CN105886645A - Primer, probe and kit for evaluating anti-wrinkle condition of skin - Google Patents

Primer, probe and kit for evaluating anti-wrinkle condition of skin Download PDF

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CN105886645A
CN105886645A CN201610367685.2A CN201610367685A CN105886645A CN 105886645 A CN105886645 A CN 105886645A CN 201610367685 A CN201610367685 A CN 201610367685A CN 105886645 A CN105886645 A CN 105886645A
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skin
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snp site
wrinkle
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纪志梁
王毛阳
杨志云
刘晓瑜
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Fujian Aiwo Health Biotechnology Co Ltd
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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Abstract

The invention discloses a primer, probe and kit for evaluating the anti-wrinkle condition of skin. The sequences of the primer and the probe are SEQ ID NO:1-4, SED ID NO:5-8, SEQ ID NO:9-12, SEQ ID NO:13-15, SEQ ID NO:16-18, SEQ ID NO:19-21 and SEQ ID NO:22-25 wherein the 5' ends and the 3'ends of SEQ ID NO:3-4, SEQ ID NO:7-8, SEQ ID NO:11-12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21 and SEQ ID NO: 24-25 are connected with different fluorescence markers respectively. The invention further discloses a method for assisting a user in selecting a correct beautifying method through gene detection. The internal cause and the external cause of wrinkles of skin of a customer are leant, a user-friendly beautifying healthy solution is provided, and the effects of anti-wrinkle beautifying products are maximized.

Description

A kind of primer for assessing skin wrinkle resisting situation and probe and test kit
Technical field
The present invention relates to beauty treatment fields, particularly relate to a kind of primer for assessing skin wrinkle resisting situation and probe and reagent Box.
Background technology
SNP (single nucleotide polymorphism) refers to the variation of single core thuja acid on genome, including conversion, transversion, disappearance And insertion, it is a kind of modal genetic marker, the frequency of carrying in crowd is more than 1%, is widely used in human diseases and examines The fields such as disconnected, medicine individuation use, Basic of Biology research.Along with different SNP site researchs are goed deep into, by multiple The combine detection of SNP site and follow-up data analysis, can formulate personalized health for the gene information of different crowd Solution.
The skin appearance of human body is affected by endogenous cause of ill and exopathogenic factor, and exopathogenic factor includes that ultraviolet, environmental pollution, improper beauty treatment are produced Product use etc., endogenous cause of ill is then embodied in the Biochemical changes relevant to skin, including protein active, hormone level etc., these changes Mostly determined by gene.Mostly the skin care method of present stage is to take according to exopathogenic factor, as used sunscreen cream, guarantor Humectant etc., but these have little effect mostly without maintenance method targetedly, cure the symptoms, not the disease.Along with growth in the living standard, Increasing for improving the demand of skin smooth and tautness, carrying out specific aim beauty and skin care in conjunction with internal and external reasons will ratio tradition side Method more advantage, the most accurately understands endogenous cause of ill and seems most important.
Be there is again multiple SNP site by multiple effect genes, each gene in the generation of wrinkle of skin, these sites can shadow Ring the enzymatic activity of related gene.The skin wrinkle resisting ability that can evaluate and test Different Individual is detected, also by the SNP site of related gene Can assess whether relevant cosmetics are applicable to tester, make product effect reach to maximize, combine daily on this basis Living habit adjusts, and makes cosmetic result promote further.For this detection method, choose Sites Combination and follow-up accurately Data analysis is particularly important.Having patent/Patent Application Publication report both at home and abroad utilizes SNP site genotype to judge cosmetics It is suitable for skin type, but there are still susceptible SNP site and choose inaccurate, testing result the most energetic total score analysis problem.When Before have no combine different genes SNP site combine detection and quantization algorithm assessment skin wrinkle resisting ability report.
Chinese utility model patent 201320009798.7 discloses a kind of gene chip, utilizes this genechip detection beautiful White and aging related gene SNP site, the SNP site of whitening gene includes SLC45A2 (re35391), KITLG (rs642742)、ASIP(rs1015362)、TYR(rs1042602)、OCA2(rs1800414)、DCT(rs2031526)、MC1R (rs885479), the SNP of aging gene include MMP1 (rs1799750), TIMP1 (rs4898), SIRT6 (rs107251), SOD2 (rs4880), NQO1 (rs1800566), TNFA (rs1800629), EPHX (rs1051740), but the base that this patent uses Because chip method is cumbersome, it is difficult to be suitable for a large amount of detection.Chinese invention patent application 201180056031.3 has screened one group The gene (LPL, ADIPOQ, PLN etc.) relevant to skin aging, and according to this group genescreen to a kind of aging resistance new material, Not mentioned gene-correlation SNP site in this patent application.Chinese patent application 201080044917.1 provides one and utilizes sky So antisense polynucleotides or compound are used for regulating and controlling FLG gene expression, thus reach prevention or treat the disease relevant to FLG.
