CN111378762B

CN111378762B - Primer combination for skin moisturizing ability gene detection and application thereof

Info

Publication number: CN111378762B
Application number: CN202010074580.4A
Authority: CN
Inventors: 李文; 郭之彬; 张欣鑫; 廖俊杰
Original assignee: Guangzhou Simpson Biotechnology Co ltd
Current assignee: Guangzhou Simpson Biotechnology Co ltd
Priority date: 2020-01-22
Filing date: 2020-01-22
Publication date: 2024-01-23
Anticipated expiration: 2040-01-22
Also published as: CN111378762A

Abstract

The invention relates to the technical field of genotyping, in particular to a skin moisturizing ability gene detection primer combination and application thereof, comprising a multiplex PCR amplification primer group and a single base extension primer group for detecting 12 SNP loci of 7 skin moisturizing ability genes of a human genome. Each SNP locus is respectively designed with a pair of multiplex PCR amplification primers and a single base extension primer, and all loci are designed into one reaction and can be detected simultaneously. The SNP genotyping method for efficiently detecting the skin moisturizing ability genes is established, molecular biological genotyping of a plurality of SNP loci can be completed at the same time, the detection sensitivity is good, the accuracy is high, the cost is low, the practicability is high, the SNP genotyping method can be used for detecting genetic factors affecting the skin moisturizing ability, the comprehensive evaluation of the skin moisturizing ability is carried out, and the development of personalized service in the beauty industry is facilitated.

Description

Primer combination for skin moisturizing ability gene detection and application thereof

Technical Field

The invention relates to the technical field of genotyping, in particular to a primer combination for skin moisturizing ability gene detection and application thereof.

Background

Skin dryness can occur at any age, in any season, and under various adverse circumstances, so skin moisturization is particularly important. Moisture retention means that water molecules are firmly locked in the deep layer of skin, and the skin is not easy to generate the problems of tightness, increased dander, dryness, relaxation, fine lines, sensitivity, wrinkles, chapping and the like. If the above situation occurs, it is possible to prevent evaporation of skin moisture, improve microcirculation, and enhance skin moisturization by using a skin care product having a moisturizing effect. At present, the demand of people on skin moisturizing is more and more focused, and the types of moisturizing skin care products in skin care products are also endless. Investigation and research show that the share of the moisturizing skin care product for Chinese people reaches 14.3%, and the moisturizing skin care product becomes a third major category of skin care. While the investigation results show that the moisture retention is mentioned as high as 58.2% in the most interesting and warmest beauty and skin care services and products for women, the third place, and the moisture retention has become one of the most interesting skin care purposes for consumers.

The internal water balance and the external water locking capability are also increasingly attracting attention as important components of moisturizing care. Normally, the skin has moisturizing capability, and is mainly a skin isolation barrier formed by epidermis horny layer and sebum membrane, and has the functions of preventing the water in the skin from dispersing and preventing the skin from being injured by external environment. However, with age, the skin suffers from various water deficiency problems. The weak ability of skin cells to absorb vitamin A can lead to unbalanced water and oil of the skin, increased secretion of grease, and the skin is prevented from being moisturized and moisturized, so that serious injury is caused to the skin; if the water-locking capacity of the cell matrix is reduced, the water loss speed of the skin is increased, the skin is dry, the skin barrier capacity is poor, the water loss in the skin is increased, and the skin becomes dry and itchy.

The differences in the phenotypic state of the skin are caused by the differences in the genetic information of human beings, and the influence of different environments on the skin plays a key role. Under the same other conditions, if the vitamin A has strong absorption capacity, the vitamin A can promote the absorption of moisture by human bodies, has an effective moisturizing effect, prevents skin mucous membrane from drying and keratinization, and ensures that the skin is moist, elastic and glossy. If the vitamin a absorption capacity is weak, skin cells cannot easily retain moisture, moisture is easily lost through the evaporation of epidermis and sweat glands, and the skin becomes dry, loose, dull and matt. The comprehensive moisture-keeping gene detection report can predict the skin state in advance, provides an important reference for keeping skin moist, is favorable for selecting a proper skin care scheme and skin care products for targeted intervention, and can better reduce the skin problems such as dryness, tightness, dryness, itching and the like.

Research progress in human genetics, molecular biology, genomics and bioinformatics has fully demonstrated that skin moisturizing ability is affected by single nucleotide polymorphisms (single nucleotide polymorphism, SNP), and patent (201810839384.4) discloses a skin-related gene locus library, a construction method and application thereof, stratum corneum hydration degree-related gene ST14, skin moisture-locking moisturizing ability gene AQP3, keratin-generation-related gene DAG and skin natural moisturizing factor-generation-related gene FLG, which have 4 genes for 5 loci in total, and the detection aspect selected by the patent does not fully meet the detection purpose of moisturizing genes, comprises other detection aspects, and the detection locus cannot cover the detection requirement. Patent (201811356181.6) discloses a method for rapidly detecting skin moisturizing ability genes, which detects skin moisturizing ability by detecting 2 sites related to moisturizing, and adopts a high-resolution melting curve analysis technology (High Resolution Melting, HRM) to analyze genotypes of each SNP site of a sample to be detected.

Therefore, the primer combination for providing the skin moisturizing ability gene detection and the application thereof have important practical significance.

