CN115486439B - Feces preservation method, use of ethanol in preparing feces preservation solution and short-chain fatty acid detection reagent or kit - Google Patents
Feces preservation method, use of ethanol in preparing feces preservation solution and short-chain fatty acid detection reagent or kit Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N1/122—Preservation or perfusion media
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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Abstract
本发明公开了粪便保存方法、乙醇在制备粪便保存液及短链脂肪酸检测试剂或试剂盒中的应用，涉及粪便保存技术领域。粪便保存液中的有效成分为乙醇。本发明提供的粪便保存液可允许在室温下收集和储存粪便样本。使用此类保存液使参与者能够通过常规邮寄方式发送样品，而无需预约，冷藏或冷链运输。本发明的保存液可以很好地保持粪便样本中短链脂肪酸的含量稳定，使得短链脂肪酸的定量检测时采样变得更加简单，可常温保存及运输，易于普及推广。本发明的提出还有助于开发相应的短链脂肪酸检测试剂或试剂盒。
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
The invention relates to the technical field of feces preservation, in particular to a feces preservation method and application of ethanol in preparation of feces preservation liquid and short-chain fatty acid detection reagent or kit.
Background
Short Chain Fatty Acids (SCFA) are produced in the gut and are critical to maintaining healthy gut microbiota, balancing lipid and carbohydrate metabolism, insulin sensitivity and natural immunity, and have important effects of maintaining water electrolyte balance, combating pathogenic microorganisms and inflammation, regulating gut microbiota balance, improving gut function, modulating immunity, combating tumors, regulating gene expression, and the like. Can be used as corresponding indexes of the post-treatment effect of treatment means (including probiotic drug treatment, flora transplantation, other treatment drugs and the like) clinically. However, since metabolism and proliferation of certain bacteria may continue, thereby affecting the microbial structure and SCFA profile, literature indicates that SCFA increases almost proportionally over time at 4 ℃ 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.], and thus it is generally not recommended to store stool samples at room temperature or at 4 ℃ for more than 12 hours. This presents an important challenge for analysis of short chain fatty acids, as the cold chain is not always maintained or guaranteed from sample collection to frozen storage. Specimens collected in the field typically take different times at room temperature and are then transported to a central laboratory for processing or storage with frozen gel packaging (4 ℃) or dry ice, i.e., the sample may have been exposed to room temperature for several hours before reaching the laboratory.
Generally, the immediate freezing of samples at-80 ℃ is considered a reference method, but such a reference method is limited by the harsh storage conditions and is not viable in many cases. In contrast, most households have a freezer at-20 ℃ and can also freeze samples at-20 ℃, but this requires that one be willing to place the sample in the freezer and have resources to transport the sample in the frozen state. Current methods of collecting fecal samples are primarily collected by participants at home to schedule reservations and store the samples in their own refrigerators or freezers on the day of collection, which, as noted above, can be uncomfortable for the participants and can present health risks. These factors may negatively impact the decision of the participants to participate in the study. Most of the time, the standard collection method, which requires immediate freezing, may not be feasible.
In summary, sample preservation and transportation difficulties are important reasons why the short-chain fatty acid detection technology has not been developed as a conventional detection project by domestic medical institutions, which limits the clinical application of short-chain fatty acids.
Therefore, the preservation research of the sample becomes a key to the accuracy of short chain fatty acid detection.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a stool sample preservation solution, which solves the problem of stool sample preservation during detection of short-chain fatty acid.
The invention is realized in the following way:
the invention provides a reagent or a kit, which contains a fecal preservation solution, wherein the active ingredient of the fecal preservation solution is ethanol.
The fecal preservation solution provided by the present invention may allow for collection and storage of fecal samples at room temperature. The use of such preservation solutions allows participants to send samples by conventional mailing without the need for reservation, refrigeration or cold chain transport. The preservation solution can well keep the content of the short-chain fatty acid in the fecal sample stable, so that the sampling becomes simpler during quantitative detection of the short-chain fatty acid, and the preservation solution can be preserved and transported at normal temperature and is easy to popularize. Is helpful to develop corresponding short chain fatty acid detection reagent or kit.
In a preferred embodiment of the present invention, the feces storage solution comprises an aqueous ethanol solution.
In one embodiment, the fecal preservation solution contains an aqueous ethanol solution with a volume concentration of 70% -90%, in one embodiment, the fecal preservation solution contains an aqueous ethanol solution with a volume concentration of 70% -75%, in one embodiment, the fecal preservation solution contains an aqueous ethanol solution with a volume concentration of 75%.
The invention provides an application of ethanol in preparing a fecal preservation solution, wherein the active ingredient in the fecal preservation solution is ethanol.
The current research of stool preservation solution mainly focuses on DNA extraction, and no special stool sample preservation solution for short-chain fatty acid detection is found.
