CN102586433B - Deafness predisposing gene 12S rRNA (ribosomal ribonucleic acid) 1494C>T fluorescence detection kit and application thereof - Google Patents
Deafness predisposing gene 12S rRNA (ribosomal ribonucleic acid) 1494C>T fluorescence detection kit and application thereof Download PDFInfo
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Abstract
The invention discloses a fluorescence detection kit for detecting deafness predisposing gene 12S rRNA (ribosomal ribonucleic acid) 1494C>T. The kit comprises a reagent before amplification and a reagent after amplification, wherein the reagent before amplification comprises a reaction mixture of a PCR (polymerase chain reaction) buffer solution, MgCl2 and dNTPs (deoxyribonucleoside triphosphate), Taq enzyme, ultrapure water, and a primer mixture for high-specific amplification of 12SrRNA1494C>T and amelogenin locus; and the reagent after amplification comprises an allelic ladder and an internal standard. According to the invention, 12SrRNA1494C>T and amelogenin locus are used as detecting objects, so that the fluorescence detection kit has important meaning to detection of deafness predisposing genes, particularly screening of newborn deafness genes.
Description
Technical field
The present invention relates to a kind of fluorescence detection reagent kit that detects deaf sick tumor susceptibility gene 12S rRNA 1494C > T and Amelogenin locus, belong to technical field of biological.
Background technology
The paathogenic factor research of 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 progressively recognize that deaf sick susceptibility gene mutation safeguarding that hearing is healthy and finding the importance of hearing in abnormal.
The ototoxicity of medicine is an important factor that causes the front hearing loss of language, part is relevant with plastosome 12S rRNA transgenation, in Chinese colony, found the relation of 12S rRNA gene 1494C > T sudden change with drug induced deafness, at least had been found that three large familys are because 1494C > T sudden change causes at present.Often hearing is normal at birth for 1494C > T sudden change person, is difficult to found in advance and predicted by universal newborn hearing screening, but has the hidden danger that hearing loss occurs because of the use of ototoxic drug.
Conventional gene tester 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, such as complex operation, result, is difficult for the problems such as interpretation, poor repeatability, false negative false positive be many.
Adopt fluorescent mark technology, capillary electrophoresis technique, by the site-specific amplification of primer pair 12S rRNA 1494C > T with fluorescent marker, and the Tm value of mixing nucleic acid analog raising primer in this primer, further strengthen the specificity of primer, and go out peak situation by capillary electrophoresis post analysis PCR product, can realize the detection to deaf gene site.The method is highly sensitive, interpretation is clear accurately only needs to take a small amount of blood sample or buccal swab sample, can detect the convenient collection that is especially applicable to newborn infants sample of sampling.
Summary of the invention
The object of the invention is to provide a kind of deaf sick tumor susceptibility gene 12S rRNA 1494C > T fluorescence detection reagent kit.
To achieve these goals, the technical scheme of taking: a kind of deaf sick tumor susceptibility gene 12S rRNA 1494C > T fluorescence detection reagent kit, this test kit comprises the gene type reagent after amplifing reagent and amplification;
Before described amplification, reagent comprises: PCR damping fluid, MgCl
2, dNTPs mixture, Taq enzyme, the 2 pairs of primer mixtures and ultrapure water;
After described amplification, reagent comprises: for detecting and mixture and the interior mark ROX-300 of the allelotrope standard substance of Amelogenin sex identification locus corresponding to 12S rRNA1494C>T of gene type;
The condition of using described test kit to carry out the reaction of PCR composite amplification: 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 2 pairs of primer mixtures is 0.2-0.4 μ M to primer final concentration.
While using described test kit to carry out pcr amplification, amplification elementary reaction in a composite amplification reaction system, increase 12S rRNA1494C>T and Amelogenin locus simultaneously.
The primer detecting for 12S rRNA1494C>T sudden change is: SEQ NO.01 and SEQ NO.02.
SEQ NO.02 is the primer (LNA nucleosides is with underlined font representation) that has mixed LNA nucleosides:
SEQ?NO.1：5’-AGTGGGTTTGGGGCTAGGTTTA-3’
SEQ?NO.2：5’-G
ACACACCGCCCGTCTCT-3’
The primer detecting for Amelogenin locus is: SEQ NO.03:5 '-CCCTGGGCTCTGTAAAGAAT-3 ', SEQ NO.04:5 '-GCAGAGCTTAAACTGGGAAGC-3 '.