Disclosure of Invention

In view of the above, the present invention provides a skin moisturizing ability gene detection primer combination and application thereof. According to the invention, molecular biology genotyping is carried out on SNP loci of skin moisturizing ability genes by using a Massarray nucleic acid mass spectrometry system, and comprehensive evaluation is carried out on skin moisturizing ability of individuals according to the genotyping result, so that the Massararay nucleic acid mass spectrometry system is expected to be used for precise skin care management.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a primer combination for detecting skin moisturizing capability, which comprises a primer group for detecting internal water balance capability and/or a primer group for detecting external water locking capability;

(1) The primer group for detecting the inherent moisture balance capability comprises a multiplex PCR amplification primer group for detecting the inherent moisture balance capability and a single base extension primer group for detecting the inherent moisture balance capability;

the multiplex PCR amplification primer set for detecting the inherent moisture balance capability has any one of the nucleotide sequences shown as follows:

(I) The nucleotide sequence shown in any one group of SEQ ID No. 1-2, 3-4, 5-6, 7-8, 9-10 and 11-12;

(II) a nucleotide sequence obtained by modifying, substituting, deleting and/or adding one or more bases to the nucleotide sequence shown by SEQ ID No. 1-2, 3-4, 5-6, 7-8, 9-10 and 11-12;

(III) a sequence having at least 80% sequence identity to the nucleotide sequences shown in SEQ ID Nos. 1-2, 3-4, 5-6, 7-8, 9-10, 11-12;

(IV), the complement of the sequence shown in (I), (II) or (III);

the single base extension primer set for detecting the inherent water balance capability has any one of the nucleotide sequences shown as follows:

(V) has a nucleotide sequence shown in any one of SEQ ID No. 25-30;

(VI) a nucleotide sequence obtained by modifying, substituting, deleting and/or adding one or more bases to the nucleotide sequence represented by SEQ ID nos. 25 to 30;

(VII) a sequence having at least 80% sequence identity to the nucleotide sequence shown in SEQ ID nos. 25-30;

(VIII), the complement of the sequence shown as (V), (VI) or (VII);

(2) The primer group for detecting the external water locking capability comprises a multiplex PCR amplification primer group for detecting the external water locking capability and a single base extension primer group for detecting the external water locking capability;

the multiplex PCR amplification primer set for detecting external water locking capability has any one of the nucleotide sequences shown as follows:

(IX) having a nucleotide sequence shown in any one of SEQ ID Nos. 13 to 14, 15 to 16, 17 to 18, 19 to 20, 21 to 22, 23 to 24;

(X) a nucleotide sequence obtained by modifying, substituting, deleting and/or adding one or more bases to a nucleotide sequence shown in any one group of SEQ ID Nos. 13 to 14, 15 to 16, 17 to 18, 19 to 20, 21 to 22 and 23 to 24;

(XI) a sequence having at least 80% sequence identity to the nucleotide sequence set forth in any one of SEQ ID Nos. 13 to 14, 15 to 16, 17 to 18, 19 to 20, 21 to 22, 23 to 24;

(XII), the complement of the sequence shown as (IX), (X) or (XI);

the single base extension primer set for detecting external water locking capability has any one of the nucleotide sequences shown as follows:

(XIII) having a nucleotide sequence as set forth in any one of SEQ ID Nos. 31 to 36;

(XIV), a nucleotide sequence having one or more bases modified, substituted, deleted and/or added to the nucleotide sequence shown in any one of SEQ ID nos. 31 to 36;

(XV), a sequence having at least 80% sequence identity to the nucleotide sequence depicted in any one of SEQ ID nos. 31 to 36;

(XVI), the complement of the sequence shown as (XIII), (XIV) or (XV).

In some embodiments of the invention, the molar ratio of the multiplex PCR amplification primer set for detecting intrinsic moisture balance capability, the single base extension primer set for detecting intrinsic moisture balance capability, the multiplex PCR amplification primer set for detecting extrinsic water lock capability, the single base extension primer set for detecting extrinsic water lock capability in the primer set is (8.57-9.21): (19.7 to 34): (8.79-8.98): (19.2-31.5).

Based on the above research, the invention provides the application of the primer combination in preparing a kit for detecting the internal water balance capability and/or external water locking capability of skin.

The invention also provides application of the primer combination in preparing a reagent and/or a kit for detecting skin moisturizing ability.

In addition, on the basis of the research, the invention also provides a detection reagent comprising the primer combination.

The invention also provides a kit comprising the primer combination or the detection reagent.

More importantly, the invention also provides application of the primer combination, the detection reagent or the kit in detecting skin moisturizing ability genes or skin moisturizing ability.

The invention also provides a method for detecting the skin moisturizing ability gene, which comprises the following steps:

step 1, obtaining nucleic acid of a sample to be detected;

step 2, taking the nucleic acid extracted in the step 1 as a template, respectively adopting the primers in the primer combination to carry out multiplex PCR amplification and single base extension, and carrying out genotyping on the SNP locus;

and step 3, obtaining a detection result according to the genotyping result.

In some embodiments of the invention, the detection result obtained according to the genotyping result in step 3 is:

。

in some embodiments of the invention, the criteria for obtaining a test result based on the genotyping result are: the influence of SNP locus genotype on phenotype is calculated as 1 score of 'strong', calculated as 0 score of 'general', calculated as-1 score of 'weak', and the final score is obtained by adding the scores of all SNP loci; and judging the intrinsic moisture balance capacity and/or the extrinsic water locking capacity according to the final score:

the final score is more than or equal to 1: "strong";

final score = 0: "general";

the final score is less than or equal to-1: "weak".

