Multi-enzyme nucleic acid cosmetics testing method

Abstract

The present invention discloses a multi-enzyme nucleic acid cosmetics inspection method, which relates to the technical field of cosmetics inspection, and includes the following specific steps: placing a liquid or solid cosmetic sample in a centrifuge tube, extracting DNA, using a Nanodrop nucleic acid detector to detect the concentration and purity of the extracted DNA, comparing the existing rDNA gene sequence in the database with the obtained DNA, selecting multiple groups of DNA with similar homology to perform a touchdown PCR program to complete amplification, purifying and recovering the PCR product, and identifying and detecting the suspicious DNA genome in the multi-enzyme nucleic acid cosmetics; selecting cells in the logarithmic growth phase, 0.3% trypsin digestion to prepare a single cell suspension, and using an optical microscope to count the cell concentration. The present invention extracts DNA from multi-enzyme nucleic acid cosmetics, completes the acquisition of enzyme nucleic acids and microbial DNA genomes in cosmetics, sets comparison and amplification to obtain suspicious DNA genomes, completes targeted identification, and improves inspection efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of cosmetics testing, in particular to a multi-enzyme nucleic acid cosmetics testing method. Background Art

[0002] Since nucleotides can promote protein synthesis, nucleotide products are also widely used in the cosmetics industry. They can be added to detergents, emulsifiers, vanishing creams, lotions, and theatrical cosmetics. They can promote skin metabolism, prevent wrinkles, promote muscle growth and moisturize, control sebum secretion, prevent UV absorption, and make the skin soft. They have strong penetration power for various skin diseases such as freckles, buckwheat skin, acne, and athlete's foot, and have significant therapeutic effects. Therefore, the application of multi-enzyme nucleic acid cosmetics in the future market will be seen increasing.

[0003] In recent years, there have indeed been many reports of skin damage caused by applying cosmetics containing toxic substances. Therefore, it is necessary to strengthen the skin toxicity test of cosmetics;

[0004] For example, a cosmetic toxicity test method disclosed in Chinese patent publication number CN113249427A, in which a plurality of ultraviolet lamps are provided in the detection mechanism used in the detection process, and the ultraviolet lamps are arranged and installed in a uniform distribution, ensuring that the ultraviolet lamps in the cover body can irradiate all the detection cups on the test base plate, the test base plate is connected to the detection mechanism in a pull-out manner by being clamped in a slide slot, which is convenient for operators to use and to place or remove the detection cups, a plurality of test slots are opened on the detection board, and are evenly distributed, which is convenient for testing multiple cosmetics at the same time and improving the test efficiency, the upper and lower ends of the test slots are arranged as magnetic pads, and a plurality of small magnets are placed in the storage slot, which can fix the PH test paper placed in the storage slot to prevent it from falling.

[0005] First of all, the above test method directly uses pH test paper to complete the acidity and alkalinity test. Secondly, light is directed to the epidermis on the cosmetics. The pH test results confirm the corrosiveness, and the epidermis test results confirm whether the cosmetics are toxic. However, there are microorganisms and multi-enzyme nucleic acids in cosmetics. For example, if Escherichia coli is present, it may cause skin infection when used, especially when the skin is cut, abraded or otherwise damaged. Bacteria can more easily invade the skin, leading to urinary tract infections such as urethritis and cystitis, which cannot be detected by pH and light tests. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides a multi-enzyme nucleic acid cosmetics testing method, which solves the problems raised in the above-mentioned background technology.

[0007] To achieve the above objectives, the present invention is implemented by the following technical scheme: a multi-enzyme nucleic acid cosmetics testing method, comprising the following specific steps:

[0008] Step 1: Preliminary extraction of DNA from multi-enzyme nucleic acid cosmetics; 300 g of liquid or solid cosmetic sample is placed in a centrifuge tube, Chelex-100 and proteinase K are added, and the mixture is kept at 60°C for 30 min, and then oscillated at high speed for 10-15 s based on an oscillating device, and then placed in a water bath at 100°C for 12 min, and then oscillated at high speed again for 10-15 s, and finally subjected to preliminary centrifugation. The obtained supernatant is transferred to a centrifuge tube, and then cell lysis solution and gDNA-Binding-Buffer are added, mixed thoroughly, and then placed in a water bath at 65°C for 15 min, and then subjected to secondary centrifugation. The residual liquid is sucked away with a hydraulic gun, and after drying at room temperature for 20 min, the supernatant is transferred to a new centrifuge tube, and the concentration and purity of the extracted DNA are detected using a Nanodrop nucleic acid detector, and then stored at -22°C for later use;

