CN102534030B - Kit for jointly detecting four deafness predisposing genes and application thereof - Google Patents
Kit for jointly detecting four deafness predisposing genes and application thereof Download PDFInfo
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Abstract
The invention discloses a fluorescent detection kit for detecting four deafness predisposing genes simultaneously. The kit comprises reagents before amplification and reagents after amplification, wherein the reagents before amplification comprise a polymerase chain reaction (PCR) buffer solution, a reaction mixture of MgCl2 and deoxyribonucleoside triphosphates (DNTPs), Taq DNA polymerase, ultrapure water, and a primer mixture for amplifying loci of GJB2235delC, 12S rRNA 1555A>G mutation, 1494C>T mutation, IVS7-2A>G and Amelogenin at high specificity; and the reagents after amplification comprise a genotyping standard and an internal standard. Deafness gene loci of the GJB2235delC, 12S rRNA 1555A>G mutation, 1494C>T mutation, IVS7-2A>G and Amelogenin are simultaneously detected at high sensitivity and high specificity by combining a fluorescent labeling technology, a linolenic acid (LNA) nucleoside monomer doping-primer modification technology and a capillary electrophoresis technology for the first time, manpower and material resources and time are greatly saved, and pollution due to multi-step operation is prevented.
Description
Technical field
The present invention relates to a kind of 4 deaf sick tumor susceptibility gene fluorescence detection reagent kits that detect simultaneously, belong to technical field of biological.
Background technology
The paathogenic factor research of the hearing and speech handicapped person being carried out in world wide shows, about 60-80% patient's the cause of disease is relevant with inherited genetic factors, and wherein the clinical study data of developed country show, hereditary hearing impairment accounts for 80% in deafness patient.Therefore in recent ten years, the pathogenesis of hereditary hearing impairment and the research of molecular epidemiology thereof become one of most important content of deaf sick research.Along with the Human Genome Project completes, the location of deaf ospc gene and clone have obtained huge progress, and the molecule genetics research of deaf disease and the data of molecular epidemiology make investigators recognize that progressively deaf sick susceptibility gene mutation safeguarding that hearing is healthy and finding the importance of hearing in abnormal.
In the deaf-related gene of having located and having cloned at present, GJB2 is modal tumor susceptibility gene, accounts for 30-50% in congenital severe deafness patient.At present, had been found that 100 various mutations types at this gene, yet, the mutant form difference that not agnate GJB2 gene is common.Common mutant form is 235delC in China, and it accounts for 74.14% in all disease cause mutations, and approximately 22.2% Chinese non-syndromic hearing loss patient is relevant with this sudden change.
The ototoxicity of medicine is an important factor that causes the front hearing loss of language, and part is relevant with plastosome 12S rRNA gene 1555A>G sudden change, and this sudden change can increase the susceptibility of cochlea to aminoglycoside drug.In the U.S., in the hearing loss patient that ototoxic drug is correlated with, 10% the 12S rRNA gene 1555A>G that has suddenlys change, and before U.S.'s language, in the hearing loss patient, 1555A>G sudden change patient's sickness rate is about 1/20000-1/40000.In Spain, 12S rRNA gene 1555A>G sudden change is relevant with the familial nonsyndromic hearing loss of 15-20%, and many old family members are even without the auditory dysesthesia that uses aminoglycoside drug also can occur so suddenly change and cause.And in China, the sickness rate of drug induced deafness has exceeded original imagination, in a series of article report, discovery is in the deafness patient that outpatient service is distributed, approximately 5% patient is because 12S rRNA gene 1555A>G sudden change causes, and, in such specific group of school for deaf-mutes, up to 12% patient, be because 12SrRNA gene 1555A>G sudden change contact aminoglycoside drug causes deaf.In Chinese colony, also found simultaneously the relation of 12S rRNA gene 1494C>T sudden change with drug induced deafness, had been found that at least that three large familys are because 1494C>T sudden change causes at present.Often hearing is normal at birth for 1555A>G sudden change and 1494C>T sudden change person, is difficult to found in advance and predict by universal newborn hearing screening, but has the hidden danger that hearing loss occurs because of the use of ototoxic drug.
