CN107267614A - The fluorescent quantificationally PCR detecting kit of chondriosome deafness A7445G mutation and its application - Google Patents
The fluorescent quantificationally PCR detecting kit of chondriosome deafness A7445G mutation and its application Download PDFInfo
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- CN107267614A CN107267614A CN201710507764.3A CN201710507764A CN107267614A CN 107267614 A CN107267614 A CN 107267614A CN 201710507764 A CN201710507764 A CN 201710507764A CN 107267614 A CN107267614 A CN 107267614A
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Abstract
The present invention provides a kind of chondriosome deafness common mutations A7445G fluorescent quantificationally PCR detecting kit, comprising quantitative PCR reaction solution, the first positive reference substance, the second positive reference substance, negative controls, specification and box body, wherein quantitative PCR reaction solution contains PCR buffer solutions, MgCl2, dNTPs, hot resistant DNA polymerase, upstream amplification primer, downstream amplification primer, the first fluorescence probe and the second fluorescence probe.The kit that the present invention is provided, for the A in the sites of mtDNA the 7445th>G single base mutations design two MGB probes, and the 7445th nucleotides of mitochondria of deaf correlation is detected by quantitative fluorescent PCR, specifies whether the site occurs A>G is mutated, and it is easy, quick, accurate to have the advantages that, can be applied to mitochondria A7445G mutation correlations deaf clinical diagnosis and preventing and treating, the prevention and treatment of chondriosome deafness provide foundation caused by being also mutated for A7445G.
Description
Technical field
The invention belongs to molecular biology and gene engineering technology field, and in particular to it is glimmering that chondriosome deafness A7445G is mutated
Fluorescent Quantitative PCR detection kit and its application.
Background technology
Deafness is the public health problem of global concern, is caused by h and E factor collective effect, wherein exceeding
50% deafness patient is caused by inherent cause.In hereditary hearing impairment, 30% is that syndrome is deaf, and 70% is non-syndrome ear
It is deaf.Deaf mode of inheritance can show autosomal dominant inheritance, autosomal recessive inheritance, X linkage inheritances and matrilinear inheritance,
Mitochondrial DNA (mtDNA) mutation is one of major reason that matrilinear inheritance deafness occurs, the incidence of disease in hereditary hearing impairment
About 1%~2%.
Deaf related mtDNA tRNA gene mutations are related to 16 kinds of tRNA, wherein tRNA altogetherSer(UCN)Gene is deaf phase
Close the hot spot region of mutation.A7445G mutation are located at tRNASer(UCN)Near the action site of the end of precursor 3 ' exonuclease, mutation
The identification of exonuclease can be influenceed during generation, causes tRNASer(UCN)Precursor manufacturing deficiency, and then influence tRNASer(UCN)Structure
Stabilization and mitochondria oneself protein translation, make cellular oxidation Phosphorylation events be damaged.Simultaneously as the mutation is in
tRNASer(UCN)/ CO1 gene junction, can cause the change of CO1 polypeptide mRNA terminator codons, and by CO1 polypeptides
C-terminal increases by 3 amino acid residues (Ser-Gln-lysine) and changes the original form of CO1 polypeptides, have impact on
The space structure and physiological function of cytochrome C oxidase, hinder the electron transfer process in respiratory chain, ultimately result in deafness
Generation.
At present, mtDNA mutation common detection method mainly have direct sequencing, micro-array chip method, PCR-RFLP methods,
Fluorescence quantitative PCR method etc..The advantage of direct sequencing is that accuracy is high, but detection cycle is longer, and needs the sequenator of specialty
With result interpretation personnel, be not suitable for clinical application.Micro-array chip method has high-throughout advantage, but there is testing cost
The shortcomings of high, cumbersome, easy cross pollution, the demand of large sample examination is not suitable for it.PCR-RFLP methods in PCR due to expanding
Need to uncap after end and carry out follow-up digestion and electrophoresis, equally exist it is cumbersome, the problems such as easily pollute.Quantitative fluorescent PCR
Technology is more and more extensive in the application of biology field in recent years, and its general principle is 5 ' -3 ' excision enzymes using Taq enzyme
Nucleic acids activity, designs double immunofluorescense probe on the basis of regular-PCR, and two ends are distinguished mark fluorescent reporter group (R) and are quenched
Group (Q)；The fluorescence signal of R group is suppressed by Q groups when probe keeps complete, once probe is cut off, the suppression of Q groups is made
With disappearance, the fluorescence signal of R group can be detected.During the technology is realized to PCR by the detection to fluorescence signal
The real-time monitoring of product amount, in addition to having the advantages that quantitatively accurate, detection is quick, maximum advantage is using the inspection of complete stopped pipe
Survey, eliminate the post processing to PCR primer, it is to avoid cross pollution.Moreover, with the further development of material and instrument, leading to
5 ' the different fluorescent dyes (FAM, HEX, ROX etc.) and multichannel quantitative real time PCR Instrument of end mark in fluorescence probe are crossed, can be with
Realize that Genotyping, detection in Gene Mutation, snp analysis etc. are studied.
