CN109517906A - A kind of skin anti-aging ability detection method and its detection primer and kit - Google Patents
A kind of skin anti-aging ability detection method and its detection primer and kit Download PDFInfo
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Abstract
The invention discloses a kind of skin anti-aging ability detection method and its detection primer and kit, a kind of detection method of skin anti-aging ability, described detection method includes the following steps: (1) extracting the DNA of sample to be tested；(2) specific primer is utilized, using DNA obtained by step (1) as template, carries out fluorescent quantitative PCR detection, wherein the specific primer is forward primer 1, forward primer 2 and reverse primer 3；(3) according to fluorescent quantitative PCR testing result obtained by step (2), skin anti-aging ability is detected.The present invention provides a kind of method of the single nucleotide polymorphism of economic quickly detection skin anti-aging gene, easy to operate, economical and efficients.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of skin anti-aging ability detection method and its detection primer
And kit.
Background technique
As social economy develops at full speed, skin health and beauty are gradually paid attention to by the public.Maintain the weight of skin health
Point is skin anti-aging, oxidation resistance and skin moisturizing anti-wrinkle ability.And these maintain the body of skin health to adjust
Inner principles are determined by a variety of key function related genes.Simultaneously also studies have found that, skin health key function gene
Single nucleotide polymorphism (SNP) determine the activity of gene, to influence the maintenance of skin health stable state.
Skin anti-aging/anti-oxidant index can reflect whether a people has maintenance " young state " potential quality.Skin is anti-ageing
Always/oxidation resistance is stronger, and " young state " potential quality is higher.There is a set of natural Antioxidative Defense System in human body, it is intracorporal
Peroxidase gene CAT, glutathione peroxidase gene GPX1, dehydrogenase gene NQO1, superoxide dismutase base
Because SOD2 cooperates with the removing and degradation of responsible interior free yl, peroxide, anti-oxidant balance in vivo is maintained.Wherein NQO1 base
Because belonging to NAD (P) H dehydrogenase (quinone) family, it is responsible for catalysis quinone Double electron reduction reaction.The enzyme is by NADH or NADPH conduct
Electron donor, the reduction reaction of quinones in catalytic body, prevents quinone that the free radical that an electron reduction process generates occurs.
There are different SNP by NQO1 in crowd, wherein crucially there are TT:CT:CC in the site gene coding region rs1800566.TT/
CT genotype oxidation resistance is general, and CC genotype oxidation resistance is strong.SOD2 is the one of ferrimanganic superoxide dismutase family
Member.It encodes a kind of mitochondrial protein for forming tetramer, and every subunit combines a manganese ion.This protein and phosphorous oxide
The superoxide radical that acidification generates combines, and is translated into hydrogen peroxide and diatomic oxygen.SOD2 exists different in crowd
SNP, wherein crucially code area rs4880 deposits CC:TC:TT in site.C allele carrier's oxidation resistance is poor, TT base
Because type anti-aging ability is stronger.Skin oxidation resistance genetic test anticipated that cutaneous sensibility speciality, take protection early
Measure avoids skin care is improper from causing the irreversible aging of skin.
Loss of moist caused by skin aging, long-term mental burden and sleep insufficiency, excessive being exposed to the sun all easily promote skin
, there is wrinkle in relaxation.Collagen is a kind of hmw protein, elastomer albumen and collagen fibrous proteins by mutually intersecting
Fork is wound a strong elastic network(s), and skin is made to keep solid and flexible state.Studies have shown that COL1A2 and skin
The wrinkling resistance of skin is related.The pro-alpha2 chain of the genes encoding Type I collagen, triple helices are by two alpha1 chains
It is formed with an alpha2 chain.Type i collagen is a kind of fibroplastic collagen, is present in most of connective tissues,
Rich content in bone, cornea, corium and tendon.There are different SNP by COL1A2 in crowd, wherein crucially gene coding region
There are CC:TC:TT in the site rs11979919.CC/TC genotype anti-light aging ability is general, and wrinkling resistance is general.TT genotype
Anti-light aging capability improving, wrinkling resistance are good.Gene unit point amino acid of differences will be such that collagen decomposition rate obviously increases
Add, skin is easy to generate wrinkle, if do not taken effective nursing, skin aging fast speed.
