A rapid genetic test to determine skin characteristics

Abstract

The present invention belongs to the field of gene detection technology, and specifically relates to a method for rapid gene detection to determine skin characteristics. The method comprises detecting 18 sites of 15 genes at the same time, formatting the detection data as needed, inputting the formatted data into a constructed skin characteristic judgment model, and drawing a conclusion of genetic detection to determine skin characteristics. This method can effectively obtain an accurate evaluation of the skin in terms of anti-oxidation, anti-glycation, collagen retention, anti-photoaging, skin barrier, oil control and moisturizing, and the detection method is simple, highly accurate and time-saving. The present invention provides technical support for preventing and improving skin problems, achieving precise skin care and scientific skin care, and preventing and treating skin diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and in particular relates to a method for quickly detecting genes to determine skin characteristics. Background Art

[0002] Skin gene testing is a method that uses advanced high-throughput sequencing biotechnology to analyze and evaluate the various capabilities of the skin. This technology can detect genetic information such as skin gene defects, mutations, and replacements early, thereby providing a scientific basis for preventing problematic skin and delaying skin aging.

[0003] In personalized skin care, skin gene testing is of great significance. Through testing, we can understand the characteristics of individual skin, such as pigmentation, sensitivity, dryness, etc., so as to formulate targeted skin care plans. In addition, genetic testing can also analyze the expression of metabolic enzymes and proteins in individual genes, and obtain an assessment of the metabolic capacity and sensitivity of certain skin care ingredients, so as to avoid the use of skin care products that are not suitable for certain individuals.

[0004] Traditional genetic testing methods, such as chip testing and DNA sequencing, need to be carried out in a central laboratory and must follow strict experimental procedures. They usually take more than 12 hours, and some even take a week. Conventional qPCR technology also takes about 2 hours to complete the test, and the sample needs to undergo DNA extraction, centrifugation, and shaking (these steps require the use of additional special equipment, such as a centrifuge). Some methods that can complete detection in a short time have been developed in the existing technology, such as loop-mediated isothermal amplification (LAMP) technology, but the current genetic detection technology generally has some or all of the following problems: First, the detection time is long: sequencing or chip sequencing takes 12 hours to 1 week; conventional qPCR takes about 2 hours; second, the above-mentioned constant temperature amplification technology has the following problems: 1) The accuracy rate is low, averaging about 80%. Once such an accuracy rate is used for infectious disease detection, its error is intolerable; 2) The resolution is low, and it is difficult to achieve single-base resolution, which is generally above 10bp; third, the operating environment requirements are high: conventional sequencing and qPCR, because they require sample extraction, centrifugation, purification and other steps, need to be carried out in a qPCR laboratory, and need to contact related equipment operation; fourth, the personnel requirements are high: because they operate in a harsh environment, there are qualification requirements for operators.

[0005] In addition, there are many types of genes for skin testing, and these genes are closely related to various characteristics and conditions of the skin. Specifically, skin gene testing can analyze and determine specific gene sequences, interpret the genetic information of local sites, and thus comprehensively analyze the characteristics of the skin. These genes include but are not limited to water-locking genes, elasticity genes, antioxidant genes, photoaging risk genes, etc. However, there is uncertainty about the reliability of skin gene DNA sequence testing itself, and the accuracy of the interpretation of test reports needs to be improved. Therefore, it is necessary to provide a method with high credibility of gene testing, accurate interpretation method, simple detection method and short time. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a method for genetically detecting skin characteristics, which is more accurate and effective in detecting skin characteristics. In addition, the detection method is simple, and even without nucleic acid extraction, genetic testing can be completed with high accuracy, which greatly saves the time of genetic testing.

[0007] In one aspect, the present invention provides a method for determining skin characteristics by genetic testing, the method comprising simultaneously testing 18 SNP sites of 15 genes; the 18 SNP sites of the 15 genes are:

[0008]

[0009] Specifically, the skin characteristics are characteristics of the skin that can be evaluated intuitively or objectively. The skin properties are artificially defined / set standards. For example, dry skin refers to skin with less than 10% moisture in the stratum corneum due to reduced sebaceous gland secretion and increased transepidermal water loss caused by skin barrier damage. The surface skin is thinner, acne and pimples rarely grow on the skin, and the pores are not obvious. Dry skin is divided into two types: dehydration and oil deficiency. Among them, skin characteristics include: (caused by reduced sebaceous gland secretion and increased transepidermal water loss caused by skin barrier damage) the moisture in the stratum corneum of the skin is less than 10%, the surface skin is thinner, acne and pimples rarely grow on the skin, and the pores are not obvious; skin characteristics include: dry skin, dehydrated dry skin, oil-deficient dry skin, etc.