The large vestibular aqueduct syndrome that SLC26A4 gene and arc are vertical and Pendred syndrome (aqueductus vestibuli enlarges or companion's inner ear malformations, nerve deafness and thyrocele) in close relations, show as clinically congenital or acquired character is deaf, deafly occur or increase the weight of relevant with wound, flu.By the temporal bone CT scan, can reach a conclusion, but at present due to equipment and professional's restriction, the hospital in most of China area can not carry out high-resolution temporal bone CT scan, causes many areas can't diagnose large vestibular aqueduct syndrome.By the detection to the SLC26A4 gene, can realize in China the syndromic Accurate Diagnosis of 80% above large vestibular aqueduct, and find and make a definite diagnosis large vestibular aqueduct syndrome prior to CT examination, this method can be avoided the radiation problem of radiograph test, more easily for family numbers of patients, accept, and, because the gene diagnosis operation can be carried out in batches, can in large sample scope, carry out examination.Quantity research shows greatly, is present in Chinese population mutantional hotspot zone and is: the IVS7-2A>G of the flanking sequence of SLC26A4 gene extron 8.
Gene tester commonly used has at present: direct Sequencing (direct sequencing, DS), Ligase detection reaction (ligase detection reaction, LDR), restriction fragment length polymorphism analysis (restriction fragment length polymorphism, RFLP), dhplc analysis (denaturing high performance liquid chromotography, DHPLC), quantitative PCR, gene chip.All there is different defects in these methods, are difficult for the problems such as interpretation, poor repeatability, false negative false positive be many such as complex operation, result.
Adopt fluorescent mark technology, capillary electrophoresis technique, multiplex PCR composite amplification, by the multipair primer with different fluorescent markers simultaneously to GJB2235delC, 12S rRNA 1555A>G sudden change, 1494C>T sudden change, the specific amplification of IVS7-2A>G, and in these primers, mix nucleic acid analog to improve the Tm value of primer, further strengthen the specificity of primer, and go out the peak situation by capillary electrophoresis post analysis PCR product, when can realize this 4 deaf gene sites, detect.The method is highly sensitive, interpretation is clear accurately, sampling is convenient, and can realize the high-throughput examination flow process of 4 critical sites diagnostic results of disposable acquisition, has greatly saved manpower and materials and time, has prevented the multistep operation and produce and pollute.
Summary of the invention
The object of the invention is to provide a kind of 4 deaf sick tumor susceptibility gene GJB2235delC, 12S rRNA 1555A>G sudden change, 1494C>T sudden change, IVS7-2A>G and Amelogenin locus fluorescence detection reagent kit of detecting simultaneously.
To achieve these goals, the technical scheme of taking: a kind of 4 deaf sick tumor susceptibility gene fluorescence detection reagent kits, gene type reagent after this test kit comprises amplifing reagent and increases of detecting simultaneously;
Before described amplification, reagent comprises: PCR damping fluid, MgCl
2, dNTPs mixture, Taq enzyme, the 5 pairs of primer mixtures and ultrapure water;
After described amplification, reagent comprises: interior mark ROX-300 and for genotyping standard thing corresponding to each gene mutation site of gene type;
Use described test kit to carry out the condition of PCR composite amplification reaction: the pH value of pcr amplification system is 8.0-9.0, magnesium ion concentration is 1.5-3.5mM, the final concentration of 4 kinds of dNTP is respectively 200-300 μ M, the consumption of Taq enzyme is 0.1-0.4U/ μ L, and the list in the 5 pairs of primer mixtures is 0.2-0.4 μ M to the primer final concentration.
While using described test kit to carry out pcr amplification, amplification elementary reaction in a composite amplification reaction system, increase simultaneously GJB2235delC, 12S rRNA 1555A>G sudden change, 1494C>T sudden change, IVS7-2A>G and Amelogenin locus.