In conclusion the relevant key function gene of skin health can react the ability of skin health maintenance well,
That is skin anti-aging ability.However current cutaneous gene method for detecting single nucleotide polymorphism, it is more multiple that there is also test procedures
It is miscellaneous, it the defects of inconvenient benefit and deficiency, therefore establishes by establishing a set of economic quickly skin appraisal procedure, passes through
Simple and quick method checks the difference of skin health maintenance ability gene vigor between individual, thus between comprehensive assessment individual
Skin health simultaneously provides the care plans changed and improved, becomes technical problem urgently to be resolved.
Summary of the invention
Therefore, at present based on fluorescent quantitative PCR technique, the cutaneous gene monokaryon of skin anti-aging ability is detected
Nucleotide polymorphism detection method, there is also test procedures it is complex, inefficient and inconvenient the defects of and deficiency, this
Invention provides a kind of new skin anti-aging ability detection method and its detection primer and kit, to solve above-mentioned skill
Art problem.
Basic technique principle of the invention are as follows: the present invention carries out the inspection of different SNP using the method that PCR product Tm is migrated
It surveys.Its brief principle is, when for SNP site design primer, using unified reverse primer, and forward primer is with SNP site knot
Tail (Fig. 1).5 ' ends of the forward primer of the different genotype of SNP adjust the Tm of different genotype product by increasing GC simultaneously
Value.It can determine whether which kind of SNP product is by the difference of Tm value between product.The key of this method is that forward primer GC is duplicate
Selection, if can play the role of distinguishing different SNP.If it is homozygote genotype, it should be single melting curve peak type.
And containing the corresponding genotype of more GC in the end of forward primer 5 ', melting curve Tm should be higher than in the end of forward primer 5 ' and contain
There is the corresponding genotype of more GC.Simultaneously if it is heterozygote genotype, theory can generate bimodal melting curve or unimodal and Tm
It is located in the middle melting curve.By interpretation melting curve, the SNP parting of related gene can be confirmed, and further interpretation skin
The anti-aging ability of skin.
In order to solve the above technical problems, one of the technical solution that the present invention takes are as follows: a kind of inspection of skin anti-aging ability
Survey method, described detection method includes the following steps:
(1) DNA of sample to be tested is extracted；
(2) specific primer is utilized, using DNA obtained by step (1) as template, carries out fluorescent quantitative PCR detection, wherein described
Specific primer is forward primer 1, forward primer 2 and reverse primer 3；
(3) according to fluorescent quantitative PCR testing result obtained by step (2), skin anti-aging ability is detected.
Wherein step (1) is to extract the DNA of sample to be tested.The sample to be tested is the sample to be tested of this field routine, packet
Serum is included, is organized, cell, mouth swab sample or other samples to be tested, preferably mouth swab sample.Wherein it is described to
The extracting method of the DNA of sample is the DNA extraction method of this field routine, as long as can extract the DNA in sample to be tested
Demand of the present invention can be met out, the DNA extraction method is preferably comprised phenol chloroform method etc., or utilizes commercially available
The DNA of DNA extraction agent box extracting sample.
Wherein step (2) are as follows: specific primer is used, using DNA obtained by step (1) as template, carries out PCR amplification detection,
Preferably, the forward primer 1 is as shown in SEQ ID NO:1 in sequence table, the forward primer 2 such as SEQ ID in sequence table
Shown in NO:2, the reverse primer 3 is as shown in SEQ ID NO:3 in sequence table.
Preferably, the forward primer 1, as shown in SEQ ID NO:4 in sequence table, the forward primer 2 is as in sequence table
Shown in SEQ ID NO:5, the reverse primer 3 is as shown in SEQ ID NO:6 in sequence table.