[0010] Specifically, the method comprises the following steps:

[0011] (1) Lyse the sample using a lysis buffer;

[0012] (2) performing qPCR detection on the sample after lysis in step (1), obtaining the correspondence between the sample and the skin characteristics, and establishing a database;

[0013] (3) Formatting the database obtained in step (2);

[0014] (4) Input the database formatted in step (3) into the skin characteristic judgment model to obtain the skin characteristic conclusion.

[0015] More specifically, the lysis solution in step (1) is lysis solution a; the lysis solution a comprises sodium dodecyl sulfate, tris(hydroxymethyl)aminomethane and silver diethyldithiocarbamate.

[0016] Preferably, the concentrations of the sodium dodecyl sulfate, tris(hydroxymethyl)aminomethane and silver diethyldithiocarbamate are 1.3%-2.1%wt, 0.1-0.2M, and 120-170mM, respectively.

[0017] In some embodiments, the concentrations of sodium dodecyl sulfate, tris(hydroxymethyl)aminomethane and silver diethyldithiocarbamate can be: 1.3%wt, 0.1M, 120mM, respectively.

[0018] In other embodiments, the concentrations of sodium dodecyl sulfate, tris(hydroxymethyl)aminomethane and silver diethyldithiocarbamate may be 2.1%wt, 0.2M, and 170mM, respectively.

[0019] More specifically, the volume of the lysate a is 3-5 times the volume of the sample.

[0020] Preferably, the volume of the lysate a is 3 times the volume of the sample.

[0021] More specifically, the lysate a is heated to 40-45° C. in a water bath before use.

[0022] More specifically, the sample in step (1) includes but is not limited to: oral swab, saliva or biological tissue.

[0023] Preferably, the sample in step (1) is an oral swab.

[0024] More specifically, the qPCR reaction system in step (2) includes reagent A, primers and probes, and reagent B; the reagent A includes Taq DNA polymerase, Tris-HCl, KCl, MgSO4, dNTP and BSA; the reagent B includes chitosan, lysine and 2,5-dimercapto-134-thiadiazole.

[0025] Preferably, the concentrations of Taq DNA polymerase, Tris-HCl, KCl, MgSO4, dNTP and BSA can be: 1.7-2.3U, 70-80 mM, 70-90 mM, 1.5-2.2 mM, 0.05-0.4 mM, 0.1-2 mg / mL, respectively.

[0026] In some embodiments, the concentrations of Taq DNA polymerase, Tris-HCl, KCl, MgSO4, dNTP and BSA can be: 1.7 U, 70 mM, 70 mM, 1.5 mM, 0.05 mM, 0.1 mg / mL, respectively.

[0027] In other embodiments, the concentrations of Taq DNA polymerase, Tris-HCl, KCl, MgSO4, dNTP and BSA can be: 2.3U, 80 mM, 90 mM, 2.2 mM, 0.4 mM, 2 mg / mL, respectively.

[0028] More specifically, the dosage of the reagent A is 1.8%-2.5%wt of the lysed sample.

[0029] Preferably, the mass ratio of chitosan, lysine and 2,5-dimercapto-1,34-thiadiazole can be 1-2:3-4:0.12-0.17.

[0030] In some embodiments, the mass ratio of chitosan, lysine and 2,5-dimercapto-1,34-thiadiazole can be 1:4:0.12.

[0031] In some other embodiments, the mass ratio of chitosan, lysine and 2,5-dimercapto-1,34-thiadiazole can be 2:3:0.17.

[0032] More specifically, the dosage of the reagent B is 1.3%-1.7%wt of the lysed sample.

[0033] More specifically, the primer and probe sequences are shown in SEQ ID NO. 1-SEQ ID NO.72.

