Feces preservation method, use of ethanol in preparing feces preservation solution and short-chain fatty acid detection reagent or kit

Abstract

The present invention discloses a stool preservation method, the use of ethanol in the preparation of a stool preservation solution and a short-chain fatty acid detection reagent or kit, and relates to the technical field of stool preservation. The effective ingredient in the stool preservation solution is ethanol. The stool preservation solution provided by the present invention allows stool samples to be collected and stored at room temperature. The use of such a preservation solution enables participants to send samples by conventional mail without the need for appointments, refrigeration or cold chain transportation. The preservation solution of the present invention can well maintain the stability of the content of short-chain fatty acids in stool samples, making sampling simpler during quantitative detection of short-chain fatty acids, and can be stored and transported at room temperature, which is easy to popularize and promote. The present invention also contributes to the development of corresponding short-chain fatty acid detection reagents or kits.

Description

Technical Field

[0001] The present invention relates to the technical field of feces preservation, and in particular to a feces preservation method, and application of ethanol in preparing a feces preservation solution and a short-chain fatty acid detection reagent or kit. Background Art

[0002] Short-chain fatty acids (SCFA) are produced in the intestines and are essential for maintaining healthy intestinal microbiota, balancing lipid and sugar metabolism, insulin sensitivity and natural immunity. They play an important role in maintaining water and electrolyte balance, resisting pathogenic microorganisms and inflammation, regulating intestinal flora balance, improving intestinal function, regulating immunity, anti-tumor and regulating gene expression. In clinical practice, they can be used as a corresponding indicator of the effect of treatment (including probiotic drug therapy, flora transplantation, other therapeutic drugs, etc.). However, since the metabolism and proliferation of some bacteria may continue, thus affecting the microbial structure and SCFA spectrum, the literature points out that SCFA increases almost proportionally with time under 4°C and room temperature conditions [Cunningham J, Bramstng L, Singh A, et al. Impact of time and temperature on gut microbiota and SCFA composition in stool samples. 2020.], so it is generally not recommended to store stool samples at room temperature or at 4°C for more than 12 hours. This poses an important challenge to the analysis of short-chain fatty acids because the cold chain is not always maintained or guaranteed from sample collection to frozen storage. Specimens collected in the field typically spend varying amounts of time at room temperature before being shipped in frozen gel packs (4°C) or on dry ice to a central laboratory for processing or storage, meaning that the specimens may have been exposed to room temperature for several hours before arriving at the laboratory.

[0003] Generally, immediate freezing of samples at -80°C is considered the reference method, but this reference method is not feasible in many cases due to the harsh storage conditions. In contrast, most households have a -20°C freezer and can also freeze samples at -20°C, but this requires people to be willing to put the samples in the freezer and have the resources to transport the samples in a frozen state. The current method of collecting stool samples mainly requires participants to collect them at home in order to arrange an appointment on the day of collection and store the samples in their own refrigerator or freezer, which can cause discomfort to participants and may pose health risks. These factors may negatively affect participants' decision to participate in the study. Most of the time, the standard collection method that requires immediate freezing may not be feasible.

[0004] In summary, the difficulty in sample preservation and transportation is an important reason why domestic medical institutions have not yet implemented short-chain fatty acid detection technology as a routine testing item, which limits the clinical application of short-chain fatty acids.

[0005] Therefore, research on sample preservation will be the key to the accuracy of short-chain fatty acid detection.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a stool sample preservation solution to solve the problem of stool sample preservation when detecting short-chain fatty acids.

[0008] The present invention is achieved in that:

[0009] The invention provides a reagent or a test kit, which contains a stool preservation solution, wherein the effective component of the stool preservation solution is ethanol.

[0010] The stool preservation solution provided by the present invention allows stool samples to be collected and stored at room temperature. The use of such a preservation solution enables participants to send samples by conventional mail without the need for appointments, refrigeration or cold chain transportation. The preservation solution of the present invention can well maintain the stability of the content of short-chain fatty acids in stool samples, making sampling easier during quantitative detection of short-chain fatty acids, and can be stored and transported at room temperature, which is easy to popularize and promote. It is helpful to develop corresponding short-chain fatty acid detection reagents or kits.

[0011] In a preferred embodiment of the present invention, the stool preservation solution comprises an ethanol aqueous solution.

[0012] In one embodiment, the stool preservation solution contains an ethanol aqueous solution with a volume concentration of 70% to 90%. In one embodiment, the stool preservation solution contains an ethanol aqueous solution with a volume concentration of 70% to 75%. In one embodiment, the stool preservation solution contains an ethanol aqueous solution with a volume concentration of 75%.

[0013] The invention provides an application of ethanol in preparing a stool preservation solution, wherein the effective component of the stool preservation solution is ethanol.

[0014] Current research on stool preservation fluid mainly focuses on extracting DNA, and no special stool sample preservation fluid for short-chain fatty acid detection has been found.

[0015] The inventors have found that adding ethanol to the stool preservation solution can effectively and low-burdenly meet the stool sample preservation requirements for short-chain fatty acid detection.

[0016] Specifically, the stool preservation solution provided by the present invention allows stool samples to be collected and stored at room temperature (RT). The use of such a preservation solution enables participants to send samples by conventional mail without the need for appointments, refrigeration or cold chain transportation. The preservation solution of the present invention can well maintain the stability of the content of short-chain fatty acids in stool samples, making sampling easier during quantitative detection of short-chain fatty acids, and can be stored and transported at room temperature, which is easy to popularize and promote.

[0017] In addition, the present invention also evaluates the reliability and efficiency of the preservation solution as a participant-friendly feces collection method (collection and storage under normal temperature conditions). Compared with fresh feces and fresh feces immediately frozen at -80°C, the short-chain fatty acid (SCFA) content in the samples after the feces samples were preserved using ethanol feces preservation solution was equivalent, with no significant difference. The proposal of the present invention shows the potential of using preservation solution to collect and store at ambient temperature, which is convenient for studies aimed at collecting large samples by enabling participants to send samples by mail, thereby reducing the difficulty of accurate quantification of short-chain fatty acids, providing a scientific basis for short-chain fatty acid detection to become a clinical application such as metabolic disease screening, early diagnosis, treatment monitoring and prognosis evaluation, and can promote the clinical application of short-chain fatty acid profiles.