Preferably, the forward primer 1, as shown in SEQ ID NO:7 in sequence table, the forward primer 2 is as in sequence table
Shown in SEQ ID NO:8, the reverse primer 3 is as shown in SEQ ID NO:9 in sequence table.
The preparation method of specific primer of the present invention is this field customary preparation methods, and preferably complete sequence is artificial
Synthesis.
Wherein the testing conditions of step (2) described fluorescent quantitative PCR are preferably: (1) 90-96 DEG C of initial denaturation 10
- 10 minutes seconds；(2) it is denaturalized -90 seconds 5 seconds for 90-96 DEG C；(3) it anneals -99 seconds 5 seconds for 45-65 DEG C；(4) extend 3 seconds -300 for 55-99 DEG C
Second；Step (2)-(4) cycle-index is 28-60；(5) extend -600 seconds 5 seconds for 60-73 DEG C, 55-99 DEG C of (6) heat preservation 3 seconds -300
Second；(7) 30-50 DEG C heat preservation 3-10 seconds.
The condition of the fluorescent quantitative PCR be more preferably (1) 95 DEG C initial denaturation 10 minutes；(2) 95 DEG C are denaturalized 10 seconds,
(3) 60 DEG C are annealed 40 seconds, and (4) 95 DEG C extend 15 seconds, and step (2)-(4) cycle-index is 35 times, and (5) 60 DEG C extend 1 minute,
(6) 95 DEG C keep the temperature 15 seconds, and (7) 40 DEG C keep the temperature 5 seconds.
Wherein the reaction system of step (2) the PCR amplification detection is preferably: 10 μ l of qPCR mixed liquor, specificity are drawn
1 solution of forward primer, 0.4 μ l, 2 solution of forward primer, 0.4 μ l, 3 solution of reverse primer, 0.4 μ l in object solution, 2 μ l of DNA profiling,
Then MiliQ water is added to supply to 20 μ l.It is not examining with disease preferably, skin anti-aging detection method of the present invention
Detection method for the purpose of disconnected and treatment.
In order to solve the above technical problems, the two of the technical solution that the present invention takes are as follows: a kind of detection skin anti-aging gene
Specific primer, the specific primer includes: forward primer 1, forward primer 2 and reverse primer 3, the forward primer 1
As shown in SEQ ID NO:1 in sequence table, the forward primer 2 is as shown in SEQ ID NO:2 in sequence table, the reverse primer
3 as shown in SEQ ID NO:3 in sequence table.
In order to solve the above technical problems, the three of the technical solution that the present invention takes are as follows: a kind of detection skin anti-aging gene
Specific primer, the specific primer includes: forward primer 1, forward primer 2 and reverse primer 3, the forward primer 1
As shown in SEQ ID NO:4 in sequence table, the forward primer 2 is as shown in SEQ ID NO:5 in sequence table, the reverse primer
3 as shown in SEQ ID NO:6 in sequence table.
In order to solve the above technical problems, the four of the technical solution that the present invention takes are as follows: a kind of detection skin anti-aging gene
Specific primer, the specific primer includes: forward primer 1, forward primer 2 and reverse primer 3, the forward primer 1
As shown in SEQ ID NO:7 in sequence table, the forward primer 2 is as shown in SEQ ID NO:8 in sequence table, the reverse primer
3 as shown in SEQ ID NO:9 in sequence table.The preparation method of specific primer of the present invention is the conventional preparation side in this field
Method, preferably complete sequence are artificial synthesized.