SNP typing detection of skin moisturizing ability genes is carried out by using a Massary ARRAY nucleic acid mass spectrometry system, and the detection is combined with a multiplex PCR technology, a Massary ARRAY iPLEX single base extension technology and a matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for typing detection. The PCR amplification is carried out on the DNA fragment containing the SNP locus, and then the single base extension reaction is carried out by utilizing the specific extension primer. Since the polymorphism site bases are different, the difference in molecular weight of the product after extension will be caused by the difference in the terminal bases of the extension product, and thus the base difference due to SNP polymorphism is manifested by the difference in molecular weight. The detection has the advantages of high sensitivity, flexible time, low error probability, low cost and cross contamination avoidance. The base difference is resolved by detecting the molecular weight, fluorescent labeling, gel electrophoresis and the like are not involved, and the accuracy is high. The method can detect 12 SNP loci of 7 genes simultaneously, and the loci cover two aspects of internal water balance and external water locking capability directly related to skin moisturizing capability, and the detection content is the most comprehensive at present. The skin moisturizing ability gene detection is used for comprehensively evaluating the skin's congenital moisturizing ability, so that scientific basis is provided for an external skin care scheme, and the skin dryness condition can be slowed down more effectively. Therefore, the gene detection with more targeted skin moisturizing ability can help people to better understand self genetic factors, know the root cause of skin problems in a deeper level, and provide scientific guidance for skin moisturizing.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows a flow chart of a time-of-flight mass spectrometry analysis mass spectrometry technique (MALDI-TOF-MS) experiment;

FIG. 2 shows an rs10792653 locus UCSC Genome Bioinformatics validation map;

FIG. 3 shows a peak diagram of the result of an rs10792653 site Massarray nucleic acid mass spectrometry system;

FIG. 4 shows a scatter plot of the results of an rs10792653 locus Massary nucleic acid mass spectrometry system;

FIG. 5 shows a photograph of the face of a subject of sample 1 in example 4;

fig. 6 shows a photograph of the face of the subject of sample 2 in example 4.

Detailed Description

The invention discloses a primer combination for skin moisturizing ability gene detection and application thereof, and a person skilled in the art can properly improve process parameters by referring to the content of the primer combination. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.

The invention provides a skin moisturizing ability gene detection primer combination and application thereof, comprising a multiplex PCR amplification primer group and a single base extension primer group of 12 SNP loci of 7 skin moisturizing ability genes of human genome DNA.

Further, the 7 skin moisturizing ability genes of the human genome DNA are Beta-carotene oxygenase 1 (Beta-carotene oxygenase 1, BCMO 1) gene, olfactory receptor family 10subfamily K member 2 (Olfactory receptor family 10subfamily Kmember 2,OR10K2) gene, prolyl carboxypeptidase (PRCP) gene, TIMELESS interaction protein (TIMELESS interacting protein, TIPIN) gene, zinc finger protein 100 (Zinc finger protein 100, ZNF100) gene, tumorigenicity 14 (Suppression of tumorigenicity, ST 14) gene, silk protein (Filaggrin, FLG) gene.

Further, the 12 SNP sites of the 7 skin moisturizing ability genes are rs12934922, rs7501331, rs58605957, rs10792653, rs9806130, rs10412066, rs137852931, rs138726443, rs150597413, rs200519781, rs397507563, rs61816761 (table 1).

Further, the multiple PCR amplification primer set and the single base extension primer set can detect skin moisturizing ability, including detecting intrinsic moisture balance and extrinsic water locking ability.

TABLE 1 SNP loci of genes

Further, the detection of the inherent moisture balance comprises the steps of detecting the inherent moisture balance gene SNP locus, wherein the multiplex PCR amplification primer group comprises primers Y1-Y12, the sequence of the multiplex PCR amplification primer group is shown as SEQ ID No. 1-12, the multiplex PCR amplification primer group comprises an upstream primer Y1 and a downstream primer Y2 at the rs12934922 locus of the BCMO1 gene, and the sequence information is shown as SEQ ID No. 1-2; comprises an upstream primer Y3 and a downstream primer Y4 at the locus rs7501331 of the BCMO1 gene, and the sequence information is shown in SEQ ID No. 3-4; the primer comprises an upstream primer Y5 and a downstream primer Y6 at the rs58605957 locus of the OR10K2 gene, and the sequence information is shown as SEQ ID No. 5-6; the primer comprises an upstream primer Y7 and a downstream primer Y8 at the rs10792653 locus of the PRCP gene, and the sequence information is shown as SEQ ID No. 7-8; the primer comprises an upstream primer Y9 and a downstream primer Y10 of a TIPIN gene rs9806130 site, and the sequence information is shown as SEQ ID No. 9-10; the primer comprises an upstream primer Y11 and a downstream primer Y12 at the rs10412066 locus of the ZNF100 gene, and the sequence information is shown as SEQ ID No. 11-12.

