CN105255999A - Method for detecting 20 mutation sites of deaf genes - Google Patents
Method for detecting 20 mutation sites of deaf genes Download PDFInfo
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Abstract
The invention relates to a method for detecting 20 hot mutation sites of four deaf genes such as GJB2, SLC26A4, GJB3 and 12S rRNA in a clinical sample, designing amplimers and single base extension primers of different sites aiming at abnormal SNP gene sites of the four genes by a matrix-assistant laser desorption/ionization flight time mass spectrum detection method and then carrying out mass spectrometry of a specific SNP site genotype.
Description
Technical field
The present invention relates to the detection method detecting deaf 20 hot mutant site in clinical sample, particularly relate to and use the detection method of carrying out SNP gene type assay with multiple PCR technique binding matrix assisted laser desorption/ionization flight time mass spectrum (being called for short MALDI-TOFMS) technology by mass-spectrometric technique and many primer extension technique and MassARRAY combine with technique.
Technical background
Deafness is one of modal inherited disease clinically.According to the Second China National Sample Survey on Disability result display in 2006, China existing hearing speech disabilities person reaches 2,780 ten thousand people, accounts for the whole nation 8,296 ten thousand Disabled personss' 33.5%, wherein hearing loss person 20,040,000, occupy first of all kinds of deformity, and increasing with the speed increasing the deaf youngster of 3-10 ten thousand every year newly.According to current rate of growth, to the year two thousand fifty, hearing loss patient will rise to 3,231 ten thousand people.Deaf reason is caused to have environmental factors, inherited genetic factors or environmental factors and inherited genetic factors acting in conjunction.Wherein inherited genetic factors is integral part, it is reported, the deafness of 60% is had to be caused by hereditary defect, in addition in a large amount of Delayed onset auditory dysesthesia patients, also there are many patients to be also because the genetic flaw of self is caused a disease, or cause a disease because genetic flaw and polymorphism cause causing the increase of deaf environmental factors susceptibility.In recent years along with to the attention of health and sociomedical development, make environmental factors as infected, deafness that acoustic trauma and other diseases cause reduces gradually, more highlight the importance of inherited genetic factors.
The Disease-causing gene quantity that deafness relates to is many, has found more than 100 gene regions and 46 Disease-causing genes up to now, and there is numerous deaf mutational site in each gene, thus had higher gene and site genetic heterogeneity.Recently, the extensive deaf molecule epidemic disease-ology research carried out at home shows, quite a few non-syndrome deafness is only caused, as GJB2, SLC26A4, mtDNA12srRNA and GJB3 etc. by several transgenations few in number.This carries out deaf gene examination on a large scale for us and diagnosis provides theoretical foundation.
GJB2 transgenation was located first in 1997, and it accounts for 20% in children's prelingual, in children's nonsyndromic sensorineural (NSHI), account for 40%.GJB2 transgenation has certain proportion in Aisa people's hereditary hearing impairment, in Sinology prelingual (deaf before 5 years old fell ill) 26%.33%, caused by GJB2 transgenation, accounts for 28% of recessive hereditary deafness.GJB2 transgenation mode is complicated and changeable, has obvious racial diversify.The most common in different pathogenic mutation in the GJB2 gene detected at present is 30delG or 35delG sudden change.And in China massive epidemiology investigation display, GJB2 gene cause deaf mutation frequency the highest be 235delC, be secondly 299_300delAT and 176del16.In mediterranean country and U.S. heredity nonsyndromic sensorineural patient, the sudden change in 30delG or 35delG site accounts for 60-85%, and China is relatively low.In the research to GJB2 transgenation mode, find the modal sudden change sporting 167delT in Jew.The people such as Dai Piao are in the examination of 1680 of 18 province schools for the deaf of China routine non-syndrome deaf case GJB2 gene 235delC sudden change, find that the allelic carrying rate of 235delC is the highest, secondly be 299-300delAT, 176dell6bp, the allelic carrying rate of other pathogenic mutation is lower.The heredity nonsyndromic prelingual that clinical and epidemiological study shows about 80% is autosomal recessive inheritance.SLC26A4 is one of deaf modal Disease-causing gene of recessive hereditary, accounts for 5% ~ 10% of prelingual infant, is only second to GJB2 gene.SLC26A4 sudden change is the common cause of American-European crowd Pendred syndrome and non-syndromic cleft lip and palate, is also one of to cause the common disease of most of China hereditary hearing impairment fierce.
GJB3 gene was located in 1998 by during the Study of Chinas such as Xia Jiahui 2 autosomal dominant inheritance nonsyndromic sensorineural familys and clones.GJB3 is positioned human chromosomal 1p33-p35, and coding has 270 amino acid whose connection protein 31s, total length 3617bp.Its sudden change is common in Chinese population, for causing one of reason of autosomal dominant and recessive inheritance nonsyndromic sensorineural.1998, Xia Jiahuis etc. report two the earliest and cause the GJB3 of dominant inheritance high-frequency balance to suddenly change, they carry out examination to the family of 42 hereditary hearing impairments, a missense mutation has been found in the deaf family in a Zhejiang, 547th bit base of GJB3 gene coding region is mutated into A by G, makes No. 183 amino acid connecting PROTEIN C x31 become Methionin 183K by L-glutamic acid.They have also found a nonsense mutation in Hunan Huaihua's family simultaneously.538th bit base of GJB3 becomes T by C, causes No. 180 amino acid to become terminator codon (Cx31R180x).