[0034] More specifically, the reaction procedure of the qPCR in step (2) is pre-denaturation at 94-96°C for 1-2 min, 1 cycle; 94-96°C for 10-15 s, 58-62°C for 25-30 s, 10-15 cycles; 94-96°C for 10-15 s, 58-62°C for 20-30 s, 25-30 cycles.

[0035] More specifically, after the reaction procedure starts, the heating rate is 6-9°C / s, and the cooling rate is 6-8°C / s.

[0036] More specifically, the corresponding relationship between the sample and the skin characteristics in step (2) is:

[0037] a) The corresponding relationship between anti-photoaging ability and skin characteristics is:

[0038] The skin's UV resistance is divided into three levels from high to low. Taking rs1800414 as the standard, CC is high-end, CT is mid-range, and TT is low-end; taking rs62543565 as the standard, TT is high-end, TC is mid-range, and CC is low-end; taking rs10733310 as the standard, TT is high-end, TG is mid-range, and GG is low-end; taking rs4911414 as the standard, GG is high-end, GT is mid-range, and TT is low-end; taking rs1015362 as the standard, TT is high-end, TC is mid-range, and CC is low-end.

[0039] Skin characteristics: The above capabilities are low-end, corresponding to the specific skin characteristics: anti-photoaging level 1; the above capabilities are mid-range, corresponding to the specific skin characteristics: anti-photoaging level 2; the above capabilities are high-end, corresponding to the specific skin characteristics: anti-photoaging level 3.

[0040] b) The corresponding relationship between anti-drying ability and skin characteristics is:

[0041] The skin's ability to regulate and delay water loss through the skin is divided into three levels from high to low. Taking rs61816761 as the standard, GG is high-end, GA is mid-range, and AA is low-end. Taking rs11103631 as an example, AA is high-end, AG is mid-range, and GG is low-end.

[0042] Skin characteristics: The above capabilities are low-end, corresponding to specific skin characteristics: anti-dryness level 1, and so on.

[0043] c) The corresponding relationship between anti-wrinkle ability and skin characteristics is:

[0044] The ability of the skin to regulate collagen synthesis in the skin was divided into three levels from high to low. Using rs1800796 as the standard, CC was high, CG was medium, and GG was low. Using rs1799750 as the standard, DD was high, DI was medium, and II was low.

[0045] Skin characteristics: The above capabilities are low-end, corresponding to specific skin characteristics: anti-wrinkle level 1, and so on.

[0046] d) The corresponding relationship between anti-glycation ability and skin characteristics is:

[0047] The ability of the receptor for advanced glycation end products protein in the skin reduction period is divided into three levels from high to low. Taking rs1800625 as the standard, TT is high-end, TC is medium-end, and CC is low-end; taking rs2070600 as the standard, CC is high-end, CT is medium-end, and TT is low-end; taking rs1049346 as the standard, GG is high-end, GA is medium-end, and AA is low-end.

[0048] Skin characteristics: The above capabilities are low-end, corresponding to specific skin characteristics: anti-glycation level 1, and so on.

[0049] e) The corresponding relationship between antioxidant capacity and skin characteristics is:

[0050] The skin's free radical scavenging ability was divided into three levels from high to low. Taking rs1800566 as the standard, GG was high-grade, GA was mid-grade, and AA was low-grade; taking rs713041 as the standard, CC was high-grade, TC was mid-grade, and TT was low-grade.

[0051] Skin characteristics: The above capabilities are low-end, corresponding to specific skin characteristics: antioxidant level 1, and so on.

[0052] f) The corresponding relationship between the ability to resist skin barrier damage and skin characteristics is:

[0053] The skin's ability to regulate / repair the skin barrier is divided into three levels from high to low. Taking rs612529 as the standard, TT is high-end, TC is mid-range, and CC is low-end; taking rs2303067 as the standard, GG is high-end, GA is mid-range, and AA is low-end.

[0054] Skin characteristics: The above capabilities are low-end, corresponding to specific skin characteristics: Level 1 resistance to skin barrier damage, and so on.

[0055] g) The corresponding relationship between oil control ability and skin characteristics is:

[0056] The skin's ability to inhibit oil secretion is divided into three levels from high to low. Taking rs174547 as the standard, TT is high-end, TC is medium-end, and CC is low-end; taking rs743572 as the standard, AA is high-end, AG is medium-end, and GG is low-end.