In order to solve the above technical problems, the five of the technical solution that the present invention takes are as follows: a kind of detection skin anti-aging gene
Detection kit, the detection kit includes: specific primer, and the specific primer includes: forward primer 1 such as sequence
In list shown in SEQ ID NO:1, the forward primer 2 is as shown in SEQ ID NO:2 in sequence table, and the reverse primer 3 is such as
In sequence table shown in SEQ ID NO:3；Or the forward primer 1 is as shown in SEQ ID NO:4 in sequence table, the forward primer
2 as shown in SEQ ID NO:5 in sequence table, and the reverse primer 3 is as shown in SEQ ID NO:6 in sequence table or the forward direction
For primer 1 as shown in SEQ ID NO:7 in sequence table, the forward primer 2 is described anti-as shown in SEQ ID NO:8 in sequence table
It further include qPCR mixed liquor to primer 3 as shown in SEQ ID NO:9 in sequence table.
QPCR mixed liquor of the present invention is the PCR reaction buffer of this field routine, can guarantee that qPCR reaction is normal
It carries out.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention
Example.
The reagents and materials used in the present invention are commercially available.
The positive effect of the present invention is that: the present invention provides a kind of economic quickly detection skin anti-aging genes
Single nucleotide polymorphism method, it is easy to operate, using Standard PCR scheme operate when, can using common PCR instrument can
To realize testing goal.Primer selected by the present invention has and efficiently synthesizes, the extremely low advantage of nonspecific reaction, while can
So that PCR reaction carries out under conditions of one more relaxed, to annealing and the temperature requirement extended no longer as previous side
Case is so stringent, thus the requirement to PCR instrument is very loose, while also loose many to the temperature requirement of detection environment, no
It needs between constant temperature.
Detailed description of the invention
Fig. 1 is the method schematic that quantitative fluorescent PCR product Tm migration detects different SNP partings.
Fig. 2 is that qPCR distinguishes NQO1 difference SNP genotype results figure.Wherein Fig. 2 (A) utilizes NQO1(C)-F1, NQO1
(T)-F1 and NQO1-R carries out qPCR amplification to three kinds of genomic DNAs that the site rs1800566 is TT, CC, CT.Fig. 2 (B)
Utilize NQO1(C)-F2, NQO1(T) the three kinds of genomic DNAs progress of-F2 and NQO1-R to the site rs1800566 for TT, CC, CT
QPCR amplification.
Fig. 3 is to distinguish SOD2 difference SNP genotype results figure using qPCR.Wherein Fig. 3 (A) utilizes SOD2(C)-F1,
SOD2(T)-F1 and SOD2-R carries out qPCR amplification to three kinds of genomic DNAs that the site rs4880 is TT, CC, CT.Fig. 3
(B) three kinds of genomic DNAs that the site rs4880 is TT, CC, CT are carried out using SOD2(C)-F2, SOD2(T)-F2 and SOD2-R
QPCR amplification.
Fig. 4 is to distinguish COL1A2 difference SNP genotype results figure using qPCR.Wherein Fig. 4 (A) is to utilize COL1A2(C)-
F1, COL1A2(T)-F1 and COL1A2-R carry out qPCR expansion to three kinds of genomic DNAs that the site rs11979919 is TT, CC, CT
Increase result.Fig. 4 (B) is to utilize COL1A2(C)-F2, COL1A2(T)-F2 and COL1A2-R to the site rs11979919 be TT,
Three kinds of genomic DNAs of CC, CT carry out qPCR amplification.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
The selection of product specification can pass through commercially available acquisition wherein the primer is complete sequence synthesis.
Embodiment 1 judges the SNP parting of NQO1 gene by fluorescence quantifying PCR method
Oral cavity sample DNA, which extracts, uses the oral cavity Suo Laibao, throat swab DNA extraction kit (D3300).Detailed process is according to explanation
Book is operated.Brief step is described as follows:
1, material processing:
A, it samples: being wiped in cheek 10 times using cotton swab.Sampling please don't be fed in first 30 minutes or u drinking-water.
B, it handles: swab being transferred in 2ml centrifuge tube, is cut cotton swab part from its bar with scissors, it is molten that 400 μ l are added
Liquid A.
2, the RNase A(10mg/ml of 20 μ L is added into solution), 55 DEG C of placement 15min.