The inventor finds that the arrangement of ethanol in the fecal preservation solution can effectively meet the fecal sample preservation requirement of short-chain fatty acid detection with low burden.
In particular, the fecal preservation solution provided by the present invention may allow for collection and storage of fecal samples at Room Temperature (RT). The use of such preservation solutions allows participants to send samples by conventional mailing without the need for reservation, refrigeration or cold chain transport. The preservation solution can well keep the content of the short-chain fatty acid in the fecal sample stable, so that the sampling becomes simpler during quantitative detection of the short-chain fatty acid, and the preservation solution can be preserved and transported at normal temperature and is easy to popularize.
In addition, the reliability and efficiency of the preservation solution as a participant excrement friendly collecting method (collecting and storing under normal temperature condition) are evaluated, and compared with fresh excrement and fresh excrement which are immediately frozen at-80 ℃, after the excrement sample is preserved by adopting the excrement preservation solution of ethanol, the content of Short Chain Fatty Acid (SCFA) in the sample is equivalent, and no obvious difference exists. The invention provides a potential for collecting and storing preservation solution at the ambient temperature, which is convenient for research aiming at collecting a large sample by the possibility that participants can send samples through mailing, thereby reducing the difficulty of accurately quantifying short-chain fatty acid, providing scientific basis for short-chain fatty acid detection to become clinical applications such as metabolic disease screening, early diagnosis, treatment monitoring, prognosis evaluation and the like, and promoting the clinical application of short-chain fatty acid spectrum.
The short chain fatty acid content in the fecal sample is changed due to the fact that the fecal sample contains more microorganisms and the metabolism speed of the microorganisms is very high, so that the short chain fatty acid content is affected, and the metabolism speed of the microorganisms in the sample can be reduced (such as instant freezing) or the microorganisms can be inactivated. After a large number of growths and screening for a long time, the ethanol is found to be used as a common organic protein precipitator, so that the metabolism speed of microorganisms is expected to be reduced. Meanwhile, when the concentration of ethanol is too high, the skin may be stimulated, and a large amount of moisture in the epidermis is absorbed, so that the skin is not suitable for direct contact of general participants. And 70% -75% of ethanol is often used for disinfection and sterilization, and if the concentration of ethanol is too high, a layer of protective film is formed on the surface of bacteria to prevent the bacteria from entering the bacteria, so that the bacteria are difficult to thoroughly kill. If the concentration of ethanol is too low, bacteria can enter, but proteins in the body cannot be coagulated, and the bacteria cannot be thoroughly killed. Based on this, 75% ethanol was selected as an active ingredient for preparing a stool sample preservation solution. Can effectively keep the content of various short-chain fatty acids in the fecal sample to be very small in variation at normal temperature
In a preferred embodiment of the present invention, the feces storage solution comprises an aqueous ethanol solution.
In one embodiment, the fecal preservation solution comprises an aqueous ethanol solution having a volume concentration of 70% -90%, such as an aqueous ethanol solution having a volume concentration of 71% -85%, such as an aqueous ethanol solution having a volume concentration of 75% -90%. In one embodiment, the fecal preservation solution contains an aqueous ethanol solution with a volume concentration of 70% -75%, and in one embodiment, the fecal preservation solution contains an aqueous ethanol solution with a volume concentration of 75%.
Experimental results show that 75% ethanol is very beneficial to the detection of the content of short-chain fatty acid in the fecal sample as the fecal preservation solution of the short-chain fatty acid, and the sample can still keep the content of the short-chain fatty acid in the sample stable after being placed for 7 days at-80 ℃ to 50 ℃. The inventors have verified that the preservation of fecal samples with the preservation solution of the present invention at preservation temperatures of-80 ℃,4 ℃, 30 ℃ and 50 ℃ is superior to the case where no preservation solution is immediately frozen to-80 ℃ (gold standard) in each SCFA in the sample, and therefore the preservation effect of 75% ethanol in the preservation solution of the present invention is superior to "gold standard", and the preservation solution has good stability, and can be used as a preservation solution for general participants.
Furthermore, the inventors found that the instant freezing of the stool sample can reduce the metabolism rate of microorganisms in the sample, but microorganisms capable of producing short-chain fatty acids can be metabolized at low temperature, and the stool preservation solution provided by the invention has better sample preservation effect.
In an alternative embodiment, the faecal management solution may also contain other substances, such as preservatives, to extend shelf life.
The invention provides a method for preserving feces, which comprises the following steps of mixing a feces sample to be preserved with a feces preserving fluid, wherein the active ingredient in the feces preserving fluid is ethanol.
In a preferred embodiment of the present invention, the feces storage solution comprises an aqueous ethanol solution.