1993, Prezant etc. proposed the dependency of plastosome A1555G point mutation and genes in aminoglycoside-induced deafness first.Some individuals has susceptibility to aminoglycoside medicaments, and namely the medicine of application normal dose or trace just can cause hearing loss, and the Drug-induced deafness that now witness line plastochondria A1555G has suddenlyd change and aminoglycoside antibiotics causes is closely related.Dai Piao etc. are in 2016 routine non-syndromic cleft lip and palate patient Mitochondrial DNA A1555G Mutation Screening of different province special-education school of China, and find positive case 57 example, recall rate is 2.83%.2004, mtDNA1494C > T sudden change was that another can apply by aminoglycoside medicaments single dose the mitochondrial gene mutation type causing deafness to have research to specify that again.
Based on a large amount of experimental studies, we have finally chosen 20 mutational sites of 4 genes (GJB2, GJB3, SLC26A4 gene and 12SrRNA): 35delG, 167delT, 176-191del16,299_300delAT and 235delC sudden change on GJB2 gene; 538C > T on GJB3 gene and 547G > A suddenlys change; 281C > T, 589G > A on SLC26A4 gene, IVS7-2A > G, 1174A > T, 1226G > A, 1229C > T, 1975G > C, 2027T > A, 2162C > T, 2168A > G and IVS15+5G > A suddenly change; And the 1494C > T of Mitochondrial DNA and 1555A > G suddenlys change.
Deaf gene detects the reason that can specify most of hereditary hearing impairment, by the analysis of deaf gene detected result, mode of inheritance can be determined, calculate deafness and send out risk again, accurate evaluation and explanation are made to the risk of sending out again of the risk of patient and kinsfolk thereof, carrier's risk, filial generation.By objective, guidance and intervening measure accurately, fundamentally preventing and block hereditary hearing impairment, is the important step and the means that realize the deaf inborn defect target of prevention.
Substance assistant laser desorpted ionized (matrixassistedlaserdesorption/ionization, MALDI) is a kind of pulsed Soft ionization techniques.Sample through ionizing is transferred in mass analyzer from ion source and analyzes, and obtains molecular weight.The ion produced due to MALDI ion source commonly uses flight time (timeofflight, TOF) mass analyzer is analyzed, use so MALDI is normal and TOF connects together, be called Matrix-assisted laser desorption ionization (MALDI-TOF-MS).Because mass-spectrometric technique and many primer extension technique and MassARRAY combine with technique use, multiple mutational site can be detected in a reaction system simultaneously, significantly reduce workload, improve detection flux, and reduce testing cost.
The present inventor applies matrix-assisted laser desorption/ionization ionization time of flight, by design PCR primer and
single-basic extension primer and carry out a kind of high accuracy of SNP gene type assay, high-throughput, low cost, fast detection method.
Summary of the invention
The object of this invention is to provide a kind of detection method of deaf mutational site, this detection method detects genomic dna with matrix-assisted laser desorption/ionization ionization time of flight, by the SNP site of 20 the deaf disease-relateds that increase, carry out single-basic extension again, make to extend 1 base in SNP site.Because the quality of ion is different, then also different according to the time length of flying in vacuum tubule again, so as to judging the size of mass of ion, thus judge the genotype in site.
In order to realize the present invention, have employed technical scheme:
(1) can SNPs site in specific detection genomic dna;
(2) primer sequence of multiple pcr amplification is carried out for a group-specific karyomit(e) SNPs site;
(3) primer sequence of multiple PCR extension is carried out for a group-specific karyomit(e) SNPs site;
(4) a group-specific karyomit(e) SNPs site is detected with Matrix-assisted laser desorption ionization method.
Based on above detection method following four step compositions again:
(1) synthetic primer sequence: the pcr amplification primer and the single-basic extension primer sequence that synthesize deaf mutator gene specificity SNPs site;
(2) multiplexed PCR amplification SNPs locus gene fragment: once increased by multiplex amplification system and cover the deaf mutated gene segment in specificity SNPs site;
(3) single-basic extension SNPs site: the SNPs site once being extended deaf mutator gene by multiple extension system;
(4) upper machine testing genotype: by Matrix-assisted laser desorption ionization method, detects data and analyzes the genotype in each mutational site.
