WO2022021927A1 - Metagenomics-based method for sampling microorganism on facial skin surface, and application - Google Patents

Abstract

Disclosed in the present invention is a metagenomics-based method for sampling a microorganism on a facial skin surface, comprising the following steps: (1) dipping a disposable medical disinfection cotton swab into sterile normal saline, and sampling an area specified by a disposable sterilization specification plate; (2) in a sterile room, collecting samples according to the sequence of left cheek, right cheek and forehead of a subject, and circling the cotton swab obtained in step (1) for 50 times in each area; and (3) putting the sampled cotton swab obtained in step (2) into a sterile Pbw solution, and storing at 4ºC for later use.

Description

Microbial sampling method and application of facial skin surface based on metagenomics

Inventors: Zhao Huabing (13010319770418182X), Yang Chen, He Xihong, Meng Xuan, Lu Fuping, Shi Tingting, Yang Liu, Tan Youlan, Zhang Bingjie, Chang Huimin

Applicant: Tianjin University of Science and Technology

technical field

The invention belongs to the technical field of microorganisms, in particular to a method and application of microorganism sampling on the surface of facial skin based on metagenomics.

Background technique

Human skin is colonized with a variety of microorganisms and maintains a dynamic balance to maintain its unique skin microecology. Metagenome takes all microbial genomes in the environment as the research object. Through high-throughput sequencing of whole genome DNA in environmental samples, the saturated data volume of a single sample is obtained, and the microbial community structure diversity is based on denovo assembly. Analysis of population gene composition and function, metabolic pathways related to specific environments, etc., so as to further explore and study the relationship between genes with application value and within the microbial community in the environment, and between microorganisms and the environment.

At present, most human microbial community characteristics analysis is designed around the study of gut microbes. The structure of skin and gut is very different. Wrong skin sampling methods can lead to contamination of samples and low microbial biomass, and increase the qualitative and quantitative analysis of microorganisms. Bias, creating obstacles for microbial research.

Through searching, no patent publications related to the patent application of the present invention have been found.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the deficiencies of the prior art, and to provide a method and application for microbial sampling on the surface of facial skin based on metagenomics.

The technical scheme adopted by the present invention to solve its technical problems is:

A metagenomics-based microbial sampling method of facial skin surface, the steps are as follows:

(1) Use a disposable medical sterile cotton swab to dip in sterile physiological saline, and sample in the area specified by the disposable sterilization specification board;

(2) In the sterile room, collect samples in the order of the subject's left cheek, right cheek and forehead, and take the cotton swab obtained in step (1) and circle each area 50 times;

(3) Put the sampled cotton swabs obtained in step (2) into sterile Pbw solution, and store at 4°C for future use.

Moreover, the disinfection in the step (1) is to use 75% alcohol for disinfection, and the sterile physiological saline is: the volume fraction is 0.9% physiological saline at 121 ° C, and is prepared by high-pressure steam sterilization for 15 minutes.

Moreover, in the step (2), the sterile room is a clean sterile room. After wiping the table top and the glass of 30 cm above the table top with disinfectant, 75% alcohol is used to wipe it once, and the sterile room is UV sterilized for one hour.

Moreover, in the step (2), the subjects are sampled at intervals of more than 1h before and after washing their faces, wash their faces with clean water, and avoid makeup and use of personal hygiene products.

Moreover, the size of the area where the sample is collected in the step (2) is 3cm×3cm.

And, the preparation method of sterile Pbw solution in described step (3) is:

Take 10 g of peptone, 3 g of beef extract, 5 g of sodium chloride, and 1000 g of distilled water, adjust the pH to 7.4, and then perform autoclaving at 121° C. for 15 minutes.

Application of the above-mentioned metagenomics-based microbial sampling method of facial skin surface in skin microbial sampling.

The advantages and positive effects obtained by the present invention are:

1. The sampling process in the present invention will not cause damage to the skin surface of the subject, avoids the stimulation of the skin caused by invasive sampling, and is safer and suitable for facial skin sampling. Subsequent microbial community sequencing verifies that the present invention can better restore the real microbial ecology of the skin compared with other skin microbial sampling methods.

2. The biomass collected by the method of the present invention is relatively large, and the concentration and quality of the total DNA obtained subsequently can meet the requirements of microbial community analysis and sequencing.

3. The materials used in the method of the present invention are cheap and easy to obtain, and the method is efficient and convenient.

4. It is verified by 16s rDNA sequencing, 18s rDNA sequencing and real-time fluorescence quantitative PCR that the method of the present invention can cover a wide variety and a large number of microorganisms on the skin surface to a greater extent.