In one embodiment, the fecal preservation solution comprises an aqueous ethanol solution having a volume concentration of 70% -90%, such as an aqueous ethanol solution having a volume concentration of 71% -85%, such as an aqueous ethanol solution having a volume concentration of 75% -90%. In one embodiment, the fecal preservation solution contains an aqueous ethanol solution with a volume concentration of 70% -75%, and in one embodiment, the fecal preservation solution contains an aqueous ethanol solution with a volume concentration of 75%.
For example, the stool sample is preserved at-4 ℃ to 50 ℃ and the stool sample is preserved at 4 ℃, 25 ℃,30 ℃ or 50 ℃ and still has good stability, and can be preserved and transported at normal temperature.
The invention also provides application of the ethanol in preparing a short-chain fatty acid detection reagent or a kit, wherein the reagent or the kit contains excrement preservation liquid, and the effective component in the excrement preservation liquid is ethanol.
The inventors evaluated the reliability and efficiency of collecting and storing feces at normal temperature using the feces-preserving fluid of the present invention, and compared with fresh feces and fresh feces immediately frozen at-80 ℃, the feces-preserving fluid of the present invention was used to preserve feces samples for several days, and the Short Chain Fatty Acid (SCFA) content in the samples was not significantly different from the SCFA content in fresh feces or in samples frozen at-80 ℃ as soon as fresh feces. The present invention proposes to demonstrate the potential of using preservation solutions for collection and storage at ambient temperature.
In one embodiment, the fecal preservation solution contains an aqueous ethanol solution with a volume concentration of 70% -90%, in one embodiment, the fecal preservation solution contains an aqueous ethanol solution with a volume concentration of 70% -75%, in one embodiment, the fecal preservation solution contains an aqueous ethanol solution with a volume concentration of 75%.
In a preferred embodiment, 0.01-0.6g of stool sample is stored in 1.8ml of stool preservation solution. For example 0.01-0.5g,0.1-0.6g,0.05-0.5g or 0.08-0.6g.
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-methylpentanoic acid, caproic acid, and isocaproic acid (ICA).
The invention also provides a sample pretreatment method for short chain fatty acid detection, which comprises the following steps:
(a) Homogenizing the fecal sample to be treated;
(b) Centrifuging, sucking supernatant, diluting with acetonitrile solution, mixing (e.g. vortex vibration), and centrifuging;
(c) Derivatizing the supernatant and centrifuging;
(d) Diluting the reaction solution obtained by centrifugation in the step (c) with an acetonitrile solution containing formic acid.
The purpose of homogenization is to reduce the size of the dispersion and to improve the uniformity of the dispersion. For example, the fecal sample is placed in a mill for 1min at 60 Hz. The derivatization reaction step is slowed down by a low-temperature centrifugation step after derivatization, and then the derivatization reaction is stopped by an acetonitrile solution containing formic acid, so that the prepared sample solution can be stably placed for 15 days under the condition of less than or equal to 4 ℃, the stability of the sample is improved, and the detection efficiency is improved.
The acetonitrile solution in the steps (b) and (d) is acetonitrile aqueous solution, the volume concentration of the acetonitrile aqueous solution is 50% -70%, and in a more preferred embodiment of the invention, the volume concentration of the acetonitrile aqueous solution is 50%.
In the step (b), the acetonitrile solution is used for dilution until 0.005-0.05 g of fecal sample is contained in each 1.8mL of acetonitrile solution. At the above dilution ratio, detection of short chain fatty acids in the sample is facilitated.
In a preferred embodiment of the present invention, the derivatizing reagent used for the derivatization in step (c) is 3-nitrophenylhydrazine hydrochloride;
In a preferred embodiment of the present invention, the centrifugation in step (C) is performed at 0-10 ℃, and in a more preferred embodiment of the present invention, the centrifugation is performed at 4 ℃ for 1min. By reducing the sample temperature to slow down the derivatization reaction step, further termination reactions are provided with advantages.
The volume concentration of formic acid in the step (d) is 0.1% -0.5%.
The stool sample may be a stool sample stored in a stool preservation solution or a stool sample without the stool preservation solution, and the effective component in the stool preservation solution is ethanol.
In a preferred embodiment of the present invention, the feces storage solution comprises an aqueous ethanol solution.
In one embodiment, the fecal preservation solution comprises an aqueous ethanol solution having a volume concentration of 70% -90%, such as an aqueous ethanol solution having a volume concentration of 71% -85%, such as an aqueous ethanol solution having a volume concentration of 75% -90%. In one embodiment, the fecal preservation solution contains an aqueous ethanol solution with a volume concentration of 70% -75%, and in one embodiment, the fecal preservation solution contains an aqueous ethanol solution with a volume concentration of 75%.
In a preferred embodiment of the present invention, the dilution factor of the reaction solution after derivatization is 15 times.
In a preferred embodiment of the invention, the reaction conditions for derivatization are derivatization at 40 ℃ for 30min.
