CN103451302A - Deafness susceptible gene mitochondrion 12SrDNA 1555A>G and 1494C>T mutant ratio detection kit - Google Patents
Deafness susceptible gene mitochondrion 12SrDNA 1555A>G and 1494C>T mutant ratio detection kit Download PDFInfo
- Publication number
- CN103451302A CN103451302A CN2013104108400A CN201310410840A CN103451302A CN 103451302 A CN103451302 A CN 103451302A CN 2013104108400 A CN2013104108400 A CN 2013104108400A CN 201310410840 A CN201310410840 A CN 201310410840A CN 103451302 A CN103451302 A CN 103451302A
- Authority
- CN
- China
- Prior art keywords
- probe
- seq
- sudden change
- plastosome
- test kit
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
The invention discloses a deafness susceptible gene mitochondrion 12SrDNA 1555A>G and 1494C>T mutant ratio detection kit which comprises amplification reagents and a series of standard substances, wherein the amplification reagents comprise a reaction mixture of a PCR (polymerase chain reaction) buffer solution, MgCl2 and dNTPs (deoxyribonucleotide triphosphates), Taq enzyme, ultrapure water, and a high-specificity amplified mitochondrion 2SrDNA:1494C>T and 1555A>G primer mixture; and the series of standard substances comprise a 1494C>T mutant ratio standard substance and a 1555A>G mutant ratio standard substance. By using the 2 deafness susceptible gene sites as the detection objects, the deafness susceptible gene sites are subjected to amplification and fluorescence quantitative detection and are compared with the mutant ratio standard substances to screen out individuals containing the site mutants and determine various mutant ratios. The kit has important meanings for screening deafness susceptible genes and especially newborn deafness genes.
Description
Technical field
The present invention relates to deaf sick tumor susceptibility gene plastosome 12SrDNA1555A > G, 1494C > T mutant proportion detection kit, 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 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 hearing loss before language, part and plastosome 12S rRNA gene 1555A > the G sudden change is relevant, and this sudden change can increase the susceptibility of cochlea to aminoglycoside drug.In the U.S., in the relevant hearing loss patient of ototoxic drug, 10% there is 12S rRNA gene 1555A the G sudden change, 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 > the G sudden change is relevant with the familial nonsyndromic hearing loss of 15-20%, the auditory dysesthesia that many old family members also can occur so suddenly change and cause even without the use aminoglycoside drug.And in China, the sickness rate of drug induced deafness has exceeded original imagination, in a series of article report, in the deafness patient that discovery is found in outpatient service, approximately 5% patient is due to 12S rRNA gene 1555A > G sudden change causes, and, in such specific group of school for deaf-mutes, up to 12% patient, be due to 12S rRNA gene 1555A G sudden change contact aminoglycoside drug causes deaf.In Chinese colony, also found 12S rRNA gene 1494C simultaneously > relation of T sudden change and drug induced deafness, at present at least had been found that three large familys are due to 1494C > T suddenlys change and causes.1555A > G sudden change and 1494C > often hearing is normal at birth for 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.Due to mitochondrial heterogeneous feature, the ratio difference of the mitochondrial mutations that different individualities shows.Large quantity research shows, 1555A > G and 1494C > there is certain dependency in the ratio height that occupies of T sudden change plastosome with the state of an illness, and the individuality that the plastosome that usually suddenlys change proportion is high shows severe deafness, and accounting is low shows slight deafness or normal.Even but the sudden change accounting is very low, still exists medicine to cause deaf danger.Therefore, need on the one hand the test kit that can tell mutant proportion for the relevant sudden change of the deaf disease of plastosome provides research means more easily, require on the other hand these reagent good Detection capability to be arranged to low mutant proportion sample.
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), gene chip.All there is different defects in these methods, such as complex operation, result, are difficult for the problems such as interpretation, poor repeatability, false negative false positive be many.For the gene type of chromosome mutation, general fluorescence quantitative kit often needs same sample is carried out to the multitube detection, could obtain the somatotype information more than 2.Mutant proportion for Mitochondrial DNA detects, and mass spectroscopy can detect the sudden change of 5% left and right, and order-checking and gene chip can only reach 20% left and right for the mitochondrial selective power of sudden change, and indistinguishable goes out the sample of lower mutant proportion.
