A method for quantitatively analyzing a greasy fur

Abstract

The application provides a method for quantitatively analyzing a greasy fur. Based on previous research, the inventors found that the essence of the greasy fur is that the tongue surface bacteria form a layer of biofilm between the tongue filiform papillae, and firmly adhere to the filiform papillae. Based on the detection method of the biofilm, the tongue fur samples are respectively taken from the greasy fur and the normal tongue fur, cultured in vitro, and the number of biofilm formation is detected, and it is found that the bacterial biofilm of the greasy fur is much higher than that of the normal tongue fur. Therefore, by detecting the biofilm formation of the tongue fur microorganism, or scraping part of the tongue fur smear, and detecting the polysaccharide components representing the sticky properties of the biofilm in the tongue fur smear by the fluorescent dye method and the AB / PAS mucopolysaccharide staining method, the strength of the greasy fur can be quantitatively detected in a high-throughput and rapid manner.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for analyzing biological samples, in particular to a method for quantitatively analyzing a greasy fur. BACKGROUND

[0002] Tongue diagnosis is an important diagnostic method in the Chinese medicine diagnosis system. In the Qing Dynasty, Zhang Nan recorded in "Medical Barbecue·Treatise on Febrile Diseases" that "the tongue can be used to verify yin and yang deficiency and excess, and the tongue coating can be used to determine the depth of cold and heat". Here, the tongue coating refers to the tongue fur. According to the theory of traditional Chinese medicine, the tongue fur is the vaporized and transformed qi of the stomach, which is formed on the tongue surface and is closely related to the function of the spleen and stomach. The greasy fur is an important basis for diagnosis in traditional Chinese medicine. The pathological nature of the greasy fur is still unclear, and there is a lack of biochemical methods for quantitative analysis.

[0003] Bacterial biofilm refers to a three-dimensional structure composed of bacteria and the viscous extracellular matrix secreted by the bacteria. The biofilm secreted by the bacteria contains a large amount of viscous polysaccharides and mucin, and forms a dense film on the tongue surface. This dense structure can improve the adhesion of bacteria and isolate the bacteria from antibiotics, which is the main reason for bacterial drug resistance. SUMMARY

[0004] The purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide a method for quantitatively analyzing a greasy fur.

[0005] The purpose of the present application is achieved by the following technical solutions:

[0006] A method for quantitatively analyzing a greasy fur is to detect the film-forming property of microorganisms in a tongue fur sample to determine whether it belongs to a greasy fur.

[0007] The film-forming property of the microorganisms is detected by at least one of crystal violet staining, fluorescent dye staining or mucin staining.

[0008] The film-forming property of the microorganisms is positively correlated with at least one of the OD value measured after the tongue fur sample is stained with crystal violet, the fluorescence intensity measured after the tongue fur sample is stained with fluorescent dye, or the staining value measured after the tongue fur sample is stained with mucin.

[0009] The criteria for the determination are that when the detection results of the tongue fur sample meet at least one of the following criteria, the tongue fur sample is determined to be a greasy fur:

[0010] a. The OD value measured after the tongue fur sample is stained with crystal violet is 0.2 or more;

[0011] b. The OD value measured after the tongue fur sample is stained with crystal violet is two times or more than that of the negative control;

[0012] c. The fluorescence intensity measured after the tongue fur sample is stained with fluorescent dye is two times or more than that of the negative control;

[0013] d. The tongue sample is negative control twice or more after the staining value of the mucin staining is detected.

[0014] The OD value is the OD value measured at 590 nm.

[0015] The OD value is the average value obtained from three repeated experiments.

[0016] The staining value is the percentage of pixels in the red (R) range of 45-255 in the total pixels.

[0017] The tongue sample is a tongue sample taken from the middle of the tongue.

[0018] The crystal violet staining has the following specific steps:

[0019] Take the tongue sample, inoculate it into the culture medium for culture, after the culture is completed, fix, stain, wash, and dissolve to detect the absorbance.