The invention also provides a short-chain fatty acid detection method, which comprises the steps of preprocessing a sample to be detected by adopting the method, and then detecting short-chain fatty acid in the preprocessed sample liquid.
The invention also provides a sample liquid which comprises excrement and excrement preservation liquid, wherein the excrement preservation liquid comprises ethanol water solution.
In one embodiment, the fecal preservation solution comprises an aqueous ethanol solution having a volume concentration of 70% -90%, such as an aqueous ethanol solution having a volume concentration of 71% -85%, such as an aqueous ethanol solution having a volume concentration of 75% -90%. In one embodiment, the fecal preservation solution contains an aqueous ethanol solution with a volume concentration of 70% -75%, and in one embodiment, the fecal preservation solution contains an aqueous ethanol solution with a volume concentration of 75%.
In one embodiment, each 0.01-0.6g of stool sample is stored in 1.8ml of stool preservation solution.
The invention has the following beneficial effects:
the fecal preservation fluid provided by the present invention may allow for collection and storage of fecal samples at Room Temperature (RT). The use of such preservation solutions allows participants to send samples by conventional mailing without the need for reservation, refrigeration or cold chain transport. The preservation solution can well keep the content of the short-chain fatty acid in the fecal sample stable, so that the sampling becomes simpler during quantitative detection of the short-chain fatty acid, and the preservation solution can be preserved and transported at normal temperature and is easy to popularize.
In addition, the reliability and efficiency of the preservation solution as a participant feces friendly collection method (collection and storage under normal temperature conditions) were evaluated, and the inventors verified that the stability of each SCFA in the sample as a feces preservation solution was superior to the case of immediately freezing to-80 ℃ without preservation solution (gold standard) at preservation temperatures of-80 ℃,4 ℃, 30 ℃ and 50 ℃ for a period of 7 days. The invention provides a potential for collecting and storing preservation solution at the ambient temperature, which is convenient for research aiming at collecting a large sample by the possibility that participants can send samples through mailing, thereby reducing the difficulty of accurately quantifying short-chain fatty acid, providing scientific basis for short-chain fatty acid detection to become clinical applications such as metabolic disease screening, early diagnosis, treatment monitoring, prognosis evaluation and the like, and promoting the clinical application of short-chain fatty acid spectrum.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is an experimental thought diagram of preservation solution effect investigation;
FIG. 2 is a chromatogram peak stack of a blank solvent, a blank sample, a fresh stool sample, and a short chain fatty acid mixed standard;
Fig. 3 is a graph of a blank solvent and high concentration short chain fatty acid mixed standard.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
The reagents and equipment used in the examples of the present invention are as follows.
Table 1 reagent consumable meter
Continuous watch
Table 2 device parameter table
Continuous watch
The following examples are respectively comparative analyses of short chain fatty acid content in fresh fecal samples, immediately frozen at-80 ℃ and samples of fresh fecal samples immediately preserved with preservation fluid at different temperatures at different times. Three time points (day 0, day 3 and day 6) were evaluated for the effect of preserving samples on SCFA content at (-80 ℃,4 ℃, 30 ℃, 50 ℃) temperature conditions, respectively. Specifically, the 0 th day is a stool sample collected on site, and a sample for short chain fatty acid detection and analysis is prepared immediately.
The analysis data shows that the results of the stool sample stored as a stool preservation solution with 75% ethanol and the immediately frozen stool sample were comparable to the results of the SCFA content in the fresh stool sample, with no significant differences, and the specific technical route is shown in FIG. 1.
Example 1 evaluation of preservative fluid Effect
1. Experiment design:
A fresh stool sample was used as a test sample, 14 samples of about 0.01g were weighed, and specific information is shown in Table 3.
TABLE 3 sample storage conditions
2. The content of each SCFA in samples under different preservation conditions is detected.
(1) After sampling, 10 samples and 4 fresh fecal samples (S7, S8, S13 and S14) without preservation solution are respectively placed in a refrigerator (-80 ℃ and 4 ℃) or an oven (30 ℃ and 50 ℃) according to the temperatures shown in Table 3;
(2) Taking out S1-S7 and S9-S13 on the 3 rd day, preparing samples according to a sample preparation method for analysis, and placing 12 samples back to the original place after sampling;
(3) And on the 6 th day, S1-S6, S8, S9-S12 and S14 are taken out, samples are prepared according to the sample preparation method and analyzed, 12 samples are replaced after sampling, and the data results of detection on the 0 th day, the 3 th day and the 6 th day are shown in Table 4, wherein the samples mainly detect AA, PA, IBA, BA, IVA, VA short chain fatty acids.
TABLE 4 results of sample short chain fatty acid content
The "-" indicates that the substance is not detected in this item, and the 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, and it is to be noted that the sample has no preservation temperature at day 0, and for the experimental group with preservation solution, the sample preparation and analysis are performed after taking a fresh sample in the preservation solution for a moment, and the labeled preservation temperature is the temperature after the sample is placed after the sample is sampled.