Above-mentioned plastosome 12SrDNA1555A is take in the present invention > G, 1494C > the T site is detected object, amplification and fluorescent quantitation by above-mentioned deaf susceptibility loci detect, contrast with the mutant proportion standard substance, can filter out the individuality that contains above-mentioned site mutation, determine the ratio of various sudden changes simultaneously.To the detection of deaf sick tumor susceptibility gene, especially significant to the examination of newborn infant's deaf gene; Utilize first the method for fluorescence quantitative PCR detection to realize 1494C > T, 1555A > target that the mutant proportion of G detects, greatly saved manpower and materials and time.The highly sensitive high specific detects simultaneously, provides the somatotype information of the relevant sudden change of karyomit(e), for plastosome 1555A > G and 1494C > selective power of T sudden change reached 0.1%.Stopped pipe detects and to have prevented the pollution of uncapping and detecting operation and producing, for clinical diagnosis and association area scientific research provide reliable method.
Summary of the invention
The object of the invention is to provide and a kind ofly can in 1 hour, detects deaf sick tumor susceptibility gene plastosome 12SrDNA1555A simultaneously > G, 1494C > the T sudden change, report the fluorescence detection reagent kit of the ratio of various sudden changes simultaneously.
To achieve these goals, the technical scheme of taking: a kind of deaf sick tumor susceptibility gene plastosome 12SrDNA1555A > G, 1494C > T mutant proportion detection kit, this test kit comprises amplifing reagent and series of standards product;
Described amplifing reagent comprises: PCR damping fluid, MgCl
2reaction mixture with dNTPs, the Taq enzyme, ultrapure water, high specific amplification plastosome 12SrDNA:1494C>T, 1555A>primer mixture of G sudden change, above-mentioned site is carried out to the probe mixture of specific detection, the general probe mixture that human mitochondrial DNA is detected, internal reference and the primer probe mixture that internal reference is detected;
Described series of standards product comprise: 1494C > T mutant proportion standard substance, 1555A > G mutant proportion standard substance.The preparation method of standard substance (with 1555A > G mutant proportion standard substance be prepared as example): screening obtains 1555A > specimen dna of G sudden change, confirm that by methods such as order-checking, mass spectrums the ratio that the DNA of its 1555G accounts for whole Mitochondrial DNAs is 100%, dilution is quantitatively to 5ng/uL, as the standard substance of 100% sudden change 1.; Get normal people's sample and extract DNA, by methods such as order-checking, mass spectrums, confirm that there is not sudden change in its 1555 site, dilution is quantitatively to 5ng/uL; Get standard substance and 1. with 5ng/uL normal people's sample, extract the mixing of DNA equal proportion, obtain the standard substance of 50% sudden change 2.; Get standard substance and 2. mix in the ratio of 1: 4 with 5ng/uL normal people's sample extraction DNA, obtain the standard substance of 10% sudden change 3.; Get standard substance and 3. mix in the ratio of 1: 9 with 5ng/uL normal people's sample extraction DNA, obtain the standard substance of 1% sudden change 4.; Get standard substance and 4. mix in the ratio of 1: 9 with 5ng/uL normal people's sample extraction DNA, obtain the standard substance of 0.1% sudden change 5..Get each standard substance 2uL joins in 20uL PCR reaction system at every turn.
Described for to plastosome 12SrDNA:1494C > T, 1555A > the G sudden change carries out the probe of specific detection, the general probe that human mitochondrial DNA is detected, the probe that internal reference is detected is divided into four groups, adopt respectively four kinds of different luminophores to carry out mark to 5 ' end of each group probe, the quenching group of 3 ' end can be identical or different fluorescence dye.
Four groups of being divided into of described probe are: for the probe that internal reference is carried out to specific detection, adopt same group of luminous-quenching group; Plastosome 12SrDNA:1494C > the T sudden change probe that carries out specific detection adopts same group of luminous-quenching group; 1555A > the G sudden change probe that carries out specific detection adopts same group of luminous-quenching group; The general probe that human mitochondrial DNA is detected adopts same group of luminous-quenching group.