2 pairs of described primers, wherein have 5 ' end of a primer to carry out fluorochrome label in every pair of primer, fluorescence dye used can be identical or different fluorescein.
Described composite amplification adopts polymerase chain reaction to realize, and adopts multiple tracks or single track capillary electrophoresis to detect.
The human gene group DNA that wherein detected is: use Chelex method, magnetic bead extraction method or phenol/chloroform extraction method to process to source sample the DNA obtaining; 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, 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.
For detecting of 12S rRNA1494C>T sudden change, use with fluorescent mark and through 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, prevented false positive and false-negative appearance.
For detecting of Amelogenin, be on the one hand the effect of internal reference: whether indication template DNA is effectively or concentration range; Can effectively indicate whether to exist PCR product pollution on the other hand, prevent the false positive producing because polluting.
accompanying drawing explanation
Fig. 1 is that the modified same male sex of primer pair confirms the somatotype result of case different concns DNA.
Fig. 2 is the somatotype result that the same male sex of the primer pair of unmodified confirms case different concns DNA.
Fig. 3 is the somatotype result of the modified same male sex's normal individual of primer pair different concns DNA.
Fig. 4 is the somatotype result of the same male sex's normal individual of the primer pair of unmodified different concns DNA.
embodiment
Below in conjunction with the drawings and specific embodiments, the invention will be further described, but not as a limitation of the invention.
Embodiment 1
Deaf sick tumor susceptibility gene 12S rRNA1494C>T fluorescence detection reagent kit detects the DNA sample of sudden change and normal individual, and the primer detecting for deaf sick tumor susceptibility gene 12S rRNA1494C>T, the primer increasing for Amelogenin locus all adopt Yellow fluorochrome TAMRA mark; Interior mark adopts red fluorescence dyestuff mark, and fluorescent marker is ROX.Wherein, the primer detecting for deaf sick tumor susceptibility gene 12S rRNA1494C>T is used: through LNA nucleoside monomers, mix primer (SEQ the NO.2:5 '-G of modification
acACACCGCCCGTCTCT-3 ') and general primer (SEQ NO.2.0:5 '-GACACACCGCCCGTCTCT-3 ') carry out the comparison of expanding effect.
1, sample to be tested is 100 parts, and the technological method that all uses " DNA extraction-pcr amplification-order-checking " carried out order-checking to 12S rRNA1494 site and detects, and 2 parts, sample wherein suddenlys change.
2, the extracting genome DNA of sample
Chelex extraction method: cut 1～3mm blood cake and be placed in 1.5mL centrifuge tube, add sdH
2o1mL, vibrates centrifugal, abandons supernatant liquor, and repeating step twice, abandons supernatant liquor, and after 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:
3.2 pcr amplification programs:
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
As shown in Figure 1, 2, for male sex's confirmed cases,
Primer SEQ NO.2 (the test kit primer of the present invention) acquired results (Fig. 1) that has mixed LNA nucleoside monomers is consistent with not modified SEQ NO.2.0 primer acquired results (Fig. 2), under each DNA concentration, all can obtain correct result of determination: interpretation position, 12S rRNA 1494C > T site goes out peak, sex goes out X, Y is bimodal, all the increase peak value with DNA concentration increases, specific as follows:
As shown in Figure 3,4, for male sex's normal individual,
Primer SEQ NO.2 (the test kit primer of the present invention) acquired results (Fig. 3) and the not modified SEQ NO.2.0 primer acquired results (Fig. 4) that have mixed LNA nucleoside monomers are inconsistent to 12S rRNA 1494C > T site: the former does not all go out peak under each DNA concentration; And the latter is when the amount of DNA is greater than 2ng, interpretation position, 1494C > T site goes out peak, inconsistent with sequencing result, uses without the SEQ NO.2.0 primer of modifying and is prone to false-positive result.Both are consistent for the judgement of sex: sex goes out X, Y is bimodal, and all the increase peak value with DNA concentration increases.Specific as follows:
Therefore, test kit primer of the present invention can detect 12S rRNA 1494C > T site mutation specifically, and result is consistent with sequencing result, does not occur false positive.