Halfway problem is quenched for quantitative fluorescent PCR tradition Taqman fluorescence probes, American AB I companies were in 2000
Release a kind of new Taqman probes --- MGB probes, its 3 ' end is absorbing reporter group using the quenching group of non-fluorescent
Energy after do not light, greatly reduce the interference of background signal.In addition, 3 ' ends of MGB probes have been also connected with a dihydro
Ring annulated indole porphyrin-tripeptides, can be greatly improved the stability that probe hybridizes with template, shorter probe is equally reached higher
Tm values.And the major advantage of short probe is the distance of fluorescent reporter group and quenching group closer to quenching effects are more preferably, glimmering
Light background is lower, and signal to noise ratio is higher；Meanwhile, short probe also simplify design and reduce cost.There is experiment to show MGB probes pair
It is more satisfactory in the differentiation of allele, it might even be possible to detect single base mutation.
The content of the invention
The present invention provides a kind of chondriosome deafness common mutations A7445G fluorescent quantificationally PCR detecting kit, by quantitative
PCR reaction solutions, the first positive reference substance, the second positive reference substance, negative controls, specification and box body composition, wherein quantitative
PCR reaction solutions contain PCR buffer solutions, MgCl2, dNTPs, hot resistant DNA polymerase, upstream amplification primer, downstream amplification primer,
One fluorescence probe and the second fluorescence probe.
Upstream amplification primer sequence is：5’-CACCCTACCACACATTCGAAGA-3’
Downstream amplification primer sequence is：5’-TGGCTTGAAACCAGCTTTGG-3’
First fluorescence probe sequence is：5’-FAM-CCGTATACATAAAATCTAGGCA-MGB-3’
Second fluorescence probe sequence is：5’-HEX-CCGTATACATAAAATCTAGACAA-MGB-3’
First positive reference substance is the DNA sample that the site of mitochondria the 7445th is A bases, and the second positive reference substance is line grain
The site of body the 7445th is the DNA sample of G bases, and negative controls are sterile water for injection sample.
Kit of the present invention should be stored in -20 DEG C, and multigelation is reduced as far as possible.
It is a further object to provide described kit in correlation deafness mitochondrial A7445G abrupt climatic changes
In application.
Kit application method of the present invention：
Detection all should set up positive control and negative control every time.
(1) human peripheral blood cell mtDNA extraction：In strict accordance with the mitochondrial DNA of BioVision companies of U.S. commercialization
Extracts kit is operated, and mtDNA is extracted from clinical periphery blood specimen.
(2) PCR augmentation detections：It is double-colored (or more) carry out on quantitative real time PCR Instrument, each PCR reaction systems cumulative volume
For 25 μ l, including 20 μ l PCR reaction solutions and 5 μ l templates (mtDNA, positive control, the negative control of extraction).Probe is examined
Survey pattern is：FAM, HEX passage are respectively used to detect the G bases and A bases in 7445 sites.PCR reaction conditions：94 DEG C 5 minutes
Pre-degeneration, 94 DEG C 20 seconds → 60 DEG C 60 seconds, totally 40 circulation.After being provided with, file, operation program are preserved.
(3) fluorescent quantitation result is reported：1. sample G base probe CT value≤35 are detected and amplification curve has obvious logarithm to increase
For a long time, A base probe CT values are more than G base probe CT values, and both difference >=5 in same reaction system, then the detection sample
7445 sites are G bases；2. sample A base probe CT value≤35 are detected and amplification curve has obvious increased logarithmic phase, it is same anti-
G base probes CT values in system are answered to be more than A base probe CT values, and both difference >=5, then the site of detection sample 7445 is A
Base；3. when two probe CT values be all higher than 35 or two probe CT values difference be less than 5 when, mtDNA need to be re-started to sample and carried
Take and PCR detections.