Described 5 ' the fluorescence dye of holding different luminophores to use can be: ALEXA Fluor350, FAM, TET, HEX/JOE/VIC, Cy3, TAMRA, ROX ,/Texas Red, Cy5; 3 ' the fluorescence dye of holding identical or different quenching group to use can be: BHQ1, BHQ2, TAMRA, DABCYL.
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 primer in the primer probe mixture, the final concentration of probe are 0.2-0.4 μ M.
While using described test kit to carry out pcr amplification, the amplification elementary reaction 12SrDNA:1494C that simultaneously increases in a composite amplification reaction system > T, 1555A > G, human mitochondrial DNA target sequence, internal reference target sequence.
For GJB2:235delC, 299delAT, SLC26A4:2168A > G, IVS7-2A > G, plastosome 12SrDNA:1494C > T, 1555A > the G detection, the primer of human mitochondrial DNA sequential detection is:
SEQ?NO.1：5’-GTAAGTTGGGTGCTTTGTGTTAAG-3’
SEQ?NO.2：5’-GCCCTGAAGCGCGTACA-3’；
For plastosome 12SrDNA1555A > probe that detects of G sudden change is:
SEQ?NO.3：5’-AGTACACTTACCATGTTACGACTTGcCTCCTC-3’
For plastosome 12SrDNA1494C > probe that detects of T sudden change is:
SEQ?NO.4：5’-GTGAAGTATACTTGAGGAGaGTGACGGGAC-3’；
For the general probe that human mitochondrial DNA is detected, be:
SEQ?NO.5：5’-TGCGTAGGGGTTTTAGTTAAATGTCCT-3’
Wherein the LNA nucleosides means with boldface type.
The sequence that internal reference is one section synthetic is loaded on the plasmid vector of pMD18-T and obtains, and the sequence of this section synthetic (SEQ NO.6) does not find high relevant homologous region through NCBI website B1ast.Extension increasing sequence and the primer probe that internal reference is detected are:
SEQ?NO.6：CACCAGCATCTCCCTTCATGTTGACTACCGTAGGCTGCCACCATTTCAGAAGAACGATGACCCCTTGCTAGCAGTGGAAGAGTTGCATGAAGGTAATATACTCGATCCGTAATTCCACTATTGAAATGGGTACATGACTGATACAGACCATC
SEQ?NO.7：5’-CACCAGCATCTCCCTTCATGT-3’
SEQ?NO.8：5’-GATGGTCTGTATCAGTCATGTACCCA-3’
SEQ?NO.9：5’-GAACGATGACCCCTTGCTAGCAGTGGAAGAG-3’
The composite amplification in described each site adopts polymerase chain reaction to realize, adopts quantitative fluorescent PCR to detect in real time, by the synchronous amplification with standard substance and analysis, draws institute's mark somatotype information or mutant proportion information originally.
The human gene group DNA that wherein detected 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; The 94-98 of 45 circulations ℃ 5-10s, 55-65 ℃ of 30-50s.
Use with fluorescent mark and mix the primer of modification through the LNA nucleoside monomers, improve the Tm value of probe, shortened probe length, strengthened the insight of probe to point mutation, thereby increased sensitivity and the specificity of probe, prevented false positive and false-negative appearance.
The accompanying drawing explanation
Fig. 1 is that test kit of the present invention is to 12S rRNA1555A under the 10ngDNA background > the detection collection of illustrative plates of G mutant proportion standard substance;
Fig. 2 is that test kit of the present invention is to 12S rRNA1494C under 10ng DNA background > the detection collection of illustrative plates of T mutant proportion standard substance;
Fig. 3 is the canonical plotting of making according to Fig. 1, Fig. 2 data;
Fig. 4 is that test kit of the present invention carrys out the detection collection of illustrative plates of source DNA (10ng/20uL system) to the normal individual blood cake;
Fig. 5 is that test kit of the present invention is to 12S rRNA1555A > the individual blood cake of G sudden change carrys out the detection collection of illustrative plates of source DNA (10ng/20uL system);
Fig. 6 is that test kit of the present invention is to 12S rRNA1494C > T mutated individual blood cake carrys out the detection collection of illustrative plates of source DNA (2.5ng/20uL system).