United States Patent (USP) US200812286033 induces related gene expression to reach to improve wrinkle by Monoterpenes Effect.United States Patent (USP) US200912512368 induces ELN and COL3A1 gene by the cosmetics containing iso-amylene isoflavone Express, thus reach to improve the effect of skin appearance.United States Patent (USP) US201514618080 and US201313932565 pass through Skin aging position uses Carlina acaulis extract and olive oil extract, makes the related gene including ELN and COL3A1 be subject to To regulation and control up-regulated expression, thus reach to improve effect of ageing skin.
Above patent/patent application all utilize foreign substance to regulate and control the intragentic expression of body, but the gene of each individuality In but there are differences SNP site, this method is difficult to reach personalized maintenance or treatment.British patent GB20110021917 is examined Survey the target gene SNP site relevant to cosmetics ingredients Biogenic approach, and then judged whether this product is suitable for use with Person, the SNP site that this patent relates to has MMP-1 (rs1799750), NQO1 (rs1800566), GSTP1 (rs1695), hGPX1 (rs10504)。
Summary of the invention
It is an object of the invention to provide a kind of primer for assessing skin wrinkle resisting situation and probe.
For achieving the above object, the present invention provides a kind of primer for assessing skin wrinkle resisting situation and probe, its feature It is, described sequence such as SEQ ID NO:1-4, and SEQ ID NO:5-8, and SEQ ID NO:9-12, and SEQ ID NO:13- 15, and SEQ ID NO:16-18, and SEQ ID NO:19-21, and SEQ ID NO:22-25；Wherein SEQ ID NO:3-4,7- 5 ' the ends of 8,11-12,15,18,21,24-25 connect different fluorescent labelinies respectively with 3 ' ends.
The present invention also provides for a kind of test kit for assessing skin wrinkle resisting situation, it is characterised in that draw containing described Thing and probe.
The present invention also provides for a kind of method utilizing the gene test auxiliary correct beauty method of customer selecting, and its feature exists In, use described primer and probe, or the test kit described in use
Further, step is:
1) use described in primer and probe or described test kit client is carried out SNP site detection；
2) the skin wrinkle resisting ability value of client is gone out according to susceptible SNP site combination calculation；Described susceptible SNP site is combined as ELNrs2071307、EMILIN1rs2011616、FLGrs146466242、rs200519781、MMP3rs3025058、 MMP1rs1799750、COL3A1rs1800255；
3) combine 1) SNP site testing result and 2) skin wrinkle resisting ability value, carry out result judgement.
Further, described step 1) be,
Extract the DNA sample of client；
Described primer and probe or described test kit is used to carry out PCR amplification；Preferably, the response procedures of PCR amplification is 50 DEG C of 2min, 95 DEG C of 10min, 95 DEG C of 15s, 60 DEG C of 1min circulate 40 times.
Further, described step 2) be,
The pathogenic risk ratio ratio of each loci gene type according to the combination of known susceptible SNP site calculates integrated risk Odds ratio OR (C), calculation equation is:
Produce average risk ratio odds (D) by known Chinese population different age group wrinkle of skin, calculate detection Individual wrinkle of skin produces value-at-risk ratio odds (X), and calculating formula is odds (X)=OR (C) × odds (D)；
Calculate integrated risk value:
According to known normal genotype crowd's value-at-risk F (N), calculate individual's skin wrinkling resistance value.
V = F ( N ) F ( X ) × 100 % .