3. The change and difference in the SCFA content of each sample was examined under the conditions of using and without using the preservation solution.
Based on the content data of 6 samples (numbers 1 to 6 in Table 4) of the 75% ethanol preservation solution group and 4 samples (numbers 7 to 10 in Table 4) of the 50% acetonitrile preservation solution group on day 0, the coefficient of variation of each SCFA content, such as the coefficient of variation CV (0 d) of AA calculated from the content data of numbers 1 to 10 under the AA column in Table 4, was calculated, and the CV values of each SCFA content detected were less than 15%, indicating that there was no significant difference between each SCFA content measured by dissolving fresh feces using 50% acetonitrile and 75% ethanol, and the data were compared with the same standard, and the stability difference results of the SCFA content in each sample are shown with reference to Table 5. .
Table 5 examination of the stability difference of 50% acetonitrile preservation solution
4. And (5) examining the change and difference analysis of the SCFA content with the preservation time under each temperature condition.
(1) Samples were stored in 75% (V/V) ethanol at-80 ℃,4 ℃, 30 ℃ and 50 ℃ respectively (as shown in table 6):
the variation coefficients of the AA, the PA and the BA content are all less than 7% within 7 days, and the variation coefficients of the AA, the PA and the BA content in the control group are 9.92%, 12.38% and 12.90% respectively.
The IBA, IVA, VA content variation coefficients are all less than 19%, and the IBA, IVA, VA content variation coefficients of the control group are 17.85%, 24.26% and 18.74% respectively.
(2) Samples were stored in 50% (V/V) acetonitrile at-80 ℃,4 ℃, 30 ℃ and 50 ℃ respectively (as shown in table 6):
the variation coefficients of the AA, the PA and the BA content are all less than 5% within 7 days, and the variation coefficients of the AA, the PA and the BA content in the control group are 6.81%, 7.89% and 6.84% respectively.
The content variation coefficients of IBA, IVA, VA are less than or equal to 13.00%, and the content variation coefficients of the control group IBA, IVA, VA are 11.66%, 17.88% and 16.11% respectively.
The sample is stored in 75% ethanol and 50% acetonitrile to meet the requirement of sample stability, namely, the variation coefficients of AA, PA and BA (short chain fatty acid with higher content) are not more than 15%, the variation coefficient of IBA, IVA, VA (short chain fatty acid close to the lower limit of quantification) is not more than 20%, the variation coefficients of 2-MBA, 3-MVA, ICA and CA (with content far lower than the lower limit of quantification) are not more than 30%, namely, when the temperature is in the range of-80 ℃ to 50 ℃, the content of the short chain fatty acid in the sample is stable in 75% ethanol or 50% acetonitrile within 7 days. In the group of samples "as 75% ethanol control" in Table 6, the coefficient of variation of each SCFA in the samples was generally higher than that of the samples preserved with 75% ethanol preservation solution in the case where no preservation solution was immediately frozen to-80 ℃, and similarly, the stability of each SCFA in the samples was better than that of the cases where no preservation solution was immediately frozen to-80 ℃ in the case where 50% acetonitrile preservation solution was used in the group of 50% acetonitrile preservation solution and the control group thereof, and the preservation temperature of either-80 ℃ or 4 ℃ or 30 ℃ or 50 ℃ was higher than that of the 50% acetonitrile, so that the preservation effect of 75% ethanol and 50% acetonitrile in the preservation solution of the present invention was better than "gold standard".
TABLE 6 analysis of sample stability data (CV) stored at different temperatures
The coefficient of variation in Table 6 was calculated based on the content data measured by sampling at a certain constant temperature at days 0, 3 and 6 in a certain preservation solution or in the absence of preservation solution, and the coefficient of variation at these 3 time points, for example, the coefficient of variation of AA at 75% ethanol preservation solution and-80℃preservation temperature was calculated based on the content data of numbers 1, 12 and 24 (corresponding to days 0, 3 and 6, respectively) in Table 4.
The Coefficient of Variation (CV) is calculated as follows:
wherein the numerator is the standard deviation and the denominator is the average value.
5. And analyzing the variation coefficient of each SCFA content in the sample under different preservation conditions when the preservation time is the same.
The content of the 4 th point shows that the short chain fatty acid content in the excrement sample is stable after the excrement sample is stored within the temperature range of-80 ℃ to 50 ℃ within 7 days. Therefore, the average value of the short chain fatty acids of each preservation solution detected on the same day is compared with the content data of the control group, and the change amount of the SCFA stored in the preservation solution can be judged to be higher or lower than the change amount of the control group.
