CN111321212B - Primer combination for detecting anti-acne capability gene of skin and application thereof - Google Patents
Primer combination for detecting anti-acne capability gene of skin and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of genotyping, and discloses a primer combination for detecting a skin anti-acne capability gene and application thereof, wherein the primer combination comprises a multiplex PCR amplification primer group and a single base extension primer group for detecting 8 SNP sites of 5 skin anti-acne capability genes of a human genome. A pair of multiplex PCR amplification primers and a single base extension primer are respectively designed for each SNP locus, and all the loci are designed into one reaction and can be simultaneously detected. The SNP typing method for efficiently detecting the anti-acne capability gene of the skin is established, molecular biological gene typing of a plurality of SNP sites can be completed at the same time, the detection sensitivity is good, the accuracy rate is high, the cost is low, the practicability is high, the SNP typing method can be used for detecting genetic factors influencing the anti-acne capability of the skin, carrying out comprehensive evaluation on the anti-acne capability of the skin, and is beneficial to promoting the development of individualized services in the beauty industry.
Description
Technical Field
The invention relates to the technical field of genotyping, in particular to a gene detection primer combination for skin anti-acne capability and application thereof.
Background
Acne, a term of acne, is a very common chronic inflammatory skin disease of pilosebaceous glands, mainly affects adolescents and young adults, has a prevalence rate of up to 40% -80%, mainly presents skin problems such as facial acne and the like, and can leave scars when severe people or patients are subjected to irregular treatment, thus affecting the appearance and mental health of patients. According to the survey data of China on partial acne patients, the method shows that: 58.8 percent of patients do not have correct knowledge on the occurrence and prevention of the acne, the self-protection is insufficient, and the treatment effect of the acne is directly influenced, and only 41.2 percent of patients with the acne select to a hospital to seek professional treatment and prevention guidance. With the increasing demand for skin care, the cost of skin care is increasing. The share of the anti-acne skin care product is also improved year by year, the anti-acne population is increased by about 400 to 600 thousands of people every year, and the investigation report of 360-year data in 2018 shows that the anti-acne skin care product accounts for 19.7 percent of the anti-acne skin care population. Thus, anti-acne has become one of the most important skin care objectives.
The anti-acne ability of the human body is mainly influenced by two aspects of the skin barrier function and the acne resistance. The skin barrier is composed of keratinized keratinocytes, a sebaceous membrane and intercellular lipids, called "epidermal brick wall", which plays a role in isolation and protection of the human body; the good living habits can increase the tolerance of the skin, strengthen the defense capability of the skin and prevent the skin barrier from being damaged easily. Part of genes are closely related to the generation of acnes and easily generate acnes on the skin, and when the genes respond to the change of the external environment and the conditions in the body, more oil is secreted or the metabolism speed of hair follicle cutin is slowed down, so that pores are blocked, a skin defense system is weakened, the invasion of bacteria cannot be resisted, and the infection of the skin surface layer causes inflammation; the gene of the skin which is not easy to produce acne has normal function of the permeable barrier of the epidermis under the conditions of external environment stimulation and poor self state, the skin keeps a good state and is not easy to grow acne.
From the perspective of genetics, the skin phenotype state difference is caused by the difference of human genetic information, and the influence of different environments on the skin plays a key role; scientific research shows that the genetic factor of acne is as high as 81%, namely acne occurs, and the influence factor of environment only accounts for 19%. The gene influences the state of the skin, and has certain influence on the regulation function of the skin barrier and the capacity of resisting acnes. The detection of the anti-acne capability of the skin is helpful for acquiring the self genetic information in advance, further improves the understanding of the pathogenesis of the common skin disease acne, and provides theoretical and practical guidance for acne prevention. By selecting a proper skin care scheme and a skin care product for targeted intervention, the generation of acne can be prevented in advance.
The research progress of human genetics, molecular biology, genomics and bioinformatics can fully illustrate the influence of Single Nucleotide Polymorphism (SNP) on the anti-acne capability of skin. The patent (201811143772.5) discloses a method for rapidly detecting hereditary skin anti-acne gene, which detects two anti-acne related gene loci rs7531806 and rs747150 by HRM method, the method can detect the difference of single basic group, but the detection range is small, the operation is complex, and the requirement of detecting more than two anti-acne related gene loci of skin can not be satisfied. The patent (201810839384.4) discloses a skin-related gene locus library, a construction method and application thereof, aiming at the detection of 5 anti-acne genes, comprising the following steps: the detection method of the patent can accurately determine the causes of skin problems, but has more classifications, low detection specificity and complex operation.