[0057] Skin characteristics: The above capabilities are low-end, corresponding to specific skin characteristics: oil control level 1, and so on.

[0058] More specifically, the correspondence between SNP sites and genes is as follows:

[0059]

[0060] Further specifically, the formatting method described in step (3) is to convert the pathway signal of the qPCR instrument into a corresponding weight value;

[0061] When the number of dangerous bases is 0, the site weight value is the wild-type weight value. When the number of dangerous bases is 1, the weight value is the heterozygous mutation weight value. When the number of dangerous bases is 2, the weight value is the homozygous mutation weight value, as shown in the following table:

[0062]

[0063] More specifically, the method for constructing the skin characteristic judgment model in step (4) is: using the EDM model to calculate the GRS value of the project, and based on the normal distribution of the GRS value, using the mean and standard deviation to perform grade division.

[0064] More specifically, the calculation formula of the GRS value is: ;

[0065] Where: OR i SNP i The weight of X i is the number of dangerous bases; the project level is calculated using the GRS, mean and sd values ​​according to the formula:

[0066] Level 1: ;

[0067] Level 2: ;

[0068] Level 3: ;

[0069] The calculation formula of threshold coefficient xs is: ;

[0070] Wherein, N is the number of sites included in the project; mean is the arithmetic mean of the GRS values ​​of all samples involved in the calculation; sd is the standard deviation of the GRS values ​​of the samples involved in the calculation.

[0071] In yet another aspect, the present invention provides application of the aforementioned method in determining skin characteristics.

[0072] The technical effects achieved by the present invention are:

[0073] (1) The detection cycle is short and can be completed in about 30 minutes (<40 minutes);

[0074] (2) High detection resolution, single-base resolution gene detection;

[0075] (3) High accuracy, reaching over 98%;

[0076] (4) Simple operation, no central laboratory required, and low requirements for operators. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 The anti-light aging fitting results of Example 1 and Comparative Examples 1-2 are shown.

[0078] Figure 2 The skin barrier fitting results of Example 1 and Comparative Examples 1-2 are shown.

[0079] Figure 3The oil control fitting results of Example 1 and Comparative Examples 1-2 are shown.

[0080] Figure 4 The moisturizing fitting results are shown in Example 1 and Comparative Examples 1-2.

[0081] Figure 5 The antioxidant fitting results of Example 1 and Comparative Examples 1-2 are shown.

[0082] Figure 6 These are the anti-glycation fitting results of Example 1 and Comparative Examples 1-2.

[0083] Figure 7 The anti-wrinkle fitting results of Example 1 and Comparative Examples 1-2 are shown. DETAILED DESCRIPTION

[0084] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0085] Example 1

[0086] 1.1 Preparation of reagents

[0087] The formula of Example 1 lysate a is shown in Table 1.

[0088] Table 1 Example 1 Lysis solution a formula

[0089] Element concentration Sodium dodecyl sulfate (SDS) 1.3%wt Tris(hydroxymethyl)aminomethane 0.1M Silver diethyldithiocarbamate (DTC) 120mM

[0090] The formula of reagent A in Example 1 is shown in Table 2.

[0091] Table 2 Formula of reagent A in Example 1

[0092] Element concentration Taq DNA Polymerase 1.7U Tris-HCl 70 mM KCl 70mM MgSO4 1.5mM dNTP 0.05mM BSA 0.1mg / mL

[0093] The formula of reagent B in Example 1 is shown in Table 3.

[0094] Table 3 Formula of reagent B in Example 1

[0095] Element Mass fraction Chitosan 1 Lysine 4 2,5-dimercapto-1,3,4-thiadiazole 0.12

[0096] The above reagents can be prepared into lyophilized preparations for storage and transportation. The preparation method of the lyophilized preparations is: freeze-drying the components of the reagents separately and then mixing them according to a proportion, or mixing the components of the reagents according to a proportion and dispersing them in a solvent and then freeze-drying them. When used, dilute them with ABS buffer to a corresponding concentration.

[0097] 1.2 Detection Method

[0098] A method for rapid genetic testing to determine skin characteristics. The genetic information detected is shown in Table 4.