[0018] The change in the content of short-chain fatty acids in fecal samples is due to the fact that there are more microorganisms in the feces and the microbial metabolism rate is very fast, which in turn affects the content of short-chain fatty acids. Therefore, the metabolic rate of the microorganisms in the sample can be reduced (such as immediate freezing) or the microorganisms can be inactivated. After a long period of extensive exploration and screening, it was found that ethanol, as a common organic protein precipitant, is expected to reduce the metabolic rate of microorganisms. At the same time, when the concentration of ethanol is too high, it may irritate the skin and absorb a large amount of moisture from the epidermis, which is not suitable for direct contact by general participants. 70% to 75% ethanol is often used for disinfection and sterilization, and if the concentration of ethanol is too high, a protective film will form on the surface of the bacteria, preventing it from entering the bacteria, making it difficult to completely kill the bacteria. If the concentration of ethanol is too low, although it can enter the bacteria, it cannot coagulate the protein in its body, nor can it completely kill the bacteria. Based on this, 75% ethanol was selected as the active ingredient for preparing fecal sample preservation solution. The content of various short-chain fatty acids in fecal samples can be effectively kept at room temperature with little change.

[0019] In a preferred embodiment of the present invention, the stool preservation solution comprises an ethanol aqueous solution.

[0020] In one embodiment, the feces preservation solution contains an ethanol aqueous solution with a volume concentration of 70% to 90%, such as an ethanol aqueous solution with a volume concentration of 71% to 85%, such as an ethanol aqueous solution with a volume concentration of 75% to 90%. In one embodiment, the feces preservation solution contains an ethanol aqueous solution with a volume concentration of 70% to 75%, and in one embodiment, the feces preservation solution contains an ethanol aqueous solution with a volume concentration of 75%.

[0021] The experimental results show that 75% ethanol as a fecal preservation solution for short-chain fatty acids is very beneficial for detecting the content of short-chain fatty acids in fecal samples. At -80°C to 50°C, the sample can still maintain the content of short-chain fatty acids in the sample stable after being placed for 7 days. The inventors verified that when the fecal samples were preserved with the preservation solution of the present invention at the preservation temperatures of -80°C, 4°C, 30°C and 50°C, the stability of each SCFA in the sample was better than that of the case where the preservation solution was not immediately frozen to -80°C (gold standard). Therefore, the preservation effect of the preservation solution of the present invention with 75% ethanol is better than the "gold standard" and has good stability, and it can be used as a preservation solution for general participants.

[0022] Furthermore, the inventors have found that although freezing the stool sample immediately can reduce the metabolic rate of the microorganisms in the sample, there are still microorganisms that can produce short-chain fatty acids that can metabolize at low temperatures, and the stool preservation solution provided by the present invention has a better effect in preserving samples.

[0023] In an optional embodiment, the stool preservation solution may also contain other substances, such as preservatives, to extend the shelf life.

[0024] The invention provides a method for preserving feces, which comprises the following steps: mixing a feces sample to be preserved with a feces preservation solution, wherein the effective component in the feces preservation solution is ethanol.

[0025] In a preferred embodiment of the present invention, the stool preservation solution comprises an ethanol aqueous solution.

[0026] In one embodiment, the feces preservation solution contains an ethanol aqueous solution with a volume concentration of 70% to 90%, such as an ethanol aqueous solution with a volume concentration of 71% to 85%, such as an ethanol aqueous solution with a volume concentration of 75% to 90%. In one embodiment, the feces preservation solution contains an ethanol aqueous solution with a volume concentration of 70% to 75%, and in one embodiment, the feces preservation solution contains an ethanol aqueous solution with a volume concentration of 75%.

[0027] For example, stool samples can be stored at -4°C to 50°C; stool samples can be stored at 4°C, 25°C, 30°C or 50°C. The samples still have good stability and can be stored and transported at room temperature.

[0028] The present invention also provides the use of ethanol in preparing a short-chain fatty acid detection reagent or a kit. The reagent or the kit contains a stool preservation solution, and the effective component in the stool preservation solution is ethanol.

[0029] The inventors evaluated the reliability and efficiency of collecting and storing feces at room temperature using the feces preservation solution of the present invention. Compared with fresh feces and fresh feces immediately frozen at -80°C, the feces samples were preserved for several days using the feces preservation solution of the present invention. The content of short-chain fatty acids (SCFA) in the samples was not significantly different from the content of SCFA in fresh feces or fresh feces immediately frozen at -80°C. The present invention shows the potential of using the preservation solution to collect and store at ambient temperature.

[0030] In one embodiment, the stool preservation solution contains an ethanol aqueous solution with a volume concentration of 70% to 90%. In one embodiment, the stool preservation solution contains an ethanol aqueous solution with a volume concentration of 70% to 75%. In one embodiment, the stool preservation solution contains an ethanol aqueous solution with a volume concentration of 75%.

[0031] In a preferred embodiment, 0.01-0.6 g of stool sample is stored in 1.8 ml of stool storage solution, for example 0.01-0.5 g, 0.1-0.6 g, 0.05-0.5 g or 0.08-0.6 g.

[0032] In a preferred embodiment of the present invention, the short-chain fatty acids include but are not limited to at least one of acetic acid, propionic acid, butyric acid, isobutyric acid, 2-methylbutyric acid, valeric acid, isovaleric acid, 3-methylvaleric acid, caproic acid and isocaproic acid (ICA).

[0033] The present invention also provides a method for sample pretreatment for short-chain fatty acid detection, which comprises the following steps:

[0034] (a) homogenizing the stool sample to be processed;

[0035] (b) after centrifugation, the supernatant is aspirated, diluted with acetonitrile solution, mixed (e.g., vortexed), and then centrifuged;

[0036] (c) taking the supernatant for derivatization and centrifuging;

[0037] (d) diluting the reaction solution obtained by centrifugation in step (c) with an acetonitrile solution containing formic acid.

[0038] The purpose of homogenization is to reduce the size of the dispersion and improve the uniformity of the dispersion. For example, a stool sample is placed in a grinder and ground at 60Hz for 1 minute. The derivatization reaction step is slowed down by a low-temperature centrifugation step after derivatization, and then the derivatization reaction is terminated with an acetonitrile solution containing formic acid. The prepared sample solution can be stably placed at ≤4°C for 15 days, which improves the sample's stability and helps improve detection efficiency.

[0039] The acetonitrile solution in steps (b) and (d) is an acetonitrile aqueous solution, and the volume concentration of the acetonitrile aqueous solution is 50% to 70%. In a more preferred embodiment of the present invention, the volume concentration of the acetonitrile aqueous solution is 50%.

[0040] In the above step (b), the acetonitrile solution is diluted to a level that every 1.8 mL of the acetonitrile solution contains 0.005 to 0.05 g of the fecal sample. At the above dilution ratio, it is easier to detect the short-chain fatty acids in the sample.

[0041] In a preferred embodiment of the present invention, the derivatization agent used in the derivatization in step (c) is 3-nitrophenylhydrazine hydrochloride;

[0042] In a preferred embodiment of the present invention, the centrifugation in step (c) is carried out at 0-10° C., and in a more preferred embodiment of the present invention, the centrifugation is carried out at 4° C. for 1 min. By lowering the sample temperature to slow down the derivatization reaction step, favorable conditions are provided for further termination of the reaction.

[0043] The volume concentration of formic acid in step (d) is 0.1% to 0.5%.