Therefore, the primer combination for detecting the anti-acne capability gene of the skin and the application thereof have important practical significance.
Disclosure of Invention
In view of the above, the invention provides a gene detection primer combination for skin anti-acne ability and application thereof. The invention utilizes a MassARRAY nucleic acid mass spectrometry system to carry out molecular biological genotyping on the SNP locus of the skin anti-acne capability gene, and carries out comprehensive evaluation on the skin anti-acne capability of an individual according to the genotyping result so as 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 the anti-acne capability of skin, which comprises a primer group for detecting the barrier function of the skin and/or a primer group for detecting the anti-acne capability;
(1) the primer group for detecting the skin barrier function comprises a multiple PCR amplification primer group for detecting the skin barrier function and a single-base extension primer group for detecting the skin barrier function;
the multiplex PCR amplification primer group for detecting the skin barrier function has any one of the following nucleotide sequences:
(I) the nucleotide sequence has any one group of SEQ ID No. 1-2, 3-4, 5-6, 7-8 and 9-10;
(II) nucleotide sequences obtained by modifying, substituting, deleting and/or adding one or more bases to the nucleotide sequences shown in SEQ ID Nos. 1-2, 3-4, 5-6, 7-8 and 9-10;
(III) a sequence having at least 80% sequence identity with the nucleotide sequence shown in SEQ ID Nos. 1-2, 3-4, 5-6, 7-8, 9-10;
(IV) a sequence complementary to the sequence of (I), (II) or (III);
the single-base extension primer group for detecting the skin barrier function has any one of the following nucleotide sequences:
(V) has a nucleotide sequence shown in any one of SEQ ID Nos. 17-21;
(VI), the nucleotide sequence is obtained by modifying, substituting, deleting and/or adding one or more bases in the nucleotide sequence shown in SEQ ID No. 17-21;
(VII) a sequence having at least 80% sequence identity with the nucleotide sequences shown in SEQ ID Nos. 17-21;
(VIII) the complement of the sequence shown in (V), (VI) or (VII);
(2) the primer group for detecting the acne resistance comprises a multiplex PCR amplification primer group for detecting the acne resistance and a single-base extension primer group for detecting the acne resistance;
the multiplex PCR amplification primer group for detecting the acne resistance has any one of the following nucleotide sequences:
(IX) has a nucleotide sequence shown in any one of SEQ ID Nos. 11-12, 13-14 and 15-16;
(X) is a nucleotide sequence obtained by modifying, substituting, deleting and/or adding one or more bases to the nucleotide sequence shown in any one group of SEQ ID Nos. 11-12, 13-14 and 15-16;
(XI) a sequence having at least 80% sequence identity with a nucleotide sequence shown in any one of SEQ ID Nos. 11-12, 13-14, 15-16;
(XII) the complement of the sequence shown In (IX), (X) or (XI);
the single-base extension primer group for detecting the capacity of resisting acne has any one of the following nucleotide sequences:
(XIII) having a nucleotide sequence shown in any one of SEQ ID Nos. 22-24;
(XIV) a nucleotide sequence obtained by modifying, substituting, deleting and/or adding one or more bases to the nucleotide sequence shown in any one of SEQ ID Nos. 22 to 24;
(XV) a sequence having at least 80% sequence identity to a nucleotide sequence as set forth in any one of SEQ ID Nos. 22 to 24;
(XVI), a sequence complementary to the sequence shown in (XIII), (XIV) or (XV).
In some embodiments of the present invention, the mole ratio of the multiple PCR amplification primer set for detecting skin barrier function, the single base extension primer set for detecting skin barrier function, the multiple PCR amplification primer set for detecting acne resistance, and the single base extension primer set for detecting acne resistance in the primer combination is (6.5-6.9): (24.3-35.9): (6.7-6.8): (17.2-28.6).
On the basis of the research, the invention also provides application of the primer combination in preparing a kit for detecting the barrier function of the skin and/or the capacity of resisting acne.
In addition, the invention also provides application of the primer combination in preparing a reagent and/or a kit for detecting the anti-acne capability of skin.
On the basis of the research, the invention also provides a detection reagent which comprises the primer combination.
In addition, the invention also provides a kit comprising the primer combination or the detection reagent.
The invention also provides application of the primer combination, the detection reagent or the kit in detecting the anti-acne capability gene of the skin or the anti-acne capability of the skin.
