CN109837349A

CN109837349A - A kind of Primer composition, kit and detection method

Info

Publication number: CN109837349A
Application number: CN201910056015.2A
Authority: CN
Inventors: 陈晓峰
Original assignee: Orange Fox (shanghai) Information Technology Co Ltd
Current assignee: Orange Fox (shanghai) Information Technology Co Ltd
Priority date: 2019-01-22
Filing date: 2019-01-22
Publication date: 2019-06-04

Abstract

The invention discloses a kind of Primer compositions.Primer composition of the invention is made of SEQ ID NO:1-14 primer.The primer pair is made of its corresponding upstream primer and downstream primer.Using Primer composition of the invention, analysis can be made with regard to the skin elasticity ability and skin oxidation resistance of detected person, auxiliary detection is provided and suggests supporting for skin nursing.Have many advantages, such as that high sensitivity, resolving power are good, result is accurate.The invention also discloses a kind of detection method of Primer composition and gene detecting kits.

Description

A kind of Primer composition, kit and detection method

Technical field

The invention belongs to field of biotechnology, and in particular to be used to a kind of Primer composition and its detection method and have be somebody's turn to do The kit of Primer composition.

Background technique

Gene is also referred to as gene, is the structure and function unit of intracellular inhereditary material.All biologies all contain him Respective gene.The hereditary information entrained by oneself is expressed by instructing the synthesis of protein, to control bion Trait expression.Single nucleotide polymorphism (single nucleotide polymorphism, SNP) is primarily referred to as in genome DNA sequence polymorphism caused by a single nucleotide variation in level.It is most common in human heritable mutation It is a kind of.Account for 90% or more of all known polymorphisms.SNP is widely present in human genome, average every 500~1000 alkali Base centering just has 1, estimates that its sum is even more up to 3,000,000.

Have a large amount of scientific researches the result shows that, there is incidence relations the composition and disease of different SNP, also with Different Individual at Long, development, aging have close association.Traditional skin managerial integration is based on inquiry, observation skin appearance, touches skin matter As a result, disturbing factor is more, being also not easy to formulate has targetedly suggestion and non-diagnosis and nursing scheme for sense etc., and effect is necessarily beaten greatly Discount.By the way that the specific SNP site of human body is detected and identified, it can fundamentally be grasped and be examined with Additional Specialty personnel The state of personnel's skin, and have more scientific understanding and understanding, refer to provide more targeted skin nursing for subject Lead suggestion.

Summary of the invention

The object of the present invention is to provide a kind of Primer composition, the detection method of the Primer composition and containing described The gene detecting kit of Primer composition.

A kind of genetic test Primer composition provided by the invention, the Primer composition include SEQ ID NO.1-2 institute Primer sequence shown in the primer sequence that shows, SEQ ID NO.3-4, primer sequence, SEQ ID shown in SEQ ID NO.5-6 Primer sequence shown in NO.7-8, primer sequence shown in primer sequence, SEQ ID NO.11-12 shown in SEQ ID NO.9-10 Primer sequence shown in column, SEQ ID NO.13-14, wherein primer sequence SEQ ID NO.1-2 is for expanding Catalase base Cause, product segment 763bp, primer sequence the SEQ ID obtained by the primer sequence SEQ ID NO.1-2 through PCR amplification NO.3-4 is for expanding GPX1 gene, the product segment obtained by the primer sequence SEQ ID NO.3-4 through PCR amplification 404bp, primer sequence SEQ ID NO.5-6 are for expanding IL6 gene, by the primer sequence SEQ ID NO.5-6 through PCR Obtained product segment 459bp is expanded, primer sequence SEQ ID NO.7-8 is for expanding MMP1 gene, by the primer sequence The product segment 458bp that SEQ ID NO.7-8 is obtained through PCR amplification, primer sequence SEQ ID NO.9-10 are for expanding MMP3 Gene, product segment 422bp, primer sequence the SEQ ID obtained by the primer sequence SEQ ID NO.9-10 through PCR amplification NO.11-12 is for expanding MMP9 gene, the product sheet obtained by the primer sequence SEQ ID NO.11-12 through PCR amplification Section 562bp, primer sequence SEQ ID NO.13-14 are for expanding NFE2L2 gene, by the primer sequence SEQ ID The product segment 496bp that NO.13-14 is obtained through PCR amplification.