The single-base extension primer set T1-T6 has a sequence shown as SEQ ID No. 25-30, and comprises a single-base extension primer T1 at the locus rs12934922 of the BCMO1 gene, and has sequence information shown as SEQ ID No. 25; comprises a BCMO1 gene rs7501331 locus single base extension primer T2, and the sequence information is shown as SEQ ID No. 26; the primer T3 comprises a single base extension primer of an OR10K2 gene rs58605957 site, and the sequence information is shown as SEQ ID No. 27; the primer T4 comprises a PRCP gene rs10792653 locus single base extension primer, and the sequence information is shown as SEQ ID No. 28; comprises a TIPIN gene rs9806130 locus single base extension primer T5, and the sequence information is shown as SEQ ID No. 29; comprises a ZNF100 gene rs10412066 locus single base extension primer T6, and the sequence information is shown as SEQ ID No. 30.

The detection of the external water locking capacity comprises detection of multiple PCR amplification primer groups Y13-Y24 of SNP loci of the external water locking capacity gene, wherein the sequences of the multiple PCR amplification primer groups are shown as SEQ ID No. 13-24, and the multiple PCR amplification primer groups comprise an upstream primer Y13 and a downstream primer Y14 of an rs137852931 locus of an ST14 gene, and the sequence information is shown as SEQ ID No. 13-14; the primer comprises an upstream primer Y15 and a downstream primer Y16 of an FLG gene rs138726443 locus, and the sequence information is shown as SEQ ID No. 15-16; the sequence information of the upstream primer Y17 and the downstream primer Y18 of the locus rs150597413 of the FLG gene is shown as SEQ ID No. 17-18; the sequence information of the upstream primer Y19 and the downstream primer Y20 of the locus rs200519781 of the FLG gene is shown as SEQ ID No. 19-20; the sequence information of the upstream primer Y21 and the downstream primer Y22 of the locus rs397507563 of the FLG gene is shown as SEQ ID No. 21-22; the primer comprises an upstream primer Y23 and a downstream primer Y24 at the locus rs61816761 of the FLG gene, and the sequence information is shown as SEQ ID No. 23-24.

The single-base extension primer set T7-T12 has a sequence shown in SEQ ID No. 31-36, and comprises a site T7 of an ST14 gene rs137852931, and has sequence information shown in SEQ ID No. 31; the gene comprises an FLG gene rs138726443 locus T8, and the sequence information is shown as SEQ ID No. 32; the gene comprises an FLG gene rs150597413 locus T9, and the sequence information is shown as SEQ ID No. 33; the gene comprises an FLG gene rs200519781 locus T10, and the sequence information is shown as SEQ ID No. 34; the gene comprises an FLG gene rs397507563 locus T11, and the sequence information is shown as SEQ ID No. 35; the gene comprises an FLG gene rs61816761 locus T12, and the sequence information is shown as SEQ ID No. 36.

The invention uses the Massary nucleic acid mass spectrometry system to carry out SNP typing detection of skin moisturizing ability genes, has the advantages of high sensitivity, flexible time, low error probability, low cost and cross contamination avoidance, can detect a plurality of SNP loci in the same time, can rapidly read genetic information, and is more suitable for batch and low-cost detection service. In recent years, the mass spectrometry technology of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) has become a hot spot for research in the industry, and has become a development trend of the technology. The related technicians can design the multiplex PCR amplification primer set and the single-base extension primer set for the detection service of the skin moisturizing ability gene through the application method of the technology.

According to the invention, through carrying out specific design on the multiplex PCR amplification primer group and the single-base extension primer group of the skin moisturizing ability genes, 12 SNP loci of 7 genes are detected, the cost is low, the accuracy is high, the time is short, the genetic information of the intrinsic moisture balance and the extrinsic water locking ability directly related to the skin moisturizing ability can be detected, and the comprehensive evaluation is carried out on the skin moisturizing ability of an individual according to the molecular biological genotyping result, so that the skin moisturizing ability is used for precise skin care management. The multiplex PCR amplification primer set and single base extension primer set combination disclosed by the invention is the skin moisturizing ability gene detection multiplex PCR amplification primer set and single base extension primer set combination with the best detection effect and the most complete detection project so far.

The primer combination for skin moisturizing ability gene detection and the raw materials and reagents used in the primer combination can be purchased from the market.

The invention is further illustrated by the following examples:

example 1 technical solution

A skin moisturizing ability gene detection primer combination and application thereof comprise a multiplex PCR amplification primer group and a single base extension primer group (table 2) for detecting 12 SNP loci of 7 skin moisturizing ability genes of human genome. Each SNP locus is designed with a pair of multiplex PCR amplification primers and a single base extension primer, and all loci are designed into a reaction (WELL) and can be detected simultaneously. The multiplex PCR amplification primer group and the single-base extension primer group are used for a Massarray nucleic acid mass spectrometry system, and the system can be used for rapidly analyzing a nucleic acid sample with high sensitivity and high accuracy, and the detection accuracy is more than or equal to 99.9%. The invention establishes the SNP genotyping method for efficiently and comprehensively evaluating the skin moisturizing ability genes, can finish molecular biological genotyping of a plurality of SNP loci at the same time, has good detection sensitivity, high accuracy, low cost and strong practicability, can be used for detecting genetic factors influencing the skin moisturizing ability, and is beneficial to promoting the development of personalized services in the beauty industry.

Step 1: multiplex PCR amplification primer group and single base extension primer group for synthesizing required SNP locus.

Step 2: and extracting human mouth cavity swab sample DNA, and performing PCR amplification.

Step 3: and detecting sample DNA by adopting a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) technology, and determining the genotype of the SNP locus of the sample.

Step 4: and comprehensively evaluating the genetic information of the skin moisturizing ability according to the SNP locus genotype detection result, and giving out result interpretation.