On the basis of the research, the invention also provides a detection method of the anti-acne capability gene of the skin, which comprises the following steps:
step 2, taking the nucleic acid extracted in the step 1 as a template, respectively adopting the primers in the primer combination according to claim 1 or 2 to carry out multiple PCR amplification and single base extension, and carrying out genotyping on the SNP locus;
and 3, obtaining a detection result according to the genotyping result.
In some embodiments of the present invention, the step 3 of obtaining the detection result according to the genotyping result is:
in some embodiments of the invention, the criteria for obtaining a test result based on the result of genotyping are: the influence of the SNP locus genotype on the phenotype is 1 point of strong score, 0 point of general score and-1 point of weak score, and the scores of the SNP loci are added to obtain a final score; and judging the skin barrier function and/or the acne resistance according to the final score:
the final score is more than or equal to 1: "Strong";
final score is 0: "general";
the final score is less than or equal to-1: "weak".
The invention uses a MassARRAY nucleic acid mass spectrometry system to carry out SNP typing detection on the skin anti-acne capability gene, and the detection combines a multiple PCR technology, a MassARRAY iPLEX single base extension technology and a matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to carry out typing detection. The method is carried out by carrying out PCR amplification on a DNA fragment containing the SNP site and then carrying out single-base extension reaction by utilizing a specific extension primer. Since the difference in molecular weight of the extended product is caused by the difference in the base at the polymorphic site and the difference in the terminal base of the extended product, the difference in base due to the SNP polymorphism is reflected by the difference in molecular weight. The detection method has the advantages of high sensitivity, flexible time, low error probability, low cost and avoidance of cross contamination. The base difference is distinguished by detecting the molecular weight, and the method does not relate to fluorescence labeling, gel electrophoresis and the like, and has high accuracy. The method can simultaneously detect 8 SNP sites of 5 genes, the sites cover two aspects of skin barrier function directly related to the anti-acne capability of skin and acne resistance, and the detection content is the most comprehensive at present. The method is used for comprehensively evaluating the innate anti-acne capability of the skin through the gene detection of the anti-acne capability of the skin, thereby providing scientific basis for an external skin care scheme, more effectively preventing acne and keeping the skin in a better state. Therefore, the targeted gene detection of the anti-acne capability of the skin can help people to better understand the genetic factors of the skin, understand the root of the skin problem more deeply and provide scientific guidance for realizing the anti-acne capability of the skin.
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 technique (MALDI-TOF-MS) experiment;
FIG. 2 shows a verification chart of UCSC Genome Bioinformatics at the site rs 6471;
FIG. 3 shows the resulting peak pattern of the rs6471 site MassARRAY nucleic acid mass spectrometry system;
FIG. 4 shows a scattergram of results of the rs6471 site MassARRAY nucleic acid mass spectrometry system;
FIG. 5 is a photograph showing the face of the subject of sample 1 in example 4;
fig. 6 is a photograph showing the face of the subject of sample 2 in example 4.
Detailed Description
The invention discloses a gene detection primer combination for skin anti-acne ability and application thereof, and a person skilled in the art can appropriately improve process parameters by referring to the contents. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention provides a primer combination for detecting skin anti-acne capability genes and application thereof, comprising a multiplex PCR amplification primer group and a single base extension primer group of 8 SNP sites of 5 skin anti-acne capability genes of human genome DNA.
Further, the 5 genes of the anti-acne capability of the skin of the human genome DNA are Cytochrome P450 family 21subfamily A member 2(Cytochrome P450 family 21subfamily A member2, CYP21A2) gene, ATP enzyme sarcoplasm/endoplasmic reticulum Ca2+Transporter 2(ATPase sarcoplasmic/endomorphic reticulum Ca)2+transport 2, ATP2a2) gene, Filaggrin (FLG) gene, Selectin L (SELL) gene, and lesion specific DNA binding protein 2 (DDB 2) gene.
Further, the 8 SNP loci of the 5 genes with the skin anti-acne capability are rs6471, rs121912734, rs61816761, rs200519781, rs121909626, rs7531806, rs747650 and rs1060573 (table 1).
Further, the multiple PCR amplification primer group and the single-base extension primer group are used for detecting the anti-acne capability of the skin, including the capability of detecting the barrier function of the skin and the capability of resisting acnes.