According to pcr amplification primer of the present invention and extension primer, can the efficiency such as a hole the multiple SNP site of amplification and extend multiple SNP site, for these 20 deaf disease-related sites, the pcr amplification primer used and single-basic extension primer be respectively: for rs80338939 (35delG) site on GJB2 gene, pcr amplification primer is:
5’-ACGTTGGATGGTCCTAGCTAGTGATTCCTG-3’(SEQIDNO：01)；
5’-ACGTTGGATGTCTGGGTTTTGATCTCCTCG-3’(SEQIDNO：02)；
Single-basic extension primer is:
5’-TGCTAGTGGAGTGTTTGTTCACACCCCC-3’(SEQIDNO：03)；
For rs80338942 (167delT) site on GJB2 gene, pcr amplification primer is:
5’-ACGTTGGATGTCTGGGTTTTGATCTCCTCG-3’(SEQIDNO：04)；
5’-ACGTTGGATGGTCCTAGCTAGTGATTCCTG-3’(SEQIDNO：05)；
Single-basic extension primer is:
5’-CCGACTTTGTCTGCAACACCC-3’(SEQIDNO：06)；
For the 176_191del16 site on GJB2 gene, pcr amplification primer is:
5’-ACGTTGGATGGTCCTAGCTAGTGATTCCTG-3’(SEQIDNO：07)；
5’-ACGTTGGATGTCTGGGTTTTGATCTCCTCG-3’(SEQIDNO：08)；
Single-basic extension primer is:
5’-GGAAGTAGTGATCGTAGC-3’(SEQIDNO：09)；
For rs80338943 (235delC) site on GJB2 gene, pcr amplification primer is,
5’-ACGTTGGATGGTCCTAGCTAGTGATTCCTG-3’(SEQIDNO：10)；
5’-ACGTTGGATGTCTGGGTTTTGATCTCCTCG-3’(SEQIDNO：11)；
Single-basic extension primer is:
5’-AAGATCAGCTGCAGG-3’(SEQIDNO：12)；
For rs111033204 (299_300delAT) site on GJB2 gene, pcr amplification primer is:
5’-ACGTTGGATGGTCCTAGCTAGTGATTCCTG-3’(SEQIDNO：13)；
5’-ACGTTGGATGTCTGGGTTTTGATCTCCTCG-3’(SEQIDNO：14)；
Single-basic extension primer is:
5’-ATGAACTTCCTCTTCTTCTC-3’(SEQIDNO：15)；
For rs74315319 (the 538C > T) site on GJB3 gene, pcr amplification primer is:
5’-ACGTTGGATGACAGATGGTGAGTACGATGC-3’(SEQIDNO：16)；
5’-ACGTTGGATGTTCCTCTACCTGCTGCACAC-3’(SEQIDNO：17)；
Single-basic extension primer is:
5’-TGGACTGCTACATTGCC-3’(SEQIDNO：18)；
For rs74315318 (the 547G > A) site on GJB3 gene, pcr amplification primer is:
5’-ACGTTGGATGTTCCTCTACCTGCTGCACAC-3’(SEQIDNO：19)；
5’-ACGTTGGATGACAGATGGTGAGTACGATGC-3’(SEQIDNO：20)；
Single-basic extension primer is:
5’-AAGTAGGTGAAGATTTTCTTCT-3’(SEQIDNO：21)；
For SLC26A4_281 (the 281C > T) site on SLC26A4 gene, pcr amplification primer is:
5’-ACGTTGGATGTGACTCTCTCCACTAAGGG-3’(SEQIDNO：22)；
5’-ACGTTGGATGTCCCCAAATACCGAGTCAAG-3’(SEQIDNO：23)；
Single-basic extension primer is:
5’-CGTCATTTCGGGAGTTAGTA-3’(SEQIDNO：24)；
For rs111033380 (the 589G > A) site on SLC26A4 gene, pcr amplification primer is:
5’-ACGTTGGATGCACTTTCTCGTATCCAGCAG-3’(SEQIDNO：25)；
5’-ACGTTGGATGCTTGTAAGTTCATTACCTG-3’(SEQIDNO：26)；
Single-basic extension primer is:
5’-GTAAGTTCATTACCTGTATAATTC-3’(SEQIDNO：27)；
For rs201562855 (the 1174A > T) site on SLC26A4 gene, pcr amplification primer is:
5’-ACGTTGGATGCTGTGTCTTTCCTCCAGTGC-3’(SEQIDNO：28)；
5’-ACGTTGGATGGTAGGATCGTTGTCATCCAG-3’(SEQIDNO：29)；
Single-basic extension primer is:
5’-AGGAATTCATTGCCTTTGGGATCAGC-3’(SEQIDNO：30)；
For rs111033305 (the 1226G > A) site on SLC26A4 gene, pcr amplification primer is:
5’-ACGTTGGATGCTGTGTCTTTCCTCCAGTGC-3’(SEQIDNO：31)；
5’-ACGTTGGATGGTAGGATCGTTGTCATCCAG-3’(SEQIDNO：32)；
Single-basic extension primer is:
5’-CACCACTGCTCTTTCCC-3’(SEQIDNO：33)；
For rs111033220 (the 1229C > T) site on SLC26A4 gene, pcr amplification primer is:
5’-ACGTTGGATGGTAGGATCGTTGTCATCCAG-3’(SEQIDNO：34)；
5’-ACGTTGGATGCTGTGTCTTTCCTCCAGTGC-3’(SEQIDNO：35)；
Single-basic extension primer is:
5’-CTTTCCTCCAGTGCTCTCCTGGACGGCC-3’(SEQIDNO：36)；