[0044] The stool sample mentioned above may be a stool sample stored in a stool preservation solution or a stool sample not containing a stool preservation solution, and the effective component in the stool preservation solution is ethanol.

[0045] In a preferred embodiment of the present invention, the stool preservation solution comprises an ethanol aqueous solution.

[0046] In one embodiment, the feces preservation solution contains an ethanol aqueous solution with a volume concentration of 70% to 90%, such as an ethanol aqueous solution with a volume concentration of 71% to 85%, such as an ethanol aqueous solution with a volume concentration of 75% to 90%. In one embodiment, the feces preservation solution contains an ethanol aqueous solution with a volume concentration of 70% to 75%, and in one embodiment, the feces preservation solution contains an ethanol aqueous solution with a volume concentration of 75%.

[0047] In a preferred embodiment of the present invention, the dilution factor of the reaction solution after derivatization is 15 times.

[0048] In a preferred embodiment of the present invention, the derivatization reaction conditions are derivatization at 40° C. for 30 minutes.

[0049] The present invention also provides a method for detecting short-chain fatty acids, comprising pre-treating a sample to be tested using the above method, and then detecting short-chain fatty acids in the pre-treated sample solution.

[0050] The present invention also provides a sample solution, which includes feces and a feces preservation solution, wherein the feces preservation solution includes an ethanol aqueous solution.

[0051] In one embodiment, the feces preservation solution contains an ethanol aqueous solution with a volume concentration of 70% to 90%, such as an ethanol aqueous solution with a volume concentration of 71% to 85%, such as an ethanol aqueous solution with a volume concentration of 75% to 90%. In one embodiment, the feces preservation solution contains an ethanol aqueous solution with a volume concentration of 70% to 75%, and in one embodiment, the feces preservation solution contains an ethanol aqueous solution with a volume concentration of 75%.

[0052] In one embodiment, each 0.01-0.6 g stool sample is stored in 1.8 ml stool storage solution.

[0053] The present invention has the following beneficial effects:

[0054] The fecal preservation solution provided by the present invention allows the collection and storage of fecal samples at room temperature (RT). The use of such a preservation solution enables participants to send samples by conventional mail without the need for appointments, refrigeration or cold chain transportation. The preservation solution of the present invention can well maintain the stability of the content of short-chain fatty acids in fecal samples, making sampling easier during quantitative detection of short-chain fatty acids, and can be stored and transported at room temperature, which is easy to popularize and promote.

[0055] In addition, the present invention also evaluates the reliability and efficiency of the preservation solution as a participant-friendly feces collection method (collection and storage under normal temperature conditions). The inventors verified that the stability of each SCFA in the sample was better than that of the sample immediately frozen to -80°C (gold standard) without a preservation solution when the 75% ethanol solution was used as a feces preservation solution at a storage temperature of -80°C, 4°C, 30°C and 50°C for 7 days. The proposal of the present invention shows the potential of using a preservation solution to collect and store at ambient temperature, which is convenient for studies aimed at collecting large samples by enabling participants to send samples by mail, thereby reducing the difficulty of accurate quantification of short-chain fatty acids, providing a scientific basis for short-chain fatty acid detection to become a clinical application for metabolic disease screening, early diagnosis, treatment monitoring and prognosis evaluation, and can promote the clinical application of short-chain fatty acid profiles. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0057] Figure 1 This is the experimental idea diagram for investigating the effect of the preservation solution;

[0058] Figure 2The chromatographic peaks of blank solvent, blank sample, fresh feces sample and mixed standard of short-chain fatty acids are overlaid;

[0059] Figure 3 Overlay of blank solvent and high concentration short-chain fatty acid mixed standard. DETAILED DESCRIPTION

[0060] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0061] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0062] The following are the reagents and equipment used in the examples of the present invention.

[0063] Table 1 Reagents and consumables

[0064]

[0065]

[0066] Continued from the table above

[0067]

[0068]

[0069] Table 2 Equipment parameters

[0070]

[0071] Continued from the table above

[0072]

[0073]

[0074] The following examples compare and analyze the short-chain fatty acid content in fresh stool samples, fresh stool samples immediately frozen at -80°C, and fresh stool samples immediately added with preservation solution and stored at different times and temperatures. The effect of storing samples at three time points (day 0, day 3, and day 6) under temperature conditions (-80°C, 4°C, 30°C, 50°C) on the SCFA content was evaluated. Specifically, day 0 is the stool sample collected on site, and the sample for short-chain fatty acid detection and analysis is immediately prepared.

[0075] Comprehensive analysis of the data showed that the SCFA content of fecal samples preserved with 75% ethanol as a fecal preservation solution and immediately frozen fecal samples was comparable to that of fresh fecal samples, with no significant difference. For specific technical routes, please refer to Figure 1 shown.

[0076] Example 1 Evaluation of the effect of the preservation solution

[0077] 1. Experimental design:

[0078] A fresh stool sample was used as the test sample, and 14 samples of approximately 0.01 g were weighed. The specific information is shown in Table 3.

[0079] Table 3 Sample storage conditions

[0080]

[0081] 2. Detect the content of each SCFA in samples under different storage conditions.

[0082] (1) Prepare samples from S1 to S6 and S9 to S12 according to the sample preparation method for analysis and record as the data of day 0; after sampling, place 10 samples and 4 fresh stool samples (S7, S8, S13, S14) without adding preservation solution in a refrigerator (-80℃ and 4℃) or an oven (30℃ and 50℃) according to the temperature shown in Table 3;

[0083] (2) On the third day, samples S1 to S7 and S9 to S13 were taken out and analyzed according to the sample preparation method. After sampling, the 12 samples were returned to their original locations;

[0084] (3) On the 6th day, samples S1-S6, S8, S9-S12, and S14 were taken out and analyzed according to the sample preparation method. After sampling, the 12 samples were returned to their original places. The data results of the tests on the 0th day, the 3rd day, and the 6th day are shown in Table 4. It can be seen from Table 4 that the six short-chain fatty acids AA, PA, IBA, BA, IVA, and VA were mainly detected in the samples.

[0085] Table 4 Results of short-chain fatty acid content of samples

[0086]

[0087] “-” means that the substance was not detected in this project. The possible reason may be that the content of the substance in the sample is lower than the detection limit of the instrument or the sample does not contain the substance. It should be noted that there is actually no storage temperature on Day 0. For the experimental group with preservation solution, the experimental group on Day 0 is to take fresh samples and put them in the preservation solution. Sample preparation and analysis are carried out after a while. The marked storage temperature is the temperature of the sample after sampling.

[0088] 3. Investigate the changes and differences in SCFA content in each sample under the conditions of using preservation solution and without preservation solution.