TABLE 1 Gene SNP sites and Gene Functions
Further, the multiple PCR amplification primer group for detecting the skin barrier function gene SNP site comprises primers Y1-Y10, the sequence of the multiple PCR amplification primer group is shown as SEQ ID No. 1-10, the multiple PCR amplification primer group comprises an upstream primer Y1 and a downstream primer Y2 of rs6471 site of CYP21A2 gene, and the sequence information is shown as SEQ ID No. 1-2; the primer sequence comprises an upstream primer Y3 and a downstream primer Y4 of an rs121912734 site of an ATP2A2 gene, and the sequence information is shown in SEQ ID Nos. 3-4; the primer comprises an upstream primer Y5 and a downstream primer Y6 at the rs61816761 site of the FLG gene, and sequence information is shown in SEQ ID No. 5-6; comprises an upstream primer Y7 and a downstream primer Y8 of site rs200519781 of FLG gene, and the sequence information is shown in SEQ ID No. 7-8; the primer comprises an upstream primer Y9 and a downstream primer Y10 of site rs121909626 of the FLG gene, and sequence information is shown in SEQ ID No. 9-10.
The single base extension primer group T1-T5 has a sequence shown in SEQ ID No. 17-21, comprises a single base extension primer T1 at the rs6471 site of a CYP21A2 gene, and has sequence information shown in SEQ ID No. 17; comprises an ATP2A2 gene rs121912734 site single base extension primer T2, and the sequence information is shown in SEQ ID No. 18; comprises FLG gene rs61816761 site single base extension primer T3, and the sequence information is shown in SEQ ID No. 19; comprises FLG gene rs200519781 site single base extension primer T4, and the sequence information is shown in SEQ ID No. 20; comprises FLG gene rs121909626 site single base extension primer T5, and the sequence information is shown in SEQ ID No. 21.
The acne resistance detection method comprises the steps of detecting multiple PCR amplification primer groups Y11-Y16 of an SNP locus of an acne resistance gene, wherein the sequence of the multiple PCR amplification primer groups is shown as SEQ ID No. 11-16, the multiple PCR amplification primer groups comprise an upstream primer Y11 and a downstream primer Y12 of an rs7531806 locus of a SELL gene, and the sequence information is shown as SEQ ID No. 11-12; comprises an upstream primer Y13 and a downstream primer Y14 at the rs747650 site of a DDB2 gene, and the sequence information is shown as SEQ ID No. 13-14; the primer sequence comprises an upstream primer Y15 and a downstream primer Y16 at the rs1060573 site of a DDB2 gene, and the sequence information is shown in SEQ ID Nos. 15-16.
The single base extension primer group T6-T8 has a sequence shown in SEQ ID No. 22-24, comprises SELL gene rs7531806 site T6, and has sequence information shown in SEQ ID No. 22; comprises DDB2 gene rs747650 site T7, and the sequence information is shown as SEQ ID No. 23; comprises DDB2 gene rs1060573 site T8, and the sequence information is shown as SEQ ID No. 24.
The invention uses the MassARRAY nucleic acid mass spectrometry system to carry out SNP typing detection on the skin anti-acne capability gene, has the advantages of high sensitivity, flexible time, low error probability, low cost and avoidance of cross contamination, can detect a plurality of SNP sites in the same time, can rapidly read genetic information, and is more suitable for batch and low-cost detection service. In recent years, matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is also gradually a hot spot of the research in the industry and becomes the development trend of the technology. The related technicians can design the multiple PCR amplification primer group and the single-base extension primer group for the detection service of the skin anti-acne capability gene by using the method of the technology.
The invention specifically designs the multiple PCR amplification primer group and the single base extension primer group of the skin anti-acne capability gene, simultaneously detects 8 SNP sites of 5 genes, has low cost, high accuracy and short time, can detect the skin barrier function directly related to the anti-acne capability of the skin and the genetic information of the anti-acne capability, and comprehensively evaluates the anti-acne capability of the skin of an individual according to the typing result of the molecular biological gene so as to be used for precise skin care management. The combination of the multiple PCR amplification primer group and the single base extension primer group disclosed by the invention is the combination of the multiple PCR amplification primer group and the single base extension primer group for detecting the skin anti-acne capability gene with the best detection effect and the most complete detection project so far.
The gene detection primer combination for the skin anti-acne capability and the raw materials and reagents used in the application thereof provided by the invention can be purchased from the market.