For rs200455203 (the 1975G > C) site on SLC26A4 gene, pcr amplification primer is:
5’-ACGTTGGATGCAGAAAACCAGAACCTTACC-3’(SEQIDNO：37)；
5’-ACGTTGGATGCTGAGCTTCCAGTCAAAGTG-3’(SEQIDNO：38)；
Single-basic extension primer is:
5’-GCCAATCCATAGCCTT-3’(SEQIDNO：39)；
For rs111033318 (the 2027T > A) site on SLC26A4 gene, pcr amplification primer is:
5’-ACGTTGGATGCTGAGCTTCCAGTCAAAGTG-3’(SEQIDNO：40)；
5’-ACGTTGGATGCAGAAAACCAGAACCTTACC-3’(SEQIDNO：41)；
Single-basic extension primer is:
5’-AACCTTACCACCCGC-3’(SEQIDNO：42)；
For rs121908363 (the 2162C > T) site on SLC26A4 gene, pcr amplification primer is:
5’-ACGTTGGATGATGGAACCTTGACCCTCTTG-3’(SEQIDNO：43)；
5’-ACGTTGGATGAATGCGGGTTCTTTGACGAC-3’(SEQIDNO：44)；
Single-basic extension primer is:
5’-AAGGACACATTCTTTTTGA-3’(SEQIDNO：45)；
For rs121908362 (the 2168A > G) site on SLC26A4 gene, pcr amplification primer is:
5’-ACGTTGGATGAATGCGGGTTCTTTGACGAC-3’(SEQIDNO：46)；
5’-ACGTTGGATGATGGAACCTTGACCCTCTTG-3’(SEQIDNO：47)；
Single-basic extension primer is:
5’-TTGGTTCTGTAGATAGAGTATAGCATCA-3’(SEQIDNO：48)；
For rs111033313 (the IVS7-2A > G) site on SLC26A4 gene, pcr amplification primer is:
5’-ACGTTGGATGCCATATGAAATGGCAGTAGC-3’(SEQIDNO：49)；
5’-ACGTTGGATGCAAAATCCCAGTCCCTATTC-3’(SEQIDNO：50)；
Single-basic extension primer is:
5’-GTTTTTAACATCTTTTGTTTTATTTC-3’(SEQIDNO：51)；
For rs192366176 (the IVS15+5G > A) site on SLC26A4 gene, pcr amplification primer is:
5’-ACGTTGGATGTTCTATGGCAATGTCGATGG-3’(SEQIDNO：52)；
5’-ACGTTGGATGGGCCTATTCCTGATTGGAC-3’(SEQIDNO：53)；
Single-basic extension primer is:
5’-AAAACAAATTTCTAGGGATAAAATA-3’(SEQIDNO：54)；
For 12SrRNArs267606619 (1494C > T) site, pcr amplification primer is:
5’-ACGTTGGATGCACTTTCCAGTACACTTACC-3’(SEQIDNO：55)；
5’-ACGTTGGATGCCCAGAAAACTACGATAGCC-3’(SEQIDNO：56)；
Single-basic extension primer is:
5’-AGCGCGTACACACCGCCCGTCAC-3’(SEQIDNO：57)；
For rs267606617 (the 1555A > G) site on 12SrRNA, pcr amplification primer is:
5’-ACGTTGGATGCCCAGAAAACTACGATAGCC-3’(SEQIDNO：58)；
5’-ACGTTGGATGCACTTTCCAGTACACTTACC-3’(SEQIDNO：59)；
Single-basic extension primer is:
5’-CTTACCATGTTACGACTTG-3’(SEQIDNO：60)
The multiplex PCR that present method is mentioned refers to: the amplification reaction system of a mixing Multiple components in single hole; Derive from the genomic dna of sample extraction as template.Wherein multiplexed PCR amplification reaction conditions is: 95 DEG C of 2min; 95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 1min, 72 DEG C of 5min, 45 circulations; 4 DEG C of preservations.Single base extension condition is:
Present method analyzes the genotype of the SNP site of chromosome specific according to the length of extension products flight time in vacuum tubule.The SNP site that present method is selected is the genetic material be present in human body cell, in crowd, there is polymorphism, and the product fragment extended is between 15-30bp.Application matrix-assisted laser desorption/ionization ionization time of flight detects SNP site, and detect the flight time of extension products in vacuum tubule, the genotypic analysis of the SNP site for chromosome specific provides foundation.
Specificity SNP site sudden change on the said GJB2 gene of present method, the catastrophe of the site relevant with deafness on GJB2 gene selecting lot of documents result of study to show: rs80338943 (235delC), 176_191del, rs111033204 (299_300delAT), rs80338939 (35delG), rs80338942 (167delT) these 5 SNP site.