Further, described step 3) result be judged as,
1) if individual's skin wrinkling resistance value is less than 100%, then it represents that the skin wrinkle resisting ability of this individuality is less than this age The normal value of Duan Renqun, wrinkling resistance is poor, belongs to the body constitution being easily generated wrinkle；Being worth the lowest, wrinkling resistance is the poorest；
2) if individual's skin wrinkling resistance value is equal to 100%, then it represents that the skin wrinkle resisting ability of this individuality and this age bracket The normal value of crowd is the same, and wrinkling resistance is general；
3) if individual's skin wrinkling resistance value is higher than 100%, then it represents that the skin wrinkle resisting ability of this individuality is higher than this age The normal value of Duan Renqun, wrinkling resistance is preferable, belongs to the body constitution being not likely to produce wrinkle；Being worth the highest, wrinkling resistance is the best；
Simultaneously；
If SNP site rs2071307 of gene ELN is A/A, show to carry homozygous mutation, weaken the elasticity of skin and draw Stretch ability, promote chronological ageing；If A/G, show to carry heterozygous mutant, weaken elasticity and the stretch capability of skin, promote slow Property is aging；If G/G, then it it is normal genotype；
If SNP site rs2011616 of gene EMILIN1 is A/A, show to carry homozygous mutation, but a crease in the skin risk Normally；If A/G, showing to carry G, elastic fiber quality reduces, dermis strength reduction, easy fold；If G/G, then For normal genotype, but owing to carrying G, elastic fiber quality reduces, dermis strength reduction, easy fold；
If SNP site rs146466242 of gene FLG is A/T, showing to carry heterozygous mutant, skin mechanical strength is more weak, Skin is easily subject to external injury；If T/T, then it it is normal genotype；
If SNP site rs200519781 of gene FLG is T/-, being shown to be deletion Genotype, silk polymeric protein activity is relatively Weak, the easy shallow stricture of vagina of skin is too much；If T/T, then it it is normal genotype；
If SNP site rs3025058 of gene M MP3 is-/A, being shown to be deletion Genotype, enzyme is lived and is reduced, and increases collagen Fibre damage and the risk of destruction, skin elasticity declines；If-/-, being then deletion Genotype, enzyme is lived and is reduced, and increases collagen fine Dimension damage and the risk destroyed, skin elasticity declines；If A/A, then it it is normal genotype；
If SNP site rs1799750 of gene M MP1 is C/C, being shown to be insertion genotype, enzyme is lived and is reduced, and increases collagen Fibre damage and the risk of destruction, skin elasticity declines；If-/C, being shown to be insertion genotype, enzyme is lived and is reduced, and increases collagen Fibre damage and the risk of destruction, skin elasticity declines；If-/-, then it it is normal genotype；
If SNP site rs1800255 of gene C OL3A1 is A/G, it is shown to be and carries heterozygous mutant, without relevant risk；If For A/A, being shown to be and carry homozygous mutation, type III collagen protein weakens or disappears so that skin is more readily formed wrinkle；If G/G, then be normal genotype.
It is clear that the present invention (1) filters out one group of molecular mechanism, can be used for the SNP position of comprehensive accurate evaluation skin wrinkle resisting ability Point combination；(2) use high specificity, highly sensitive TaqMan-MGB Probe-detection methods to detect, can detect many simultaneously Individual sample；(3) by algorithm precise quantification wrinkle generation risk to assess skin wrinkle resisting ability, personalization is provided for tester Skin wrinkle resisting scheme.
The technical solution used in the present invention is: binding molecule Mechanism Study, literature search and clinical data screening, obtains one The susceptible SNP site combination that group skin wrinkle resisting is relevant, described combination site is respectively ELN (rs2071307), EMILIN1 (rs2011616)、FLG(rs146466242、rs200519781)、MMP3(rs3025058)、MMP1(rs1799750)、 COL3A1 (rs1800255), the site information of described combination is shown in Table 1, and this combination site can be with the skin of accurate evaluation Different Individual Skin wrinkling resistance.
Table 1 susceptible SNP site information table
In order to detect described site, applicant devises the primer needed for detection and probe, described primer and fluorescent probe As follows (underscore is SNP site):
ELN (rs2071307) primer and fluorescent probe sequence:
EMILIN1 (rs2011616) primer and fluorescent probe sequence:
FLG (rs146466242) primer and fluorescent probe sequence:
FLG (rs200519781) primer and fluorescent probe sequence:
Forward primer GATCCTTGTCTTCGTCCAGT SEQ ID NO:13；
Reverse primer AGCAGAGCCACCAAGAGTCC SEQ ID NO:14；
Fluorescent probe VIC-TCCAGACCTTCCCCCTGACCAGT-MGB SEQ ID NO:15；
MMP3 (rs3025058) primer and fluorescent probe sequence:
Forward primer TCATATCAATGTGGCCAAATATTT SEQ ID NO:16；
Reverse primer CCTGCCTCAACCTCTCAAAGTGCT SEQ ID NO:17；
Fluorescent probe VIC-CAAGACATGGTTTTTTCCCCC-MGB SEQ ID NO:18；
MMP1 (rs1799750) primer and fluorescent probe sequence:
Forward primer TACCCTCTTGAACTCACATGTTAT SEQ ID NO:19；
Reverse primer TTACTCATAAACAATACTTCAGTA SEQ ID NO:20；
Fluorescent probe VIC-AATTAGAAAGGATATGACTTA-MGB SEQ ID NO:21；
COL3A1 (rs1800255) primer and fluorescent probe sequence:
Described loci detection method specifically comprises the following steps that
1, human saliva sample collection.Using non-injurious salivary automatically to flow out acquisition mode, before saliva gathers, 30min needs to use Foreign material in drinking water cleaning oral cavity, collection 2mL saliva is deposited in saliva preservation liquid and (is caused kind biotechnology share purchased from Xiamen limited Company) for detection.