The invention is further illustrated by the following examples:
example 1
A primer combination for detecting skin anti-acne ability gene and application thereof comprise a multiplex PCR amplification primer group and a single base extension primer group (table 2) for detecting 8 SNP sites of 5 skin anti-acne ability genes of human genome. A pair of multiplex PCR amplification primers and a single base extension primer are respectively designed for each SNP locus, and all the loci are designed into one reaction and can be simultaneously detected. The multiplex PCR amplification primer group and the single base extension primer group are used for a MassARRAY nucleic acid mass spectrometry system, the system can be used for quickly analyzing nucleic acid samples with high sensitivity and high accuracy, and the detection accuracy is more than or equal to 99.9%. The invention establishes an efficient SNP typing method for comprehensively evaluating the anti-acne capability gene of the skin, can finish the molecular biological gene typing of a plurality of SNP sites at the same time, has good detection sensitivity, high accuracy, low cost and strong practicability, can be used for detecting genetic factors influencing the anti-acne capability of the skin, and is beneficial to promoting the development of individualized services in the beauty industry.
Step 1: synthesizing a multiplex PCR amplification primer set and a single base extension primer set of the needed SNP locus.
Step 2: human buccal swab sample DNA is extracted and PCR amplification is carried out.
And step 3: and (3) carrying out sample DNA detection by adopting matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to determine the genotype of the SNP locus of the sample.
And 4, step 4: and comprehensively evaluating the genetic information of the anti-acne capability of the skin according to the SNP locus genotype detection result, and reading the result.
TABLE 2 multiplex PCR amplification primers and Single base extension primer sequences
EXAMPLE 2 sample testing
In order to verify the design accuracy and practicability of the multiplex PCR amplification primer set and the single-base extension primer set for detecting the SNP locus of the skin anti-acne capability gene, 30 oral swab samples are collected for detection in the embodiment, and the specific contents are as follows:
1. designing a primer:
according to the SNP locus sequence information, the multiple PCR amplification primer and the single base extension primer of the SNP locus to be detected are designed by utilizing the Assay Design Suite V2.0 of Agena company, the multiple PCR amplification primer group and the single base extension primer group of 8 SNP loci of 5 genes are designed into one reaction (WELL), and UCSC Genome Bioinformatics is used for verification.
The result of designing the quality value of the software primer combination shows that the quality of the primer combination disclosed by the invention is good (Table 3).
Verified by on-line software UCSC Genome Bioinformatics, the primer combination amplification fragment disclosed by the invention meets the requirement that only or each amplification sequence contains a detection site, and has good specificity (figure 3).
The multiplex PCR amplification primer set and the single-base extension primer set were synthesized by Biotechnology engineering (Shanghai) Ltd.
TABLE 3 primer combination Mass values
2. Collecting samples:
30 buccal swab samples were selected for DNA extraction and information on the age, sex, skin condition etc. of the subjects was recorded (table 5). The skin condition evaluation method comprises the following steps: the face of the subject corresponding to 30 oral swab samples was photographed, and 10 skin detection technicians scored the skin status of the photographs according to the item, with a score of 1-9. And adding the scores of the items to obtain an average value, then taking the average value of the scores of 10 technicians, taking the result as an integer, and finally determining the skin state score of the detected person, wherein the skin state is better when the score is higher.
TABLE 4 skin status score criteria
TABLE 5 sample Collection information
3. Oral swab DNA extraction:
extracting DNA from a buccal swab by using a nucleic acid extraction and purification kit of the Meiji gene, and adding a certain amount of absolute ethyl alcohol into Buffer BW1 according to the instruction. The buccal swab collection tip was cut into a 2mL EP tube, 400mL Buffer ATL and 20. mu.L protease K were added, the swab was transferred to a 2mL centrifuge tube, and a portion of the swab handle was removed. Incubating the mixture for 15-30 minutes at 55 ℃ with shaking. Add 25. mu.L Magpure and 600. mu.L Buffer GXP2 to the centrifuge tube. Transferring 250-300 mu L of supernatant into a centrifuge tube filled with magnetic beads/GXP 2, reversing and mixing uniformly for 15-30 times, standing at room temperature for 5-8 minutes, and reversing and mixing uniformly for several times. Transferring to a magnetic frame for adsorption for 2 minutes, and absorbing and discarding or dumping the solution. Add 500. mu.L Buffer BW1 and vortex for 15 seconds to break up the beads. Transferring to a magnetic frame for adsorption for 1 minute, and absorbing and discarding the solution. Add 700. mu.L of 75% ethanol and vortex for 15 seconds to break up the beads. Transferring to a magnetic frame for adsorption for 1 minute, and absorbing and discarding the solution. Add 700. mu.L of 75% ethanol and vortex for 15 seconds to break up the beads. Transferring to a magnetic frame for adsorption for 1 minute, and absorbing and discarding the solution. And (4) centrifuging for a short time, completely sucking residual liquid, and drying in air for 10-15 minutes to remove ethanol. Adding 30-100 mu L of Elution Buffer into a centrifuge tube, and carrying out shaking incubation for 5-10 minutes at 55 ℃. Transferring the DNA solution to a magnetic frame for adsorption for 3-5 minutes, transferring the DNA solution to a new centrifugal tube, performing quality inspection on the DNA solution by using an ultramicro spectrophotometer, taking the volume of the qualified DNA for quality inspection to be 30 mu L, transferring the DNA solution to a 384-pore plate for sample loading, and storing the DNA solution at the temperature of-20 ℃ for later use.