The present invention is directed to the A in the sites of mtDNA the 7445th>G single base mutations design two MGB probes, develop the site and dash forward
The fluorescent quantificationally PCR detecting kit of change, it is intended to accurate, quick, easily detection mtDNA A7445G mutation, so as to be applied to
The deaf clinical diagnosis of the mutation correlation, prevention and treatment.The present invention uses Real-Time Fluorescent Quantitative PCR Technique and Two Colour Fluorescence
Probe, has developed the detection kit for chondriosome deafness common mutations A7445G.The invention kit can pass through one
Whether footwork detection mtDNA the 7445th sites occur A>G is mutated, can meet it is clinical accurate, quick, easily diagnose the mutation phase
The need for closing property is deaf, while the prevention and treatment of chondriosome deafness provide foundation caused by being mutated for A7445G.
Brief description of the drawings
Fig. 1 is the structural representation of kit of the present invention.
Fig. 2 is the amplification curve that the first positive reference substance detects that 7445 sites are A bases.
Fig. 3 is the amplification curve that the second positive reference substance detects that 7445 sites are G bases.
Embodiment
The present invention is described further with accompanying drawing in conjunction with the embodiments.It should be understood that these embodiments are for illustration purposes only,
Rather than limitation the scope of the present invention.
Embodiment 1
Referring to Fig. 1, the fluorescent quantificationally PCR detecting kit for the chondriosome deafness A7445G mutation that the present invention is provided, by fixed
Measure PCR reaction solutions (1), the first positive reference substance (2), the second positive reference substance (3), negative controls (4), specification (5) and
Box body (6) is constituted, and wherein quantitative PCR reaction solution contains PCR buffer solutions, MgCl2, dNTPs, hot resistant DNA polymerase, upstream amplification
Primer, downstream amplification primer, the first fluorescence probe and the second fluorescence probe.
Upstream amplification primer sequence is：5’-CACCCTACCACACATTCGAAGA-3’
Downstream amplification primer sequence is：5’-TGGCTTGAAACCAGCTTTGG-3’
First fluorescence probe sequence is：5’-FAM-CCGTATACATAAAATCTAGGCA-MGB-3’
Second fluorescence probe sequence is：5’-HEX--MGB-3’
First positive reference substance is the DNA sample that the site of mitochondria the 7445th is A bases, and the second positive reference substance is line grain
The site of body the 7445th is the DNA sample of G bases, and negative controls are sterile water for injection sample.
Kit of the present invention should be stored in -20 DEG C, and multigelation is reduced as far as possible.
The chondriosome deafness A7445G of embodiment 2 is mutated the application of fluorescent quantificationally PCR detecting kit
(1) sample is detected：
Phonosensitive nerve deafness infant 35 is diagnosed as in The Children's Hospital, Zhejiang University School of Medicine, all cases are adopted
With the mitochondrial DNA extracts kit of BioVision companies of the U.S., extraction mitochondrial DNA is used as inspection from its periphery blood specimen
Test sample product.
(2) fluorescence quantitative PCR detection
Detection sample, positive reference substance or the μ l of negative controls 5 are separately added into quantitative PCR reaction liquid pipe, double-colored
(or more) amplification of quantitative real time PCR Instrument enterprising performing PCR, FAM, HEX passage is respectively used to detect the G bases and A in 7445 sites
Base.PCR reaction conditions are 94 DEG C of 5 minutes pre-degenerations, 94 DEG C 20 seconds → 60 DEG C 60 seconds, totally 40 circulations.
(3) testing result
The site of first positive reference substance testing result 7445 is A bases (Fig. 2), the second positive reference substance testing result 7445
Site is G bases (Fig. 3).7445 sites have 33 for A bases in clinical detection sample, account for 94.3%；7445 sites are G alkali
Base has 2, accounts for 5.7%.The pcr amplification products of all detection samples through direct Sequencing, find the base in 7445 sites with
Fluorescence quantitative PCR detection result is consistent, and shows that kit detection A7445G mutation have good accuracy.
Present invention combination most preferred embodiment is described, but after the above of the present invention has been read, ability
Field technique personnel can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims
The scope of institute's limit.