Description of drawings

Fig. 1 is the Krona pie chart of 16s rDNA sequencing species in the present invention;

Fig. 2 is the Krona pie chart of 18s rDNA sequencing species in the present invention;

Fig. 3 is the bacterial real-time fluorescence quantitative PCR result figure in the present invention;

FIG. 4 is a graph showing the results of real-time fluorescent quantitative PCR of fungi in the present invention.

detailed description

The present invention will be further described below with reference to the examples. The following examples are descriptive and not restrictive, and the protection scope of the present invention cannot be limited by the following examples.

The raw materials used in the present invention, unless otherwise specified, are conventional commercial products, the methods used in the present invention, unless otherwise specified, are conventional methods in the field, and the quality of each substance used in the present invention is conventionally used quality.

A metagenomics-based microbial sampling method of facial skin surface, the steps are as follows:

(1) Use a disposable medical sterile cotton swab to dip in sterile physiological saline, and sample in the area specified by the disposable sterilization specification board;

(2) In the sterile room, collect samples in the order of the subject's left cheek, right cheek and forehead, and take the cotton swab obtained in step (1) and circle each area 50 times;

(3) Put the sampled cotton swabs obtained in step (2) into sterile Pbw solution, and store at 4°C for future use.

Preferably, the disinfection in the step (1) is to use 75% alcohol for disinfection, and the sterile physiological saline is: the volume fraction is 0.9% physiological saline at 121 ° C, and is prepared by high pressure steam sterilization for 15 minutes.

Preferably, in the step (2), the sterile room is a clean sterile room. After wiping the table top and the glass 30 cm above the table top with disinfectant, wipe it once with 75% alcohol, and the sterile room after UV sterilization for one hour. .

Preferably, in the step (2), the subjects take samples at intervals of more than 1 hour before and after washing their faces, wash their faces with clean water, and avoid makeup and use of personal hygiene products.

Preferably, the size of the area where the sample is collected in the step (2) is 3cm×3cm.

Preferably, the preparation method of sterile Pbw solution in the step (3) is:

Take 10 g of peptone, 3 g of beef extract, 5 g of sodium chloride, and 1000 g of distilled water, adjust the pH to 7.4, and then perform autoclaving at 121° C. for 15 minutes.

The metagenomics-based microbial sampling method of the facial skin surface as described above can be applied in the field of skin microbial sampling.

Specifically, the relevant preparation and detection are as follows:

1. Microbiological sampling of the subject's facial skin

Healthy subjects aged 21-28 years were selected. Subject exclusion criteria: obvious skin diseases, smoking, hormone disorders, hormone therapy within one month, taking antibiotics, taking antifungal drugs, etc. The above sampling steps can only be performed at intervals of more than one hour after the subjects wash their faces, wash their faces with clean water, and avoid makeup and use of personal hygiene products such as skin care products.

(1) Collection of microorganisms on the skin surface

1.1 Use a disposable medical sterilized cotton swab to dip an appropriate amount of sterile saline, and sample in the area specified by the disposable sterilization specification board;

1.2 In the sterile room, collect samples in the order of the subject's left cheek, right cheek and forehead, take the cotton swab obtained in step 1.1 and circle each area 50 times with appropriate pressure, and use the cotton swab obtained in 1.1 that has not been in contact with the skin as negative control;

The sterile room refers to a clean sterile room. After wiping the table top and the glass 30cm above the table with disinfectant water, wipe it once with 75% alcohol, and sterilize it with UV light for one hour;

1.3 Put the cotton swabs obtained in 1.2 into sterile Pbw solution and store at 4°C for later use.

The Pbw solution was prepared as follows: 10 g of peptone, 3 g of beef extract, 5 g of sodium chloride, and 1000 g of distilled water. After adjusting the pH value to 7.4, autoclaving was performed at 121° C. for 15 minutes.

(2) Sample pretreatment

After the Pbw solution and cotton swab obtained in step (1) were shaken on a vortex shaker for 2 min, the cotton swab was taken out and centrifuged in a high-speed centrifuge for 1 min at a speed of 14000 r/min.

(3) DNA extraction from different skin samples

Extract bacterial DNA and fungal DNA respectively according to the operating instructions of the DNA extraction kit, and store the extracted DNA in a refrigerator at -20°C until use.

(4) PCR amplification

The 20 μL PCR reaction system for bacteria is: 1 μL DNA extracted in step (3), 1 μL primer ccsF (5’-CGGTGAATACGTTCTCGG-3’), 1 μL primer ccsR (5’-GGTTACCTTGTTACGACTT-3’), 7 μL sterile ultrapure water, 10 μL GoldStar TaqMan Mixture Enzyme

The reaction conditions were: pre-denaturation at 95 °C for 10 min; then 94 °C for 30 s, 55 °C for 30 s, 72 °C for 8 s, a total of 40 cycles; finally, 72 °C extension for 5 min, and 10 °C incubation to complete the reaction.