[0009] In step 1, the purpose of multiple centrifugation and oscillation is to completely crush and dissolve the cosmetics. The currently commonly used nucleic acid lysis solution with a pH of 8.0-8.5, which is 1-3 times the volume of the cosmetic sample, is not completely dissolved automatically, and in the actual extraction process, there are still cases where the sample sticks to the wall or precipitates unevenly.

[0010] Step 2: Comparison and amplification; compare the obtained DNA with the existing rDNA gene sequence in the database, select multiple groups of DNA with similar homology for touchdown PCR program to complete amplification, purify and recover the PCR products, and sequence them through ABI3730-XLDNA sequencer to identify the suspicious DNA genome in the multi-enzyme nucleic acid cosmetics.

[0011] Step 3: Detection: Select cells in logarithmic growth phase, digest them with 0.3% trypsin to make a single-cell suspension, adjust the cell concentration to 12 / ml and inoculate them into each well of an 8-well plate, culture them in an incubator at 36°C and 2% CO, change the medium every 2-3 days, take 3 wells every day, digest them with 0.25% trypsin to make a single-cell suspension, transfer the supernatant obtained in step 1 to the single-cell suspension, and count the cell concentration using a light microscope.

[0012] A further specific method is to place 300 g of liquid or solid cosmetic sample in a centrifuge tube, add Chelex-100 and enzyme K, keep warm at 60°C for 30 minutes, and oscillate at high speed for 10-15 seconds based on an oscillating device, then water bath at 100°C for 12 minutes, oscillate at high speed again for 12 seconds, and finally perform preliminary centrifugation. After the obtained supernatant is transferred to a centrifuge tube, cell lysate and gDNA-Binding-Buffer are added, the mixture is fully mixed, and water bathed at 65°C for 15 minutes, and then centrifuged again. The residual liquid is sucked away with a hydraulic gun. After drying at room temperature for 20 minutes, the supernatant was transferred to a new centrifuge tube. The initial centrifugation time in step 1 was set to 5 minutes and the speed was 10000 r / min. The secondary centrifugation time in step 1 was set to 1 minute and the speed was 4000 r / min. The desired DNA genome was obtained. At this time, there was a suspicious DNA genome. The cosmetic sample was applied to 155 groups of substitute skins, and the substitute skins were immediately rinsed with phosphate buffer. After absorbing the excess phosphate buffer, the substitute skins were immersed in 220uL, 3mg / ml MTT solution. The environment was set to, The light-proof condition is 20-28℃, wash off the MTT solution on the substitute skin, rinse repeatedly with phosphate buffer, add 2mL of acidified isopropanol, shake for 3h, and keep overnight in the dark. Pipette 200uL of liquid and add it to a 96-well plate, with 4 replicate wells for each sample. The OD value is read at 550nm by an automatic microplate reader. The acidified isopropanol solution is used as a blank control. The method for calculating the relative cell viability is: relative cell viability percentage - (average OD value of a chemical / average OD value of the negative control) x 100%, among which the relative cell viability of the exposure group <15% is judged to be corrosive, and the relative cell Viability ≥15% is judged as non-corrosive, relative cell viability after exposure <50% is judged as irritating, relative cell viability after exposure ≥50% is judged as non-irritating, and in the experiment of this embodiment, after the cosmetic sample and the test cells are inoculated, the test cells adhere to the wall and stretch, and after 2 days, the test cells are spindle-shaped, and protrude with the extension of culture time. After 24 hours of inoculation, the test cells can be seen to adhere to the wall and stretch, and finally the test cells are dense and short round in shape, and obvious halos can be seen around the test cells. It is judged that the test cells are in good condition, non-toxic and non-irritating, and the test cells are selected as epidermal cells.