The present invention also protects a kind of detection method using the Primer composition, includes the following steps: from biological sample Middle extraction sample to be tested DNA carries out PCR amplification with the primer pair respectively using sample to be tested DNA as template, obtains PCR amplification Product；The pcr amplification product is subjected to electrophoresis, dyeing and identification.

The above method is during PCR amplification, according to 30 μ L general reaction system computings, the reactant of the PCR amplification It is as follows:

Reagent name	Usage amount ul	Final concentration
			2×pfu-PCR mastermix	15	1×
Primer F(10uM)	0.75	0.3uM
			Primer R(10uM)	0.75	0.3uM
DNA	3	100ng
			ddwater	10.5	-

Response procedures of above method during PCR amplification are as follows:

In the detection method that the present invention is protected, the identification can assess skin oxidation resistance, and appraisal and evaluation is as follows:

In the detection method that the present invention is protected, the identification can assess skin elasticity ability, and identification method is as follows:

In the detection method that the present invention is protected, the biological sample is human saliva, buccal swab sample, hair sample This.

The present invention also protects a kind of gene inspection that the Primer composition that the application present invention is protected is nursed for aid in skin Test agent box.It further include the reagent for extracting genomic DNA or/and the reagent for PCR amplification in the kit.Institute The each component stated in kit can be liquid, can also be freeze-dried powder.

The kit that the present invention is protected, as further scheme, the detection method that can be protected through the invention, identification Assess the oxidation resistance of skin.

The kit that the present invention is protected, as further scheme, the detection method that can be protected through the invention, identification Assess the elasticity capacity of skin.

Detailed description of the invention

Present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

Fig. 1 is the PCR product electricity of Catalase, GPX1, IL6, MMP1, MMP3, MMP9, NFE2L2 gene in embodiment Swimming figure.

Fig. 2 is the sequencing peak figure in the site Rs1001179 of Catalase gene in embodiment.

Fig. 3 is the sequencing peak figure in the site Rs1050450 of GPX1 gene in embodiment.

Fig. 4 is the sequencing peak figure in the site Rs1800795 of IL6 gene in embodiment.

Fig. 5 is the sequencing peak figure in the site Rs1799750 of MMP1 gene in embodiment.

Fig. 6 is the sequencing peak figure in the site Rs3025058 of MMP3 gene in embodiment.

Fig. 7 is the sequencing peak figure in the site Rs3918242 of MMP9 gene in embodiment.

Fig. 8 is the sequencing peak figure in the site Rs35652124 of NFE2L2 gene in embodiment.

Fig. 9 is the sequencing peak figure in the site Rs6706649 of NFE2L2 gene in embodiment.

Figure 10 is the sequencing peak figure in the site Rs6721961 of NFE2L2 gene in embodiment.

Specific embodiment

Below with reference to the specific embodiment in the present invention, technical solution in the embodiment of the present invention carries out clear, complete Ground description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on this Embodiment in invention, every other reality obtained by those of ordinary skill in the art without making creative efforts Example is applied, shall fall within the protection scope of the present invention.

Embodiment:

One, it samples

It obtains tested personnel oral cavity sample using buccal swab not feeding or drink water 30 minutes before sampling, by swab cotton Label are put in oral cavity, are wiped repeatedly at the buccal mucosa of inside 20 times or more, until cotton head is sufficiently infiltrated by saliva, take out oral cavity Swab is put into collection tube and fractures handle along swab folding line, covers specimen collection tube lid at random, completes sample.

One, DNA is extracted

1) appropriate sample liquid is extracted from the collection tube for be completed sampling, adds 200ul buffer GA, and oscillation is hanged to thorough It is floating；

2) add 20ul Proteinase K Solution, mix, 56 DEG C of appropriate hot digestions；

3) brief centrifugation is added 200ul buffer GB, is sufficiently mixed by inversion, and 70 DEG C are placed 10 minutes, and solution strain is clear Clearly, brief centrifugation removes tube wall droplet, and solution becomes clarification；

4) 200ul dehydrated alcohol is added, mixing 15 seconds, brief centrifugation fullys shake；

5) previous step acquired solution is transferred in adsorption column CB3 (adsorption column is put into 2ml collecting pipe), 12000rpm from The heart 30 seconds, waste liquid is discarded, adsorption column is put back in collecting pipe；

6) 500ul buffer GD, 12000rpm is added to be centrifuged 30 seconds, discard waste liquid, adsorption column is put back to receipts to adsorption column In collector；

7) 600ul buffer PW is added, 12000rpm is centrifuged 30 seconds and rinses, and is repeated once this operation；

8) adsorption column is put into 2ml collecting pipe and is centrifuged 2 minutes；

9) adsorption column is put into a clean 1.5ml EP pipe and is placed at room temperature for 3 to 5 minutes；

10) add 80ul elution buffer 80ul to adsorption column filter membrane center, place 2 to 5 minutes, 12000rpm is centrifuged 2 points Clock；

11) adsorption column is discarded, measurement DNA concentration carries out next step experiment.