Specificity SNP site sudden change on the said GJB3 gene of present method, the catastrophe of the site relevant with deafness on GJB3 gene selecting lot of documents result of study to show: rs74315318 (547G → A), rs74315319 (538C → T) these 2 SNP site.
Specificity SNP site sudden change on SLC26A4 gene described in present method, the site relevant with deafness on SLC26A4 gene selecting lot of documents result of study to show: rs111033220 (1229C > T), rs201562855 (1174A > T), rs200455203 (1226G > A), rs192366176 (IVS15+5G > A), rs111033318 (2027T > A), rs200455203 (1975G > C), rs111033380 (589G > A), rs121908363 (2162C > T), SLC26A4_281 (281C > T), rs111033313 (IVS7-2A > G), the catastrophe of rs121908362 (2168A > G) these 11 SNP site.
Specificity SNP site sudden change on 12SrRNA gene described in present method, the catastrophe of the site relevant with deafness on 12SrRNA gene selecting lot of documents result of study to show: rs267606617 (m.1555A > G), rs267606619 (m.1494C > T) these 2 SNP site.
In brief, present method comprises multiplexed PCR amplification primer and extends primer; Select sample genomic dna as template; The SNP site of chromosome specific is increased; PCR is utilized to carry out single-basic extension to amplified production; Application matrix-assisted laser desorption/ionization ionization time of flight carries out the length distinguishing the flight time, and analytical data.
Be worth special instruction, present method can detect 11 sites of 5 sites of GJB2 gene, 2 sites of GJB3 gene, 2 sites of 12SrRNA and SLC26A4 gene simultaneously.By the optimization to present method, the SNP site of close together can be carried out in two holes increase and extend, therefore present method can be tested simultaneously the SNP site sudden change in the same gene that may exist in exceptional sample and analyzes.
Accompanying drawing explanation
Figure 1A, Fig. 2 A, Fig. 3 A, Fig. 4 A shows four normal sample essential spectrum analysis charts.Sample type is whole blood.
Figure 1B shows the abnormal mass spectroscopy figure of a GJB2 (GJB2_176-191del16).Sample type is whole blood.
Fig. 2 B shows the abnormal mass spectroscopy figure of a GJB3 (rs74315318/rs74315319).Sample type is whole blood.
Fig. 3 B shows the abnormal mass spectroscopy figure of a SLC26A4 (rs121908362).Sample type is whole blood.
Fig. 4 B shows the abnormal mass spectroscopy figure of a 12SrRNA (rs267606617).Sample type is whole blood.
Embodiment
Embodiment one: the use of detection method in normal people
1, used in the method reagent: OMEGAE.Z.N.A.
tMbloodDNAKit, AgenaComplete
goldGenotypingReagentSet10x384, PrimerP1P2Mix (500uMeach), PrimeP3Mix, dehydrated alcohol.
2, collection of specimens, transport and preservation:
(1) collection of specimens: sample is whole blood.Blood is conventional extracting vein blood 5ml, EDTA anti-freezing process.
(2) preserve: can detect immediately, preserve one week for 4 DEG C.
(3) transport: sample transports should adopt 0 DEG C of curling stone.
3, detecting step and interpretation of result:
(1) whole blood extracts genomic dna:
1)≤250 μ L blood are shifted to EP pipe, as hematopenia 250 μ L then supplies 250 μ L with ElutionBuffer.
2) 25 μ LOB proteolytic enzyme and 250 μ LBufferBL are added, high speed whirlpool concussion 25s mixing.
3) 65 DEG C of water-bath 10min, in water-bath process, whirlpool mixing in every 5min minute once.
4) add the dehydrated alcohol of 260 μ L, high speed whirlpool 20s mixes, of short duration centrifugal after with collection tube lid drop.
5) aforesaid liquid is proceeded to the HibindDNA pillar being enclosed within 2mL collection tube, the centrifugal 1min of 8000rpm, outwells waste liquid, changes collection tube.
6) add the centrifugal 1min of 500 μ LBufferHB8000rpm, outwell waste liquid.
7) add 700 μ LDNAWashBuffer, the centrifugal 1min of 8000rpm, outwells waste liquid.
8) add 700 μ LDNAWashBuffer, the centrifugal 1min of 8000rpm, outwells waste liquid.
9) pillar is reinstalled collection tube, the centrifugal 2min of 12000rpm.
10) pillar is put into clean 1.5mLEP pipe, add 100 μ LElutionBuffer (65 DEG C), after left at room temperature 5min, the centrifugal 2min of 12000rpm, retains DNA elutriant.
(2) pcr amplification:
Upper PCR instrument carries out amplified reaction, and its reaction conditions is as follows:
(3) SAP process:
Upper PCR instrument is reacted, and reaction conditions is as follows:
37℃for40min
85℃for5min
4℃forever
(4) single-basic extension:
Upper PCR instrument is reacted, and reaction conditions is:
(5) upper mass spectrograph data analysis
1, resin desalination reaction is carried out to single base extension product, centrifugal, make pitch deposition bottom 384 orifice plates;
2, be placed on auto sample applicator by 384 orifice plates after desalination, chip is placed on corresponding position;
3, the calibrate that 60 μ L are got in every hole is added to the corresponding position of chip, for the quality detecting this experiment chip used and point sample;
4, the sample processed in 384 holes is added to one to one the corresponding position of chip;
5, the chip after application of sample is placed in a mass spectrometer, the hole that editor's need detects, click START, until all complete;
6, open software MassARRAYTyper4.0, check experimental test data and carry out data analysis.