TABLE 2 multiplex PCR amplification primers and Single base extension primer sequences

Example 2 sample detection procedure

In order to verify the design accuracy and practicality of the multiplex PCR amplification primer set and the single base extension primer set for detecting the SNP locus of the skin moisturizing ability gene, 30 oral swab samples are collected for detection in the embodiment, and the specific contents are as follows:

1. primer design:

according to the sequence information of SNP loci, multiplex PCR amplification primers and single base extension primers of the SNP loci to be detected are designed by using Agena company design software Assay Design Suite V2.0.0, and multiplex PCR amplification primer sets and single base extension primer sets of 12 SNP loci of 7 genes are designed into one reaction (WELL) and verified by UCSC Genome Bioinformatics.

The results of designing the quality values of the primer combinations of the software show that the quality of the primer combinations disclosed by the invention is good (Table 3).

The primer combination amplified fragment disclosed by the invention meets the requirement that only or each amplified sequence contains a detection site through verification of online software UCSC Genome Bioinformatics, and has good specificity (shown in figure 3).

Multiplex PCR amplification primer sets and single base extension primer sets were synthesized by Biotechnology (Shanghai) Inc.

TABLE 3 primer combination Mass values

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2. Sample collection:

30 oral swab samples were selected for DNA extraction and information on age, sex, skin condition, etc. of the subjects were recorded (table 5). The skin state evaluation method comprises the following steps: subjects corresponding to 30 oral swab samples were photographed for their faces, and the skin conditions of the photographs were scored by 10 skin test technicians for each item, with scores ranging from 1 to 9. The item scores are added to average, then the average of the scores of 10 technicians is taken, the result is taken as an integer, and finally the skin state score of the testee is determined, wherein the higher the score is, the better the skin state is.

TABLE 4 skin State scoring criteria

Table 5 sample acquisition information

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3. Oral swab DNA extraction:

the DNA extraction of the oral swab is carried out by using a nucleic acid extraction and purification kit of the Meiya gene, and a certain amount of absolute ethyl alcohol is added into Buffer BW1 according to instructions. The oral swab collection head was cut down into a 2mL EP tube, 400mL Buffer ATL and 20. Mu.L Proteinase K were added, the swab was transferred to a 2mL centrifuge tube, and a portion of the swab handle was removed. Incubation was carried out at 55℃for 15-30 min with shaking. 25. Mu.L Magpure and 600. Mu.L Buffer GXP2 were added to the centrifuge tube. Transferring 250-300 mu L of supernatant to a centrifuge tube filled with magnetic beads/GXP 2, reversely and uniformly mixing for 15-30 times, standing for 5-8 minutes at room temperature, and reversely and uniformly mixing for several times. Transferring to a magnetic rack for adsorption for 2 minutes, and sucking or pouring out the solution. 500. Mu.L Buffer BW1 was added and vortexed for 15 seconds to break up the beads. Transferring to a magnetic rack for adsorption for 1 min, and sucking or pouring out the solution. 700 μL of 75% ethanol was added and vortexed to break up the beads for 15 seconds. Transferring to a magnetic rack for adsorption for 1 min, and sucking or pouring out the solution. 700 μL of 75% ethanol was added and vortexed to break up the beads for 15 seconds. Transferring to a magnetic rack for adsorption for 1 min, and sucking or pouring out the solution. Centrifuging briefly, sucking out residual liquid, and air drying for 10-15 min to remove ethanol. Add 30-100. Mu.L of the solution Buffer into the centrifuge tube and incubate with shaking at 55℃for 5-10 minutes. Transferring to a magnetic rack for adsorption for 3-5 min, transferring DNA to a new centrifuge tube, performing quality inspection on the DNA solution by using an ultra-micro spectrophotometer, taking 30 mu L of qualified DNA, transferring to a 384-well plate for sampling, and storing at-20 ℃ for later use.

The quality inspection standard of genome DNA is that the total amount of DNA is more than or equal to 0.5 mug, the concentration is more than or equal to 15 ng/. Mu.L, and the purity OD _260/280 1.7-2.1, no protein or RNA pollution, clear band of integrity, molecular weight greater than 10Kb and no obvious degradation.

TABLE 6 sample DNA quality test results

4. PCR amplification reaction:

multiplex PCR was performed in 384 well plates, with a total volume of 5. Mu.L per reaction system.

A. A new 1.5ml EP tube was prepared with a PCR reaction system solution using 384 well plates with 38% reagent loss.

TABLE 7 PCR reaction System solution

B. The volume of the sample was adjusted to 4. Mu.L using a multichannel sample applicator, and a PCR reaction system solution was added to each well of the 384-well plate. The 384 well plate is the PCR reaction plate.

C. The prepared DNA sample 384-well plate was removed, and the sample volume was adjusted to 1. Mu.L using a multichannel applicator so that each 5. Mu.L of the PCR reaction system contained 20 to 50ng,Hotstar Taq 0.5U template DNA, 0.5pmol of each amplification primer, and 0.1. Mu.L of 25mM dNTPs.

D. The following reaction conditions were set on a PCR apparatus to perform PCR amplification.

TABLE 8 PCR amplification reaction cycle parameters

5. Alkaline phosphatase treatment of PCR amplified products:

A. after the end of the PCR reaction, the PCR amplification product was treated with SAP (shrimp alkaline phosphatase ) to remove free dNTPs in the system.