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 (4)
1. a deaf sick tumor susceptibility gene 12S rRNA1494C>T fluorescence detection reagent kit, is characterized in that comprising genotyping standard thing and the interior mark of primer, gene mutation site 12S rRNA1494C>T and the Amelogenin locus of archaeal dna polymerase and buffered soln, amplification 12S rRNA1494C>T and Amelogenin locus; The primer that described mutantional hotspot 12S rRNA1494C>T detects is: SEQ ID NO.1 and SEQ ID NO.2,
SEQ?NO.1：5’-AGTGGGTTTGGGGCTAGGTTTA-3’
SEQ?NO.2：5’-G
ACACACCGCCCGTCTCT-3’
Wherein LNA nucleosides is with underlined font representation; The detection primer of described 12S rRNA1494C>T and Amelogenin, has 5 ' end of a primer to carry out fluorochrome label in every pair of primer.
2. test kit according to claim 1, is characterized in that the primer that described Amelogenin locus detects is:
SEQ?NO.3：5’-CCCTGGGCTCTGTAAAGAAT-3’
SEQ?NO.4：5’-GCAGAGCTTAAACTGGGAAGC-3’。
3. test kit according to claim 1, is characterized in that: the primer that described 12S rRNA1494C>T detects and the primer increasing for Amelogenin locus can adopt identical or different fluorochrome label; Interior mark is selected and is different from above-mentioned fluorochrome label.
4. test kit according to claim 1, it is characterized in that: the composite amplification of described 12S rRNA1494C>T and Amelogenin locus adopts polymerase chain reaction to realize, adopt multiple tracks or single track capillary electrophoresis to detect.
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| CN102796822A (en) * | 2012-08-30 | 2012-11-28 | 山东百福基因科技有限公司 | Motion function gene ACTN3 (alpha-Actinin) fluorescent detection kit and detection method |
| CN102796823A (en) * | 2012-08-30 | 2012-11-28 | 山东百福基因科技有限公司 | Fluorescence detection kit and detection method for motor function related gene CKMM |
| CN104419757A (en) * | 2013-09-04 | 2015-03-18 | 中南大学湘雅医院 | Novel deafness related gene mutation detection system and kit |
| CN103451302A (en) * | 2013-09-11 | 2013-12-18 | 步迅 | Deafness susceptible gene mitochondrion 12SrDNA 1555A>G and 1494C>T mutant ratio detection kit |
Citations (5)
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| CN1332805A (en) * | 1998-11-25 | 2002-01-23 | 普罗梅加公司 | Multiplex amplification of short tandem repeat loci |
| CN1733917A (en) * | 2004-09-17 | 2006-02-15 | 中国人民解放军总医院 | Human phonosensitive nerve deafness related WFS1 mutant gene and its uses in diagnosing genetic deafness |
| CN101597638A (en) * | 2008-06-04 | 2009-12-09 | 博奥生物有限公司 | A kind of method and dedicated kit thereof that detects gene multi-mutant site |
| CN101684497A (en) * | 2008-09-23 | 2010-03-31 | 中国人民解放军总医院 | Deafness susceptibility gene screen test kit |
| CN102304584A (en) * | 2011-09-15 | 2012-01-04 | 上海交通大学医学院附属新华医院 | GJB2 gene p.V37I mutation diagnostic kit for diagnosing late-onset deafness |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| AU3274500A (en) * | 1999-03-18 | 2000-10-09 | Exiqon A/S | Detection of mutations in genes by specific lna primers |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1332805A (en) * | 1998-11-25 | 2002-01-23 | 普罗梅加公司 | Multiplex amplification of short tandem repeat loci |
| CN1733917A (en) * | 2004-09-17 | 2006-02-15 | 中国人民解放军总医院 | Human phonosensitive nerve deafness related WFS1 mutant gene and its uses in diagnosing genetic deafness |
| CN101597638A (en) * | 2008-06-04 | 2009-12-09 | 博奥生物有限公司 | A kind of method and dedicated kit thereof that detects gene multi-mutant site |
| CN101684497A (en) * | 2008-09-23 | 2010-03-31 | 中国人民解放军总医院 | Deafness susceptibility gene screen test kit |
| CN102304584A (en) * | 2011-09-15 | 2012-01-04 | 上海交通大学医学院附属新华医院 | GJB2 gene p.V37I mutation diagnostic kit for diagnosing late-onset deafness |
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