Embodiment
Below in conjunction with the drawings and specific embodiments, the invention will be further described, but not as a limitation of the invention.
For 12S rRNA1494C > the probe 5 ' end that detects of T sudden change adopts the FAM fluorochrome label, for 1555A > the probe 5 ' end that detects of G sudden change adopts the HEX fluorochrome label, probe 5 ' the end detected for human mitochondrial DNA adopts the Cy5 fluorochrome label, and the probe 5 ' end detected for internal reference adopts the ROX fluorochrome label.
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, 12S rRNA1555A > 10 parts, G sudden change sample, 1494C > 1 part, T sudden change sample.
2, the extracting genome DNA of sample
Chelex extraction method: cut 1～3mm blood cake (sample comes from the XX hospital laboratory) and be placed in the 1.5mL centrifuge tube, add sdH
2o1mL, vibrate centrifugal, abandons supernatant liquor, and repeating step twice, abandon supernatant liquor, and draw 200 μ L with the rifle head of cutting fast after the 5%Chelex-100 concussion is suspended 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
(1) pcr amplification system:
(2) amplification program on ABI StepOne Plus type quantitative real time PCR Instrument: 95C3min; 95C5s, 60C35s (collection fluorescent signal), be 45cycles.
(3) detect and analyze
1555 mutant proportion standard substance (1555 different mutant proportions under 10ng DNA background), 1494 mutant proportion standard substance (1494 different mutant proportions under 10ng DNA background) are got respectively to 1uL and joined in amplification system as template, carry out fluorescent quantitation and collect data obtaining Fig. 1, Fig. 2 (unrisen signal all omits), be figure according to each concentration and corresponding Ct value, obtain the typical curve of Fig. 3.
Fig. 4 is that test kit of the present invention carrys out the detection collection of illustrative plates of source DNA (10ng/20uL system) to the normal individual blood cake: obtain internal reference probe (ROX) passage and signal and sigmoid curve detected, the Ct value is 23.12, shows that this amplified reaction is effective; Obtain two passages of human mitochondrial DNA probe (Cy5) and signal and sigmoid curve detected, the Ct value is 19.95, shows that the concentration of this DNA sample is about 10ng/uL; 1555A > G probe (FAM) and 1494C > two sense channels of T probe (HEX) do not have sigmoid curve, show that this sample is the normal individual source that there is not sudden change in 1555 and 1494 sites;
Fig. 5 is that test kit of the present invention is to 12S rRNA1555A > the individual blood cake of G sudden change carrys out the detection collection of illustrative plates of source DNA (10ng/20uL system): obtains internal reference probe (ROX) passage and signal and sigmoid curve detected, the Ct value is 23.07, shows that this amplified reaction is effective; Obtain two passages of human mitochondrial DNA probe (Cy5) and signal and sigmoid curve detected, the Ct value is 20.01, shows that the DNA background concentration of this sample is about 10ng/uL; 1494C > T probe (FAM) sense channel do not have sigmoid curve, shows that there is not sudden change in these sample 1494 sites; 1555A > G probe (HEX) passage detects signal and sigmoid curve, the Ct value is 23.19, contrast Fig. 3 determines that this sample should be 1555A > the G mutant proportion is 100%.
Fig. 6 is that test kit of the present invention is to 12S rRNA1494C > T mutated individual blood cake carrys out the detection collection of illustrative plates of source DNA (2.5ng/20uL system): obtains internal reference probe (ROX) passage and signal and sigmoid curve detected, the Ct value is 23.07, shows that this amplified reaction is effective; Obtain two passages of human mitochondrial DNA probe (Cy5) and signal and sigmoid curve detected, the Ct value is 21.97, and 10ng/uL DNA background concentration Ct value is about 20, shows that the concentration of this DNA sample is about 2.5ng/uL; 1555A > G probe (HEX) sense channel do not have sigmoid curve, shows that there is not sudden change in these sample 1555 sites; 1494C > T probe (FAM) passage detects signal and sigmoid curve, the Ct value is 23.02, contrast Fig. 3 (mutant proportion that under the 10ng background, Ct value is 23 left and right correspondences is 10%) shows the 1494C of this sample > the T content that suddenlys change is about 40%.