B. Preparing an alkaline phosphatase treatment reaction SAP Mix system solution, wherein a 384-well plate is adopted as a reaction system, and 38% of reagent loss is set.

TABLE 9 SAP Mix System solution

C. The SAP Mix was added to 384 well plates using a multichannel applicator with a control volume of 2. Mu.L. For each alkaline phosphatase-treated reaction well, the total volume of the reaction system was 7. Mu.L, with 5. Mu.L of PCR product and 2. Mu.L of SAP mix.

D. The 384-well plate was placed on a PCR instrument, and alkaline phosphatase treatment was performed under the following reaction conditions.

TABLE 10 alkaline phosphatase treatment cycle parameters

6. Single base extension reaction:

A. after the completion of the alkaline phosphatase treatment, a single base extension reaction was carried out, and the total volume of the reaction system was 9. Mu.L.

B. Preparing a single-base extension reaction solution EXTEND Mix system solution, wherein a 384-hole plate is adopted as a reaction system, and 38% of reagent loss is set.

TABLE 11 EXTEND Mix System solution

C. The EXTEND Mix was added to 384 well plates using a multichannel applicator with a control volume of 2. Mu.L. For each reaction well, the single base extension reaction system contained 7. Mu.L of SAP-treated PCR product and 2. Mu.L of EXTEND Mix (where each extension reaction primer mixture was 0.94. Mu.L, iPLEX enzyme was 0.041. Mu.L, extension mixture was 0.2. Mu.L).

D. A384-well plate was placed on a PCR instrument, and single base extension reaction was performed under the following PCR reaction conditions.

TABLE 12 Single base extension reaction cycle parameters

7. And (3) purifying resin:

A. the reaction product (9. Mu.L in total) was diluted 3-fold, desalted with a resin, and the desalted sample was spotted on a sample target and naturally crystallized.

B. Clean Resin was tiled into 6mg Resin plates.

C. Add 16. Mu.L of water to the corresponding wells of the extension product.

D. Pouring the dried resin into an extension product plate, sealing the film, and vertically rotating at a low speed for 30min to enable the resin to be fully contacted with reactants.

E. Centrifuging to allow the resin to sink into the bottom of the well.

8. Chip sample application:

the MassARRAY Nanodispenser RS spotter was started and the resin purified extension product was transferred to 384 well SpectroCHIP (Sequenom) chips.

9. Mass spectrometry detection:

the spotted SpectroCHIP was analyzed using MassARRAY Analyzer System, and the detection results were typed using TYPER 4.0 software (sequenom) and the results were output.

Example 3 primer comparison

The multiplex PCR amplification primer set and the single base extension primer set designed by the invention and the common multiplex PCR amplification primer set and the single base extension primer set (Table 13) are used for respectively detecting the oral swab DNA samples, the specific implementation method and the steps of sample extraction and detection are as in the above-mentioned example 1 and example 2, and the result data are as in Table 14.

TABLE 13 common multiplex PCR amplification primers and Single base extension primer sequences

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The data of the detection result of the comparison experiment show that the multiplex PCR amplification primer set and the single base extension primer set designed by the invention are more suitable for SNP locus detection of skin moisturizing ability genes than the common multiplex PCR amplification primer set and the single base extension primer set (Table 14). The positive detection rate of the multiplex PCR amplification primer set and the single-base extension primer set designed by the invention is 100 percent, which is far higher than that of the common multiplex PCR amplification primer set and the single-base extension primer set, thus realizing the purpose of experiments.

TABLE 14 comparison of the results of the detection of the present invention with the combination of ordinary primers

Example 4 test result statistics and interpretation

The results of detection of 12 SNP sites of 7 skin moisturizing genes for 30 oral swab samples tested in example 3 were counted (table 15), and SNP site molecular biology genotyping results were interpreted according to the detection result interpretation criteria (table 16) (tables 17, 18), and the genetic information of the skin moisturizing ability of the subject was comprehensively evaluated for guiding accurate skin care management.

TABLE 15 SNP site detection results

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Table 15 shows that the molecular biological genotyping results of 12 SNP loci of 7 genes for detecting skin moisturizing ability according to the present invention can be obtained by the detection method disclosed by the present invention. Detecting that the homozygous genotype is identical letter repetition, such as "AA"; two different letters, such as "AG", indicate heterozygous genotypes "AG" or "GA", "D" indicates that the single base is "del" (deleted) and "-" indicates undetected.

Table 16 interpretation criteria for test results

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The method for determining the comprehensive evaluation result comprises the following steps: the effect of SNP locus genotype on phenotype is calculated as "strong" for 1 score, "general" for 0 score, "weak" for-1 score, and the final score is obtained by adding the SNP locus scores. And judging the capability of each item to be 'strong' (the final score is more than or equal to 1), 'general' (the final score=0), 'weak' (the final score is less than or equal to-1) according to the final score.

The final conclusion overall moisturizing ability assessment method comprises the following steps: according to the "strong", "general" and "weak" ratings of each item capability, by "strong+strong=strong, strong+general=strong+weak=general, general+general=general+weak=weak, weak+weak=weak", where "strong+general=strong" is consistent with "general+strong=strong" results.