[0013] A further improvement of the technical solution of the present invention is that if the result of step 2 shows that there is a suspicious DNA genome, the following specific inspection steps are also included:

[0014] Ⅰ: First, take 10 ml of the prepared solution of the cosmetic sample to be tested prepared in step 1 and add it to the dilution bottle of 90 ml SCDLP enrichment solution to make a 1:100 sample preparation solution;

[0015] Ⅱ: Take a sterile dish after drying and cleaning, add 20-30ml of distilled water, then add lecithin and heat to dissolve, add Tween 80 and protein to the distilled water, shake evenly until the added substances are dissolved, pour into a sterile dish, control the pH value at 7.0-7.6, add agar to the sterile dish, autoclave at 100Kpa, 100℃ for 10min, prepare the culture medium, add the sample preparation solution respectively, and select the suspicious bacteria and colonies after the culture is completed;

[0016] III: Use crystal violet dye to initially stain the suspected bacterial flora and colonies, add iodine solution to form a complex with crystal violet, treat with ethanol or acetone, and use safranin for re-staining. If there is a staining result, the suspicious DNA sequence in step 2 is identified by 2% agarose gel electrophoresis to confirm whether any one or more of Pseudomonas aeruginosa, Staphylococcus aureus and fecal coliforms are present.

[0017] The further improvement of the technical solution of the present invention is that: the amount of Chelex-100 added is 300uL, and polystyrene / divinylbenzene=5:95, the amount of proteinase K added is 30mg / mL, the amount of cell lysate added is 6mol / L, and the amount of gDNA-Binding-Buffer added is 600uL.

[0018] A further improvement of the technical solution of the present invention is that the detection in step 3 further includes:

[0019] i: Apply the cosmetic sample with suspicious DNA genome obtained in step 2 to the substitute skin for 1 hour, and then immediately rinse the substitute skin with phosphate buffer;

[0020] ii: After removing the excess phosphate buffer, immerse the substitute skin in 220uL, 3mg / ml MTT solution, and set the environment to 20-28℃ in the dark;

[0021] ⅲ: Wash off the MTT solution on the substitute skin, rinse repeatedly with phosphate buffer, add 2 mL of acidified isopropanol, shake for 3 hours, and keep overnight in the dark;

[0022] iv: Pipette 200uL of liquid into a 96-well plate, with 4 replicate wells for each sample. Read the OD value at 550nm using an automatic microplate reader. Use acidified isopropanol solution as a blank control to complete the corrosion test.

[0023] A further improvement of the technical solution of the present invention is that the production of the substitute skin includes the following specific steps:

[0024] Acidified rat tail collagen was mixed with DMEM culture medium, and the pH value was adjusted. Adult fibroblast suspension in good growth state was placed in it, and finally placed in a 24-well plate, dried at 37°C for 30 minutes, and then DMEM culture medium was added for continuous culture for 4 days. When epidermal keratinocytes were formed, the culture medium was replaced with K-SFM and cultured for 2 days. After air-liquid surface culture was performed for 6 days, the keratinized surface cells were obtained to obtain a substitute skin for detection.

[0025] A further improvement of the technical solution of the present invention is that the solution added to adjust the pH value is NaOH, the NaOH is 1 mol / L, and the pH value is controlled to be 7.4.

[0026] A further improvement of the technical solution of the present invention is that the initial centrifugation time in step 1 is set to 5 minutes and the rotation speed is 10000 r / min.

[0027] A further improvement of the technical solution of the present invention is that the secondary centrifugation time in step 1 is set to 1 min and the rotation speed is 4000 r / min.

[0028] A further improvement of the technical solution of the present invention is that in order to ensure that the purity and concentration can meet the requirements of subsequent PCR detection, the DNA genome OD260 / 0D280 in step 2 is between 1.2-2.1, and the DNA concentration is controlled at 13.2-31.8ng / uL.

[0029] The present invention provides a multi-enzyme nucleic acid cosmetics testing method. Compared with the prior art, the method has the following beneficial effects: the method extracts the DNA of the multi-enzyme nucleic acid cosmetics to obtain the enzyme nucleic acid and microbial DNA genome in the cosmetics, and the purpose of multiple centrifugation and oscillation during the extraction process is to completely crush and dissolve the cosmetics, which solves the problem that the commonly used pH 8.0-8.5 nucleic acid lysis solution does not completely dissolve automatically and that the sample still hangs on the wall or precipitates unevenly during the actual extraction process, and sets comparison and amplification to obtain the suspected DNA genome, completes targeted identification, and improves the inspection efficiency;