First, AVERAGE in table 7 refers to the AVERAGE value of the contents of all samples at each preservation temperature in the group of the preservation solution at day 0 or 3 or 6, and for example, AVERAGE value of 0d, 75% ethanol group is the AVERAGE value of a certain SCFA content measured in the experimental group numbered 1-6 in table 4.
Second, CV (0 d, 3d, 6 d) are coefficients of variation calculated based on the average value of the content of SCFA in each group at each time point (including 0 day, 3 day, 6 day) in Table 7 and the result of the content of the control group, and CV (0 d, 3d, 6 d) values of AA are coefficients of variation calculated based on all values under "AA column" in the table. The CV (0 d, 3d, 6 d) values of the short-chain fatty acids detected in the experiment were less than 15% except IVA, and the difference in short-chain fatty acid content was not large among the preservation solution 75% ethanol, the preservation solution 50% acetonitrile, and the no preservation solution in the experiment as a whole. And the content of each SCFA in each control sample (fresh feces is frozen immediately at-80 ℃) detected on the 3 rd and the 6 th days is higher than the average content of each SCFA in a sample containing 75% ethanol preservation solution or 50% acetonitrile preservation solution at the same time point, which shows that the instant freezing can reduce the metabolism speed of microorganisms in the sample, but microorganisms capable of producing short-chain fatty acid can be metabolized at low temperature, and the feces preservation solution provided by the invention has better sample preservation effect.
TABLE 7 analysis of sample stability data (CV) at different times
The lower limit of IVA (isovaleric acid) was 0.1ug/ml (Table 13), i.e., about 0.02mg/g. The average amount or content of IVA (isovaleric acid) in Table 7, 0.01 or 0.02mg/g, has been less than or equal to the lower limit of quantitation, so that a CV approaching 20% is acceptable.
Example 2
This example provides a method for detecting the content of short chain fatty acids in the sample of example 1.
1. Sample preparation method.
(1) Sample collection (1 metal grinding bead in collection tube):
① A sample with preservation solution is prepared by taking about 0.01g of fresh feces or intestinal contents to be preserved in 1.8ml of preservation solution, and turning the solution upside down for tens of times, wherein preservation solution 1 is 50% (V/V) acetonitrile aqueous solution, and preservation solution 2 is 75% (V/V) ethanol aqueous solution.
② Control samples without preservation solution were collected directly from feces and immediately frozen for preservation at-80 ℃.
③ The fresh feces sample without preservation liquid is obtained by directly collecting feces and is used immediately.
(2) Sample pretreatment:
a) The samples were weighed, about 0.01g of fresh feces or intestinal contents were taken into a 2mL centrifuge tube for a control sample without preservation solution and a fresh feces sample without preservation solution, 1.8mL of 50% ACN was added, 1 particle of metal grinding beads was added, and the sample containing preservation solution could be directly homogenized.
B) Sample homogenization ①、②、③ samples were all handled identically by placing them in a tissue mill for 1min at 60Hz and then in a 4℃centrifuge at 13000rpm for 10min (4 ℃).
C) Sample derivatization 10. Mu.L of 200mM 3-NPH. HCl (3-nitrophenylhydrazine hydrochloride), 10. Mu.L of 200mM EDC. HCl (1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride), 80. Mu.L of 50% (V/V) acetonitrile, 50. Mu.L of 7% Pyridine (pyridine), 1. Mu.L of internal standard solution (ACETIC ACID-13C2 concentration 50. Mu.g/ml, isobutyric acid-d6 concentration 12. Mu.g/ml, propionic acid-d5, butanonic acid-d7, isovaleric acid-d9, VALERIC ACID-d3, caproic acid-d5 concentrations 15. Mu.g/ml) were each added, vortexed for 1min, centrifuged for 1min, and derivatization at 40℃for 30min.
(3) Sample dilution loading machine
Mu.L of the derivatized reaction solution was added to 280. Mu.L of 50% (V/V) acetonitrile aqueous solution, vortexed for 30s, centrifuged at 13000rpm for 10min (4 ℃) and the supernatant was aspirated into a sample bottle for LC-MS/MS analysis.
2. And determining parameters in the detection method.
The inventors compared various chromatographic columns such as waters-ACQUITY UPLC BEH C1.7μm,2.1mm X 100mm),Phenomenex(Kinetex 2.6μC18,100x3.00mm),Phenomenex LC Column(1.7μm C18 50×2.1 Mm), and the final integrated chromatographic peak separation effect, and the analytical duration was chosen from Phenomnex (Kinetex 2.6.6 μC18,100×3.00 mm).
3. The system parameters are shown with reference to tables 8 and 9.
Lc 30 parameters
Table 9.SCIEX QTRAP 5500 triple quadrupole/linear ion trap tandem mass spectrometry parameters
Example 3
This example evaluates the detection method provided in example 2.