[0099] Table 4

[0100]

[0101] The specific steps of the test are as follows:

[0102] (1) Sample lysis: Add the sample (oral swab) into lysis solution a and mix thoroughly to obtain a lysis sample; the lysis solution a is heated to 40-45° C. in a water bath before use; the amount of lysis solution a added is sufficient to cover the sample, and in this embodiment 1, its volume is preferably 3 times the volume of the sample.

[0103] (2) Take reagent A, lysed sample, primers and probes, and reagent B in turn and add them to the qPCR reaction tube, vortex mix for 5-10 seconds, let stand for 5-10 seconds, and then vortex mix for 5-10 seconds to fully mix; the amount of reagent A is 1.8%wt of the mass of the lysed sample; the amount of reagent B is 1.3%wt of the mass of the lysed sample; the sequences of the primers and probes are shown in Table 5.

[0104] Table 5

[0105]

[0106] Table 5

[0107]

[0108] Table 5

[0109]

[0110] (3) Amplification: The obtained qPCR reaction tube is placed in a qPCR device and reacted according to the PCR program; the PCR program is: pre-denaturation at 95°C for 2 min, 1 cycle; 95°C for 10 s, 60°C for 25 s, 10 cycles; 95°C for 10 s, 60°C for 20 s, 28 cycles.

[0111] In the PCR process, the heating rate is 6-9°C / s and the cooling rate is 6-8°C / s after the program starts. The qPCR collects fluorescence signals at 60°C, and the collected fluorescence amplification signals include FAM, VIC and CY5, and the internal reference is ROX.

[0112] (4) Collecting test result data on a qPCR instrument, obtaining the relationship between specific skin characteristics of the sample and related test genes, and establishing a database; the relationship between the specific skin characteristics and related test genes is as follows:

[0113] a) Antioxidation: The skin's free radical scavenging ability was divided into three levels from high to low. Taking rs1800566 as the standard, GG was high-grade, GA was mid-grade, and AA was low-grade; taking rs713041 as the standard, CC was high-grade, TC was mid-grade, and TT was low-grade.

[0114] Skin characteristics: The above capabilities are low-level, corresponding to the specific skin characteristics: anti-oxidation level 1; the above capabilities are mid-level, corresponding to the specific skin characteristics: anti-oxidation level 2; the above capabilities are high-level, corresponding to the specific skin characteristics: anti-oxidation level 3.

[0115] b) Anti-glycation: The ability of the receptor protein for advanced glycation end products in the skin reduction period is divided into three levels from high to low. Taking rs1800625 as the standard, TT is high-end, TC is medium-end, and CC is low-end; taking rs2070600 as the standard, CC is high-end, CT is medium-end, and TT is low-end; taking rs1049346 as the standard, GG is high-end, GA is medium-end, and AA is low-end.

[0116] Skin characteristics: The above capabilities are low-end, corresponding to specific skin characteristics: anti-glycation level 1, and so on.

[0117] c) Anti-wrinkle: The skin's ability to regulate collagen synthesis in the skin is divided into three levels from high to low. Taking rs1800796 as the standard, CC is high-end, CG is mid-range, and GG is low-end. Taking rs1799750 as the standard, DD is high-end, DI is mid-range, and II is low-end.

[0118] Skin characteristics: The above capabilities are low-end, corresponding to specific skin characteristics: anti-wrinkle level 1, and so on.

[0119] d) Anti-photoaging: (1) The skin's ability to resist ultraviolet rays is divided into three levels from high to low. Based on rs1800414, CC is high-grade, CT is medium-grade, and TT is low-grade. Based on rs62543565, TT is high-grade, TC is medium-grade, and CC is low-grade. Based on rs10733310, TT is high-grade, TG is medium-grade, and GG is low-grade. Based on rs4911414, GG is high-grade, GT is medium-grade, and TT is low-grade. Based on rs1015362, TT is high-grade, TC is medium-grade, and CC is low-grade.

[0120] Skin characteristics: The above capabilities are low-end, corresponding to specific skin characteristics: anti-photoaging level 1, and so on.

[0121] e) Skin barrier: The skin's ability to regulate / repair the skin barrier is divided into three levels from high to low. Taking rs612529 as the standard, TT is high-end, TC is mid-range, and CC is low-end; taking rs2303067 as the standard, GG is high-end, GA is mid-range, and AA is low-end.

[0122] Skin characteristics: The above capabilities are low-end, corresponding to specific skin characteristics: Level 1 resistance to skin barrier damage, and so on.