[0089] Based on the content data of 6 samples in the 75% ethanol preservation solution group (numbered 1-6 in Table 4) and 4 samples in the 50% acetonitrile preservation solution group (numbered 7-10 in Table 4) on day 0, the coefficient of variation of each SCFA content was calculated. For example, the coefficient of variation CV (0d) of AA is the coefficient of variation calculated based on the content data of numbers 1-10 under the AA column in Table 4. The CV values ​​of the detected SCFA contents are all less than 15%, indicating that there is no significant difference in the SCFA content measured by using 50% acetonitrile and using 75% ethanol to dissolve fresh feces. The data of the two can be compared with the same standard. The stability difference results of the SCFA content in each sample are shown in Table 5. 。

[0090] Table 5: Stability differences of 50% acetonitrile storage solution

[0091]

[0092] 4. Investigate the changes in SCFA content with storage time under different temperature conditions and conduct differential analysis.

[0093] (1) Samples were stored in 75% (V / V) ethanol at -80°C, 4°C, 30°C and 50°C (as shown in Table 6):

[0094] The coefficients of variation of AA, PA and BA contents within 7 days were all less than 7%; those of the control group were 9.92%, 12.38% and 12.90%, respectively.

[0095] The coefficients of variation of IBA, IVA and VA contents were all less than 19%; those of the control group were 17.85%, 24.26% and 18.74%, respectively.

[0096] (2) Samples were stored in 50% (V / V) acetonitrile at -80°C, 4°C, 30°C and 50°C (as shown in Table 6):

[0097] The coefficients of variation of AA, PA and BA contents within 7 days were all less than 5%; those of the control group were 6.81%, 7.89% and 6.84%, respectively.

[0098] The coefficients of variation of IBA, IVA and VA contents were all ≤13.00%; those of the control group were 11.66%, 17.88% and 16.11%, respectively.

[0099] The samples stored in 75% ethanol and 50% acetonitrile met the sample stability requirements, that is, the coefficient of variation of (short-chain fatty acids with higher content) AA, PA, and BA shall not exceed 15%; the coefficient of variation of (short-chain fatty acids close to the quantitative limit) IBA, IVA, and VA shall not exceed 20%; the coefficient of variation of (short-chain fatty acids with content far below the quantitative limit) 2-MBA, 3-MVA, ICA, and CA shall not exceed 30%, that is, when the temperature is in the range of -80°C to 50°C, the content of short-chain fatty acids in the samples stored in 75% ethanol or 50% acetonitrile is stable within 7 days. Moreover, for the group of samples "used as 75% ethanol control" in Table 6, when the samples were immediately frozen to -80°C without a preservation solution, the coefficient of variation of each SCFA in the samples was generally higher than that of the samples preserved in 75% ethanol preservation solution; similarly, in the 50% acetonitrile preservation solution group and its control group, when 50% acetonitrile preservation solution was also used, whether at a preservation temperature of -80°C, 4°C, 30°C or 50°C, the stability of each SCFA in the samples was better than that of the samples immediately frozen to -80°C without a preservation solution. Therefore, the preservation effects of the preservation solutions of 75% ethanol and 50% acetonitrile of the present invention are better than the "gold standard".

[0100] Table 6 Stability data analysis of samples stored at different temperatures (CV)

[0101]

[0102] Note: The coefficient of variation in Table 6 is based on the content data of samples taken and tested in a certain preservation solution or in the absence of preservation solution, at a certain fixed temperature, on the 0th, 3rd and 6th days, so as to calculate the coefficient of variation of these three time points. For example, the coefficient of variation of AA in 75% ethanol preservation solution and -80°C preservation temperature is calculated based on the content data of numbers 1, 12, and 24 in Table 4 (corresponding to the 0th, 3rd and 6th days, respectively).

[0103] The coefficient of variation (CV) is calculated as follows:

[0104]

[0105] Here, the numerator is the standard deviation and the denominator is the mean.

[0106] 5. Analyze the coefficient of variation of the SCFA content in samples under different storage conditions with the same storage time.

[0107] The content of the above point 4 shows that after the fecal sample is stored in the temperature range of -80℃ to 50℃ for 7 days, the content of short-chain fatty acids in the sample is stable. Therefore, by comparing the average value of short-chain fatty acids in each preservation solution tested on the same day with the content data of the control group, it can be determined whether the change in SCFA in the preservation solution is higher or lower than that in the control group.

[0108] First, AVERAGE in Table 7 refers to the average value of the content of all samples in the group of the preservation solution at each preservation temperature on day 0, 3 or 6. For example, the AVERAGE value of the 0d, 75% ethanol group is the average value of the SCFA content measured in the experimental groups numbered 1-6 in Table 4.

[0109] Secondly, CV (0d, 3d, 6d) is the coefficient of variation calculated based on the average value of the SCFA content in each group at each time point (including 0 days, 3 days, and 6 days) in Table 7 and the content result of the control group, such as the CV (0d, 3d, 6d) value of AA is the coefficient of variation calculated based on all the values ​​under the "AA column" in the table. The CV (0d, 3d, 6d) values ​​of the short-chain fatty acids detected in this experiment are all less than 15%, except for IVA. On the whole, the short-chain fatty acid content between the preservation solution 75% ethanol, the preservation solution 50% acetonitrile, and the preservation solution without preservation solution in this experiment is not much different. And the content of each SCFA in each control sample (fresh feces immediately frozen at -80°C) detected on the 3rd and 6th days is higher than the average content of each SCFA in the sample containing 75% ethanol preservation solution or 50% acetonitrile preservation solution at the same time point, indicating that although immediate freezing can reduce the metabolic rate of microorganisms in the sample, there are still microorganisms that can produce short-chain fatty acids that can metabolize at low temperatures, and the feces preservation solution provided by the present invention has a better effect on preserving samples.

[0110] Table 7 Stability data analysis of samples stored at different times (CV)

[0111]

[0112] Note: The lower limit of quantification of IVA (isovaleric acid) is 0.1 ug / ml (as shown in Table 13), which is about 0.02 mg / g. The average content or content of IVA (isovaleric acid) in Table 7 is 0.01 or 0.02 mg / g, which is less than or equal to the lower limit of quantification, so its CV close to 20% is acceptable.

[0113] Example 2

[0114] This embodiment provides a method for detecting the content of short-chain fatty acids in the sample of Example 1.

[0115] 1. Sample preparation method.

[0116] (1) Sample collection (the collection tube contains 1 metal grinding bead):

[0117] ① Samples with preservation solution: Take about 0.01g of fresh feces or intestinal contents and preserve it in 1.8ml of preservation solution. Invert it upside down dozens of times. Preservation solution 1 is 50% (V / V) acetonitrile aqueous solution, and preservation solution 2 is 75% (V / V) ethanol aqueous solution.

[0118] ② Control sample without preservation solution: Feces were collected directly and immediately frozen at -80℃.

[0119] ③Fresh stool samples without preservation fluid: Collect stool directly and use immediately.