2, human body complete genome DNA extracts.Use potassium iodide method, with reference to concretely comprising the following steps: take 0.1mL saliva in 1.5mL In centrifuge tube, adding 500 μ L 0.01mol/L PBS solution and repeatedly blow and beat, 10000rpm is centrifuged 5min；Supernatant, precipitation is gone to add 50 μ L5mol/L KI solution, concussion mixing, add 100 μ L 0.9%NaCl solution, 150 μ L phenol/chloroform (25: 24, V/V) Mixed liquor, concussion 30s, 10000rpm are centrifuged 5min；Taking 200 μ L of supernatant, add 200 μ L isopropanols, mixing, 10000rpm is centrifuged 5min；Going supernatant, precipitation to add 1mL absolute ethanol washing, 10000rpm is centrifuged 5min；Abandon dehydrated alcohol, be deposited in super-clean bench In dry up, add 50 μ L TE buffer solution ,-20 DEG C save backup.
3, PCR reaction system and response procedures.10 μ L reaction systems containUniversal PCR Master Mix (Applied Biosystems, Applied Biotechnology company limited of the U.S.) 5 μ L, DNA profiling 1 μ L, fluorescent probe (each SNP The detection correspondence probe in site mixes) (2.5 μMs) and primer mixed liquor (its corresponding primer of the detection of each SNP site Mix) 1.5 μ L, ddH2O 2.5μL.Response procedures is 50 DEG C of 2min, 95 DEG C of 10min, and 95 DEG C of 15s, 60 DEG C of 1min follow Ring 40 times.Detecting instrument is StepOnePlus real-time fluorescence quantitative PCR instrument (Applied Biosystems, U.S.'s application biology Technology Co., Ltd.).
4, interpretation.Use StepOnePlusTMSoftware v2.3 (Applied Biosystems, the U.S. Applied Biotechnology company limited) it is analyzed, according to different fluorescence signals, draw the genotype of corresponding SNP site.
Skin wrinkle resisting ability quantization method is as follows: 1, wrinkle of skin produces integrated risk ratio than calculating.Known detection institute The pathogenic risk ratio of each loci gene type obtained is than respectively OR1、OR2…ORm(literature query is learnt, the results are shown in Table 3), Can calculate individual's skin wrinkle produce integrated risk ratio than OR (C), calculation equation is as follows:
O R ( C ) = Π k = 1 n OR m .
2, the individual's skin wrinkle calculated by step 1 produces integrated risk ratio than OR (C), by document or other Statistical approach can obtain Chinese population different age group wrinkle of skin and produce average risk ratio odds (D) (known Chinese Group different age group wrinkle of skin produce average risk ratio odds (D) be respectively as follows: 20-35 year: 0.08,36-45 years old: 0.30, 46-55 year: 0.62), by odds ratio computing formula can calculate detection individual's skin wrinkle produce value-at-risk ratio odds (X), calculation equation is as follows:
I.e. odds (X)=OR (C) * odds (D).
3, the individual's skin wrinkle calculated by step 2 produces risk ratio odds (X), i.e. can be pushed away by value-at-risk Calculating formula and can calculate integrated risk value F (X), calculation equation is as follows:
F ( X ) = o d d s ( X ) o d d s ( X ) + 1 .
4, the skin wrinkle resisting ability of Different Individual can be assessed by the calculated integrated risk value of step 3, it is known that just Often genotype crowd's value-at-risk F (N) (known normal genotype crowd's value-at-risk F (N) be respectively as follows: 20-35 year: 7.4%, 36-45 Year: 23%, 46-55 year: 38.3%), individual's skin wrinkling resistance V can be calculated, calculation equation is as follows:
V = F ( N ) F ( X ) × 100 % .