The 20 μL PCR reaction system for fungi was: 1 μL DNA extracted in step (3), 1 μL primer ccsF (5’-AAGGAAGGCAGCAGGCG-3’), 1 μL primer ccsR (5’-CACCAGACTTGCCCTCTAAT-3’), 7 μL sterile ultrapure water, 10 μL GoldStar TaqMan Mixture Enzyme

The reaction conditions were: pre-denaturation at 95 °C for 10 min; then 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 7 s, a total of 40 cycles; finally, 72 °C for 5 min, and 10 °C incubation to complete the reaction.

(5) Sequencing

The PCR product obtained in step (4) is sent to a sequencing company for 16s rDNA and 18s rDNA sequencing, and the partial results obtained are shown in Figure 1 and Figure 2.

Fig. 1 shows the bacterial species distribution map obtained by performing 16s rDNA sequencing after the skin sampling method of the present invention;

Figure 2 shows the bacterial species distribution map obtained by performing 18s rDNA sequencing after the skin sampling method of the present invention.

2. Real-time PCR assay of microorganisms in skin samples

The 20 μL fluorescent quantitative PCR reaction system of bacteria is: 2 μL DNA extracted in step (3), 10 μL TB Green Premix Ex Taq ii enzyme, 0.8 μL primer ccsF (5'-CGGTGAATACGTTCTCGG-3'), 0.8 μL primer ccsR (5'- GGTTACCTTGTTACGACTT-3'), 6μL sterile ultrapure water, 0.4μLRox Reference Dye dye.

The 20 μL fluorescent quantitative PCR reaction system of the fungus is: 2 μL DNA extracted in step (3), 10 μL TB Green Premix Ex Taq ii enzyme, 0.8 μL primer ccsF (5'-AAGGAAGGCAGCAGGCG-3'), 0.8 μL primer ccsR (5'- CACCAGACTTGCCCTCTAAT-3'), 6μL sterile ultrapure water, 0.4μLRox Reference Dye dye.

Fluorescence quantitative PCR reaction conditions for fungi and bacteria are as follows: first heat up to 95°C for 30s; then 95°C for 10s, 55°C for 30s, 55°C for 20s, 72°C for 30s, a total of 40 cycles; finally enter the dissolution curve stage, 95°C for 15s , 60s at 60℃, 15s at 95℃.

The results of microbiological analysis on the skin surface of different subjects are shown in Figures 3 and 4. Different swabs all generate amplification products, and the obtained biomass is relatively large.

Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, therefore , the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims (7)

1. A method for microbial sampling on the surface of facial skin based on metagenomics, characterized in that: the steps are as follows:

   (1) Use a disposable medical sterile cotton swab to dip in sterile physiological saline, and sample in the area specified by the disposable sterilization specification board;

   (2) In the sterile room, collect samples in the order of the subject's left cheek, right cheek and forehead, and take the cotton swab obtained in step (1) and circle each area 50 times;

   (3) Put the sampled cotton swabs obtained in step (2) into sterile Pbw solution, and store at 4°C for future use.
2. The method for microbial sampling on the surface of facial skin based on metagenomics according to claim 1, wherein the disinfection in the step (1) is to use 75% alcohol for disinfection, and the sterile physiological saline is: the volume fraction is 0.9% Physiological saline was prepared by autoclaving at 121°C for 15 minutes.
3. The method for microbial sampling on the surface of facial skin based on metagenomics according to claim 1, characterized in that: in the step (2), the sterile room is a clean sterile room, and disinfected water is used to wipe the desktop and the glass of 30 cm above the desktop. After that, use 75% alcohol to wipe the sterile room again after one hour of UV sterilization.
4. The method for microbial sampling on the surface of facial skin based on metagenomics according to claim 1, characterized in that: in the step (2), the subject performs sampling at intervals of more than 1h before and after washing his face, washes his face with clean water, and avoids makeup and the use of personal hygiene products .
5. The method for microbial sampling on the surface of facial skin based on metagenomics according to claim 1, wherein the size of the area where the sample is collected in the step (2) is 3cm×3cm.
6. The microorganism sampling method of the facial skin surface based on metagenomics according to claim 1, is characterized in that: the preparation method of sterile Pbw solution in described step (3) is:

   Take 10 g of peptone, 3 g of beef extract, 5 g of sodium chloride, and 1000 g of distilled water, adjust the pH to 7.4, and then perform autoclaving at 121° C. for 15 minutes.
7. Application of the metagenomics-based microbial sampling method of facial skin surface according to any one of claims 1 to 6 in skin microbial sampling.

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