[0120] (2) Sample pretreatment:

[0121] a) Weigh the sample: For the control sample without preservation solution and the fresh stool sample without preservation solution, take about 0.01g of fresh stool or intestinal contents into a 2mL centrifuge tube, add 1.8mL 50% ACN, and add 1 metal grinding bead. The sample containing preservation solution can be directly homogenized.

[0122] b) Sample homogenization: The samples of ①, ②, and ③ were subjected to the same operation: they were placed in a tissue grinder and ground at 60 Hz for 1 min, and then placed in a 4°C centrifuge and centrifuged at 13,000 rpm for 10 min (4°C).

[0123] c) Sample derivatization: 20 μL of the supernatant was taken and added with 10 μL of 200 mM 3-NPH·HCl (3-nitrophenylhydrazine hydrochloride), 10 μL of 200 mM EDC·HCl (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride), 80 μL of 50% (V / V) acetonitrile, 50 μL of 7% Pyridine, and 1 μL of internal standard solution (Acetic acid-13C2 concentration was 50 μg / ml, Isobutyric acid-d6 concentration was 12 μg / ml, Propionic acid-d5, Butanonic acid-d7, Isovaleric acid-d9, Valeric acid-d3, and Caproic acid-d5 concentrations were all 15 μg / ml), vortexed for 1 min, centrifuged for 1 min, and derivatized at 40°C for 30 min.

[0124] (3) Sample dilution and loading

[0125] 20 μL of the derivatized reaction solution was added to 280 μL of 50% (V / V) acetonitrile aqueous solution, vortexed for 30 seconds, centrifuged at 13000 rpm for 10 minutes (4° C.), and then the supernatant was pipetted into a sampling bottle for LC-MS / MS analysis.

[0126] 2. Determine the parameters in the above detection method.

[0127] The inventors compared different chromatographic columns such as waters-ACQUITY UPLC BEH C18 ( 1.7μm, 2.1mmX 100mm), Phenomenex (Kinetex 2.6μC18, 100x3.00mm), Phenomenex LC Column (1.7μmC18 ,50x2.1mm), and finally Phenomenex (Kinetex 2.6μC18,100x3.00mm) was selected based on the comprehensive chromatographic peak separation effect and analysis time.

[0128] 3. Refer to Table 8 and Table 9 for system parameters.

[0129] Table 8. LC 30 parameters

[0130]

[0131] Table 9. SCIEX QTRAP 5500 triple quadrupole / linear ion trap tandem mass spectrometer parameters

[0132]

[0133]

[0134] Example 3

[0135] This example evaluates the detection method provided in Example 2.

[0136] 1. Exclusivity

[0137] Fresh feces samples were used as test samples, mixed short-chain fatty acid standards were used as reference samples, 50% (V / V) acetonitrile was used to prepare blank control samples, and 50% (V / V) acetonitrile was used as double blanks for detection. The results are shown in Figure 2 The blank control peak data analysis is shown in Table 10 and Table 11. Figure 2 It can be seen that in 50% (V / V) acetonitrile, only acetic acid has an interference peak, and the ratio of its peak area to the quantitative lower limit peak area is 0.06%. The SA / IA ratio of the blank control and the quantitative lower limit of each short-chain fatty acid is in the range of 1% to 20%, which meets the acceptable standard of specificity, and the response of the interfering component is no higher than 20% of the quantitative lower limit response of the analyte.

[0138] Table 10 Blank control-50% (V / V) acetonitrile data analysis

[0139]

[0140] Note: SA is the peak area of ​​the analyte (Sample Area), IA is the peak area ratio of the internal standard (Internal Standards Area); "-" means that the substance was not detected in this project, which may be because the content of the substance in the sample is lower than the instrument detection limit or the sample does not contain the substance.

[0141] Table 11 Blank control data analysis

[0142] AA PA IBA BA 2-MBA IVA VA 3-MVA ICA CA Blank Sample SA / IA 0.33 0.14 0.02 0.05 0.30 0.01 0.04 0.02 0.00 0.14 Quantitation limit SA / IA 1.61 1.38 0.70 2.36 1.86 0.32 0.72 0.23 0.41 2.84 percentage(%) 20％ 10％ 2％ 2％ 16％ 2％ 5％ 7％ 1％ 5％

[0143] Note: SA / IA=Sample Area / Internal Standards Area.

[0144] 2. Residue analysis.

[0145] The blank solvent 50% (V / V) acetonitrile and high-concentration short-chain fatty acid mixed standard were used as test samples. A blank solvent was injected before the sequence started, and a blank solvent was injected again after the high-concentration short-chain fatty acid mixed standard was tested to verify the residual situation. The results are shown in Figure 3 And Table 12. From Table 12 and Figure 3 It can be seen that in the first injection of 50% (V / V) acetonitrile, there are interfering peaks at acetic acid and butyric acid, and the ratios of their peak areas to the quantitative lower limit peak area are 0.08% and 0.04%, respectively, and the other target peaks have no interference; in the second injection of 50% (V / V) acetonitrile, there is only an interfering peak at acetic acid, and the ratio of its peak area to the quantitative lower limit peak area is 0.11%, and the other target peaks have no interference, all <2%, which meets the acceptable standard for residues, and the response of the interfering component is lower than 20% of the quantitative lower limit response of the analyte.

[0146] Table 12 Residue data analysis.

[0147]

[0148] 3. Linear

[0149] Accurately weigh each short-chain fatty acid reference substance, dissolve and dilute with 50% (V / V) acetonitrile to obtain single stock solutions of 10 standards with concentrations of AA 11.6 mg / mL, PA 11.3 mg / mL, IBA 11.3 mg / mL, BA 10.1 mg / mL, 2-MBA 10.1 mg / mL, IVA 10.5 mg / mL, VA 10.9 mg / mL, 3-MVA 12.1 mg / mL, ICA 10.6 mg / mL, and CA 11.6 mg / mL. Take appropriate amount of each standard stock solution, dilute to 10mL with 50% acetonitrile (V / V) to obtain mixed standard solution, the concentrations are: AA 77.33μg / mL, PA 28.25μg / mL, IBA 18.83μg / mL, BA 30.30μg / mL, 2-MBA 7.58μg / mL IVA 17.75μg / mL, VA 27.25μg / mL, 3-MVA 30.25μg / mL, ICA 15.75μg / Ml, CA 24.17μg / mL, and dilute stepwise with 50% (V / V) acetonitrile solution to obtain a series of mixed standard solutions with different concentrations. Prepare 3 samples at each concentration level, and inject 1 injection each. Linear regression is performed with sample concentration as the horizontal axis and the peak area ratio y of the standard to the internal standard as the vertical axis to obtain the linear determination coefficient R 2 , investigate the linearity of the method. As shown in Table 13, AA, PA, IBA, BA, 2-MBA, IVA, VA, 3-MVA, ICA and CA have good linear relationships within the corresponding concentration ranges, and the correlation coefficients are all greater than 0.996. The linear investigation meets the acceptable standards.