<110>Zhejiang University
<120>The fluorescent quantificationally PCR detecting kit of chondriosome deafness A7445G mutation and its application
<160> 4
<210> 1
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>Expand human mitochondrion tRNA upstream primer sequence
<400> 1
CACCCTACCA CACATTCGAA GA 22
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Expand human mitochondrion tRNA downstream primer sequence
<400> 2
TGGCTTGAAA CCAGCTTTGG 20
<210> 3
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>The site of human mitochondrion the 7445th is the fluorescence probe sequence of G bases
<400> 3
CCGTATACAT AAAATCTAGG CA 22
<210> 4
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<223>The site of human mitochondrion the 7445th is the fluorescence probe sequence of A bases
<400> 4
CCGTATACAT AAAATCTAGA CAA 23
Claims (4)
1. a kind of fluorescent quantificationally PCR detecting kit of chondriosome deafness A7445G mutation, it is characterised in that anti-by quantitative PCR
Liquid, the first positive reference substance, the second positive reference substance, negative controls composition are answered, wherein quantitative PCR reaction solution contains PCR and delayed
Fliud flushing, MgCl2, dNTPs, hot resistant DNA polymerase, upstream amplification primer, downstream amplification primer, the first fluorescence probe and second glimmering
Light probe；
Upstream amplification primer sequence is：5’-CACCCTACCACACATTCGAAGA-3’
Downstream amplification primer sequence is：5’-TGGCTTGAAACCAGCTTTGG-3’
First fluorescence probe sequence is：5’-FAM-CCGTATACATAAAATCTAGGCA-MGB-3’
Second fluorescence probe sequence is：5’-HEX-CCGTATACATAAAATCTAGACAA-MGB-3’.
2. a kind of fluorescent quantificationally PCR detecting kit of chondriosome deafness A7445G mutation according to claim 1, it is special
Levy and be, the first positive reference substance is the DNA sample that the site of mitochondria the 7445th is A bases, the second positive reference substance is line grain
The site of body the 7445th is the DNA sample of G bases, and negative controls are sterile water for injection sample.
3. a kind of fluorescent quantificationally PCR detecting kit of chondriosome deafness A7445G mutation according to claim 1, it is special
Levy and be, kit of the present invention is stored in -20 DEG C, reduce multigelation.
4. the fluorescent quantificationally PCR detecting kit of chondriosome deafness A7445G mutation according to claim 1 is in correlation
Application in deafness mitochondrial A7445G abrupt climatic changes.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710507764.3A CN107267614A (en) | 2017-06-28 | 2017-06-28 | The fluorescent quantificationally PCR detecting kit of chondriosome deafness A7445G mutation and its application |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710507764.3A CN107267614A (en) | 2017-06-28 | 2017-06-28 | The fluorescent quantificationally PCR detecting kit of chondriosome deafness A7445G mutation and its application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111621556A (en) * | 2019-12-31 | 2020-09-04 | 安徽医科大学第一附属医院 | Kit and method for detecting human mitochondrial ATP6 gene 8993 locus genotype |
| CN112111568A (en) * | 2019-12-31 | 2020-12-22 | 安徽医科大学第一附属医院 | Kit and method for detecting mitochondrial 3243 locus genotype |
| CN112746105A (en) * | 2021-01-26 | 2021-05-04 | 上海博奥颐和医学检验所有限公司 | Genetic deafness gene detection method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101875938A (en) * | 2009-04-29 | 2010-11-03 | 复旦大学附属眼耳鼻喉科医院 | Gene relevant mutational site of mitochondrial deaf and application |
-
2017
- 2017-06-28 CN CN201710507764.3A patent/CN107267614A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101875938A (en) * | 2009-04-29 | 2010-11-03 | 复旦大学附属眼耳鼻喉科医院 | Gene relevant mutational site of mitochondrial deaf and application |
Non-Patent Citations (1)
| Title |
|---|
| 刘玉和等: ""国人无综合征性耳聋患者的线粒体基因组7445A→G突变263例调查"", 《临床耳鼻咽喉科杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111621556A (en) * | 2019-12-31 | 2020-09-04 | 安徽医科大学第一附属医院 | Kit and method for detecting human mitochondrial ATP6 gene 8993 locus genotype |
| CN112111568A (en) * | 2019-12-31 | 2020-12-22 | 安徽医科大学第一附属医院 | Kit and method for detecting mitochondrial 3243 locus genotype |
| CN112746105A (en) * | 2021-01-26 | 2021-05-04 | 上海博奥颐和医学检验所有限公司 | Genetic deafness gene detection method |
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Application publication date: 20171020 |