3, the Proteinase K (10mg/ml) of 20 μ L is added, is sufficiently mixed by inversion, 55 DEG C of water-baths digest 60min, during which suggest
Every ten minutes, reverse centrifuge tube mixed primary.
4,400 μ L bulk solution B are added, is sufficiently mixed by inversion, white precipitate such as occurs, 75 DEG C of 15- can be placed in
30min, precipitating can disappear, and not influence subsequent experimental.
5, solution is transferred to new centrifuge tube, 400 μ L dehydrated alcohols are added, mixes well, is likely to occur at this time cotton-shaped
Precipitating, does not influence the extraction of DNA, and solution and flocculent deposit can be added in adsorption column.
6,12000rpm is centrifuged 1min, abandons waste liquid, adsorption column is put into collecting pipe.
7,600 μ L rinsing liquids are added into adsorption column, 12000rpm is centrifuged 1min, abandons waste liquid, adsorption column is put into collection
Guan Zhong.
8,600 μ L rinsing liquids are added into adsorption column, 12000rpm is centrifuged 1min, abandons waste liquid, adsorption column is put into collection
Guan Zhong.
9,12000rpm is centrifuged 2min, and adsorption column opening is placed in several minutes of room temperature.
10, adsorption column is put into a clean centrifuge tube, to the hanging 50-200 μ L that is added in adsorbed film center through 65 DEG C
The eluent of water-bath preheating, room temperature 5min, 12000rpm are centrifuged 2min.
11, centrifugation gained eluent is added in adsorption column, 12000rpm is centrifuged 2min, and the base of high quality can be obtained
Because of a group DNA.
The reaction system of fluorescent quantitative PCR is as shown in table 1.
1 fluorescent quantitative PCR reaction system of table
| Ingredient | Volume (μ L) |
| DNA profiling | 2 |
| SNP(C) forward primer 1 | 0.4 |
| SNP(T) forward primer 2 | 0.4 |
| Reverse primer 3 | 0.4 |
| QPCR mixed liquor | 10 |
| Water | 6.8 |
| Total volume | 20 |
Fluorescent quantitative PCR reaction condition is as follows:
(1) 95 DEG C initial denaturation 10 minutes；(2) 95 DEG C are denaturalized 10 seconds, and (3) 60 DEG C are annealed 40 seconds, and (4) 95 DEG C extend 15 seconds, step
(2)-(4) cycle-index is 35 times, and (5) 60 DEG C extend 1 minute, and (6) 95 DEG C keep the temperature 15 seconds, and (7) 40 DEG C keep the temperature 5 seconds.
For the SNP site rs1800566 of NQO1, design obtains forward primer 1 are as follows: NQO1(C)-F1, in sequence table
Shown in SEQ ID NO:1；
Forward primer 2 are as follows: NQO1(T)-F1, as shown in SEQ ID NO:2 in sequence table；
Reverse primer 3 are as follows: NQO1-R, when as shown in SEQ ID NO:3 in sequence table, fluorescent quantitative PCR reaction system is such as
Shown in table 1, reaction condition is as described above.
Acquired results are as shown in Figure 2 (A) shows, should the result shows that, using above-mentioned three kinds of primers, using the DNA of extraction as template, into
Row fluorescent quantitative PCR, melting curve shows that three kinds of genotype can be clearly distinguished after amplification, can be very good to distinguish
The different SNP types of NQO1 gene.Wherein with NQO1(C)-F1 5 ' end because G/C content height lead to homozygote CC genotype
Product Tm is maximum, NQO1(T) 5 ' ends of-F1 are because G/C content relatively rarely causes the product Tm of homozygote TT relatively small.And when miscellaneous
In the presence of zygote CT genotype, the amplified production of existing C genotype has the amplified production of T genotype again, so CT amplified production
Tm between CC and TT.And forward primer 1 are as follows: NQO1(C)-F2(is as shown in SEQ ID NO:10 in sequence table) and NQO1
(T)-F2(is as shown in SEQ ID NO:11 in sequence table), with same reverse primer 3 and reaction system and reaction condition, institute
Obtaining amplified production then cannot be distinguished three kinds of genotype.Content shown in (B) according to fig. 2, melting curve shows three kinds of genes after amplification
Type cannot be distinguished.