[0020] The culture medium is tryptone soy broth medium.

[0021] The culture is at 35-38°C for 18-36h; preferably at 37°C for 24h.

[0022] The fixation is using methanol.

[0023] The staining is using 0.5-2% crystal violet solution; preferably using 1% crystal violet solution.

[0024] The dissolution is using 30-35% ice acetic acid solution; preferably using 33% ice acetic acid solution.

[0025] The fluorescent dye staining has the following specific steps:

[0026] Take the tongue sample, add the fluorescent dye, and after the staining is completed, detect the fluorescence intensity.

[0027] The fluorescent dye is Ebba Biolight 680.

[0028] The mucin staining has the following specific steps:

[0029] Take the tongue sample, use the mucin staining kit to stain, and after the staining is completed, detect the staining value.

[0030] The mucin staining kit is AB-PAS staining kit.

[0031] A kit for quantitatively analyzing coating of tongue fur, which can determine whether the tongue fur sample belongs to coating of tongue fur by detecting the film-forming property of microorganisms in the tongue fur sample.

[0032] The kit comprises reagents for detecting the film-forming property of microorganisms.

[0033] The kit comprises reagents for at least one of crystal violet staining, fluorescent dye staining or mucin staining.

[0034] The kit determines the tongue fur as coating of tongue fur when the detection result of the tongue fur sample meets at least one of the following conditions:

[0035] a. The OD value of the tongue fur sample after crystal violet staining is 0.2 or higher;

[0036] b. The OD value of the tongue fur sample after crystal violet staining is twice or higher than that of the negative control;

[0037] c. The fluorescence intensity of the tongue fur sample after fluorescent dye staining is twice or higher than that of the negative control;

[0038] d. The staining value of the tongue fur sample after mucin staining is twice or higher than that of the negative control.

[0039] The OD value is the OD value measured at 590 nm.

[0040] The staining value is the percentage of pixels in the red range of 45-255 in the total pixels.

[0041] The tongue fur sample is a tongue fur sample taken from the middle of the tongue.

[0042] The kit for quantitatively analyzing coating of tongue fur is used in identifying coating of tongue fur.

[0043] The present application has the following advantages and effects compared with the prior art:

[0044] Based on previous research, it is found that the essence of coating of tongue fur is that the tongue surface bacteria form a layer of biofilm between the filamentous papillae, firmly adhering to the filamentous papillae. Based on the detection method of biofilm, the tongue fur samples are respectively taken from coating of tongue fur and normal tongue fur, cultured in vitro and the number of biofilm formation is detected. It is found that the bacterial biofilm of coating of tongue fur is much higher than that of normal tongue fur. Therefore, by detecting the film-forming property of microorganisms in the tongue fur, coating of tongue fur can be qualitatively distinguished.

[0045] By dividing the film-forming property interval, coating of tongue fur can be qualitatively distinguished, and the standard is as follows:

[0046] 1. The overall film-forming property of the coating of tongue fur bacteria is 0.2, which is the boundary for distinguishing coating of tongue fur from normal tongue fur

[0047] 2. The culture solution without inoculation of bacteria is used as negative control, and 2 times of the value of the negative control is used as the limit value (Dc). The D value of single bacteria isolated from the greasy fur is greater than 2 x Dc.

[0048] 3. After the greasy fur tongue smear is dyed with fluorescent dye, the fluorescence brightness is more than 2 times of that of normal tongue fur.

[0049] 4. After the greasy fur tongue smear is dyed with AB / PAS, the mucin dyeing value is more than 2 times of that of normal tongue fur. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 is a picture of normal tongue fur and greasy fur.

[0051] Figure 2 is a box plot of the film-forming property determined in Example 1.

[0052] Figure 3 is a picture of the 96-well plate after dyeing in Example 1.

[0053] Figure 4 is a picture of the filter paper dipped in the centrifuge tube after dipping the tongue fur in Example 1.