Testing result personality analysis and solution:
According to wrinkling resistance quantum chemical method result, in conjunction with function and the molecular mechanism of related locus, it is judged that skin problem Probability, assessment tester can use the cosmetics containing which kind of composition, and adjust the daily life being conducive to skin protection for it Dietary habit alive.The molecular mechanism of related gene is as follows: if ELN carries sudden change, then skin easily relaxes；FLG carries sudden change, Easily cause the symptom such as dermatitis, too much, the allergy of the shallow stricture of vagina of skin；MMP1 and MMP3 carries sudden change, easily causes skin aging； EMILIN1 carries sudden change, easily causes cutis laxa；COL3A1 carries sudden change, then type III collagen protein weakens or disappears so that Skin is more readily formed wrinkle.
Beneficial effect: can be fully understood by endogenous cause of ill and exopathogenic factor that wrinkle of skin produces, provides the user personalized beauty care and is good for Health solution, makes anti-wrinkle cosmetic product using effect maximize.Further, it is also possible to adjust the day being conducive to skin protection for tester Often Diet lifestyle.
Detailed description of the invention
Embodiments of the invention are described below in detail, and the example of described embodiment is being intended to for explaining the present invention, and not It is understood that as limitation of the present invention.In embodiment, unreceipted concrete technology or condition person, retouched according to the document in this area The technology stated or condition or carry out according to product description.Agents useful for same or instrument unreceipted production firm person, be permissible By city available from conventional products.
Embodiment 1: primer and probe design
1, design and synthesize following primer and probe, synthesize unit: Applied Biosystems, the U.S. applies biological skill Art company limited.
ELN (rs2071307) primer and fluorescent probe sequence:
EMILIN1 (rs2011616) primer and fluorescent probe sequence:
FLG (rs146466242) primer and fluorescent probe sequence:
FLG (rs200519781) primer and fluorescent probe sequence:
Forward primer GATCCTTGTCTTCGTCCAGT SEQ ID NO:13；
Reverse primer AGCAGAGCCACCAAGAGTCC SEQ ID NO:14；
Fluorescent probe VIC-TCCAGACCTTCCCCCTGACCAGT-MGB SEQ ID NO:15；
MMP3 (rs3025058) primer and fluorescent probe sequence:
Forward primer TCATATCAATGTGGCCAAATATTT SEQ ID NO:16；
Reverse primer CCTGCCTCAACCTCTCAAAGTGCT SEQ ID NO:17；
Fluorescent probe VIC-CAAGACATGGTTTTTTCCCCC-MGB SEQ ID NO:18；
MMP1 (rs1799750) primer and fluorescent probe sequence:
Forward primer TACCCTCTTGAACTCACATGTTAT SEQ ID NO:19；
Reverse primer TTACTCATAAACAATACTTCAGTA SEQ ID NO:20；
Fluorescent probe VIC-AATTAGAAAGGATATGACTTA-MGB SEQ ID NO:21；
COL3A1 (rs1800255) primer and fluorescent probe sequence:
2, SNP site detection
1) sample collection.Detection sample in the present embodiment uses human saliva, and before saliva gathers, 30min need to be with drinking Foreign material in water cleaning oral cavity, gather 2mL saliva and deposit in saliva preservation liquid (purchased from the kind limited public affairs of biotechnology share of Xiamen cause Department) for detection.
2) human body complete genome DNA extracts.Use potassium iodide method, with reference to concretely comprising the following steps: take 0.1mL saliva in 1.5mL In centrifuge tube, adding 500 μ L 0.01mol/L PBS solution and repeatedly blow and beat, 10000rpm is centrifuged 5min；Supernatant, precipitation is gone to add 50 μ L5mol/L KI solution, concussion mixing, add 100 μ L 0.9%NaCl solution, 150 μ L phenol/chloroform (25: 24, V/V) Mixed liquor, concussion 30s, 10000rpm are centrifuged 5min；Taking 200 μ L of supernatant, add 200 μ L isopropanols, mixing, 10000rpm is centrifuged 5min；Going supernatant, precipitation to add 1mL absolute ethanol washing, 10000rpm is centrifuged 5min；Abandon dehydrated alcohol, be deposited in super-clean bench In dry up, add 50 μ L TE buffer solution ,-20 DEG C save backup.
3) reaction system and response procedures: 10 μ L reaction systems containUniversal PCR Master Mix (Applied Biosystems, Applied Biotechnology company limited of the U.S.) 5 μ L, DNA profiling 1 μ L, fluorescent probe (each SNP The detection correspondence probe in site mixes) (2.5 μMs) and primer mixed liquor 1.5 μ L, ddH2O 2.5μL.Response procedures is 50 DEG C of 2min, 95 DEG C of 10min, 95 DEG C of 15s, 60 DEG C of 1min circulate 40 times.Detecting instrument is StepOnePlus real time fluorescent quantitative PCR instrument (Applied Biosystems, Applied Biotechnology company limited of the U.S.).