The above results show that NQO1(C)-F1, NQO1(T)-F1 and NQO1-R be preferred primer combination, can pass through
Clear easily three kinds of the site the NQO1 rs1800566 SNP genotype in differentiation crowd of qPCR method, and further judge skin
Anti-aging ability.
Embodiment 2 judges the SNP parting of superoxide dismutase gene SOD2 by fluorescence quantifying PCR method
For the SNP site rs4880(TT:CT:CC of SOD2), first by 1 the method for embodiment, by buccal swab side
Method extracts the genomic DNA of multiple people.Then by the method and system of above-mentioned fluorescent quantitative PCR, fluorescent quantitation is carried out
The DNA profiling as the optimization of subsequent SNP primer for three kinds of genotype that rs4880 is TT, CT, CC is found in PCR amplification sequencing.Hair
Bright people continuously devises the forward primer of different G/C contents for SOD2.
Forward primer 1 are as follows: SOD2(C)-F1, as shown in SEQ ID NO:4 in sequence table；
Forward primer 2 are as follows: SOD2(T)-F1, as shown in SEQ ID NO:5 in sequence table；
Reverse primer 3 are as follows: SOD2-R, when as shown in SEQ ID NO:6 in sequence table, fluorescent quantitative PCR reaction system is such as
Shown in table 1, reaction condition is as described in Example 1.
Fluorescent quantitative PCR acquired results, melting curve shows that three kinds of genotype can be clearly distinguished after amplification.It can
To distinguish different SNP(Fig. 3 A well).Wherein with SOD2(C)-F1 5 ' end because G/C content height lead to homozygote CC gene
The product Tm of type is maximum, SOD2(T) 5 ' ends of-F1 are because G/C content relatively rarely causes the product Tm of homozygote TT relatively small.And
In the presence of heterozygote CT genotype, the amplified production of existing C genotype has the amplified production of T genotype again, so CT is expanded
The Tm of product is between CC and TT.And another set of primer SOD2(C)-F2(is as shown in SEQ ID NO:12 in sequence table) and
SOD2(T)-F2(is as shown in SEQ ID NO:13 in sequence table) amplified production if cannot be distinguished three kinds of genotype (Fig. 3 B).
The above results show that SOD2(C)-F1, SOD2(T)-F1 and SOD2-R be preferred primer combination, can pass through
Clear easily three kinds of the site the SOD2 rs4880 SNP genotype in differentiation crowd of qPCR method, and further interpretation skin is anti-
Aging ability.
Embodiment 3 judges the SNP parting of COL1A2 gene by fluorescence quantifying PCR method
For the SNP site rs11979919(CC:TC:TT of COL1A2), it is mentioned first by buccal swab method described in embodiment 1
Take the genomic DNA of multiple people.Then by the amplification sequencing of fluorescence quantifying PCR method described in embodiment 1, rs11979919 is found
For the DNA profiling as the optimization of subsequent SNP primer of three kinds of genotype of TT, CT, CC.Inventor continuously designs for COL1A2
The forward primers of different G/C contents.
Forward primer 1 are as follows: COL1A2(C)-F1, as shown in SEQ ID NO:7 in sequence table；
Forward primer 2 are as follows: COL1A2(T)-F1, as shown in SEQ ID NO:8 in sequence table；
Reverse primer 3 are as follows: COL1A2-R, when as shown in SEQ ID NO:9 in sequence table, fluorescent quantitative PCR reaction system
As shown in table 1, reaction condition is as described in Example 1.