[0123] f) Oil control: (7) The skin's ability to inhibit oil secretion is divided into three levels from high to low. Based on rs174547, TT is high-end, TC is medium-end, and CC is low-end; based on rs743572, AA is high-end, AG is medium-end, and GG is low-end.

[0124] Skin characteristics: The above capabilities are low-end, corresponding to specific skin characteristics: oil control level 1, and so on.

[0125] g) Anti-drying: The skin's ability to regulate and delay water loss through the skin is divided into three levels from high to low. Taking rs61816761 as the standard, GG is high-end, GA is mid-range, and AA is low-end. Taking rs11103631 as the standard, AA is high-end, AG is mid-range, and GG is low-end.

[0126] Skin characteristics: The above capabilities are low-end, corresponding to specific skin characteristics: anti-dryness level 1, and so on.

[0127] (5) Formatting the detection data as needed, wherein the gene data is formatted to convert the pathway signal of the qPCR instrument into a corresponding weight value.

[0128] The qPCR instrument has four channel signals, namely ROX, CY5, FAM, and VIC. Each site corresponds to two of the channels. After the instrument analysis is completed, each channel will output a Boolean value to indicate negative or positive. Negative means not detected, and positive means detected.

[0129] Taking the site rs1800566 as an example, the genotype of this site corresponds to the test results of channels FAM and VIC. When FAM is positive and VIC is positive, the genotype is GA. When FAM is positive and VIC is negative, the genotype is GG. When FAM is negative and VIC is positive, the genotype is AA.

[0130] After obtaining the site genotype, referring to Table 6, when the number of dangerous bases is 0, the site weight value is the wild type weight value, when the number of dangerous bases is 1, the weight value is the heterozygous mutation weight value, and when the number of dangerous bases is 2, the weight value is the homozygous mutation weight value.

[0131] Table 6

[0132]

[0133] (6) Based on the formatted sample genetic data, a machine learning model is selected to construct a skin characteristic judgment model based on the sample genetic data to judge specific skin characteristics; specifically, the GRS value of the project is calculated using the EDM model, and based on the normal distribution of the GRS value, the mean and standard deviation are used for grading; the calculation formula of the GRS value is: (where OR i SNP i The weight of (see Table 7); X i is the number of dangerous bases. ); the project level is calculated according to the formula using GRS, mean and sd values, where the mean is the arithmetic mean of the GRS values ​​of all samples involved in the calculation; the sd is the standard deviation of the GRS values ​​of the samples involved in the calculation.

[0134] Level 1:

[0135] Level 2:

[0136] Level 3:

[0137] The calculation formula of the threshold coefficient xs is: (N is the number of sites included in the project).

[0138] Table 7

[0139]

[0140] The above gene detection method and the Moji MAX skin detector were used to test the oral swab samples of 75 volunteers. The 75 people were divided into three groups, 15 in group one with natural skin condition, 30 in group two with bad living habits, and 30 in group three with careful skin care.

[0141] Example 2

[0142] 2.1 Preparation of reagents

[0143] The formula of Example 2 lysate a is shown in Table 8.

[0144] Table 8 Example 2 Lysis solution a formula

[0145] Element concentration SDS 2.1%wt Tris(hydroxymethyl)aminomethane 0.2M DTC 170mM

[0146] The formula of reagent A of Example 2 is shown in Table 9.

[0147] Table 9 Example 2 Reagent A formula

[0148] Element concentration Taq DNA Polymerase 2.3U Tris-HCl 80 mM KCl 90mM MgSO4 2.2mM dNTP 0.4mM BSA 2mg / mL

[0149] The formula of reagent B in Example 2 is shown in Table 10.

[0150] Table 10: Formula of reagent B in Example 2

[0151] Element Mass fraction Chitosan 2 Lysine 3 2,5-dimercapto-1,3,4-thiadiazole 0.17

[0152] 2.2 Detection methods

[0153] A method for rapid gene detection to determine skin characteristics, the difference between this method and Example 1 is step (2), and the rest is the same as Example 1.

[0154] (2) Take reagent A, lysed sample, primers and probes, and reagent B in sequence and add them to the qPCR reaction tube, vortex mix for 5-10 seconds, let stand for 5-10 seconds, and vortex mix again for 5-10 seconds to fully mix; the amount of reagent A used is 2.5%wt of the mass of the lysed sample; the amount of reagent B used is 1.7%wt of the mass of the lysed sample.