1. Specialization of
A blank control sample is prepared by taking a fresh fecal sample as a test sample and a short chain fatty acid mixed standard as a reference sample and 50% (V/V) acetonitrile, and 50% (V/V) acetonitrile is taken as a double blank for detection, and the result is shown in figure 2. The data analysis of the blank peaks is shown in tables 10 and 11. As can be seen from tables 10, 11 and FIG. 2, only acetic acid in 50% (V/V) acetonitrile has an interference peak, the ratio of the peak area to the quantitative lower limit peak area is 0.06%, the SA/IA ratio of the blank control to each short chain fatty acid quantitative lower limit is in the range of 1% -20%, the special acceptable standard is met, and the response of the interference component is not higher than 20% of the response of the analyte quantitative lower limit.
TABLE 10 blank-50% (V/V) acetonitrile data analysis
Note that SA is the analyte peak area (SAMPLE AREA), IA is the peak area ratio of the internal standard (INTERNAL STANDARDS AREA), and "-" indicates that the substance was not detected in this item, possibly because the substance was below the instrument detection limit in the sample or the sample was free of the substance.
Table 11 blank data analysis
| 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 |
| Quantitative lower 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% |
Note that SA/ia= SAMPLE AREA/INTERNAL STANDARDS AREA.
2. And (5) residual analysis.
The test is carried out by taking a mixed standard of 50% (V/V) acetonitrile of a blank solvent and high-concentration short-chain fatty acid as a test sample, a needle of blank solvent is advanced before the sequence starts, and a needle of blank solvent is advanced after the high-concentration short-chain fatty acid mixed standard is detected, so that the residual condition is verified, and the results are shown in fig. 3 and table 12. As can be seen from Table 12 and FIG. 3, the first needle had interference peaks at acetic acid and butyric acid in 50% (V/V) acetonitrile, the ratio of the peak area to the quantitative lower limit peak area was 0.08% and 0.04%, respectively, the remaining target peaks were undisturbed, the second needle had interference peaks at acetic acid alone in 50% (V/V) acetonitrile, the ratio of the peak area to the quantitative lower limit peak area was 0.11%, the remaining target peaks were undisturbed, both <2%, and met the residual acceptable standards, and the response of the interfering component was less than 20% of the analyte quantitative lower limit response.
Table 12 residual data analysis.
3. Linearity of
Accurately weighing each short chain fatty acid reference substance, dissolving with 50% (V/V) acetonitrile and fixing the volume to obtain single stock solutions of 10 standard substances, respectively taking a proper amount of each standard substance stock solution with the concentration of AA 11.6mg/mL、PA 11.3mg/mL、IBA 11.3mg/mL、BA 10.1mg/mL、2-MBA 10.1mg/mL、IVA 10.5mg/mL、VA 10.9mg/mL、3-MVA 12.1mg/mL、ICA 10.6mg/mL、CA 11.6mg/mL., fixing the volume to 10mL with 50% acetonitrile (V/V), obtaining mixed standard substance solutions, respectively obtaining mixed standard substance solutions with the concentration of ：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 gradually diluting with 50% (V/V) acetonitrile solutions to obtain a series of mixed standard substance solutions with different concentrations. 3 samples were prepared for each concentration level, each with 1 needle. And (3) carrying out linear regression by taking the sample concentration as an abscissa and the peak area ratio y of the standard substance and the internal standard substance as an ordinate to obtain a linear determination coefficient R 2, and examining the linearity of the method. As can be seen from Table 13, AA, PA, IBA, BA, 2-MBA, IVA, VA, 3-MVA, ICA and CA have good linear relationships in the corresponding concentration ranges, the correlation coefficients are all >0.996, and the linear examination meets the acceptable standards.
Table 13 linear equation, correlation coefficient and quantitative range of short chain fatty acids.
| ID | Eqμation | Weighting | R2 | 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 |
Note that Eq mu ation is a linear equation, R2 is a determination coefficient, WEIGHTING is a weight, LLOQ (μg/mL) and ULOQ (μg/mL) are a lower limit of quantification and an upper limit of quantification, respectively, and accurate quantification can be performed in the two ranges.
4. Stability of
4.1 Stability of sample solution at 4 ℃ or less.
The mixed standard (the mixed standard is a standard sample containing the 10 short chain fatty acids, the difference between the mixed standard and a real sample is no complex matrix interference, and only 50% acetonitrile is taken as a solvent) is taken as a sample, samples with high, medium and low concentration levels of 3 are respectively prepared, the samples are placed under the condition of less than or equal to 4 ℃, and the samples are respectively subjected to sample injection and needle detection at 0 time 00,0 time 09, 0 time 19, 0 time 28, 0 time 38, 0 time 48, 3 time 00 and 6 time 00. The concentrations (ug/ml) at AA, PA, IBA, BA, 2-MBA, VA, IVA, -3MVA, ICA, and CA were counted in the samples and placed at 4 ℃ or less for 0 hour 00 minutes, 0 hour 09 minutes, 0 hour 19 minutes, 0 hour 28 minutes, 0 hour 38 minutes, 0 hour 48 minutes, 3 hours, and 6 hours, and the relative difference percentage values (RD) of the concentrations detected at each time point and the concentrations detected at 0 hour 00 minutes were calculated as shown in tables 14-1 and 14-2.