[0150] Table 13 Linear equations, correlation coefficients and quantitative ranges of short-chain fatty acids.

[0151] ID Eqμation Weighting <![CDATA[R 2 ]]> LLOQ ULOQ AA y=0.32790x+0.72644 1 / x 0.99733 3.02 77.33 PA y=1.56216x+0.33984 1 / x 0.99785 0.74 28.25 IBA y=1.29364x+0.24597 1 / x 0.99738 0.37 18.83 BA y=1.24018x+0.51706 1 / x 0.99648 1.58 30.30 2-MBA y=3.55389x+0.45081 1 / x 0.99937 0.39 7.58 IVA y=1.41211x+0.17910 1 / x 0.99842 0.10 15.75 VA y=0.73881x+0.10776 1 / x 0.99913 0.85 27.25 3-MVA y=0.72697x+0.04758 1 / x 0.99943 0.24 30.25 ICA y=1.49530x+0.08759 1 / x 0.99887 0.21 15.9 CA y=1.34369x+1.02125 1 / x 0.99707 1.51 24.17

[0152] Note: Eqμation is the linear equation, R2 is the determination coefficient, Weighting is the weight, LLOQ (μg / mL) and ULOQ (μg / mL) are the lower limit of quantification and the upper limit of quantification respectively. Accurate quantification can be achieved within the range of the two.

[0153] 4. Stability

[0154] 4.1 Stability of sample solution at ≤4°C.

[0155] Using mixed standard (mixed standard is a standard sample containing these 10 short-chain fatty acids, which is different from the real sample in that there is no complex matrix interference and only 50% acetonitrile is used as the solvent) as the test sample, three samples with high, medium and low concentration levels were prepared respectively. The samples were placed at ≤4℃, and one injection was made at 0:00, 0:09, 0:19, 0:28, 0:38, 0:48, 3:00 and 6:00 respectively. The concentrations (ug / ml) of AA, PA, IBA, BA, 2-MBA, VA, IVA, -3MVA, ICA and CA in the samples placed at ≤4℃ at 0:00, 0:09, 0:19, 0:28, 0:38, 0:48, 3:00 and 6:00 were recorded, and the relative difference percentage values ​​(RD) between the concentrations detected at each time point and the concentration detected at 0:00 were calculated as shown in Tables 14-1 and 14-2.

[0156] The relative difference percentage of each SCFA in the high-concentration mixed standard within 6 hours was ≤7.1%; the relative difference percentage of AA and CA detected at 0:00 after 6:00 and 0:00 under ≤4℃ was 16.2% and 27.2% respectively, and the RD values ​​of other SCFA were ≤10.7%; the RD of AA and CA after 3:00 of the low-concentration mixed standard was 39.9% and 38.0% respectively, and the RD of AA, VA and CA after 6:00 was 87.7%, 17.2% and 72.6% respectively, and the RD values ​​of other SCFA were ≤11.4%. According to the acceptable standard of stability resistance: the coefficient of variation shall not exceed 15%, it can be known that the high-concentration sample solution can be placed at ≤4℃ for 6h, the medium-concentration sample solution can be placed at ≤4℃ for 3h, and the low-concentration sample needs to be detected and analyzed as soon as possible.

[0157] The calculation formula of RD (Relative Percent Difference) is:

[0158]

[0159] For this application, the calculation formula is: (concentration measured at other time points - concentration measured at 0:00) / concentration measured at 0:00 * 100%

[0160] Table 14 Stability data analysis of sample solutions at ≤4°C

[0161]

[0162]

[0163] Table 14-2 Stability data analysis of sample solutions at ≤4°C

[0164]

[0165] 4.2 Stability of sample solution at ≤-80℃

[0166] Using the mixed standard as the test sample, three concentration levels of high, medium and low were prepared, and 6 injections were made at 0 days. The samples were placed at ≤-80℃ for 1 day, 2 days, 3 days, and 6 days. After being taken out at each time point, they were restored to room temperature and placed in a 4℃ injector for 3 injections. The average SCFA concentrations of AA, PA, IBA, BA, 2MBA, VA, IVA, 3MVA, ICA and CA in the samples were calculated at ≤-80℃ for 0 days, 1 day, 2 days, 3 days, and 6 days, and the coefficient of variation of the three concentration levels for 7 days was calculated.

[0167] The results are shown in Table 15. The concentration coefficients of variation of the sample solutions at high, medium and low concentration levels tested at ≤-80℃ for 7 days were all less than 6%. All three sets of data met the acceptable stability standard: the coefficient of variation should not exceed 15%. Therefore, the sample solutions can be stably placed at ≤-80℃ for 7 days.

[0168] Table 15 Stability data analysis of sample solutions at ≤-80°C

[0169]

[0170] 5. Accuracy

[0171] Take 3 portions of 10 μL of the fresh fecal sample solution collected in step (1) to test the content of each SCFA in the samples under different storage conditions in Example 2 and add appropriate amounts of short-chain fatty acid standard solution to prepare 3 concentration levels for spike recovery experiments. Prepare 3 samples for each concentration level, and inject 1 injection for each. Substitute the average value of each concentration level into the linear equation to obtain the concentration of each concentration level, and take the amount of each short-chain fatty acid added plus the background value as the theoretical value. The results are shown in Table 16.

[0172] It can be seen from Table 16 that, except for the low concentration level CA recovery rate of 75% (combining Table 14-1 and Table 14-2, Table 15 and Table 16, it can be seen that CA was not detected in the sample, and the CA added at a low concentration level was close to the quantitative limit of 1.51 μg / mL), the recovery rates of the other three concentration levels were all in the range of 95% to 110%, which met the acceptable standards.

[0173] Table 16 Accuracy recovery rate investigation

[0174]

[0175] 6. Repeatability

[0176] Fresh fecal samples were used as test samples, and 6 test sample solutions were prepared, each with 3 injections. The repeatability data analysis is shown in Table 17. As shown in Table 17, the CV values ​​of AA, PA, BA, IBA, IVA, VA, 2-MBA, and ICA in the 6 test sample solutions were 5.14%, 4.44%, 6.00%, 14.66%, 12.47%, 9.28%, 19.74%, and 11.39%, respectively; 3-MVA and CA were not detected. The acceptable repeatability standard was met, that is, the coefficient of variation of AA, PA, and BA should not exceed 15%; the coefficient of variation of IBA, IVA, and VA should not exceed 20%; the coefficient of variation of 2-MBA, 3-MVA, ICA, and CA should not exceed 30%.

[0177] Table 17 Repeatability data analysis

[0178]

[0179] Example 4 Requirements for sampling amount in sample detection method

[0180] Take a stool sample as the test sample and prepare 9 test sample solutions (the sampling volume is about 0.01g, 0.03g, and 0.09g for 3 samples respectively).