[0030] In addition, by manufacturing skin substitutes that are similar to skin, the actual effect test of cosmetic samples can be completed. Based on the vitality of epidermal cells and the total number and growth rate of tissue cells, the corrosiveness and irritation of cosmetic samples and whether they hinder normal cell growth can be accurately judged, which greatly improves the effect and accuracy of multi-enzyme nucleic acid cosmetic testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is an overall schematic diagram of the multi-enzyme nucleic acid cosmetics testing method;

[0032] Figure 2This is a schematic diagram of the testing steps when a suspicious DNA genome is present in the multi-enzyme nucleic acid cosmetics testing method;

[0033] Figure 3 This is a schematic diagram of some of the testing steps in the multi-enzyme nucleic acid cosmetics testing method;

[0034] Figure 4 This is a table showing normal data of the toxic cosmetics hindering the activity of epidermal cells in Example 1;

[0035] Figure 5 The figure is a flow chart of the multi-enzyme nucleic acid cosmetics testing method. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Reference Figure 1 - Figure 5 The multi-enzyme nucleic acid cosmetics testing method of the present invention comprises the following specific steps:

[0038] Step 1: Preliminary extraction of DNA from multi-enzyme nucleic acid cosmetics; 300 g of liquid or solid cosmetic sample is placed in a centrifuge tube, Chelex-100 and proteinase K are added, and the mixture is kept at 60°C for 30 min, and then oscillated at high speed for 10-15 s based on an oscillating device, and then placed in a water bath at 100°C for 12 min, and then oscillated at high speed again for 10-15 s, and finally subjected to preliminary centrifugation. The obtained supernatant is transferred to a centrifuge tube, and then cell lysis solution and gDNA-Binding-Buffer are added, mixed thoroughly, and then placed in a water bath at 65°C for 15 min, and then subjected to secondary centrifugation. The residual liquid is sucked away with a hydraulic gun, and after drying at room temperature for 20 min, the supernatant is transferred to a new centrifuge tube, and the concentration and purity of the extracted DNA are detected using a Nanodrop nucleic acid detector, and then stored at -22°C for later use;

[0039] The amount of Chelex-100 added was 300uL, and polystyrene / divinylbenzene=5:95, the amount of proteinase K added was 30mg / mL, the amount of cell lysate added was 6mol / L, and the amount of gDNA-Binding-Buffer added was 600uL.

[0040] The initial centrifugation time in step 1 is set to 5 min and the speed is 10000 r / min. The secondary centrifugation time in step 1 is set to 1 min and the speed is 4000 r / min. In step 1, the purpose of multiple centrifugation and oscillation is to completely crush and dissolve the cosmetics. The currently commonly used nucleic acid lysis solution with a pH of 8.0-8.5, which is 1-3 times the volume of the cosmetic sample, is not completely dissolved automatically, and there is still a situation in which the sample hangs on the wall or precipitates unevenly during the actual extraction process.

[0041] Step 2: Comparison and amplification; compare the obtained DNA with the existing rDNA gene sequence in the database, select multiple groups of DNA with similar homology for touchdown PCR program to complete amplification, purify and recover the PCR products, and sequence them through ABI3730-XLDNA sequencer to identify the suspicious DNA genome in the multi-enzyme nucleic acid cosmetics.

[0042] To ensure that the purity and concentration can meet the requirements of subsequent PCR detection, the DNA genome OD260 / OD280 in step 2 is between 1.2-2.1, and the DNA concentration is controlled at 13.2-31.8 ng / uL.

[0043] If the result of step 2 shows a suspicious DNA genome, the following specific testing steps are also included:

[0044] Ⅰ: First, take 10 ml of the prepared solution of the cosmetic sample to be tested prepared in step 1 and add it to the dilution bottle of 90 ml SCDLP enrichment solution to make a 1:100 sample preparation solution;

[0045] Ⅱ: Take a sterile dish after drying and cleaning, add 20-30ml of distilled water, then add lecithin and heat to dissolve, add Tween 80 and protein to the distilled water, shake evenly until the added substances are dissolved, pour into a sterile dish, control the pH value at 7.0-7.6, add agar to the sterile dish, autoclave at 100Kpa, 100℃ for 10min, prepare the culture medium, add the sample preparation solution respectively, and select the suspicious bacteria and colonies after the culture is completed;

[0046] III: Use crystal violet dye to initially stain the suspected bacterial flora and colonies, add iodine solution to form a complex with crystal violet, treat with ethanol or acetone, and use safranin for re-staining. If there is a staining result, the suspicious DNA sequence in step 2 is identified by 2% agarose gel electrophoresis to confirm whether any one or more of Pseudomonas aeruginosa, Staphylococcus aureus and fecal coliforms are present.