4, after detection program end of run, StepOnePlus is usedTMSoftware v2.3(Applied Biosystems, Applied Biotechnology company limited of the U.S.) testing result is analyzed, draw corresponding SNP genotype (see Table 2).
Table 2SNP site testing result table
Detection gene SNP_ID Genotype
ELN rs2071307 G/G
EMILIN1 rs2011616 A/A
FLG rs200519781 T/-
FLG rs146466242 T/T
MMP3 rs3025058 -/-
MMP1 rs1799750 -/C
COL3A1 rs1800255 A/G
3, wrinkling resistance quantitative evaluation and personalized solution
1) wrinkle produces integrated risk value calculating.Being learnt by literature query, the ratio of different loci is as shown in table 3.
Risk ratio ratio (OR) table of table 3 related locus genotype
(1) individual's skin wrinkle produces integrated risk ratio than OR (C) calculating.The OR value of different loci is as shown in table 2, then
(2) calculate individual's skin wrinkle by step (1) and produce integrated risk ratio than OR (C), comprehensive literature and tune Look into data and learn that the ratio odds (D) of Chinese population 20-35 age bracket wrinkle of skin is about 0.08 (the detection sample of the present embodiment Belong to this age bracket, therefore select the data of this age bracket), detection individual's skin can be calculated by odds ratio computing formula and wrinkle Stricture of vagina produces value-at-risk ratio odds (X)=OR (C) × odds (D)=10.13 × 0.08=0.81.
(3) the detection individual's skin wrinkle calculated by step (2) produces risk ratio odds (X), integrated risk value
F ( X ) = o d d s ( X ) o d d s ( X ) + 1 = 0.81 0.81 + 1 = 44.75 % .
(4) known normal genotype crowd's value-at-risk F (N) is 7.4%, can calculate skin wrinkle resisting ability value
V = F ( N ) F ( X ) × 100 % = 7.4 % 44.75 % × 100 % = 16.53 % .
2), testing result is comprehensively analyzed.The skin wrinkle resisting ability value of this individuality is only 16.53%, and skin wrinkle resisting ability is relatively Difference, belongs to the body constitution being easily generated wrinkle.In combination with the molecular mechanism of related gene, as shown in Table 2 in this detection individuality EMILIN1, MMP3 carry susceptibility loci homozygous mutation, and FLG, MMP1 carry susceptibility loci heterozygous mutant, and these sudden changes can affect Silk polymeric protein, matrix metal proteinase activity, affect skin elasticity, it means that the skin elasticity of this individuality relatively common people Difference, skin easily relaxes.
3), personalized solution: this individual's skin wrinkling resistance is poor, and the sudden change of multiple related genes makes this individuality Congenital it is easily generated wrinkle, the selection of cosmetics need to be should be specifically noted that, use and can improve a polymeric protein, the change of elastin laminin activity Cosmetic；Keeping skin clean clean, makeup removing should be thorough, reduces skin pore blocking.Note supplementing natural complex C, as western red Fructus Kaki, Radix Dauci Sativae etc.；Optional collagen protein and the food of elastin laminin rich content, avoid eating and drinking too much at one meal；Fat-reducing of excessively going on a diet also can Cause the too fast loss of skin-nourishing to can not get effectively supplementing, cause skin depressions to relax；Unhealthy emotion and expression can cause skin Skin can not get effectively having a rest because overworking and formed lax.
Although above it has been shown and described that embodiments of the invention, it is to be understood that above-described embodiment is example Property, it is impossible to be interpreted as limitation of the present invention, those of ordinary skill in the art is without departing from the principle of the present invention and objective In the case of above-described embodiment can be changed within the scope of the invention, revise, replace and modification.

Claims (7)

1. primer and the probe being used for assessing skin wrinkle resisting situation, it is characterised in that described sequence such as SEQ ID NO:1- 4, and SEQ ID NO:1-4, and SEQ ID NO:5-8, and SEQ ID NO:9-12, and SEQ ID NO:13-15, and SEQ ID NO:16-18, and SEQ ID NO:19-21, and SEQ ID NO:22-25；Wherein SEQ ID NO:3-4,7-8,11-12,15, 5 ' the ends of 18,21,24-25 connect different fluorescent labelinies respectively with 3 ' ends.