[0054] Figure 5 is a picture of the staining of the carbohydrate components of the biofilm of the normal tongue fur smear.

[0055] Figure 6 is a picture of the staining of the carbohydrate components of the biofilm of the greasy fur smear. DETAILED DESCRIPTION

[0056] The present application will be further described in conjunction with the examples and drawings, but the embodiments of the present application are not limited thereto.

[0057] In the following implementation, if the specific test conditions are not specified, the general test conditions or the test conditions recommended by the reagent company are usually used. If not specified, the materials, reagents, etc. used are reagents and materials obtained from commercial channels.

[0058] Example 1: Analysis of the overall film-forming property of the greasy fur sample

[0059] Experimental materials:

[0060] The samples come from 24 college student volunteers, which are divided according to the tongue appearance identification standard of traditional Chinese medicine, among which 11 individuals have greasy fur and 13 individuals do not have greasy fur.

[0061] Experimental method:

[0062] (1) The tongue coating of the above 24 people was sampled by using about 1 / 8 of a circular sterilized filter paper, and the filter paper was put into a 1.5 ml sterile centrifuge tube, 1 ml PBS buffer was added, and the filter paper was soaked for 1 hour. After centrifugation at 3000 rpm for 5 minutes, the supernatant was discarded, 10 μL of normal saline was added, and after resuspension, 5 μL of bacteria was taken to a 96-well plate containing 95 μL of tryptone soya broth medium (TSB) in each well, 3 replicates for each sample, and incubated at 37°C for 24 hours.

[0063] (2) After the culture was completed, the culture solution was aspirated, and the filter paper was washed with sterile PBS for 3 times. After 100 μL of methanol was added to each well for 15 minutes, the methanol was aspirated, and the filter paper was naturally air-dried. Then, 100 μL of 1% crystal violet solution was added to each well, and the filter paper was dyed at room temperature for 5 minutes. After the dye was aspirated, each well was uniformly washed until the control well was colorless, and the filter paper was naturally air-dried. 100 μL of 33% glacial acetic acid solution was added to each well, and the filter paper was dissolved at 37°C for 30 minutes. The OD value of each well was measured at 590 nm by using an enzyme-labeled instrument.

[0064] The comparison picture and film-forming property of the normal tongue coating and the greasy tongue coating obtained under the above experimental conditions are as follows: Figure 1 The statistical results of the two groups are shown in Table 1. The dense and slippery film of the greasy tongue coating is located in the middle of the tongue. Figure 1 ).

[0065] The statistical method of non-parametric analysis was used for analysis, and the crystal violet quantification of the film-forming property showed that the median of the OD value (film-forming property) of the greasy tongue coating group was more than 2 times that of the normal group, and the p value was less than 0.01. The A590 of the normal group was between 0.095 and 0.176, and the A590 of the greasy tongue coating group was between 0.248 and 0.928, as shown in Table 1. Figure 2 The experimental results prove that the film-forming property of microorganisms can be used to distinguish the greasy tongue coating and the normal tongue coating.

[0066] Table 1: Absorbance determination results

[0067]

[0068] Example 2: Single-bacterium film-forming property determination of the greasy tongue coating sample

[0069] The tongue coating samples of the greasy tongue coating group and the normal tongue coating group were taken by using the same size of fan-shaped sterilized filter paper, and the filter paper was soaked in 1 ml of sterile PBS buffer for 1 hour. After centrifugation at 3000 rpm for 5 minutes, the sample suspension was streaked on tryptone soya broth medium (TSB), and cultured at 37°C. The colonies were picked and streaked on a new culture plate. The above steps were repeated, and finally the single colonies after purification were obtained. The single colonies were subjected to PCR amplification and sequencing analysis.

[0070] The colonies finally isolated and purified from the greasy tongue coating group were compared with the normal tongue coating group, and two strains of bacteria not present in the normal tongue coating group were identified as Acinetobacter and Streptococcus anginosus.