The primer detected for GJB2235delC is:
SEQ?NO.1：5’-CGCATTATGATCCTCGTTGTGG-3’
SEQ?NO.2：5’-GCTTGATGAACTTCCTCTTCTTCTC-3’
For the primer that 12S rRNA 1555A>the G sudden change detects, be:
SEQ?NO.3：5’-AGTGGGTTTGGGGCTAGGTTTAG-3’
SEQ?NO.4：5’-G?
ACGCATTTATATAGAGTAGC-3’
For the primer that 12S rRNA 1494C>the T sudden change detects, be:
SEQ?NO.5：5’-AGTGGGTTTGGGGCTAGGTTTA-3’
SEQ?NO.6：5’-G?
CACACCGCCCGTCTCT-3’
The primer detected for IVS7-2A>G is:
SEQ?NO.7：5’-CCAGCATTGTAATTTTTTTCCAGG-3’
SEQ?NO.8：5’-G?
TTTTAACATCTTTTGTTTTATTTAG-3’
Wherein the LNA nucleosides means with boldface type.
The primer detected for the Amelogenin locus is:
SEQ?NO.9：5’-CCCTGGGCTCTGTAAAGAAT-3’
SEQ?NO.10：5’-GCAGAGCTTAAACTGGGAAGC-3’。
Described 5 pairs of primers, wherein have 5 ' end of a primer to carry out fluorochrome label in every pair of primer, and fluorescence dye used is different fluorescein.
Described composite amplification adopts polymerase chain reaction to realize, adopts multiple tracks or single track capillary electrophoresis to detect.
The human gene group DNA who wherein detects is: use Chelex method, magnetic bead extraction method or phenol/chloroform extraction method to process to the source sample DNA obtained; Described source sample is: derive from the mankind: filter paper blood cake/buccal swab sample, FTA card blood cake/buccal swab sample, buccal swab sample, blood/trace, tissue, amniotic fluid.
While using described test kit to carry out pcr amplification, the amplification elementary reaction carries out on the PCR of any model instrument, amplification program: 94-98 ℃ 1-5min; 94-98 ℃ of 15-60s of 26-32 circulation, 55-65 ℃ of 15-60s, 72 ℃ of 15-60; 60 ℃ of 30-60min.
Use with fluorescent mark and through the LNA nucleoside monomers, mix the primer of modification, improved the Tm value of primer, shortened primer length, thereby increased sensitivity and the specificity of primer, prevent false positive and false-negative appearance.
For detecting of Amelogenin, be on the one hand the effect of internal reference: whether the indication template DNA is effectively or concentration range; Can effectively indicate whether to exist the PCR product pollution on the other hand, prevent the false positive produced because polluting.
The accompanying drawing explanation
Fig. 1 is that test kit of the present invention carrys out the somatotype result after source DNA (2.0ng/25uL system) increases to 12S rRNA 1494C>T mutated individual blood cake;
Fig. 2 is that test kit of the present invention carrys out the somatotype result after source DNA (0.2ng/25uL system) increases to GJB2235delC mutated individual blood cake;
Fig. 3 is that the individual blood cake that test kit of the present invention suddenlys change to 12S rRNA 1555A>G carrys out the somatotype result after source DNA (1.0ng/25uL system) increases;
Fig. 4 is that test kit of the present invention carrys out the somatotype result after source DNA (0.5ng/25uL system) increases to IVS7-2A>G mutated individual blood cake;
Fig. 5 is that test kit of the present invention carrys out the somatotype result after source DNA (5.0ng/25uL system) increases to the normal individual blood cake;
Fig. 6 is the somatotype result of the normal individual blood cake source DNA sample of not modified primer pair same concentration (5.0ng/25uL system).
Embodiment
The invention will be further described below in conjunction with the drawings and specific embodiments, but not as a limitation of the invention.
Embodiment 1
Test kit of the present invention detects the DNA sample of sudden change and normal individual.The primer detected for deaf sick tumor susceptibility gene SLC26A4 mutantional hotspot IVS7-2A>G adopts the blue fluorescent dyes mark, the primer that GJB2235delC detects adopts the green fluorescence dye marker, for the primer employing Yellow fluorochrome mark that Amelogenin locus, 12S rRNA 1494C>T, 1555A>G sudden change detect, interior mark adopts the red fluorescence dyestuff mark.