With sequence measurement, compare: the homologous genes seat somatotype result to the sample in same source is consistent.
10 * 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 (11)
1. deaf sick tumor susceptibility gene plastosome 12SrDNA1555A>G, 1494C>T mutant proportion detection kit, it is characterized in that comprising amplifing reagent and series of standards product; Described amplifing reagent comprises: PCR damping fluid, MgCl
2reaction mixture with dNTPs, the Taq enzyme, ultrapure water, high specific amplification plastosome 12SrDNA:1494C>T, 1555A>primer mixture of G, above-mentioned site is carried out to the probe mixture of specific detection, the general probe mixture that human mitochondrial DNA is detected, internal reference and the primer probe mixture that internal reference is detected; Described series of standards product comprise: 1494C>T mutant proportion standard substance, 1555A>G mutant proportion standard substance.
2. test kit according to claim 1 is characterized in that:
For plastosome 12SrDNA:1494C > T, 1555A > the G detection, the primer of human mitochondrial DNA sequential detection is:
SEQ?NO.1：5’-GTAAGTTGGGTGCTTTGTGTTAAG-3’
SEQ?NO.2：5’-GCCCTGAAGCGCGTACA-3’；
For plastosome 12SrDNA1555A > probe that detects of G sudden change is:
SEQ?NO.3：5’-AGTACACTTACCATGTTACGACTTGCCTCCTC-3’
For plastosome 12SrDNA1494C > probe that detects of T sudden change is:
SEQ?NO.4：5’-GTGAAGTATACTTGAGGAGaGTGACGGGAC-3’；
For the general probe that human mitochondrial DNA is detected, be:
SEQ?NO.5：5’-TGCGTAGGGGTTTTAGTTAAATGTCCT-3’
Wherein the LNA nucleosides means with boldface type.
3. test kit according to claim 1 and 2, it is characterized in that: the sequence that described internal reference is one section synthetic is loaded on the plasmid vector of pMD18-T and obtains, and the sequence of this section synthetic (SEQ NO.6) does not find high relevant homologous region through NCBI website Blast.Extension increasing sequence and the primer probe that internal reference is detected are:
SEQ?NO.6：CACCAGCATCTCCCTTCATGTTGACTACCGTAGGCTGCCACCATTTCAGAAGAACGATGACCCCTTGCTAGCAGTGGAAGAGTTGCATGAAGGTAATATACTCGATCCGTAATTCCACTATTGAAATGGGTACATGACTGATACAGACCATC
SEQ?NO.7：5’-CACCAGCATCTCCCTTCATGT-3’
SEQ?NO.8：5’-GATGGTCTGTATCAGTCATGTACCCA-3’
SEQ?NO.9：5’-GAACGATGACCCCTTGCTAGCAGTGGAAGAG-3’
4. test kit according to claim 1 and 2, it is characterized in that: described for plastosome 12SrDNA:1494C T, 1555A the probe that detects of G sudden change, the general probe that human mitochondrial DNA is detected, the internal reference probe, 5 ' end and the 3 ' end of every probe all carry out fluorochrome label, and 5 ' end is held as quenching group for luminophore and 3 '.
5. test kit according to claim 1 and 2, it is characterized in that: described for to plastosome 12SrDNA:1494C T, 1555A the G sudden change carries out the probe of specific detection, the general probe that human mitochondrial DNA is detected, the internal reference probe is divided into four groups, adopt respectively four kinds of different luminophores to carry out mark to 5 ' end of each group probe, the quenching group of 3 ' end can be identical or different fluorescence dye.