[0071] The two isolated bacteria were cultured in 3 ml TSB medium at 220 rpm and 37°C for 18 hours, respectively. Then, 10 ul of the bacterial solution was taken and added to 90 ul TSB medium in a 96-well plate. The film-forming property was tested by referring to the method of Example 1.

[0072] Result determination: The average value of three duplicate wells (D value) was taken. The culture solution without inoculation of bacteria was used as negative control, and the value twice that of the negative value was used as the limit value (Dc). Based on the D value, the bacterial strains were divided into three categories: (1) strong biofilm-forming strains (D > 2 x Dc); (2) weak biofilm-forming strains (Dc < D ≤ 2 x Dc); and (3) non-biofilm-forming strains (D ≤ Dc).

[0073] The film-forming property of the isolated strains under the above experimental conditions is shown in Table 2. It can be seen that the film-forming property of the isolated strains is strong.

[0074] Table 2 Film-forming property of isolated strains of tongue fur

[0075]

[0076] Example 3 Use of fluorescent dye to show the intensity of tongue fur biofilm

[0077] A clean and sterilized glass slide was used to gently scrape the tongue fur and normal tongue fur from the middle of the tongue to the tip of the tongue, and then the smears were made on another glass slide. One drop of fluorescent dye Ebba Biolight 680 (1:500 dilution) was added, which was purchased from AID Chemical Co., Ltd. and had good affinity with various polysaccharides in the biofilm. After incubation at room temperature for 10 minutes in the dark, the fluorescence was analyzed under a fluorescence microscope. It was found that the fluorescence value of the tongue fur glass slide was much higher than that of the normal tongue fur. In this example, it was found that the fluorescence brightness was more than 2 times higher than that of the normal.

[0078] Example 4 Use of mucin staining technique to detect the composition of mucin in tongue fur

[0079] The mucin staining technique is commonly used as a means for extensive detection of mucin. It is first stained with standard Alcian blue (pH = 2.5) and then subjected to PAS technique. Alcian blue can stain salivary mucin, sulfur mucin and proteoglycans blue. PAS technique can stain neutral mucin deep red or red-purple, and can stain tissues and cells containing both neutral mucin and acid mucin different shades of purple.

[0080] A clean and sterilized glass slide was used to gently scrape the tongue fur and normal tongue fur from the middle of the tongue to the tip of the tongue, and then the smears were made on another glass slide. One drop of fluorescent dye Ebba Biolight 680 (1:500 dilution) was added, which was purchased from AID Chemical Co., Ltd. and had good affinity with various polysaccharides in the biofilm. After incubation at room temperature for 10 minutes in the dark, the fluorescence was analyzed under a fluorescence microscope. It was found that the fluorescence value of the tongue fur glass slide was much higher than that of the normal tongue fur. In this example, it was found that the fluorescence brightness was more than 2 times higher than that of the normal.

[0081] 1. Fix with 95% ethanol for 5 min, wash with distilled water for 2 min.

[0082] 2. Stain with Alcian blue staining solution for 10-20 min.

[0083] 3. Wash with distilled water for 3 times, each for 1-2 min.

[0084] 4. Oxidize with periodic acid solution for 5 min.

[0085] 5. Stain with Schiff Reagent for 10-20 min.

[0086] 6. Discard the Schiff Reagent and wash with running water for 10 min.

[0087] 7. Stain with hematoxylin staining solution for 1-2 min.

[0088] 8. Differentiate with acid differentiation solution for 2-5 s, and wash with water.

[0089] 9. Blue with Scott blueing solution, and wash with water for 3 min.

[0090] 10. Dehydrate with graded regular ethanol, xylene transparent, and seal with neutral balsam.

[0091] The results are shown in Figure 5 and Figure 6 The slime coating of the tongue coating smear in Figure 6 represents that the mucin content of the biofilm adhesion is much higher than that of the normal tongue coating smear in Figure 5 , proving that the mucin content in the slime sample is significantly increased compared with the normal group, mainly from the strains with higher film-forming ability, therefore, the slime and normal tongue coating can also be effectively distinguished by detecting the mucin content.