According to the genotype of the signal value analysis SNP site that mass spectrograph detects.Result collection of illustrative plates as shown in Figure 1.Wherein Figure 1A, Fig. 2 A, Fig. 3 A, Fig. 4 A is the interpretation of result collection of illustrative plates of the specific SNP site of normal people.
Embodiment 2: it is abnormal that application present method detects GJB2SNP site
Choose 1 part of blood sample, the doubtful deaf symptom of its patient, the test kit of Application Example 1 carries out extraction DNA according to above-mentioned standard program, then the SNP site of pcr amplification chromosome specific is carried out, utilize the process of SAP enzyme that the dNTP in step PCR step is lost activity, and then carry out single base extension, finally go up mass spectrograph and detect and data analysis.This site (GJB2_176-191del16) of GJB2 there occurs heterozygous deletion.It is abnormal that present method can realize realizing detecting GJB2SNP site fast.Wherein Figure 1B is the mass spectroscopy figure that GJB2 (GJB2_176-191del16) is abnormal.
Embodiment 3: it is abnormal that application present method detects GJB3SNP site
Choose 1 part of blood sample, the doubtful deaf symptom of its patient, the test kit of Application Example 1 carries out extraction DNA according to above-mentioned standard program, then the SNP site of pcr amplification chromosome specific is carried out, utilize the process of SAP enzyme that the dNTP in step PCR step is lost activity, and then carry out single base extension, finally go up mass spectrograph and detect and data analysis.This site (rs74315318) of GJB3 there occurs heterozygous mutant.It is abnormal that present method can realize realizing detecting GJB3SNP site fast.Wherein Fig. 2 A is the mass spectroscopy figure that GJB3 (rs74315318) is abnormal.
Embodiment 4: it is abnormal that application present method detects SLC26A4SNP site
Choose 1 part of blood sample, the doubtful deaf symptom of its patient, the test kit of Application Example 1 carries out extraction DNA according to above-mentioned standard program, then the SNP site of pcr amplification chromosome specific is carried out, utilize the process of SAP enzyme that the dNTP in step PCR step is lost activity, and then carry out single base extension, finally go up mass spectrograph and detect and data analysis.This site (rs121908362) of SLC26A4 there occurs heterozygous mutant.It is abnormal that present method can realize realizing detecting SLC26A4SNP site fast.Wherein Fig. 3 B is the mass spectroscopy figure that SLC26A4 (rs121908362) is abnormal.
Embodiment 5: it is abnormal that application present method detects 12SrRNASNP site
Choose 1 part of blood sample, the doubtful deaf symptom of its patient, the test kit of Application Example 1 carries out extraction DNA according to above-mentioned standard program, then the SNP site of pcr amplification chromosome specific is carried out, utilize the process of SAP enzyme that the dNTP in step PCR step is lost activity, and then carry out single base extension, finally go up mass spectrograph and detect and data analysis.This site (rs267606617) of 12SrRNA there occurs heterozygous mutant.It is abnormal that present method can realize realizing detecting 12SrRNASNP site fast.Wherein Fig. 4 B is the mass spectroscopy figure that 12SrRNA (rs267606617) is abnormal.
Claims (5)
1. detect a method for deaf gene 20 hot mutant site, it is characterized in that the method detecting 20 hot mutant site in people's deaf gene GJB2, SLC26A4, GJB3,12SrRNA, detection method is as follows:
(1) synthetic primer sequence: the pcr amplification primer and the single-basic extension primer sequence that synthesize deaf mutator gene specificity SNPs site;
(2) multiplexed PCR amplification SNPs locus gene fragment: once increased by multiplex amplification system and cover the deaf mutated gene segment in specificity SNPs site;
(3) single-basic extension SNPs site: the SNPs site once being extended deaf mutator gene by multiple extension system;
(4) upper machine testing genotype: by Matrix-assisted laser desorption ionization method, detects data and analyzes the genotype in each mutational site.
2. detection method according to claim 1, is further characterized in that deaf gene 20 hot spot mutations are respectively:
(1) rs80338943 (235delC) on GJB2 gene, 176_191del, rs111033204 (299_300delAT), rs80338939 (35delG), rs80338942 (167delT) 5 SNP mutational sites;
(2) on GJB3 gene: rs74315318 (547G → A) and rs74315319 (538C → T) 2 SNP mutational sites;
(3) 2rs111033220 (1229C > T) on SLC26A4 gene, rs201562855 (1174A > T), rs200455203 (1226G > A), rs192366176 (IVS15+5G > A), rs111033318 (2027T > A), rs200455203 (1975G > C), rs111033380 (589G > A), rs121908363 (2162C > T), SLC26A4_281 (281C > T), rs111033313 (IVS7-2A > G), rs121908362 (2168A > G) 11 SNP mutational sites,
(4) rs267606617 (m.1555A > G) on 12SrRNA gene and rs267606619 (m.1494C > T) 2 SNP mutational sites.