[0181] The sample size was 0.01 g of the test sample, and the sample preparation method was as described in "1. Sample preparation method" in Example 2. The sample size was 0.03 g and 0.09 g. After homogenization, the sample dilution step was added to keep the ratio unchanged (0.01 g sample: 1.8 mL acetonitrile solution), and the other parameters remained unchanged. The specific dilution and content results are shown in Table 18:

[0182] Table 18 SCFA content in samples measured at different sampling volumes

[0183]

[0184]

[0185] Note: “-” indicates that the CV value of the short-chain fatty acid was not calculated in this project because the content of the substance in the sample was below the linear quantification limit.

[0186] As can be seen from the table above, the sample mainly contains AA (acetic acid) and PA (propionic acid), and the results of other short-chain fatty acid contents are all below the quantitative limit, so only the coefficient of variation of acetic acid and propionic acid is calculated. From the results, it can be seen that the coefficient of variation of acetic acid and propionic acid detected with a sampling volume of about 0.01g to 0.09g is less than 15%, indicating that there is no significant change in the content of short-chain fatty acids in this value range, and the sampling range can be set to 0.01g to 0.09g.

[0187] Example 5 Optimization of sample preparation method in sample detection

[0188] As can be seen from 4.1 in Example 3, in order to ensure the accuracy of the sample content determination, the sample solution is preferably tested as soon as possible, the high-concentration sample solution can be placed at ≤4°C for 6 hours, the medium-concentration sample solution can be placed at ≤4°C for 3 hours, and the low-concentration sample needs to be tested and analyzed as soon as possible. This condition limits the detection efficiency. To solve this problem, the inventor optimized the sample preparation method, adding a step of slowing down the derivatization reaction (after the derivatization reaction, centrifuge at low temperature for 1 minute) and a step of terminating the derivatization reaction (using 50% ACN containing 0.1% formic acid as the diluent for dilution).

[0189] 1. The optimized sample detection method is as follows:

[0190] (1) Sample collection (the collection tube contains 1 metal grinding bead):

[0191] ① Samples with preservation solution: Take a certain amount of fresh feces or intestinal contents and preserve them in preservation solution, turn them upside down dozens of times. Preservation solution 1 is 50% ACN, and preservation solution 2 is 75% (V / V) ethanol aqueous solution.

[0192] ② Control sample without preservation solution: Feces were collected directly and immediately frozen at -80℃.

[0193] ③Fresh stool samples without preservation fluid: Collect stool directly and use immediately.

[0194] (2) Sample pretreatment:

[0195] a) Weighing samples: For ① control samples without preservation solution and ② fresh stool samples without preservation solution, weigh a certain weight of sample (0.01g-0.09g) into a 2mL centrifuge tube, add 1mL 50% ACN, and add 1 metal grinding bead. Samples containing preservation solution can be directly homogenized.

[0196] b) Sample homogenization: The samples of ①, ②, and ③ were subjected to the same operation: they were placed in a tissue grinder and ground at 60 Hz for 1 min, and then placed in a 4°C centrifuge and centrifuged at 13,000 rpm for 10 min (4°C).

[0197] c) Sample dilution: Take a certain amount of supernatant, dilute the sample in proportion, dilute to 0.01g sample per 1.8mL 50% ACN solution, vortex for 30s, centrifuge for 30s. For example, the operation for taking 0.03g feces sample is: weigh 0.0298g sample, dissolve it with 1ml 50% ACN solution, take 100ul supernatant after homogenization, add 436uL50% ACN (equivalent to 0.0298g + 5.364mL 50% ACN), and finally 1.8mL 50% ACN solution contains 0.01g sample.

[0198] d) Derivatization: 20 μL of the supernatant was taken and added with 10 μL of 200 mM 3-NPH·HCl, 10 μL of 200 mM EDC·HCl, 80 μL of 50% ACN, 50 μL of 7% Pyridine and 1 μL of internal standard solution (Acetic acid-13C2 concentration was 50 μg / ml, Isobutyric acid-d6 concentration was 12 μg / ml, Propionic acid-d5, Butanonic acid-d7, Isovaleric acid-d9, Valeric acid-d3 and Caproic acid-d5 concentrations were all 15 μg / ml), vortexed for 1 min, centrifuged for 1 min, derivatized at 40°C for 30 min, and centrifuged at 4°C, 13,000 rpm for 1 min.

[0199] (3) Dilution and loading

[0200] First, prepare an acetonitrile solution containing 0.1% formic acid: 1 mL of formic acid is dissolved in 999 mL of 50% acetonitrile aqueous solution. Take 280 μL of the solution, add 20 μL of the derivatized reaction solution, vortex for 30 seconds, centrifuge at 13000 rpm for 10 minutes (4°C), and then draw the supernatant into the injection bottle for LC-MS / MS analysis.

[0201] 2. Comparison of the optimized sample preparation method and the sample preparation method shown in Example 2

[0202] Four stool samples were prepared according to "1. Sample preparation method" (hereinafter referred to as "original method") and the optimized method (as follows) in Example 2, respectively. The results are shown in Table 18 for data comparison of the original method and the optimized method. It can be seen from Table 19 for data comparison of the original method and the optimized method that the contents of IBA, 2-MBA, IVA, VA, 3-MVA, ICA, and CA in the four stool samples are relatively low, and the concentrations do not reach the lower limit of quantification. The data results are for reference only, and the difference between the original method and the optimized method is not calculated; while the contents of AA, PA, and BA in the samples are relatively high, and the relative deviation range of AA, PA, and BA detected by the optimized method and AA, PA, and BA detected by the original method is 0.7% to 6.5% (<15%), that is, there is no significant difference in the results of short-chain fatty acids detected by the two methods. From the stability results of the samples prepared by the optimized method at ≤4°C, Table 20 shows that the coefficient of variation of the samples prepared by the optimized method placed at ≤4°C for 15 days ranges from 0.2% to 1.8%. According to the acceptable standard for stability: the coefficient of variation shall not exceed 15%, it can be seen that the sample solution prepared by the optimized method can be stably placed at ≤4°C for 15 days.

[0203] Table 19 Data comparison between the method of Example 2 and the optimized method

[0204]

[0205] Note: A is the original method, B is the optimized method; ① is the calculated concentration (ug / ml), ② is the relative deviation of the two methods, and the calculation formula is: Since the concentration results of IBA, 2-MBA, IVA, VA, 3-MVA, ICA, and CA did not reach the quantitative limit, the data results are for reference only, and the difference between the two was not calculated.

[0206] Table 20 Stability of various short-chain fatty acids in samples prepared by the optimized method at ≤4°C.

[0207]

[0208]

[0209] Note: ① is Calculated Concentration (ug / ml), ② is the coefficient of variation of the short-chain fatty acid content detected on the 1st, 2nd, 3rd, 4th and 15th days of the optimized method; since the concentration results of IBA, 2-MBA, IVA, VA, 3-MVA, ICA and CA did not reach the quantitative limit, the data results are for reference only and their coefficient of variation was not calculated.