Three, PCR reacts

1) PCR amplification site sequence is searched:

2) design primer:

3) PCR amplification:

PCR reaction system (30ul)

5) it mixes, upper machine amplification.

Four, experimental result

1) DNA concentration result:

Sample name	Result(ng/ul)	A260/280
			Buccal swab 1	37.19	1.75

2) Testing and appraisal result

3) simple conclusion

Tested personnel's skin elasticity ability with higher still has the oxidation resistance of non-significant advantage, in tested person Member time, energy, in the case where having quite modest financial resources, the mechanisms such as beauty parlor can be emphatically for providing specific aim in terms of oxidation resistance Opinion is nursed, tested personnel can also increase rich in ascorbic food more.

Claims

1. a kind of genetic test Primer composition, which is characterized in that the Primer composition includes shown in SEQ ID NO.1-2 Primer sequence shown in primer sequence, SEQ ID NO.3-4, primer sequence, SEQ ID NO.7- shown in SEQ ID NO.5-6 Primer sequence shown in 8, primer sequence shown in primer sequence, SEQ ID NO.11-12 shown in SEQ ID NO.9-10, Primer sequence shown in SEQ ID NO.13-14, wherein primer sequence SEQ ID NO.1-2 is used to expand Catalase gene, The product segment 763bp obtained by the primer sequence SEQ ID NO.1-2 through PCR amplification, primer sequence SEQ ID NO.3-4 For expanding GPX1 gene, the product segment 404bp obtained by the primer sequence SEQ ID NO.3-4 through PCR amplification, primer Sequence SEQ ID NO.5-6 is for expanding IL6 gene, the production obtained by the primer sequence SEQ ID NO.5-6 through PCR amplification Object segment 459bp, primer sequence SEQ ID NO.7-8 are for expanding MMP1 gene, by the primer sequence SEQ ID NO.7-8 The product segment 458bp obtained through PCR amplification, primer sequence SEQ ID NO.9-10 are drawn for expanding MMP3 gene by described The product segment 422bp that object sequence SEQ ID NO.9-10 is obtained through PCR amplification, primer sequence SEQ ID NO.11-12 are used for Expand MMP9 gene, the product segment 562bp obtained by the primer sequence SEQ ID NO.11-12 through PCR amplification, primer sequence Column SEQ ID NO.13-14 is obtained by the primer sequence SEQ ID NO.13-14 through PCR amplification for expanding NFE2L2 gene The product segment 496bp arrived.

2. a kind of detection method using genetic test Primer composition described in claim 1, which is characterized in that including walking as follows It is rapid: to extract sample to be tested DNA from biological sample and carry out PCR expansion with the primer pair respectively using sample to be tested DNA as template Increase, obtains pcr amplification product；The pcr amplification product is subjected to electrophoresis, dyeing and identification.

3. detection method according to claim 2, it is characterised in that: totally anti-according to 30 μ L during the PCR amplification System computing is answered, the reaction system of the PCR amplification is as follows:

4. detection method according to claim 2, which is characterized in that the response procedures of the PCR amplification are as follows:

5. detection method according to claim 2, which is characterized in that the biological sample is human saliva, buccal swab Sample.

6. detection method according to claim 2, which is characterized in that the identification can assess skin oxidation resistance, mirror Accepted opinion is estimated as follows:

7. detection method according to claim 2, it is characterised in that the identification can assess skin elasticity ability, and identification is commented Estimate as follows:

8. a kind of gene detecting kit, which is characterized in that it contains Primer composition described in claim 1.

9. a kind of gene detecting kit according to claim 8, which is characterized in that use inspection as claimed in claim 6 Survey method, appraisal and evaluation skin oxidation resistance.

10. a kind of gene detecting kit according to claim 8, which is characterized in that use inspection as claimed in claim 7 Survey method, appraisal and evaluation skin elasticity ability.