[0047] Step 3: Detection: Select cells in logarithmic growth phase, digest them with 0.3% trypsin to make a single-cell suspension, adjust the cell concentration to 12 / ml and inoculate them into each well of an 8-well plate, culture them in an incubator at 36°C and 2% CO, change the medium every 2-3 days, take 3 wells every day, digest them with 0.25% trypsin to make a single-cell suspension, transfer the supernatant obtained in step 1 to the single-cell suspension, and count the cell concentration using a light microscope.

[0048] The detection in step 3 further comprises:

[0049] i: Apply the cosmetic sample with suspicious DNA genome obtained in step 2 to the substitute skin for 1 hour, and then immediately rinse the substitute skin with phosphate buffer;

[0050] ii: After removing the excess phosphate buffer, immerse the substitute skin in 220uL, 3mg / ml MTT solution, and set the environment to 20-28℃ in the dark;

[0051] ⅲ: Wash off the MTT solution on the substitute skin, rinse repeatedly with phosphate buffer, add 2 mL of acidified isopropanol, shake for 3 hours, and keep overnight in the dark;

[0052] iv: Pipette 200uL of liquid into a 96-well plate, with 4 replicate wells for each sample. Read the OD value at 550nm using an automatic microplate reader. Use acidified isopropanol solution as a blank control to complete the corrosion test.

[0053] The production of alternative skin includes the following specific steps:

[0054] After acidified rat tail collagen is mixed with DMEM culture medium, the pH value is adjusted, and the adult fibroblast suspension in good growth state is placed in it, and finally placed in a 24-well plate, dried at 37°C for 30 minutes, and then DMEM culture medium is added and cultured continuously for 4 days. When epidermal keratinocytes are formed, the culture medium is replaced with K-SFM and cultured for 2 days. After air-liquid surface culture is performed for 6 days, the keratinized surface cells are obtained to obtain a substitute skin for detection.

[0055] In Example 1, 300 g of a liquid or solid cosmetic sample was placed in a centrifuge tube, Chelex-100 and white enzyme K were added, and the mixture was kept at 60° C. for 30 min, and then oscillated at high speed for 10-15 s based on an oscillating device, and then placed in a water bath at 100° C. for 12 min, and then oscillated at high speed for 12 s again, and finally subjected to preliminary centrifugation. The obtained supernatant was transferred to a centrifuge tube, and then cell lysate and gDNA-Binding-Buffer were added, mixed thoroughly, and then placed in a water bath at 65° C. for 15 min, and then subjected to secondary centrifugation. The residual liquid was sucked away using a hydraulic gun, and then dried at room temperature for 20 min later, the supernatant was transferred to a new centrifuge tube, the initial centrifugation time in step 1 was set to 5 min, the speed was 10000 r / min, the secondary centrifugation time in step 1 was set to 1 min, the speed was 4000 r / min, and the desired DNA genome was obtained. At this time, there was a suspicious DNA genome. The cosmetic sample was applied to the substitute skin, and the substitute skin was immediately rinsed with phosphate buffer. After absorbing the excess phosphate buffer, the substitute skin was immersed in 220uL, 3mg / ml MTT solution. The environment was set to, and the light-proof condition was 20 -28℃, wash off the MTT solution on the substitute skin, rinse repeatedly with phosphate buffer, add 2mL of acidified isopropanol, shake for 3h, keep overnight in the dark, aspirate 200uL of liquid and add it to a 96-well plate, 4 replicate wells for each sample, read the OD value at 550nm with an automatic microplate reader, and use the acidified isopropanol solution as a blank control. The method for calculating the relative cell viability is: relative cell viability percentage - (average OD value of a chemical / average OD value of the negative control) x 100%, where the relative cell viability of the exposure group <15% is judged to be corrosive, and the relative cell viability ≥15 % is judged as non-corrosive, relative cell viability after exposure <50% is judged as irritating, and relative cell viability after exposure ≥50% is judged as non-irritating. In the experiment of this embodiment, after the cosmetic sample and the test cells are inoculated, the test cells adhere to the wall and stretch. After 2 days, the test cells are spindle-shaped, and protrusions are seen as the culture time increases. After 24 hours of inoculation, the test cells can be seen to adhere to the wall and stretch. Finally, the test cells are dense and short round in shape. Obvious halos can be seen around the test cells. It is judged that the test cells are in good condition and are not toxic and irritating. The test cells are selected as epidermal cells, such as attached Figure 4 Shown are toxic cosmetics that hinder the activity of epidermal cells.