The quality inspection standard of the genome DNA is that the total amount of the DNA is more than or equal to 0.5 mug, the concentration is more than or equal to 15 ng/mug, the purity OD 260/2801.7-2.1 is free from protein and RNA pollution, the integrity reaches clear bands, the molecular weight is more than 10Kb, and no obvious degradation is caused.
TABLE 6 sample DNA quality test results
4. PCR amplification reaction:
the multiplex PCR technique was performed in 384-well plates, with a total volume of 5. mu.L per reaction system.
A. A PCR reaction system solution was prepared in a new 1.5ml EP tube using 384 well plates with a reagent loss of 38%.
TABLE 7 PCR reaction System solution
B. Using a multichannel applicator, the application volume was adjusted to 4. mu.L, and the PCR reaction system solution was applied to each application well of a 384-well plate. The 384-well plate is a PCR reaction plate.
C. The prepared DNA sample 384-well plate was taken out, and the sample volume was adjusted to 1. mu.L using a multichannel sample applicator, so that each 5. mu.L PCR reaction system contained 20 to 50ng of template DNA, 0.5U of Hotstar Taq, 0.5pmol of each amplification primer, and 0.1. mu.L of 25mM dNTPs.
D. The following reaction conditions were set on the PCR instrument to perform PCR amplification.
TABLE 8 PCR amplification reaction cycling parameters
5. Alkaline phosphatase treatment of PCR amplification product:
A. after the PCR reaction is completed, the PCR amplification product is treated with SAP (shrimp alkaline phosphatase) to remove free dNTPs from the system.
B. Alkaline phosphatase treatment reaction SAP Mix system solution is prepared, a 384-well plate is adopted in a reaction system, and 38% reagent loss is set.
TABLE 9 SAP Mix system solution
C. Using a multichannel applicator, the loading volume was adjusted to 2. mu.L and SAP Mix was added to 384 well plates.
The total reaction volume was 7. mu.L for each alkaline phosphatase treated well, 5. mu.L of PCR product and 2. mu.L of SAP mixture.
D. The 384-well plate was set 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 alkaline phosphatase treatment was completed, the single-base extension reaction was carried out in a total volume of 9. mu.L.
B. Prepare single base extension reaction solution EXTEND Mix system solution, the reaction system adopts 384-well plate, sets up 38% reagent loss.
TABLE 11 EXTEND Mix system solution
C. The EXTEND Mix was added to 384-well plates using a multichannel applicator with an adjusted sample 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 primer Mix was 0.94. mu.L, iPLEX enzyme 0.041. mu.L, extension Mix was 0.2. mu.L).
D. The 384-well plate was set on a PCR apparatus, and the following PCR reaction conditions were set to conduct a single base extension reaction.
TABLE 12 Single base extension reaction cycle parameters
7. Resin purification:
A. the reaction product (9 ul in total) was diluted 3 times, desalted using a resin, and the desalted sample was spotted on a sample target and naturally crystallized.
B. Clean Resin was spread into a 6mg Resin plate.
C. Add 16. mu.L of water to the corresponding well of the extension product.
D. Pouring the dried resin into the extension product plate, sealing the film, and vertically rotating at low speed for 30min to make the resin fully contact with the reactant.
E. The resin was allowed to settle to the bottom of the well by centrifugation.
8. Chip spotting:
the MassARRAY Nanodispenser RS1000 spotting instrument was started and the resin purified extension product was transferred to 384-well SpectroCHIP (sequenom) chips.
9. Mass spectrum detection:
the spotted SpectroCHIP chip was analyzed using MassARRAY Analyzer System, and the assay results were typed using TYPER 4.0 software (sequenom) and output.