6. require described test kit according to right 4, it is characterized in that: four groups of being divided into of probe are: for the probe that internal reference is carried out to specific detection, adopt same group of luminous-quenching group; Plastosome 12SrDNA:1494C > the T sudden change probe that carries out specific detection adopts same group of luminous-quenching group; 1555A > the G sudden change probe that carries out specific detection adopts same group of luminous-quenching group; The general probe that human mitochondrial DNA is detected adopts same group of luminous-quenching group.
7. require described test kit according to right 4, it is characterized in that: the 5 ' fluorescence dye of holding different luminophores to use can be: ALEXA Fluor350, FAM, TET, HEX/JOE/VIC, Cy3, TAMRA, ROX ,/Texas Red, Cy5; 3 ' the fluorescence dye of holding identical or different quenching group to use can be: BHQ1, BHQ2, TAMRA, DABCYL.
8. according to right 1, require described test kit, it is characterized in that: comprise series of standards product: 1494C T mutant proportion standard substance, 1555A G mutant proportion standard substance.
9. require described test kit according to right 1 or 7, it is characterized in that: 1494C T mutant proportion standard substance are: the people DNA sample of 7 pipe 10ng/uL, the 1494C of each pipe > the T mutant proportion is 100%, 50%, 20%, 10%, 5%, 1%, 0.5%, its preparation method is for by 10ng/uL100%1494C > the DNA sample of T sudden change mixes by a certain percentage 10ng/uL and do not contain 1494C > in the DNA sample of T sudden change.
10. require described test kit according to right 1 or 7, it is characterized in that: 1555A G mutant proportion standard substance are: the people DNA sample of 7 pipe 10ng/uL, the 1555A of each pipe > the G mutant proportion is 100%, 50%, 20%, 10%, 5%, 1%, 0.5%, its preparation method is for by 10ng/uL100%1555A > the DNA sample of G sudden change mixes by a certain percentage 10ng/uL and do not contain 1555A > in the DNA sample of G sudden change.
11. the described test kit of claim 1 is applied to the detection of deaf sick tumor susceptibility gene, it is characterized in that by the deaf sick tumor susceptibility gene plastosome 12SrDNA:1494C of quantitative fluorescent PCR specific detection > T, 1555A > G; For 12S rRNA1555A > the primer probe that detects of G sudden change is: SEQ NO.1, SEQ NO.2, SEQ NO.3; For 12S rRNA1494C > the primer probe that detects of T sudden change is: SEQ NO.1, SEQ NO.2, SEQ NO.4; For the primer probe that human mitochondrial DNA is detected, be: SEQ NO.1, SEQ NO.2, SEQ NO.5; Primer probe for internal control test is: SEQ NO.5, SEQ NO.6, SEQ NO.6.The composite amplification in described each site adopts polymerase chain reaction to realize, adopts quantitative fluorescent PCR to detect in real time, by the synchronous amplification with standard substance and analysis, draws institute's mark mutant proportion information originally.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013104108400A CN103451302A (en) | 2013-09-11 | 2013-09-11 | Deafness susceptible gene mitochondrion 12SrDNA 1555A>G and 1494C>T mutant ratio detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013104108400A CN103451302A (en) | 2013-09-11 | 2013-09-11 | Deafness susceptible gene mitochondrion 12SrDNA 1555A>G and 1494C>T mutant ratio detection kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103451302A true CN103451302A (en) | 2013-12-18 |
Family
ID=49734135
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013104108400A Pending CN103451302A (en) | 2013-09-11 | 2013-09-11 | Deafness susceptible gene mitochondrion 12SrDNA 1555A>G and 1494C>T mutant ratio detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103451302A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103937875A (en) * | 2014-01-21 | 2014-07-23 | 芮宝生物医药科技（厦门）有限公司 | Primers and probes used for human drug-induced deafness gene mutation screening, and application methods thereof |