It according to fluorescent quantitative PCR acquired results, can be very good to distinguish different SNP(Fig. 4 A).Wherein with COL1A2
(C) 5 ' ends of-F1 are because G/C content height causes the product Tm of homozygote CC genotype maximum, COL1A2(T) 5 ' ends of-F1 because
G/C content relatively rarely causes the product Tm of homozygote TT relatively small.And in the presence of heterozygote CT genotype, existing C genotype
Amplified production has the amplified production of T genotype again, so CT amplified production forms bimodal melting curve.And another set of primer
COL1A2(C)-F2(is as shown in SEQ ID NO:14 in sequence table) and COL1A2(T)-F2(such as SEQ ID NO:15 in sequence table
It is shown) amplified production then cannot be distinguished three kinds of genotype (Fig. 4 B).
The above results show that COL1A2(C)-F1, COL1A2(T)-F1 and COL1A2-R be preferred primer combination, it can be with
By clear easily three kinds of the site the COL1A2 rs11979919 SNP genotype in differentiation crowd of qPCR method, and further sentence
The anti-aging ability of disconnected skin.
It should be understood that those skilled in the art can make the present invention various after having read above content of the invention
Change or modification, these equivalent forms also fall within the scope of the appended claims of the present application.
Sequence table
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<213>artificial sequence (Artificial Sequence)
<400> 9
tactgataag gaggcataac tcaaa 25
<210> 10
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
gggcggcgct tccaagtctt agaac 25
<210> 11
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
ggcggcttcc aagtcttaga at 22
<210> 12
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
gcagggcggc caggcagctg gctccggc 28
<210> 13
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
ggcgcaggca gctggctccg gt 22
<210> 14
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
gcgggcgggc ggcttaatta gcaccactct c 31
<210> 15
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
gggccttaat tagcaccact ctt 23
Claims (10)
1. a kind of detection method of skin anti-aging ability, which is characterized in that described detection method includes the following steps:
(1) DNA of sample to be tested is extracted；
(2) specific primer is utilized, using DNA obtained by step (1) as template, carries out fluorescent quantitative PCR detection, wherein described
Specific primer is forward primer 1, forward primer 2 and reverse primer 3；
(3) according to fluorescent quantitative PCR testing result obtained by step (2), skin anti-aging ability is detected.
2. detection method as described in claim 1, which is characterized in that detection method step (2) forward primer 1 is such as
In sequence table shown in SEQ ID NO:1, the forward primer 2 is as shown in SEQ ID NO:2 in sequence table, the reverse primer 3
As shown in SEQ ID NO:3 in sequence table.
3. detection method as described in claim 1, which is characterized in that detection method step (2) forward primer 1 is such as
In sequence table shown in SEQ ID NO:4, the forward primer 2 is as shown in SEQ ID NO:5 in sequence table, the reverse primer 3
As shown in SEQ ID NO:6 in sequence table.
4. detection method as described in claim 1, which is characterized in that detection method step (2) forward primer 1 is such as
In sequence table shown in SEQ ID NO:7, the forward primer 2 is as shown in SEQ ID NO:8 in sequence table, the reverse primer 3
As shown in SEQ ID NO:9 in sequence table.
5. the detection method of skin anti-aging ability as described in claim 1, which is characterized in that wherein step (2) fluorescence is fixed
Measure the testing conditions of PCR amplification are as follows: (1) 90-96 DEG C initial denaturation -10 minutes 10 seconds；(2) it is denaturalized -90 seconds 5 seconds for 90-96 DEG C；(3)
45-65 DEG C is annealed -99 seconds 5 seconds；(4) extend -300 seconds 3 seconds for 55-99 DEG C；Step (2)-(4) cycle-index is 28-60；(5) 60-
73 DEG C extend -600 seconds 5 seconds, 55-99 DEG C of (6) heat preservation -300 seconds 3 seconds；(7) 30-50 DEG C heat preservation 3-10 seconds.
6. the detection method of anti-aging ability as described in any one in claim 1-5, which is characterized in that the detection method is
Detection method not for the purpose of the diagnosing and treating of disease.