The skin moisturizing ability was detected by the multiple PCR amplification primer set and the single base extension primer set disclosed by the invention, and finally the comprehensive evaluation result of the skin moisturizing ability of sample 1 was obtained (Table 17). The result shows that the sample has strong water balance capability, can effectively promote skin cells to absorb vitamin A and maintain water-oil balance, fully absorb water and make skin moist, elastic and glossy; in addition, the water locking capability is general, free water cannot be tightly locked by keratinocytes, water is easy to run off through the evaporation of epidermis and sweat glands, and the skin becomes dry, yellow, dull and matt. By observing that the actual face state of the sample 1 accords with the detection result, the slightly water-deficient state is presented, and the overall skin health condition is good. The final conclusion is that the skin of sample 1 has strong overall moisturizing ability, but care should be taken to perform daily moisturizing and skin care, make sun protection, and actively promote self internal moisture balance and external water locking ability.

Table 17 sample 1 test results statistics and interpretation

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The skin moisturizing ability was detected by the multiplex PCR amplification primer set and the single base extension primer set disclosed by the invention, and finally the comprehensive evaluation result of the skin moisturizing ability of sample 2 was obtained (Table 18). The result shows that the sample has strong inherent moisture balance capability, can effectively promote skin cells to absorb vitamin A and maintain water-oil balance, fully absorb moisture, and make skin moist, elastic and glossy; the external water locking capability is strong, and the keratinocytes can tightly lock the free water, so that the free water is changed into combined water and is not easy to evaporate and lose, and the moisturizing effect is achieved. Through observation, the actual facial state of the sample 2 accords with the detection result, and the skin presents a tender, smooth and bright health state. The final conclusion is that the skin of the sample 2 has strong overall moisturizing capability, can keep the daily normal skin care habit, and properly supplements water, moisturizes and protects the skin.

Table 18 sample 2 test result statistics and interpretation

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Sequence listing

<110> Guangzhou Citizen Biotech Co., ltd

<120> primer combination for skin moisturizing ability gene detection and application thereof