Example 3 primer comparison
The specific implementation method and steps of sample extraction and detection are as in example 1 and example 2 above, and the result data are shown in Table 14, wherein the oral swab DNA sample is detected by using the multiplex PCR amplification primer set and single base extension primer set designed by the present invention and the common multiplex PCR amplification primer set and single base extension primer set (Table 13).
TABLE 13 common multiplex PCR amplification primers and Single base extension primer sequences
The data of the detection results of the comparative experiment show that the multiplex PCR amplification primer group and the single-base extension primer group designed by the invention are more suitable for detecting the SNP locus of the anti-acne capability gene of the skin than the common multiplex PCR amplification primer group and the single-base extension primer group (Table 14). The positive detection rate of the multiple PCR amplification primer group and the single base extension primer group designed by the invention is 100 percent, which is far more than that of the common multiple PCR amplification primer group and the single base extension primer group, thereby realizing the purpose of experiment.
TABLE 14 comparison of the test results of the present invention with those of the common primer combinations
Example 4 test result statistics and interpretation
The results of detection of 8 SNP sites of 5 skin anti-acne genes in 30 buccal swab samples tested in example 3 were counted (table 15), molecular biological genotyping results of SNP sites were interpreted according to interpretation criteria (table 16) (tables 17 and 18), and genetic information of anti-acne ability of skin of the subjects was comprehensively evaluated for guidance of precise skin care management.
TABLE 15 SNP site detection results
Table 15 shows that the molecular biological genotyping results of 8 SNP loci of 5 genes for detecting anti-acne ability of skin can be obtained by the detection method disclosed by the invention. The homozygous genotype of the detection result is the same letter repetition, such as AA; two different letters, such as "AG", indicate a heterozygous genotype of "AG" or "GA", "D" indicates that the single base is "del" (deleted), "-" indicates undetected.
TABLE 16 interpretation of test results
The method for determining the comprehensive evaluation result comprises the following steps: the influence of SNP locus genotype on phenotype is counted as 1 point of 'strong', 0 point of 'general' and-1 point of 'weak', and the scores of the SNP loci are added to obtain a final score. And judging the capability of each item is 'strong' (the final score is more than or equal to 1), 'common' (the final score is 0) and 'weak' (the final score is less than or equal to-1) according to the final score.
The final conclusion is that the evaluation method of the total anti-acne capability is as follows: the term "strong", "weak", and "strong" is used to identify the term "strong", "weak", and the term "strong", "weak", and the term "weak", and the term "strong" is used to identify the term "strong", "weak", and "weak".
The skin anti-acne capability is detected by the multiplex PCR amplification primer group and the single base extension primer group disclosed by the invention, and finally the comprehensive evaluation result of the skin anti-acne capability of the sample 1 is obtained (Table 17). The result shows that the skin barrier function of the sample is general, the skin cells are damaged by external harmful substances, irritants and ultraviolet rays, and inflammatory reaction is easy to occur; the acne resistance is weak, the sebum secreted by the facial skin is more, and the pilosebaceous glands are easy to block and easily infected by bacteria, so that the facial skin is easy to have acnes. Through observation, the actual facial state of the sample 1 is consistent with the detection result, and skin problems such as a small amount of acne and seborrhea, large pores, dark skin color and the like are presented. The final conclusion is that the skin of sample 1 has a weak overall anti-acne ability, and special attention should be paid to selecting a mild way for daily care, reducing skin irritation, enhancing skin barrier function, avoiding squeezing or scratching skin lesions by hands, and enhancing anti-acne care of the skin.
TABLE 17 statistics and interpretation of sample 1 test results
The anti-acne capability of the skin is detected by the multiple PCR amplification primer group and the single base extension primer group disclosed by the invention, and finally, the comprehensive evaluation result of the anti-acne capability of the skin of the sample 2 is obtained (Table 18). The result shows that the skin barrier function of the sample is strong, the regulation capacity of lipid barriers among keratinocytes, sebaceous membranes and cells is strong, and the inflammation state is less; the acne resistance is strong, and the skin is not easy to have the problems of excessive sebum secretion, blockage of pilosebaceous ducts, bacterial infection, inflammatory reaction and the like. By observation, the actual facial state of sample 2 coincided with the test results, and the skin was in a clean and refreshing state. The final conclusion was that the skin of sample 2 was overall strong in anti-acne capacity, but the subjects were still advised not to carelessly attend to carelessness and to keep their face clean daily, taking care to protect the skin.