2. the test kit being used for assessing skin wrinkle resisting situation, it is characterised in that containing the primer described in claim 1 and spy Pin.
3. the method utilizing the gene test auxiliary correct beauty method of customer selecting, it is characterised in that use claim 1 Primer and probe, or use claim 2 test kit.
4. the method utilizing the gene test auxiliary correct beauty method of customer selecting described in claim 3, it is characterised in that step For:
1) use the primer of claim 1 and the test kit of probe or claim 2 that client is carried out SNP site detection；
2) the skin wrinkle resisting ability value of client is gone out according to susceptible SNP site combination calculation；Described susceptible SNP site is combined as ELNrs2071307、EMILIN1rs2011616、FLGrs146466242、rs200519781、MMP3rs3025058、 MMP1rs1799750、COL3A1rs1800255；
3) combine 1) SNP site testing result and 2) skin wrinkle resisting ability value, carry out result judgement.
5. the method utilizing the gene test auxiliary correct beauty method of customer selecting described in claim 4, it is characterised in that described Step 1) be,
Extract the DNA sample of client；
Test kit described in primer described in claim 1 and probe or claim 2 is used to carry out PCR amplification；Preferably, PCR expands The response procedures increased is 50 DEG C of 2min, 95 DEG C of 10min, and 95 DEG C of 15s, 60 DEG C of 1min circulate 40 times.
6. the method utilizing the gene test auxiliary correct beauty method of customer selecting described in claim 4, it is characterised in that described Step 2) be,
The pathogenic risk ratio ratio of each loci gene type according to the combination of known susceptible SNP site calculates integrated risk ratio Ratio OR (C), calculation equation is:
Produce average risk ratio odds (D) by known Chinese population different age group wrinkle of skin, calculate detection individuality Wrinkle of skin produce value-at-risk ratio odds (X), calculating formula is odds (X)=OR (C) × odds (D)；
Calculate integrated risk value:
According to known normal genotype crowd's value-at-risk F (N), calculate individual's skin wrinkling resistance value.
7. the method utilizing the gene test auxiliary correct beauty method of customer selecting described in claim 4, it is characterised in that described Step 3) result be judged as,
1) if individual's skin wrinkling resistance value is less than 100%, then it represents that the skin wrinkle resisting ability of this individuality is less than this age bracket people The normal value of group, wrinkling resistance is poor, belongs to the body constitution being easily generated wrinkle；Being worth the lowest, wrinkling resistance is the poorest；
2) if individual's skin wrinkling resistance value is equal to 100%, then it represents that the skin wrinkle resisting ability of this individuality and this age bracket crowd Normal value the same, wrinkling resistance is general；
3) if individual's skin wrinkling resistance value is higher than 100%, then it represents that the skin wrinkle resisting ability of this individuality is higher than this age bracket people The normal value of group, wrinkling resistance is preferable, belongs to the body constitution being not likely to produce wrinkle；Being worth the highest, wrinkling resistance is the best；
Simultaneously；
If SNP site rs2071307 of gene ELN is A/A, show to carry homozygous mutation, weaken elasticity and the stretching energy of skin Power, promotes chronological ageing；If A/G, show to carry heterozygous mutant, weaken elasticity and the stretch capability of skin, promote chronic always Change；If G/G, then it it is normal genotype；
If SNP site rs2011616 of gene EMILIN1 is A/A, shows to carry homozygous mutation, but a crease in the skin risk is normal； If A/G, showing to carry G, elastic fiber quality reduces, dermis strength reduction, easy fold；If G/G, then it is normal Genotype, but owing to carrying G, elastic fiber quality reduces, dermis strength reduction, easy fold；
If SNP site rs146466242 of gene FLG is A/T, showing to carry heterozygous mutant, skin mechanical strength is more weak, skin It is easily subject to external injury；If T/T, then it it is normal genotype；
If SNP site rs200519781 of gene FLG is T/-, being shown to be deletion Genotype, silk polymeric protein activity is more weak, skin The easy shallow stricture of vagina of skin is too much；If T/T, then it it is normal genotype；
If SNP site rs3025058 of gene M MP3 is-/A, being shown to be deletion Genotype, enzyme is lived and is reduced, and increases collagen fiber Damage and the risk destroyed, skin elasticity declines；If-/-, being then deletion Genotype, enzyme is lived and is reduced, and increases collagen fiber and damages Wound and the risk destroyed, skin elasticity declines；If A/A, then it it is normal genotype；
If SNP site rs1799750 of gene M MP1 is C/C, being shown to be insertion genotype, enzyme is lived and is reduced, and increases collagen fiber Damage and the risk destroyed, skin elasticity declines；If-/C, being shown to be insertion genotype, enzyme is lived and is reduced, and increases collagen fiber Damage and the risk destroyed, skin elasticity declines；If-/-, then it it is normal genotype；
If SNP site rs1800255 of gene C OL3A1 is A/G, it is shown to be and carries heterozygous mutant, without relevant risk；If A/ A, is shown to be and carries homozygous mutation, and type III collagen protein weakens or disappears so that skin is more readily formed wrinkle；If G/G, It it is then normal genotype.