<130> MP1933792

<160> 72

<170> SIPOSequenceListing 1.0

<210> 1

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

acgttggatg ttctcctccc tgtggaaagc 30

<210> 2

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

acgttggatg tcaagatggc aaccgcatac 30

<210> 3

<211> 29

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

acgttggatg ttcagaatgc agaagtggg 29

<210> 4

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

acgttggatg cagtagactt ggccatcttc 30

<210> 5

<211> 29

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

acgttggatg ccaggctggt cttgaagtc 29

<210> 6

<211> 29

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

acgttggatg aggtgcagtt tctcacgac 29

<210> 7

<211> 31

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 7

acgttggatg ggaatctaaa aagtactggt c 31

<210> 8

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 8

acgttggatg tgggagaagg ggtataaaag 30

<210> 9

<211> 29

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 9

acgttggatg agttatctta tcagcttgg 29

<210> 10

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 10

acgttggatg tttcctgact ctggtgaaac 30

<210> 11

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

acgttggatg tagccggagc tccaggtttt 30

<210> 12

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 12

acgttggatg ccaatatctg caggtcagag 30

<210> 13

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 13

acgttggatg atcttccagg ccggtgtggt 30

<210> 14

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 14

acgttggatg ctttgatcca gtcccgaaac 30

<210> 15

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 15

acgttggatg tgtccatgaa tggtgtcctg 30

<210> 16

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 16

acgttggatg gacaaggatc ccaccacaag 30

<210> 17

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 17

acgttggatg tgtcttcgtg atgggacctg 30

<210> 18

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 18

acgttggatg tctgcttcca gaaaccatcg 30

<210> 19

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 19

acgttggatg atcagcagag ccaccaagag 30

<210> 20

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 20

acgttggatg gagtgctcac ctggtagatg 30

<210> 21

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 21

acgttggatg tagagctgtc agcccaagag 30

<210> 22

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 22

acgttggatg ggacaaacaa tcaggagacg 30

<210> 23

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 23

acgttggatg tgtccacgaa tggtgtcctg 30

<210> 24

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 24

acgttggatg ctggaggaag acaaggatcg 30

<210> 25

<211> 16

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 25

ccgcatacat ccggag 16

<210> 26

<211> 17

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 26

ccgtggctgt tgtagat 17

<210> 27

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 27

actgtaatcc cagcattttg g 21

<210> 28

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 28

tgtgaagaat aaatgatctg taaag 25

<210> 29

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 29

cttcctctgg tgaaacaaac cac 23

<210> 30

<211> 27

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 30

gcccctagga gcagaagaca cagagaa 27

<210> 31

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 31

cctgagcgca gccgtctc 18

<210> 32

<211> 28

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 32

gttacaagga tcccaccaca agcaggca 28

<210> 33

<211> 17

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 33

atctgttcag gagcagt 17

<210> 34

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 34

gaccctgaac gtccagacct tccccc 26

<210> 35

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 35

cccttctcca ggcactcagg 20

<210> 36

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 36

gaggctcgca ccacgagcag gca 23

<210> 37

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 37

acgttggatg cttctcctcc ctgtggaaag 30

<210> 38

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 38

acgttggatg tggagtcacc gagaactatg 30

<210> 39

<211> 17

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 39

accgcataca tccggag 17

<210> 40

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 40

acgttggatg tgtttgctct ggattacacg 30

<210> 41

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 41

acgttggatg atcttcttcc ttcagggccg 30

<210> 42

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 42

ggcgtggctg ttgtagat 18

<210> 43

<211> 29

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 43

acgttggatg ccaggctggt cttgaagtc 29

<210> 44

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 44

acgttggatg ttctggccag gtgcagtttc 30

<210> 45

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 45

cacggtaatc ccagcatttt gg 22

<210> 46

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 46

acgttggatg gcctgggaat agaatcacac 30

<210> 47

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 47

acgttggatg tcatgggatt ctccagttgc 30

<210> 48

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 48

gtgaagaata aatgatctgt aaag 24

<210> 49

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 49

acgttggatg tccatttcct gactctggtg 30

<210> 50

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 50

acgttggatg aaaaggtgtc ccatgtttag 30

<210> 51

<211> 27

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 51

actcatttaa gtacagtaat actgaat 27

<210> 52

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 52

acgttggatg gctgtggatc tcccaatatc 30

<210> 53

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 53

acgttggatg gtctttgtct ctcgctgtag 30

<210> 54

<211> 26

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 54

ctcctgagct ccaggttttg ctctca 26

<210> 55

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 55

acgttggatg tgtctccgcc cagggtgatt 30

<210> 56

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 56

acgttggatg ttgatccagt cccgaaacag 30

<210> 57

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 57

gatcggagcg cagccgtctc 20

<210> 58

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 58

acgttggatg tgaccctctt gggacgttga 30

<210> 59

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 59

acgttggatg tcatgaacag tctgagtccg 30

<210> 60

<211> 15

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 60

caccacaagc aggca 15

<210> 61

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 61

acgttggatg tgcgtgacga gtgcctgatt 30

<210> 62

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 62

acgttggatg aaaccatcgt ggatctgttc 30

<210> 63

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 63

ctggctctgt tcaggagcag t 21

<210> 64

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 64

acgttggatg agacacacag tcagtgtcag 30

<210> 65

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 65

acgttggatg gagtgctcac ctggtagatg 30

<210> 66

<211> 15

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 66

tccagacctt ccccc 15

<210> 67

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 67

acgttggatg gcagacaaac tcgtaaggac 30

<210> 68

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 68

acgttggatg tggaccccga tgattgttcc 30

<210> 69

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 69

aaacctcatg gtgacgtgac c 21

<210> 70

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 70

acgttggatg accctcttgg gacgctgaat 30

<210> 71

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 71

acgttggatg aggtgagcac tcatgaacag 30

<210> 72

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 72

cccccaccac gagcaggca 19

Claims

1. A primer set for detecting the moisture retention capacity of skin, characterized by comprising a primer set for detecting the internal moisture balance capacity and a primer set for detecting the external water retention capacity;

the multiplex PCR amplification primer group for detecting the inherent water balance capability is a nucleotide sequence shown as SEQ ID No. 1-2, 3-4, 5-6, 7-8, 9-10 and 11-12;

the single-base extension primer group for detecting the inherent water balance capability is a nucleotide sequence shown as SEQ ID No. 25-30;

the multiplex PCR amplification primer group for detecting the external water locking capability is a nucleotide sequence shown as SEQ ID No. 13-14, 15-16, 17-18, 19-20, 21-22 and 23-24;

the single-base extension primer group for detecting the external water locking capability is a nucleotide sequence shown as SEQ ID No. 31-36;

the molar ratio of the multiplex PCR amplification primer set for detecting the intrinsic moisture balance capability to the single base extension primer set for detecting the intrinsic moisture balance capability to the multiplex PCR amplification primer set for detecting the extrinsic water locking capability to the single base extension primer set for detecting the extrinsic water locking capability in the primer combination is (8.57-9.21): (19.7-34): (8.79-8.98): (19.2 to 31.5);

the detected SNP loci are rs12934922, rs7501331, rs58605957, rs10792653, rs9806130, rs10412066, rs137852931, rs138726443, rs150597413, rs200519781, rs397507563 and rs61816761.

2. Use of a primer combination according to claim 1 for the preparation of a kit for detecting the intrinsic moisture balance capacity and the extrinsic water locking capacity of skin.

3. Use of a primer combination according to claim 1 for the preparation of a reagent and/or kit for detecting skin moisturization capacity.

4. A detection reagent comprising the primer set according to claim 1.

5. A kit comprising the primer combination of claim 1 or the detection reagent of claim 4.

6. Use of the primer combination according to claim 1, the detection reagent according to claim 4 or the kit according to claim 5 for detecting a skin moisturizing ability gene or skin moisturizing ability for non-disease diagnosis purposes.

7. The method for detecting the skin moisturizing ability gene for the purpose of non-disease diagnosis is characterized by comprising the following steps:

step 1, obtaining nucleic acid of a sample to be detected;

step 2, using the nucleic acid extracted in the step 1 as a template, respectively adopting the primer in the primer combination of claim 1 to carry out multiplex PCR amplification and single base extension, and carrying out genotyping on the SNP locus;

step 3, obtaining a detection result according to the genotyping result;

the detection result obtained according to the genotyping result in the step 3 is as follows:

；

the standard for obtaining the detection result according to the genotyping result is as follows: the influence of SNP locus genotype on phenotype is calculated as 1 score of 'strong', calculated as 0 score of 'general', calculated as-1 score of 'weak', and the final score is obtained by adding the scores of all SNP loci; and judging the internal water balance capacity and the external water locking capacity according to the final score:

the final score is more than or equal to 1: "strong";

final score = 0: "general";

the final score is less than or equal to-1: "weak".