3. detection method according to claim 1, is further characterized in that the primer sequence described in detection method step (1) is for designing for 20 deaf hot mutant site, is respectively:
(1) for rs80338939 (35delG) site on GJB2 gene, pcr amplification primer is:
5’-ACGTTGGATGGTCCTAGCTAGTGATTCCTG-3’(SEQIDNO：01)
5’-ACGTTGGATGTCTGGGTTTTGATCTCCTCG-3’(SEQIDNO：02)
Single-basic extension primer is:
5’-TGCTAGTGGAGTGTTTGTTCACACCCCC-3’(SEQIDNO：03)
(2) for rs80338942 (167delT) site on GJB2 gene, pcr amplification primer is:
5’-ACGTTGGATGTCTGGGTTTTGATCTCCTCG-3’(SEQIDNO：04)
5’-ACGTTGGATGGTCCTAGCTAGTGATTCCTG-3’(SEQIDNO：05)
Single-basic extension primer is:
5’-CCGACTTTGTCTGCAACACCC-3’(SEQIDNO：06)
(3) for the 176_191del16 site on GJB2 gene, pcr amplification primer is:
5’-ACGTTGGATGGTCCTAGCTAGTGATTCCTG-3’(SEQIDNO：07)
5’-ACGTTGGATGTCTGGGTTTTGATCTCCTCG-3’(SEQIDNO：08)
Single-basic extension primer is:
5’-GGAAGTAGTGATCGTAGC-3’(SEQIDNO：09)
(4) for rs80338943 (235delC) site on GJB2 gene, pcr amplification primer is,
5’-ACGTTGGATGGTCCTAGCTAGTGATTCCTG-3’(SEQIDNO：10)
5’-ACGTTGGATGTCTGGGTTTTGATCTCCTCG-3’(SEQIDNO：11)
Single-basic extension primer is:
5’-AAGATCAGCTGCAGG-3’(SEQIDNO：12)
(5) for rs111033204 (299_300delAT) site on GJB2 gene, pcr amplification primer is:
5’-ACGTTGGATGGTCCTAGCTAGTGATTCCTG-3’(SEQIDNO：13)
5’-ACGTTGGATGTCTGGGTTTTGATCTCCTCG-3’(SEQIDNO：14)
Single-basic extension primer is:
5’-ATGAACTTCCTCTTCTTCTC-3’(SEQIDNO：15)
(6) for rs74315319 (the 538C > T) site on GJB3 gene, pcr amplification primer is:
5’-ACGTTGGATGACAGATGGTGAGTACGATGC-3’(SEQIDNO：16)
5’-ACGTTGGATGTTCCTCTACCTGCTGCACAC-3’(SEQIDNO：17)
Single-basic extension primer is:
5’-TGGACTGCTACATTGCC-3’(SEQIDNO：18)
(7) for rs74315318 (the 547G > A) site on GJB3 gene, pcr amplification primer is:
5’-ACGTTGGATGTTCCTCTACCTGCTGCACAC-3’(SEQIDNO：19)
5’-ACGTTGGATGACAGATGGTGAGTACGATGC-3’(SEQIDNO：20)
Single-basic extension primer is:
5’-AAGTAGGTGAAGATTTTCTTCT-3’(SEQIDNO：21)
(8) for SLC26A4_281 (the 281C > T) site on SLC26A4 gene, pcr amplification primer is:
5’-ACGTTGGATGTGACTCTCTCCACTAAGGG-3’(SEQIDNO：22)
5’-ACGTTGGATGTCCCCAAATACCGAGTCAAG-3’(SEQIDNO：23)
Single-basic extension primer is:
5’-CGTCATTTCGGGAGTTAGTA-3’(SEQIDNO：24)
(9) for rs111033380 (the 589G > A) site on SLC26A4 gene, pcr amplification primer is:
5’-ACGTTGGATGCACTTTCTCGTATCCAGCAG-3’(SEQIDNO：25)
5’-ACGTTGGATGCTTGTAAGTTCATTACCTG-3’(SEQIDNO：26)
Single-basic extension primer is:
5’-GTAAGTTCATTACCTGTATAATTC-3’(SEQIDNO：27)
(10) for rs201562855 (the 1174A > T) site on SLC26A4 gene, pcr amplification primer is:
5’-ACGTTGGATGCTGTGTCTTTCCTCCAGTGC-3’(SEQIDNO：28)
5’-ACGTTGGATGGTAGGATCGTTGTCATCCAG-3’(SEQIDNO：29)
Single-basic extension primer is:
5’-AGGAATTCATTGCCTTTGGGATCAGC-3’(SEQIDNO：30)
(11) for rs111033305 (the 1226G > A) site on SLC26A4 gene, pcr amplification primer is:
5’-ACGTTGGATGCTGTGTCTTTCCTCCAGTGC-3’(SEQIDNO：31)
5’-ACGTTGGATGGTAGGATCGTTGTCATCCAG-3’(SEQIDNO：32)
Single-basic extension primer is:
5’-CACCACTGCTCTTTCCC-3’(SEQIDNO：33)
(12) for rs111033220 (the 1229C > T) site on SLC26A4 gene, pcr amplification primer is:
