transgenation appears in SLC26A4IVS7-2A>G heterozygote as shown in Figure 7; Fig. 8 is SLC26A42168A>G heterozygote Sequencing chromatogram; The collection of illustrative plates that base pair in Fig. 8 in circle is answered occurs bimodal, and this base is gene mutation site, then transgenation appears in SLC26A42168A>G heterozygote as shown in Figure 8; Fig. 9 is SLC26A41174A>T homozygote Sequencing chromatogram; The collection of illustrative plates that base pair in Fig. 9 in circle is answered is base T, and this base is gene mutation site, then transgenation appears in SLC26A41174A>T homozygote as shown in Figure 9; Figure 10 is SLC26A41226G>A homozygote Sequencing chromatogram; The collection of illustrative plates that base pair in Figure 10 in circle is answered is base A, and this base is gene mutation site, then transgenation appears in SLC26A41226G>A homozygote as shown in Figure 10.
Figure 11 is SLC26A41229C>T homozygote Sequencing chromatogram; The collection of illustrative plates that base pair in Figure 11 in circle is answered is base T, and this base is gene mutation site, then transgenation appears in SLC26A41229C>T homozygote as shown in Figure 11.
Figure 12 is SLC26A42027T>A homozygote Sequencing chromatogram; The collection of illustrative plates that base pair in Figure 12 in circle is answered is base A, and this base is gene mutation site, then transgenation appears in SLC26A42027T>A homozygote as shown in Figure 12.
According to the detected result of clinical sample, the rfu value of this test kit order-checking peak figure raw data should be no less than 100rfu.Each experiment all will arrange quality control product, and quality control product reaches the interpretation that quality control standard can carry out detected result; By the template concentrations research to different genotype, determine that genomic dna minimum detectable concentration is 10ng; Test kit and other gene no cross reaction of human genome.
Result judges: adopt SeqScanner software to analyze, if the sequences match that each fragment sequence is corresponding with HL quality control product, be then judged to be the wild-type not detecting this site.Other are then saltant type.When there is deletion mutantion, the order-checking summit of wild-type and saltant type mixes, and causes from mutated site, and order-checking summit is chaotic.The sequencing result of each sample is 18 fragments comprising object site, and other mutational sites in each sequence also can detect simultaneously.
In sum, hereditary hearing impairment gene mutation detection kit provided by the invention has the advantage that detection site is many, wide coverage, positive rate are high, Clinical practice value is high, highly sensitive, accuracy is high, detection specificity is high, repeatability is high and detect good stability.
The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every equivalents utilizing specification sheets of the present invention and accompanying drawing content to do, or be directly or indirectly used in relevant technical field, be all in like manner included in scope of patent protection of the present invention.
Claims (4)
1. a hereditary hearing impairment gene mutation detection kit, is characterized in that, comprises sequencing primer, and described sequencing primer comprises following sequence:
According to upstream primer F1 and the downstream primer R1 of GJB2 gene design, the sequence of described F1 is as SEQID NO:1, and the sequence of described R1 is as SEQ ID NO:2;
According to upstream primer F2 and the downstream primer R2 of GJB3 gene design, the sequence of described F1 is as SEQID NO:3, and the sequence of described R1 is as SEQ ID NO:4;
According to upstream primer F3-F16 and the downstream primer R3-R16 of SLC26A4 gene design, the sequence of described F3-F16 is if the sequence of SEQ ID NO:5-18, described R3-R16 is as SEQ ID NO:19-32;
According to upstream primer F17-18 and the downstream primer R17-18 of 12S rRNA gene design, the sequence of described F17-F18 is if the sequence of SEQ ID NO:33-34, described R17-R18 is as SEQ ID NO:35-36.
2. hereditary hearing impairment gene mutation detection kit according to claim 1, is characterized in that, also comprises PCR reaction solution, and described PCR reaction solution comprises MgCl
2, PCR Buffer, dNTP mixed solution and archaeal dna polymerase.
3. hereditary hearing impairment gene mutation detection kit according to claim 1, is characterized in that, also comprises PCR sequencing reagent, and described PCR sequencing reagent comprises BigDye and 5 × Sequencing buffer.
4. hereditary hearing impairment gene mutation detection kit according to claim 1, is characterized in that, also comprise quality control product, and described quality control product is normal people's genomic dna.
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| CN111378736A (en) * | 2018-12-28 | 2020-07-07 | 迈基诺（重庆）基因科技有限责任公司 | Deafness related gene capturing kit and application thereof |
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| CN106367491A (en) * | 2016-09-23 | 2017-02-01 | 大连晶泰生物技术有限公司 | Kit for detecting deafness susceptibility genes |
| CN109504753A (en) * | 2017-09-13 | 2019-03-22 | 上海伯豪生物技术有限公司 | Hereditary hearing impairment related gene detection chip kit |
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| CN108753948A (en) * | 2018-06-11 | 2018-11-06 | 奥维森基因科技（天津）有限公司 | A kind of primer pair and its kit and method of detection hereditary hearing impairment |
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| CN111378736A (en) * | 2018-12-28 | 2020-07-07 | 迈基诺（重庆）基因科技有限责任公司 | Deafness related gene capturing kit and application thereof |
| CN109777867A (en) * | 2018-12-29 | 2019-05-21 | 广州凯普医药科技有限公司 | A kind of method that Overlap extension PCR combination Sanger sequencing detects deaf susceptibility gene mutation |
| CN109777867B (en) * | 2018-12-29 | 2019-12-20 | 广州凯普医药科技有限公司 | Method for detecting deafness susceptibility gene mutation by combining overlap extension PCR with Sanger sequencing |
| CN110229880A (en) * | 2019-07-02 | 2019-09-13 | 杭州艾迪康医学检验中心有限公司 | Detect primer, kit and the method for hereditary hearing impairment gene mutation site |
| CN111979313A (en) * | 2020-09-21 | 2020-11-24 | 郑州桑林生物科技有限公司 | Specific primer and kit for detecting pathogenic variation of deafness gene based on multiplex PCR and high-throughput sequencing technology and application |
| CN111979313B (en) * | 2020-09-21 | 2023-05-26 | 郑州桑林生物科技有限公司 | Specific primer, kit and application for detecting pathogenic variation of deafness gene based on multiplex PCR and high-throughput sequencing technology |
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