TABLE 18 statistics and interpretation of sample 2 test results
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Pusen Biotechnology Ltd, Guangzhou City
Primer combination for detecting skin anti-acne capability gene and application thereof
<130> MP1934402
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acgttggatg tcagtgggtt gtcatgagtg 30
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<213> Artificial sequence (Artificial sequence)
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acgttggatg cttggtcgga tccctttatg 30
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<213> Artificial sequence (Artificial sequence)
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<213> Artificial sequence (Artificial sequence)
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<212> DNA
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<210> 18
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
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cgtccagacc ttccccc 17
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<213> Artificial sequence (Artificial sequence)
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<213> Artificial sequence (Artificial sequence)
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<212> DNA
<213> Artificial sequence (Artificial sequence)
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acgttggatg tcagtgggtt gtcatgagtg 30
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<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 29
acgttggatg tgtctgtcat tcgagagtgg 30
<210> 30
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 30
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<210> 31
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
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<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
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<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 33
gtgggcgctg aatgcctgga gctgtctc 28
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<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
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<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
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<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 36
tgaccgtcca gaccttcccc c 21
<210> 37
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 37
acgttggatg atccccagtt cctgcttgtc 30
<210> 38
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 38
acgttggatg tcaccatgaa gcttcctctc 30
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<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 39
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<210> 40
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<212> DNA
<213> Artificial sequence (Artificial sequence)
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acgttggatg aaatgaggac aataccatc 29
<210> 41
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 41
acgttggatg gagatggtac ctgacatctg 30
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<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 42
cctcatagca tttaatccc 19
<210> 43
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 43
acgttggatg tttcctgtct ctagtccctc 30
<210> 44
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 44
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<210> 45
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 45
tcggatccct ttatgagtca ga 22
<210> 46
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 46
acgttggatg aaaaaggtct ccgcagacac 30
<210> 47
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 47
acgttggatg gactatttac tcagtgacgc 30
<210> 48
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequence)
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gtgacgctag agattatata tca 23
Claims (6)
1. The primer combination for detecting the anti-acne capability of the skin is characterized by comprising a primer group for detecting the barrier function of the skin and a primer group for detecting the anti-acne capability;
(1) the primer group for detecting the skin barrier function comprises a multiple PCR amplification primer group for detecting the skin barrier function and a single-base extension primer group for detecting the skin barrier function;
the multiple PCR amplification primer group for detecting the skin barrier function is a nucleotide sequence shown as SEQ ID Nos. 1-2, 3-4, 5-6, 7-8 and 9-10;
the single base extension primer group for detecting the skin barrier function is a nucleotide sequence shown in SEQ ID No. 17-21;
(2) the primer group for detecting the acne resistance comprises a multiplex PCR amplification primer group for detecting the acne resistance and a single-base extension primer group for detecting the acne resistance;
the multiplex PCR amplification primer group for detecting the acne resistance is a nucleotide sequence shown in SEQ ID Nos. 11-12, 13-14 and 15-16;
the single base extension primer group for detecting the acne resistance is a nucleotide sequence shown in SEQ ID No. 22-24.
2. The primer combination according to claim 1, wherein the molar ratio of the multiple PCR amplification primer set for detecting skin barrier function, the single base extension primer set for detecting skin barrier function, the multiple PCR amplification primer set for detecting acne resistance and the single base extension primer set for detecting acne resistance in the primer combination is (6.5-6.9): (24.3-35.9): (6.7-6.8): (17.2-28.6).
3. Use of a primer combination according to claim 1 or 2 for the preparation of a kit for the detection of skin barrier function and comedo resistance.
4. Use of the primer combination according to claim 1 or 2 for the preparation of a reagent for measuring the anti-acne ability of skin.
5. A detection reagent comprising the primer combination according to claim 1 or 2.
6. A kit comprising the primer combination of claim 1 or 2.
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| 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 |
| KR20170124014A (en) * | 2016-04-29 | 2017-11-09 | (주)아모레퍼시픽 | Method for predicting skin sensitivity |
| CN108950013A (en) * | 2018-07-27 | 2018-12-07 | 江颖纯 | A kind of skin-related gene site library and its construction method and application |
| CN109554457A (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 anti-acne gene |
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| KR20170124014A (en) * | 2016-04-29 | 2017-11-09 | (주)아모레퍼시픽 | Method for predicting skin sensitivity |
| 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 |
| CN108950013A (en) * | 2018-07-27 | 2018-12-07 | 江颖纯 | A kind of skin-related gene site library and its construction method and application |
| CN109554457A (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 anti-acne gene |
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