[0210] Linearity evaluation of the optimized method.

[0211] According to the content of short-chain fatty acids in the actual samples and the results in Table 13, the linear standard solution was reconfigured (see Table 21 below), and 3 samples were prepared at each concentration level, and 1 injection was made for each sample. Linear regression was performed with the sample concentration as the horizontal axis and the peak area ratio y of the standard and the internal standard as the vertical axis to obtain the linear determination coefficient R 2 , investigate the linearity of the optimization method. From the results in Table 22, it can be seen that AA, PA, IBA, BA, 2-MBA, IVA, VA, 3-MVA, ICA and CA have good linear relationships within the corresponding concentration ranges, and the correlation coefficients are all greater than 0.997. The linear investigation meets the acceptable standards.

[0212] Table 21 Standard sample concentration (μg / mL)

[0213] AA PA BA IBA 2-MBA IVA VA 3-MVA ICA CA STD 58.00 24.86 24.24 11.30 10.10 14.70 15.26 9.68 14.84 16.24 1 48.33 20.72 20.20 9.42 8.42 12.25 12.72 8.07 12.37 13.53 2 24.17 10.36 10.10 4.71 4.21 6.13 6.36 4.03 6.18 6.77 3 12.08 5.18 5.05 2.35 2.10 3.06 3.18 2.02 3.09 3.38 4 6.04 2.59 2.53 1.18 1.05 1.53 1.59 1.01 1.55 1.69 5 3.02 1.29 1.26 0.59 0.53 0.77 0.79 0.50 0.77 0.85 6 1.51 0.65 0.63 0.29 0.26 0.38 0.40 0.25 0.39 0.42 7 0.76 0.32 0.32 0.15 0.13 0.19 0.20 0.13 0.19 0.21 8 0.38 0.16 0.16 0.07 0.07 0.10 0.10 0.06 0.10 0.11 9 0.19 0.08 0.08 0.04 0.03 0.05 0.05 0.03 0.05 0.05 10

[0214] Table 22 Linear equation, correlation coefficient and quantitative range of short-chain fatty acids (optimized method)

[0215]

[0216]

[0217] Note: Eqμation is a linear equation, R 2 is the determination coefficient, Weighting is the weight, LLOQ (μg / mL) and ULOQ (μg / mL) are the lower limit of quantification and the upper limit of quantification, respectively. Accurate quantification can be achieved within the range of the two.

[0218] In summary, the present invention tested two preservation solutions that allow stool samples to be collected and stored at room temperature (RT). The use of such preservation solutions enables participants to send samples by regular mail without the need for appointments, refrigeration or cold chain transportation. The present invention also evaluated the reliability and efficiency of the collection and storage of the preservation solution under room temperature conditions, and there was no significant difference in SCFA content compared with fresh feces and fresh feces immediately frozen at -80°C. The proposal of the present invention highlights the potential for collection and storage at ambient temperature using a preservation solution, which is convenient for studies aimed at collecting large samples by enabling participants to send samples by mail, thereby reducing the difficulty of accurate quantification of short-chain fatty acids, and providing a scientific basis for short-chain fatty acid detection to become a clinical application such as metabolic disease screening, early diagnosis, treatment monitoring and prognosis evaluation, which can promote the clinical application of short-chain fatty acid profiles.

[0219] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of ethanol in the preparation of fecal preservation solution, It is characterized in that The stool preservation solution contains only an ethanol aqueous solution with a volume concentration of 75%, and the stool preservation solution is used to maintain the stability of the short-chain fatty acid content in the stool sample.

2. Application of fecal preservation solution in the preparation of short-chain fatty acid detection reagents or kits, It is characterized in that The stool preservation solution contains only an ethanol aqueous solution with a volume concentration of 75%, and the stool preservation solution is used to maintain the stability of the short-chain fatty acid content in the stool sample.

3. A method for preserving feces, It is characterized in that The method comprises the following steps: mixing a stool sample to be preserved with a stool preservation solution, wherein the stool preservation solution contains only an ethanol aqueous solution with a volume concentration of 75%, and the stool preservation solution is used to maintain the stability of the short-chain fatty acid content in the stool sample.

4. The method for preserving feces according to claim 3, It is characterized in that Each 0.01-0.6 g stool sample was stored in 1.8 ml stool preservation solution.

5. The method for preserving feces according to claim 3, It is characterized in that The stool samples were stored at -4°C to 50°C.

6. The method for preserving feces according to claim 5, It is characterized in that Stool samples were stored at 4°C, 25°C, 30°C, or 50°C.

7. A method for sample pretreatment for short-chain fatty acid detection, It is characterized in that It includes the following steps: (a) homogenizing the stool sample to be processed; (b) After centrifugation, the supernatant was aspirated, diluted with acetonitrile solution, mixed and then centrifuged; (c) taking the supernatant for derivatization and centrifuging; (d) diluting the reaction solution obtained by centrifugation in step (c) with an acetonitrile solution containing formic acid; The stool sample is a stool sample preserved in a stool preservation solution or a stool sample without a stool preservation solution, and the stool preservation solution only contains an ethanol aqueous solution with a volume concentration of 75%.

8. The method for sample pretreatment for short-chain fatty acid detection according to claim 7, It is characterized in that The acetonitrile solution in steps (b) and (d) is an acetonitrile aqueous solution, and the volume concentration of the acetonitrile aqueous solution is 50% to 70%.

9. The method for sample pretreatment for short-chain fatty acid detection according to claim 8, It is characterized in that The volume concentration of the acetonitrile aqueous solution is 50%.

10. The method for sample pretreatment for short-chain fatty acid detection according to claim 7, It is characterized in that In step (b), the stool sample is diluted with acetonitrile solution until each 1.8 mL of acetonitrile solution contains 0.005-0.05 g of stool sample.

11. The method for sample pretreatment for short-chain fatty acid detection according to claim 7, It is characterized in that The derivatization reagent used in the derivatization in step (c) is 3-nitrophenylhydrazine hydrochloride.

12. The method for sample pretreatment for short-chain fatty acid detection according to claim 7, It is characterized in that The centrifugation in step (c) is performed at 0-10°C.

13. The method for sample pretreatment for short-chain fatty acid detection according to claim 12, It is characterized in that Centrifuge at 4 °C for 30-60 s.

14. The method for sample pretreatment for short-chain fatty acid detection according to claim 7, It is characterized in that The volume concentration of formic acid in the step (d) is 0.1% to 0.5%.

15. A method for detecting short-chain fatty acids, It is characterized in that The method comprises pre-treating the sample to be tested by the method described in any one of claims 7 to 14, and then detecting short-chain fatty acids in the pre-treated sample solution.

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