| CN104711366A (en) * | 2015-04-03 | 2015-06-17 | 济南英盛生物技术有限公司 | Drug-induced deafness gene multichannel fluorescence PCR (polymerase chain reaction) detection kit |
| EP3004389A4 (en) * | 2013-05-30 | 2017-02-01 | CapitalBio Corporation | Kits and methods for detecting drug-induced deafness susceptibility |
| CN107287303A (en) * | 2017-06-28 | 2017-10-24 | 浙江大学 | The fluorescent quantificationally PCR detecting kit of chondriosome deafness A1555G mutation and its application |
| CN112852948A (en) * | 2021-02-08 | 2021-05-28 | 广东药科大学 | Method and kit for detecting drug-induced deafness related gene locus |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101684497A (en) * | 2008-09-23 | 2010-03-31 | 中国人民解放军总医院 | Deafness susceptibility gene screen test kit |
| CN101684498A (en) * | 2008-09-27 | 2010-03-31 | 董大光 | Method for detecting 1494 C-T and 1555 A-G mutation of maternally inherited deafness mitochondrial genes, and kit thereof |
| CN101768637A (en) * | 2009-11-20 | 2010-07-07 | 温州医学院 | Kit for simultaneously detecting mutations in mitochondria DNA A1555G and C1494T and using method thereof |
| CN102154481A (en) * | 2011-02-11 | 2011-08-17 | 智海生物工程（北京）有限公司 | Method for detecting mitochondrial mutations and kit thereof |
| CN102586433A (en) * | 2012-02-14 | 2012-07-18 | 北京科聆金仪生物技术有限公司 | Deafness predisposing gene 12S rRNA (ribosomal ribonucleic acid) 1494C>T fluorescence detection kit and application thereof |
| CN102660636A (en) * | 2012-02-14 | 2012-09-12 | 北京科聆金仪生物技术有限公司 | A fluorescence detection kit for detecting deafness susceptibility gene 12S rRNA 1555A>G and application thereof |
| CN102787169A (en) * | 2012-08-22 | 2012-11-21 | 郭丽敏 | Mixed liquor, kit and detection system for detecting A1555G and C1494T mutation of mitochondria DNA (deoxyribonucleic acid) |
| CN103276082A (en) * | 2013-05-30 | 2013-09-04 | 博奥生物有限公司 | Kit for detecting mutation sites of drug-induced deafness susceptible gene |
-
2013
- 2013-09-11 CN CN2013104108400A patent/CN103451302A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101684497A (en) * | 2008-09-23 | 2010-03-31 | 中国人民解放军总医院 | Deafness susceptibility gene screen test kit |
| CN101684498A (en) * | 2008-09-27 | 2010-03-31 | 董大光 | Method for detecting 1494 C-T and 1555 A-G mutation of maternally inherited deafness mitochondrial genes, and kit thereof |
| CN101768637A (en) * | 2009-11-20 | 2010-07-07 | 温州医学院 | Kit for simultaneously detecting mutations in mitochondria DNA A1555G and C1494T and using method thereof |
| CN102154481A (en) * | 2011-02-11 | 2011-08-17 | 智海生物工程（北京）有限公司 | Method for detecting mitochondrial mutations and kit thereof |
| CN102586433A (en) * | 2012-02-14 | 2012-07-18 | 北京科聆金仪生物技术有限公司 | Deafness predisposing gene 12S rRNA (ribosomal ribonucleic acid) 1494C>T fluorescence detection kit and application thereof |
| CN102660636A (en) * | 2012-02-14 | 2012-09-12 | 北京科聆金仪生物技术有限公司 | A fluorescence detection kit for detecting deafness susceptibility gene 12S rRNA 1555A>G and application thereof |
| CN102787169A (en) * | 2012-08-22 | 2012-11-21 | 郭丽敏 | Mixed liquor, kit and detection system for detecting A1555G and C1494T mutation of mitochondria DNA (deoxyribonucleic acid) |
| CN103276082A (en) * | 2013-05-30 | 2013-09-04 | 博奥生物有限公司 | Kit for detecting mutation sites of drug-induced deafness susceptible gene |
Non-Patent Citations (1)
| Title |
|---|
| HUI ZHAO ET AL: "Maternally Inherited Aminoglycoside-Induced and Nonsyndromic Deafness Is Associated with the Novel C1494T Mutation in the Mitochondrial 12S rRNA Gene in a Large Chinese Family", 《AM. J. HUM. GENET.》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3004389A4 (en) * | 2013-05-30 | 2017-02-01 | CapitalBio Corporation | Kits and methods for detecting drug-induced deafness susceptibility |