[0155] Comparative Example

[0156] A comparative example was set up according to Example 1, and the differences between the comparative example and Example 1 are shown in Table 11.

[0157] Table 11

[0158] Comparative Example Differences from Example 1 Comparative Example 1 The number of gene detection sites in Comparative Example 1 is 25, and the number of increased detection sites is shown in Table 12. Comparative Example 2 The number of gene detection sites was reduced. The detection sites of comparative example 2 were 12, and the detection sites were rs62543565, rs10733310, rs1015362, rs4911414, rs612529, rs174547, rs11103631, rs713041, rs1800625, rs2070600 and rs1800796. Comparative Example 3 The chitosan in the reagent B in Example 1 was replaced with an equal amount of 2,5-dimercapto-1,34-thiadiazole Comparative Example 4 The 2,5-dimercapto-1,3,4-thiadiazole in the reagent B in Example 1 was replaced with an equal amount of chitosan Comparative Example 5 The reagent B in Example 1 was modified without adding chitosan and 2,5-dimercapto-1,34-thiadiazole.

[0159] Table 12 Comparative Example 1 Increased detection sites and primer and probe sequences

[0160]

[0161] Table 13 Site weights increased in comparative example 1

[0162]

[0163] Table 14 Correspondence between the gear position and genotype of the increased sites in comparative example 1

[0164]

[0165] Performance data:

[0166] 1. Test results of Example 1

[0167] The gene detection results of Example 1 are shown in Table 15 (level 1-3, the larger the number, the stronger the ability):

[0168] Table 15

[0169]

[0170] Table 15

[0171]

[0172] Table 15

[0173]

[0174] The test results of the skin detector are shown in Figure 16 (0-100 points, the higher the score, the stronger the level):

[0175] Table 16

[0176]

[0177] Table 16

[0178]

[0179] Table 16

[0180]

[0181] The condition level is calculated based on the current status test (skin tester), with less than 50 points as level 1, 50 to 80 points as level 2, and more than 80 points as level 3. The results are shown in Table 17.

[0182] Table 17

[0183]

[0184] Table 17

[0185]

[0186] Table 17

[0187]

[0188] The seven-dimensional genetic and current status test results of each volunteer tested were compared. The comparison results are shown in Table 18, which shows the consistency rate of the seven-dimensional results of the current status test and genetic test of 75 samples.

[0189] Table 18

[0190]

[0191] 2. Test results of Example 2

[0192] The consistency between the 75 samples tested using the detection method of Example 2 and the current test results is shown in Table 19.

[0193] Table 19

[0194]

[0195] 3. Test results of comparative examples 1-5

[0196] The consistency between the 75 samples tested using the detection methods of Comparative Examples 1-5 and the current test results is shown in Table 20.

[0197] Table 20

[0198]

[0199] The normal distribution diagram of the experimental data of Comparative Example 1 and Comparative Example 2 is as follows: Figure 1-Figure 7 As shown, frequency (the frequency of the risk value appearing in the sample); risk value is (odds ratio, which is a ratio that measures the likelihood of an event occurring in two different groups. The odds ratio is a commonly used effect size measure, and the larger the value, the greater the probability of the event occurring in the group). The higher the curve, the denser the probability of this interval. The steeper the normal curve and the smaller the standard deviation, the shorter the distance between most variable values ​​and the mean, so most values ​​are closely clustered around the mean. Judging criteria: The peak is close to the center, and the curve is high and steep, which is the best.