[0056] Example 2. With respect to the above test results, a further approach is to exclude the toxicity of cosmetics and avoid adverse reactions between the microbial composition of cosmetics and the human body. Therefore, the DNA extracts preserved for later use are compared with the obtained DNA according to the existing rDNA gene sequences in the database, and multiple groups of DNA with similar homology are selected for touchdown PCR program to complete amplification. The PCR products are purified and recovered, and sequenced by ABI3730-XLDNA sequencer to identify the suspicious DNA genome in the multi-enzyme nucleic acid cosmetics.

[0057] During the identification process, take the cosmetic sample for primary staining, add iodine solution and crystal violet to form a complex, treat with ethanol or acetone, and use safranin for counterstaining. If there is a staining result, the suspicious DNA sequence in step 2 is identified by 2% agarose gel electrophoresis to confirm whether there is any one or more of Pseudomonas aeruginosa, Staphylococcus aureus and fecal coliform bacteria;

[0058] The method for culturing and testing the total number of sample colonies and bacterial flora required for primary staining specifically includes taking a sterile plate after drying and cleaning, taking out 20-30 ml of distilled water from a glass container and adding it, then adding lecithin and heating it to dissolve, adding Tween 80 and protein to the distilled water in the remaining glass container, shaking evenly until the added substances are dissolved, pouring into a sterile plate, controlling the pH value at 7.0-7.6, adding agar to the sterile plate, and sterilizing it under high pressure at 100Kpa and 100°C for 10 minutes, after preparing the culture medium, adding the sample test solution respectively, and selecting the suspicious bacterial flora and colonies after the culture is completed;

[0059] First observe with the naked eye, count the number of colonies, and then check with a magnifying glass of 5-10 times to prevent omissions. After recording the number of colonies on each plate, calculate the average number of colonies grown on each plate at the same dilution. First count the number of mold and yeast colonies growing on each plate, and calculate the average number of colonies for each dilution. At this time, use crystal violet dye to initially stain the flora and colonies, add iodine solution to form a complex with crystal violet, treat with ethanol or acetone, and use safranin for re-staining. If there is a staining result, the suspicious DNA sequence in step 2 is identified by 2% agarose gel electrophoresis to confirm whether there is any one or more of Pseudomonas aeruginosa, Staphylococcus aureus, and fecal coliforms.

[0060] Meanwhile, the contents not described in detail in this specification belong to the prior art known to those skilled in the art, and the model parameters of each electrical appliance are not specifically limited, and conventional equipment can be used.