[0092] The microscope photos of the smears, using image analysis software, calculate the staining value of the gray value of the specific color. In this embodiment, the Figure 5 and Figure 6 with similar cell numbers under the same magnification are calculated, using software Image J, calculating the gray value in the red (R) range of 45-255 (including both endpoints), the positive pixels falling within this range, dividing the number of positive pixels by the total pixels to obtain the staining value. Figure 5 The positive area ratio of the staining part of the first picture in Figure 6 is 4.355%, obtaining a staining value of 4.355, with a resolution of 429*343; the ratio of the second picture is 60.640%, obtaining a staining value of 60.640, with a resolution of 423*340, Figure 5 The staining value of is 13.92 times that of

[0093] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A quantitative analysis method for greasy skin, characterized in that: By detecting the film-forming properties of microorganisms in tongue coating samples, it can be determined whether the coating is greasy. The film-forming property of the microorganisms is detected by at least one of crystal violet staining, fluorescent dye staining, or mucin staining; The film-forming properties of the microorganisms are positively correlated with at least one of the following: the OD value of the tongue coating sample after crystal violet staining, the fluorescence intensity of the tongue coating sample after fluorescent staining, or the staining value of the tongue coating sample after mucin staining. The criteria for judgment are as follows: a tongue coating sample is judged to be a greasy tongue coating if the test results meet at least one of the following: a. The OD value of the tongue coating sample after staining with crystal violet is greater than 0.2; b. The OD value of the tongue coating sample after crystal violet staining was more than twice that of the negative control; c. The fluorescence intensity of the tongue coating sample after fluorescent staining was more than twice that of the negative control; d. The staining value of the tongue coating sample after mucin staining was more than twice that of the negative control; The OD value mentioned is the OD value measured at 590 nm; The coloring value is defined as the percentage of pixels in the red range of 45 to 255 out of the total number of pixels.

2. The method according to claim 1, characterized in that: The specific steps for the crystal violet staining are as follows: Tongue coating samples were collected, inoculated into culture medium, and after culture, fixed, stained, washed, dissolved, and their absorbance was measured. The culture medium is tryptone soybean broth medium; The culture was carried out at 35–38°C for 18–36 hours. The fixation mentioned above is achieved using methanol fixation; The staining method involves using a 0.5–2% crystal violet solution. The dissolution is performed using a 30-35% glacial acetic acid solution.

3. The method according to claim 1, characterized in that: The specific steps for the fluorescent dye staining are as follows: Take a tongue coating sample, add fluorescent dye, and after staining, detect its fluorescence intensity; The fluorescent dye mentioned is EbbaBiolight 680.

4. The method according to claim 1, characterized in that: The specific steps for mucin staining are as follows: Tongue coating samples were collected and stained using a mucin staining kit. After staining, the staining values ​​were measured. The mucin staining kit mentioned is the AB-PAS staining kit.

5. A kit for quantitative analysis of greasy tongue coating, characterized in that: It can determine whether a tongue coating is greasy by detecting the film-forming properties of microorganisms in the sample; The kit includes reagents for detecting microbial film-forming properties; The kit includes reagents for at least one of crystal violet staining, fluorescent dye staining, or mucin staining; The aforementioned kit determines a tongue coating sample to be greasy if the test results meet at least one of the following criteria: a. The OD value of the tongue coating sample after staining with crystal violet is greater than 0.2; b. The OD value of the tongue coating sample after crystal violet staining was more than twice that of the negative control; c. The fluorescence intensity of the tongue coating sample after fluorescent staining was more than twice that of the negative control; d. The staining value of the tongue coating sample after mucin staining was more than twice that of the negative control; The OD value mentioned is the OD value measured at 590 nm; The coloring value is defined as the percentage of pixels in the red range of 45 to 255 out of the total number of pixels.

6. The application of the quantitative analysis kit for greasy moss as described in claim 5 in the identification of greasy moss.

Images