1, sample to be tested is 1000 parts, all uses the technological method of " DNA extraction-pcr amplification-order-checking " to carry out the order-checking detection to each site.Wherein, IVS7-2A>10 parts, G sudden change sample, 10 parts, GJB2235delC sudden change sample, 12SrRNA 1555A>10 parts, G sudden change sample, 1 part, 1494C>T, sudden change sample.
2, the extracting genome DNA of sample
Chelex extraction method: cut 1～3mm blood cake and be placed in the 1.5mL centrifuge tube, add sdH
2O 1mL, vibrate centrifugal, abandons supernatant liquor, and repeating step twice, abandon supernatant liquor, and after the 5%Chelex-100 concussion is suspended, with the rifle head of cutting, draw 200 μ L fast and add in centrifuge tube, the vibration several seconds.After 56 ℃ of water bath heat preservation 30min, the vibration several seconds.95 ℃ of boiling water bath 10min, slightly vibrate the several seconds.The centrifugal 5min of 2000rpm, the DNA for extracting in supernatant.
3, the detection analysis of amplification and amplified production
3.1PCR amplification system:
3.2PCR amplification program:
4, amplified production fluoroscopic examination on genetic analyzer
By mark in deionized formamide and molecular weight, form loading mixture ((mark in 0.2 μ L molecular weight) * (sample introduction number)+(9.8 μ L deionized formamide) * (sample introduction number)).10 μ L loading mixtures are mixed with 1 μ L amplified production or alleles analysis standard substance, avoid producing bubble.95 ℃ of sex change 5min, ice bath 5min, and electrophoresis as early as possible.With genetic analyzer, detect and analyze.
5, conclusion
It is in full accord with the result of using sequence measurement that use the present invention tests 1000 increments result originally.Legend of the present invention is that result is given an example:
Fig. 1: 12S rRNA 1494C>T confirmed cases, female.The detected peaks of peak value 3700H appears in 1494C>interpretation position, T site (170bp); Sex interpretation position goes out X unimodal (105bp), peak value 4000H; The detected peaks of peak value 8000 appears in G2 position (238bp).
Fig. 2: GJB2235delC confirmed cases, female.The detected peaks of peak value 400H appears in interpretation position, 235delC site (237bp), and the detected peaks of peak value 400H appears in G2 position (238bp), and it is unimodal that sex goes out X, peak value 1400H.
Fig. 3: 12S rRNA 1555A>G confirmed cases, man.The detected peaks of peak value 1900H appears in 1555A>G interpretation position (225bp); The detection of peak value 1800H appears in G2 position (238bp); Sex goes out X, Y is bimodal: 105bp, peak value 1600H and 111bp, peak value 1600H.
Fig. 4: IVS7-2A>G confirmed cases, the female.The detected peaks of peak value 1800H appears in IVS7-2A>G interpretation position (88bp), and the detected peaks of peak value 2400H appears in G2 position (238bp), and sex interpretation position goes out X unimodal (105bp), peak value 2300H.
Fig. 5: normal individual, man.Each Bu Chu peak, interpretation position, site, the detection of peak value 8000H appears in G2 position (238bp); Sex goes out X, Y is bimodal: 105bp, peak value 2400H and 111bp, peak value 2450H.
With sequence measurement, compare: the homologous genes seat somatotype result to the sample in same source is consistent.
Embodiment 2
The normal individual blood cake source DNA sample of not modified primer pair same concentration (5.0ng/25uL system) detects.Labeled primer is with embodiment 1.The non-marked primer detected for deaf sick susceptibility loci is respectively the general primer without the LNA modification that embodiment 1 each primer pair is answered.
1, sample to be tested is with the normal individual blood cake in embodiment 1.
2, the extracting genome DNA of sample
Adopt the Chelex method to carry out the extraction of genomic dna.
3, the detection analysis of amplification and amplified production
3.1PCR amplification system:
3.2PCR amplification program: with embodiment 1.