5’-ACGTTGGATGGTAGGATCGTTGTCATCCAG-3’(SEQIDNO：34)
5’-ACGTTGGATGCTGTGTCTTTCCTCCAGTGC-3’(SEQIDNO：35)
Single-basic extension primer is:
5’-CTTTCCTCCAGTGCTCTCCTGGACGGCC-3’(SEQIDNO：36)
(13) for rs200455203 (the 1975G > C) site on SLC26A4 gene, pcr amplification primer is:
5’-ACGTTGGATGCAGAAAACCAGAACCTTACC-3’(SEQIDNO：37)
5’-ACGTTGGATGCTGAGCTTCCAGTCAAAGTG-3’(SEQIDNO：38)
Single-basic extension primer is:
5’-GCCAATCCATAGCCTT-3’(SEQIDNO：39)
(14) for rs111033318 (the 2027T > A) site on SLC26A4 gene, pcr amplification primer is:
5’-ACGTTGGATGCTGAGCTTCCAGTCAAAGTG-3’(SEQIDNO：40)
5’-ACGTTGGATGCAGAAAACCAGAACCTTACC-3’(SEQIDNO：41)
Single-basic extension primer is:
5’-AACCTTACCACCCGC-3’(SEQIDNO：42)
(15) for rs121908363 (the 2162C > T) site on SLC26A4 gene, pcr amplification primer is:
5’-ACGTTGGATGATGGAACCTTGACCCTCTTG-3’(SEQIDNO：43)
5’-ACGTTGGATGAATGCGGGTTCTTTGACGAC-3’(SEQIDNO：44)
Single-basic extension primer is:
5’-AAGGACACATTCTTTTTGA-3’(SEQIDNO：45)
(16) for rs121908362 (the 2168A > G) site on SLC26A4 gene, pcr amplification primer is:
5’-ACGTTGGATGAATGCGGGTTCTTTGACGAC-3’(SEQIDNO：46)
5’-ACGTTGGATGATGGAACCTTGACCCTCTTG-3’(SEQIDNO：47)
Single-basic extension primer is:
5’-TTGGTTCTGTAGATAGAGTATAGCATCA-3’(SEQIDNO：48)
(17) for rs111033313 (the IVS7-2A > G) site on SLC26A4 gene, pcr amplification primer is:
5’-ACGTTGGATGCCATATGAAATGGCAGTAGC-3’(SEQIDNO：49)
5’-ACGTTGGATGCAAAATCCCAGTCCCTATTC-3’(SEQIDNO：50)
Single-basic extension primer is:
5’-GTTTTTAACATCTTTTGTTTTATTTC-3’(SEQIDNO：51)
(18) for rs192366176 (the IVS15+5G > A) site on SLC26A4 gene, pcr amplification primer is:
5’-ACGTTGGATGTTCTATGGCAATGTCGATGG-3’(SEQIDNO：52)
5’-ACGTTGGATGGGCCTATTCCTGATTGGAC-3’(SEQIDNO：53)
Single-basic extension primer is:
5’-AAAACAAATTTCTAGGGATAAAATA-3’(SEQIDNO：54)
(19) for 12SrRNArs267606619 (1494C > T) site, pcr amplification primer is:
5’-ACGTTGGATGCACTTTCCAGTACACTTACC-3’(SEQIDNO：55)
5’-ACGTTGGATGCCCAGAAAACTACGATAGCC-3’(SEQIDNO：56)
Single-basic extension primer is:
5’-AGCGCGTACACACCGCCCGTCAC-3’(SEQIDNO：57)
(20) for rs267606617 (the 1555A > G) site on 12SrRNA, pcr amplification primer is:
5’-ACGTTGGATGCCCAGAAAACTACGATAGCC-3’(SEQIDNO：58)
5’-ACGTTGGATGCACTTTCCAGTACACTTACC-3’(SEQIDNO：59)
Single-basic extension primer is:
5’-CTTACCATGTTACGACTTG-3’(SEQIDNO：60)。
4. detection method according to claim 1, is further characterized in that the reaction conditions of pcr amplification in described detection method step (2) is: 95 DEG C of 2min; 95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 1min, 72 DEG C of 5min, 45 circulations; 4 DEG C of preservations.
5. detection method according to claim 1, is further characterized in that in described detection method step (3), single base extension condition is:
。
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| CN115678976A (en) * | 2022-10-31 | 2023-02-03 | 广州凯普医药科技有限公司 | Kit for simultaneously detecting 26 deafness susceptibility gene mutation sites based on time-of-flight mass spectrometry and application thereof |
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