[0200] Figure 1 (Anti-light aging) The results show that, compared with Example 1, the distribution of Example 1 is basically unchanged, while the peak value of the curve of Example 2 becomes lower. Conclusion: Example 1 is the best. Figure 2 (Skin Barrier) The results show that compared with Example 1, the curve of Comparative Example 1 deviates from the center; for Comparative Example 2, the peak value of the curve becomes lower. Conclusion: Example 1 is the best. Figure 3 (Oil control) The results show that compared with Example 1, Example 1 has uneven distribution and the curve deviates from the center; while the peak value of the curve of Example 2 becomes lower. Conclusion: Example 1 is the best. Figure 4 (Moisturizing) The results show that compared with Example 1, the curve of Example 1 deviates from the center, and the peak of the curve of Example 2 becomes lower. Conclusion: Example 1 is the best. Figure 5 (Antioxidation) The results show that compared with Example 1, the curve of Example 1 deviates from the center, and the peak of the curve of Example 2 becomes lower. Conclusion: Example 1 is the best. Figure 6 (Anti-glycation) The results show that compared with Example 1: For Example 1, the curve deviates from the center; for Example 2, the curve deviates from the center. Conclusion: Example 1 is the best. Figure 7 (Collagen) The results show that compared with Example 1: For Example 1, the curve deviates from the center; For Example 2, the peak value of the curve becomes lower. Conclusion: Example 1 is the best.

[0201] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining skin characteristics by genetic testing for non-disease diagnosis purposes, characterized in that: The method comprises simultaneously detecting 18 SNP sites of 15 genes; the 18 SNP sites of the 15 genes are: The method comprises the following steps: (1) Lyse the sample using a lysis buffer; (2) performing qPCR detection on the sample after lysis in step (1), obtaining the correspondence between the sample and the skin characteristics, and establishing a database; (3) Formatting the database obtained in step (2); (4) Inputting the database formatted in step (3) into the skin characteristic judgment model to obtain the skin characteristic conclusion; The formatting method described in step (3) is to convert the pathway signal of the qPCR instrument into a corresponding weight value; When the number of dangerous bases is 0, the site weight value is the wild-type weight value. When the number of dangerous bases is 1, the weight value is the heterozygous mutation weight value. When the number of dangerous bases is 2, the weight value is the homozygous mutation weight value, as shown in the following table: The method for constructing the skin characteristic judgment model in step (4) is: using the EDM model to calculate the GRS value of the project, and based on the normal distribution of the GRS value, using the mean and standard deviation to perform grade division; The calculation formula of the GRS value is: ; Where: OR i SNP i The weight of X i is the number of dangerous bases; the project level is calculated using the GRS, mean and sd values ​​according to the formula: Level 1: ; Level 2: ; Level 3: ; The calculation formula of threshold coefficient xs is: ; Where N is the number of sites included in the project.

2. The method according to claim 1, characterized in that The lysis solution in step (1) is lysis solution a; the lysis solution a comprises sodium dodecyl sulfate, tris(hydroxymethyl)aminomethane and silver diethyldithiocarbamate.

3. The method according to claim 2, characterized in that The concentrations of the sodium dodecyl sulfate, tris(hydroxymethyl)aminomethane and silver diethyldithiocarbamate are respectively: 1.3%-2.1%wt, 0.1-0.2M, 120-170mM.

4. The method according to claim 2, characterized in that: The volume of the lysate a is 3-5 times the volume of the sample.

5. The method according to claim 1, characterized in that The sample in step (1) is an oral swab, saliva or biological tissue.

6. The method according to claim 1, characterized in that The qPCR reaction system in step (2) includes reagent A, primers and probes and reagent B; the reagent A includes Taq DNA polymerase, Tris-HCl, KCl, MgSO4, dNTP and BSA; the reagent B includes chitosan, lysine and 2,5-dimercapto-134-thiadiazole.

7. The method according to claim 6, characterized in that The concentrations of Taq DNA polymerase, Tris-HCl, KCl, MgSO4, dNTP and BSA are 1.7-2.3U, 70-80 mM, 70-90 mM, 1.5-2.2 mM, 0.05-0.4 mM, and 0.1-2 mg / mL, respectively.

8. The method according to claim 6 or 7, characterized in that: The dosage of the reagent A is 1.8%-2.5%wt of the lysed sample.

9. The method according to claim 6, characterized in that The mass ratio of chitosan, lysine and 2,5-dimercapto-1,34-thiadiazole is 1-2:3-4:0.12-0.

17.

10. The method according to claim 6 or 9, characterized in that: The dosage of the reagent B is 1.3%-1.7%wt of the lysed sample.

11. The method according to claim 6, characterized in that The primer and probe sequences are shown in SEQ ID NO. 1-SEQ ID NO.

72.

12. Use of the method according to any one of claims 1 to 11 in determining skin characteristics, characterized in that: The applications described are for non-disease diagnosis and treatment purposes.

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