| CN103937875A (en) * | 2014-01-21 | 2014-07-23 | 芮宝生物医药科技（厦门）有限公司 | Primers and probes used for human drug-induced deafness gene mutation screening, and application methods thereof |
| CN104711366A (en) * | 2015-04-03 | 2015-06-17 | 济南英盛生物技术有限公司 | Drug-induced deafness gene multichannel fluorescence PCR (polymerase chain reaction) detection kit |
| CN107287303A (en) * | 2017-06-28 | 2017-10-24 | 浙江大学 | The fluorescent quantificationally PCR detecting kit of chondriosome deafness A1555G mutation and its application |
| CN112852948A (en) * | 2021-02-08 | 2021-05-28 | 广东药科大学 | Method and kit for detecting drug-induced deafness related gene locus |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102534031B (en) | High-specificity kit for detecting deafness predisposing genes and uses | |
| CN104651488B (en) | Detect the Amplification thing and quick detection kit of chromosome aneuploid numerical abnormality | |
| CN105177115B (en) | A kind of UGT1A1 Polymorphisms site fluorescence detection reagent kit for being used to instruct Irinotecan based chemotherapy drug individualized treatment | |
| CN103451302A (en) | Deafness susceptible gene mitochondrion 12SrDNA 1555A>G and 1494C>T mutant ratio detection kit | |
| CN102146475A (en) | Method and kit for detecting southeast Asia deletion alpha-thalassemia | |
| CN110055312B (en) | Primer, probe and kit for detecting cis-trans mutation of EGFR gene C797S and T790M | |
| CN106591473A (en) | Human MTHFR and MTRR gene polymorphism detection primer, probe, test kit and method | |
| CN104830852B (en) | One kind detection HLA B*15:The multiple real time fluorescence PCR method of 02 allele | |
| CN106399479A (en) | SNP typing kit used for detecting susceptibility genes of type-II diabetes | |
| CN113846191B (en) | Primer and probe for detecting novel coronavirus and application of primer and probe | |
| CN107513577A (en) | A kind of method of efficient detection EGFRT790M mutant and probe and kit for detection | |
| CN102586433B (en) | Deafness predisposing gene 12S rRNA (ribosomal ribonucleic acid) 1494C>T fluorescence detection kit and application thereof | |
| CN109182493A (en) | The primer and kit and its detection method of people's 16p11.2 microdeletion syndrome detection | |
| CN107641649B (en) | Primer pair, kit and method for detecting stability of NR27 locus of microsatellite | |
| CN112111609A (en) | Universal nucleic acid detection kit for enteroviruses | |
| CN102534030B (en) | Kit for jointly detecting four deafness predisposing genes and application thereof | |
| CN106755530B (en) | MGB probe real-time fluorescence PCR method for detecting HLA-A31: 01 allele and primer probe combination thereof | |
| CN102660636A (en) | A fluorescence detection kit for detecting deafness susceptibility gene 12S rRNA 1555A>G and application thereof | |
| CN104232774A (en) | Primers for detecting breast cancer susceptibility gene SNP (single nucleotide polymorphism), fluorescent probes and applications | |
| CN102021228B (en) | Specific primers for tissue or whole blood EGFR gene mutation detection | |
| CN103451301B (en) | Deaf sick tumor susceptibility gene SLC26A4 2168A > G, IVS7-2A > G mutation detection kit | |
| CN103451300B (en) | Deaf sick tumor susceptibility gene GJB2 235delC, 299delAT mutation detection kit | |
| CN103436625B (en) | 6 deaf sick susceptibility loci somatotype/mutant proportion detection kit | |
| CN109385487B (en) | Recombinase-mediated amplification isothermal detection method and kit for American ginseng as Chinese medicinal herb | |
| CN108998528B (en) | Lung cancer diagnosis molecular marker lncRNA LINC00516, kit and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20131218 |
|
| WD01 | Invention patent application deemed withdrawn after publication |