[0061] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0062] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A multi-enzyme nucleic acid cosmetics testing method, characterized in that: The specific steps include: Step 1: Preliminary extraction of DNA from multi-enzyme nucleic acid cosmetics; 300 g of liquid or solid cosmetic sample is placed in a centrifuge tube, Chelex-100 and proteinase K are added, and the mixture is kept at 60°C for 30 min, and then oscillated at high speed for 10-15 s based on an oscillating device, and then placed in a water bath at 100°C for 12 min, and then oscillated at high speed again for 10-15 s, and finally subjected to preliminary centrifugation. The obtained supernatant is transferred to a centrifuge tube, and then cell lysis solution and gDNA-Binding-Buffer are added, mixed thoroughly, and then placed in a water bath at 65°C for 15 min, and then subjected to secondary centrifugation. The residual liquid is sucked away with a hydraulic gun, and after drying at room temperature for 20 min, the supernatant is transferred to a new centrifuge tube, and the concentration and purity of the extracted DNA are detected using a Nanodrop nucleic acid detector, and then stored at -22°C for later use; Step 2: Comparison and amplification: Compare the obtained DNA with the existing rDNA gene sequence in the database, select multiple groups of DNA with similar homology for touchdown PCR program to complete amplification, purify and recover the PCR product, and sequence it through ABI3730-XLDNA sequencer to identify the suspicious DNA genome in the multi-enzyme nucleic acid cosmetics; Step 3: Detection: Select cells in logarithmic growth phase, digest with 0.3% trypsin to prepare single cell suspension, adjust the cell concentration to 12 / ml and inoculate in each well of an 8-well plate, culture in an incubator at 36°C and 2% CO, change the medium every 2-3 days, take 3 wells every day, digest with 0.25% trypsin to prepare single cell suspension, transfer the supernatant obtained in step 1 to the single cell suspension, and count the cell concentration using a light microscope; If the result of step 2 shows a suspicious DNA genome, the following specific testing steps are also included: Ⅰ: First, take 10 ml of the prepared solution of the cosmetic sample to be tested prepared in step 1 and add it to the dilution bottle of 90 ml SCDLP enrichment solution to make a 1:100 sample preparation solution; Ⅱ: Take a sterile dish after drying and cleaning, add 20-30ml of distilled water, then add lecithin and heat to dissolve, add Tween 80 and protein to the distilled water, shake evenly until the added substances are dissolved, pour into a sterile dish, control the pH value at 7.0-7.6, add agar to the sterile dish, autoclave at 100Kpa, 100℃ for 10min, prepare the culture medium, add the sample preparation solution respectively, and select the suspicious bacteria and colonies after the culture is completed; III: Use crystal violet dye to initially stain the bacterial flora and colonies, add iodine solution to form a complex with crystal violet, treat with ethanol or acetone, and use safranin for counterstaining. If there is a staining result, the suspicious DNA sequence in step 2 is identified by 2% agarose gel electrophoresis to confirm whether there is any one or more of Pseudomonas aeruginosa, Staphylococcus aureus and fecal coliforms; The detection in step 3 also includes: i: Apply the cosmetic sample with suspicious DNA genome obtained in step 2 to the substitute skin for 1 hour, and then immediately rinse the substitute skin with phosphate buffer; ii: After removing the excess phosphate buffer, immerse the substitute skin in 220uL, 3mg / ml MTT solution, and set the environment to 20-28℃ in the dark; ⅲ: Wash off the MTT solution on the substitute skin, rinse repeatedly with phosphate buffer, add 2 mL of acidified isopropanol, shake for 3 hours, and keep overnight in the dark; iv: Pipette 200uL of liquid into a 96-well plate, with 4 replicate wells for each sample. Read the OD value at 550nm using an automatic microplate reader. Use acidified isopropanol solution as a blank control to complete the corrosion test.

2. The multi-enzyme nucleic acid cosmetics testing method according to claim 1, characterized in that: The amount of Chelex-100 added was 300uL, the amount of proteinase K added was 30mg / mL, the amount of cell lysate added was 6mol / L, and the amount of gDNA-Binding-Buffer added was 600uL.

3. The multi-enzyme nucleic acid cosmetics testing method according to claim 1, characterized in that: The production of alternative skin includes the following specific steps: Acidified rat tail collagen was mixed with DMEM culture medium, and the pH value was adjusted. Adult fibroblast suspension in good growth state was placed in it, and finally placed in a 24-well plate, dried at 37°C for 30 minutes, and then DMEM culture medium was added for continuous culture for 4 days. When epidermal keratinocytes were formed, the culture medium was replaced with K-SFM and cultured for 2 days. After air-liquid surface culture was performed for 6 days, the keratinized surface cells were obtained to obtain a substitute skin for detection.

4. The multi-enzyme nucleic acid cosmetics testing method according to claim 3, characterized in that: The solution added for adjusting the pH value is NaOH, the NaOH concentration is 1 mol / L, and the pH value is controlled to be 7.

4.

5. The multi-enzyme nucleic acid cosmetics testing method according to claim 1, characterized in that: In the step 1, the initial centrifugation time is set to 5 min and the rotation speed is 10000 r / min.

6. The multi-enzyme nucleic acid cosmetics testing method according to claim 5, characterized in that: In the step 1, the secondary centrifugation time is set to 1 min and the rotation speed is 4000 r / min.

7. The multi-enzyme nucleic acid cosmetics testing method according to claim 1, characterized in that: In the step 2, the DNA genome OD260 / OD280 is between 1.2-2.1, and the DNA concentration is controlled at 13.2-31.8 ng / uL.

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