4, amplified production fluoroscopic examination on genetic analyzer
With embodiment 1.
5, conclusion
As shown in Figure 6, for male sex's normal individual, the detected peaks of peak value 8000 appears to result in G2 position (238bp), for normally going out peak; Sex goes out X, Y is bimodal: 105bp, and peak value 1600H and 111bp, peak value 1600H, for normally going out peak.But other each sites occur small peak without the primer amplification of modifying on a plurality of sites, false positive namely occurs.Be specially: the detected peaks of peak value 800H appears in 1494C>interpretation position, T site (170bp); The detected peaks of peak value 700H appears in 1555A>G interpretation position (225bp); The detected peaks of peak value 400H appears in IVS7-2A>G interpretation position (88bp).If it is not read, consistent with sequencing result.
2.5 * PCR damping fluid of different pH used in above embodiment, with the Tris-HCl damping fluid preparation of different pH values, the Tris-HCl concentration in 1 * PCR damping fluid is 10mM, KCl concentration is 50mM; In the present invention, Taq polysaccharase used and other reagent and material are commercially available prod.
Claims (3)
1.4 individual deaf sick tumor susceptibility gene combined detection kit, it is characterized in that comprising archaeal dna polymerase and buffered soln thereof, the primer of increase each tumor susceptibility gene and Amelogenin locus, and interior mark and genotyping standard thing corresponding to each gene mutation site, described deaf sick susceptibility loci is GJB2235delC, 12S rRNA1555A > G, 1494C > T, and IVS7-2A G;
The primer detected for GJB2235delC is:
SEQ?NO.1：5’-CGCATTATGATCCTCGTTGTGG-3’
SEQ?NO.2：5’-GCTTGATGAACTTCCTCTTCTTCTC-3’；
For 12S rRNA1555A > primer that detects of G sudden change is:
SEQ?NO.3：5’-AGTGGGTTTGGGGCTAGGTTTAG-3’
SEQ?NO.4：5’-G?
ACGCATTTATATAGAGTAGC-3’；
For 12S rRNA1494C > primer that detects of T sudden change is:
SEQ?NO.5：5’-AGTGGGTTTGGGGCTAGGTTTA-3’
SEQ?NO.6：5’-G
CACACCGCCCGTCTCT-3’；
For IVS7-2A > primer that detects of G is:
SEQ?NO.7：5’-CCAGCATTGTAATTTTTTTCCAGG-3’
SEQ?NO.8：5’-G
TTTTAACATCTTTTGTTTTATTTAG-3’；
Wherein the LNA nucleosides means with italics;
The primer detected for the Amelogenin locus is:
SEQ?NO.9：5’-CCCTGGGCTCTGTAAAGAAT-3’
SEQ?NO.10：5’-GCAGAGCTTAAACTGGGAAGC-3’；
Described GJB2235delC, 12S rRNA1555A > G, 1494C > T, IVS7-2A > the detection primer of G and Amelogenin locus, in every pair of primer, there is 5 ' end of a primer to carry out fluorochrome label.
2. test kit according to claim 1, is characterized in that: described GJB2235delC, 12SrRNA1555A > G, 1494C > T, IVS7-2A > the detection primer of G and Amelogenin locus adopts different fluorochrome labels from interior mark.
3. test kit according to claim 1, it is characterized in that: described GJB2235delC, 12S rRNA1555A > G, 1494C > T, IVS7-2A > composite amplification of G and Amelogenin locus adopts polymerase chain reaction to realize, adopts multiple tracks or single track capillary electrophoresis to detect.
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| CN103436625B (en) * | 2013-09-11 | 2016-03-30 | 步迅 | 6 deaf sick susceptibility loci somatotype/mutant proportion detection kit |
| CN103451301B (en) * | 2013-09-11 | 2016-04-06 | 步迅 | Deaf sick tumor susceptibility gene SLC26A4 2168A > G, IVS7-2A > G mutation detection kit |
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| CN101684497A (en) * | 2008-09-23 | 2010-03-31 | 中国人民解放军总医院 | Deafness susceptibility gene screen test kit |
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