7. a kind of specific primer for detecting skin anti-aging gene, which is characterized in that the specific primer includes: that forward direction is drawn
Object 1, forward primer 2 and reverse primer 3, the forward primer 1 is as shown in SEQ ID NO:1 in sequence table, the forward primer 2
As shown in SEQ ID NO:2 in sequence table, the reverse primer 3 is as shown in SEQ ID NO:3 in sequence table.
8. a kind of specific primer for detecting skin anti-aging gene, which is characterized in that the specific primer includes: that forward direction is drawn
Object 1, forward primer 2 and reverse primer 3, the forward primer 1 is as shown in SEQ ID NO:4 in sequence table, the forward primer 2
As shown in SEQ ID NO:5 in sequence table, the reverse primer 3 is as shown in SEQ ID NO:6 in sequence table.
9. a kind of specific primer for detecting skin anti-aging gene, which is characterized in that the specific primer includes: that forward direction is drawn
Object 1, forward primer 2 and reverse primer 3, the forward primer 1 is as shown in SEQ ID NO:7 in sequence table, the forward primer 2
As shown in SEQ ID NO:8 in sequence table, the reverse primer 3 is as shown in SEQ ID NO:9 in sequence table.
10. a kind of detection kit for detecting skin anti-aging gene, which is characterized in that the detection kit includes: spy
Specific primer, the specific primer include: forward primer 1 as shown in SEQ ID NO:1 in sequence table, the forward primer 2
As shown in SEQ ID NO:2 in sequence table, the reverse primer 3 is as shown in SEQ ID NO:3 in sequence table；And/or it is described just
To primer 1 as shown in SEQ ID NO:4 in sequence table, the forward primer 2 is described as shown in SEQ ID NO:5 in sequence table
Reverse primer 3 is as shown in SEQ ID NO:6 in sequence table and/or the forward primer 1 such as SEQ ID NO:7 institute in sequence table
Show, the forward primer 2 is as shown in SEQ ID NO:8 in sequence table, the reverse primer 3 such as SEQ ID NO:9 in sequence table
It is shown, it further include qPCR mixed liquor.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111455035A (en) * | 2020-01-22 | 2020-07-28 | 广州市普森生物科技有限公司 | Primer combination and kit for detecting skin anti-aging capability gene and application of primer combination and kit |
| WO2021235551A1 (en) * | 2020-05-22 | 2021-11-25 | 株式会社 資生堂 | Method for determining resistance against skin aging using genetic polymorphism |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108251521A (en) * | 2018-03-15 | 2018-07-06 | 北京天平永达科技发展有限公司 | For detecting the primer set of tumor susceptibility gene SNP related to skin aging and its application |
-
2018
- 2018-12-29 CN CN201811630156.2A patent/CN109517906A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108251521A (en) * | 2018-03-15 | 2018-07-06 | 北京天平永达科技发展有限公司 | For detecting the primer set of tumor susceptibility gene SNP related to skin aging and its application |
Non-Patent Citations (2)
| Title |
|---|
| JUN WANG 等: "High-throughput SNP genotyping by single-tube PCR with Tm-shift primers", 《BIOTECHNIQUES》, vol. 39, no. 6, pages 885, XP001208019, DOI: 10.2144/000112028 * |
| WENSHAN GAO 等: "Genetic variants associated with skin aging in the Chinese Han population", 《JOURNAL OF DERMATOLOGICAL SCIENCE》, vol. 86, pages 21, XP029943499, DOI: 10.1016/j.jdermsci.2016.12.017 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111455035A (en) * | 2020-01-22 | 2020-07-28 | 广州市普森生物科技有限公司 | Primer combination and kit for detecting skin anti-aging capability gene and application of primer combination and kit |
| WO2021235551A1 (en) * | 2020-05-22 | 2021-11-25 | 株式会社 資生堂 | Method for determining resistance against skin aging using genetic polymorphism |
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