CN201610367685.2A 2016-05-27 2016-05-27 Primer, probe and kit for evaluating anti-wrinkle condition of skin Pending CN105886645A (en)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106435001A (en) * 2016-12-07 2017-02-22 山东省皮肤病性病防治研究所 Application of single nucleotide polymorphism rs146466242 to screening to leprosy patients
CN106498071A (en) * 2016-11-14 2017-03-15 天津市康婷生物工程有限公司 The system of skin skin quality is detected and is assessed from gene level
CN108018346A (en) * 2018-01-15 2018-05-11 无锡中德美联生物技术有限公司 Composite amplification reagent kit and its application of female skin and hair quality SNP genetic polymorphisms site are detected at the same time
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
CN109234406A (en) * 2018-10-30 2019-01-18 深圳市万众基因转化医学研究院 A kind of quick detection combination primer of mankind's skin quality and its application
CN109554480A (en) * 2018-09-21 2019-04-02 广州益养生物科技有限公司 Method for rapidly detecting hereditary skin elasticity gene
CN110904207A (en) * 2019-06-14 2020-03-24 陕西九州医学检验有限公司 Susceptible gene detection panel related to skin aging characterization and application thereof
CN114686578A (en) * 2020-12-30 2022-07-01 北京本真工坊生物科技有限公司 Skin type assessment method, application and system

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN203174095U (en) * 2013-01-08 2013-09-04 百岳特生物科技（上海）有限公司 Whitening and aging gene detection chip
WO2015131110A1 (en) * 2014-02-27 2015-09-03 Igenomx International Genomics Corporation Methods for analysis of somatic mobile elements, and uses thereof
CN105603111A (en) * 2016-03-29 2016-05-25 博奥颐和健康科学技术（北京）有限公司 Application of SNP site of CNTN4 gene and detection primer and kit thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN203174095U (en) * 2013-01-08 2013-09-04 百岳特生物科技（上海）有限公司 Whitening and aging gene detection chip
WO2015131110A1 (en) * 2014-02-27 2015-09-03 Igenomx International Genomics Corporation Methods for analysis of somatic mobile elements, and uses thereof
CN105603111A (en) * 2016-03-29 2016-05-25 博奥颐和健康科学技术（北京）有限公司 Application of SNP site of CNTN4 gene and detection primer and kit thereof

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106498071A (en) * 2016-11-14 2017-03-15 天津市康婷生物工程有限公司 The system of skin skin quality is detected and is assessed from gene level
CN106435001A (en) * 2016-12-07 2017-02-22 山东省皮肤病性病防治研究所 Application of single nucleotide polymorphism rs146466242 to screening to leprosy patients
CN106435001B (en) * 2016-12-07 2019-09-06 山东省皮肤病性病防治研究所 Application of the single nucleotide polymorphism rs146466242 in screening leper
CN108018346A (en) * 2018-01-15 2018-05-11 无锡中德美联生物技术有限公司 Composite amplification reagent kit and its application of female skin and hair quality SNP genetic polymorphisms site are detected at the same time
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
CN108251521B (en) * 2018-03-15 2021-08-13 北京天平永达科技发展有限公司 Complete set of primers for detecting susceptibility gene SNP related to skin aging and application thereof
CN109554480A (en) * 2018-09-21 2019-04-02 广州益养生物科技有限公司 Method for rapidly detecting hereditary skin elasticity gene
CN109234406A (en) * 2018-10-30 2019-01-18 深圳市万众基因转化医学研究院 A kind of quick detection combination primer of mankind's skin quality and its application
CN110904207A (en) * 2019-06-14 2020-03-24 陕西九州医学检验有限公司 Susceptible gene detection panel related to skin aging characterization and application thereof
CN110904207B (en) * 2019-06-14 2022-07-29 陕西九州医学检验有限公司 Susceptible gene detection panel related to skin aging characterization and application thereof
CN114686578A (en) * 2020-12-30 2022-07-01 北京本真工坊生物科技有限公司 Skin type assessment method, application and system

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