The relative difference percentage of the concentration of each SCFA in the high concentration mixed standard substance within 6 hours is less than or equal to 7.1 percent, the relative difference percentage of the concentration of AA and CA in the medium concentration mixed standard substance detected by 00 minutes and 0 hour after being placed for 6 hours at the temperature of less than or equal to 4 ℃ is respectively 16.2 percent and 27.2 percent, the RD value of other SCFAs is less than or equal to 10.7 percent, the RD of AA and CA after being placed for 3 hours in the low concentration mixed standard substance is respectively 39.9 percent and 38.0 percent, the RD of AA, VA and CA after being placed for 6 hours is respectively 87.7 percent, 17.2 percent and 72.6 percent, and the RD value of the rest SCFAs is less than or equal to 11.4 percent. According to the acceptable standard of stability, the variation coefficient cannot exceed 15%, the high-concentration sample solution can be placed for 6 hours at the temperature of less than or equal to 4 ℃, the medium-concentration sample solution can be placed for 3 hours at the temperature of less than or equal to 4 ℃, and the low-concentration sample needs to be detected and analyzed as soon as possible.
Calculation formula of RD (relative difference percentage, RELATIVE PERCENT DIFFERENCE):
For the present application, the calculation formula is (other time point measured concentration-0.00 measured concentration)/0.00 measured concentration 100%
TABLE 14 analysis of stability data for sample solutions at 4 ℃ or less
TABLE 14-2 analysis of stability data for sample solutions at 4 ℃ or less
4.2 Stability of sample solutions at less than or equal to-80 ℃
Taking a mixed standard product as a test product, respectively preparing high, medium and low 3 concentration level samples, detecting by a needle at 0d, placing the samples for 1d, 2d, 3d and 6d at the temperature of less than or equal to-80 ℃, taking out the samples at each time point, recovering to room temperature, and placing the samples in a 4 ℃ sampler for detecting by a needle at 3 times. Average values of SCFA concentrations detected in AA, PA, IBA, BA, 2MBA, VA, IVA, 3MVA, ICA and CA at-80℃for 0d, 1d, 2d, 3d and 6d were calculated, respectively, and 3 concentration levels were calculated for 7 days.
The results are shown in Table 15, and the concentration variation coefficients of the sample solutions with the high, medium and low concentration levels, which are detected by standing for 7 days at the temperature of less than or equal to-80 ℃, are all less than 6%. All three sets of data meet the stability acceptable standard, and the variation coefficient is not more than 15%. Thus, the sample solution can be stably left for 7 days at a temperature of less than or equal to-80 ℃.
TABLE 15 analysis of stability data of sample solutions at-80℃or less
5. Accuracy of
3 Standard solutions of short-chain fatty acid were added to 3 samples of the fresh fecal sample solution collected in step (1), 3 samples of the standard solution of short-chain fatty acid were prepared and subjected to standard recovery experiments. 3 samples were prepared for each concentration level, each with 1 needle. The average value of the respective concentration levels was substituted into the concentration of each concentration level obtained by the linear equation, and the result was shown in Table 16, with the addition of each short chain fatty acid plus the background value as the theoretical value.
As can be seen from Table 16, the recovery rates of the other 3 concentration levels were in the range of 95% -110% except that the recovery rate of CA at the low concentration level was 75% (as can be seen from tables 14-1 and 14-2, and tables 15 and 16, CA was not detected in the samples, and CA added at the low concentration level was close to the lower limit of quantitation of 1.51. Mu.g/mL), and all the recovery rates were in the range of 95% -110% and met the acceptable standards.
Table 16 recovery investigation of accuracy
6. Repeatability of
The fresh fecal sample is a sample, 6 sample solutions are prepared, and 3 needles are introduced. The repetitive data analysis is shown in table 17. As is clear from Table 17, the CV values of AA, PA, BA, IBA, IVA, VA,2-MBA, ICA in 6 parts of the sample solutions were 5.14%, 4.44%, 6.00%,14.66%, 12.47%, 9.28%,19.74%, 11.39%, respectively, and 3-MVA and CA were not detected. Meets the acceptable repeatability standards, namely, the variation coefficient of AA, PA and BA is not more than 15%, the variation coefficient of IBA, IVA, VA is not more than 20%, and the variation coefficients of 2-MBA, 3-MVA, ICA and CA are not more than 30%.
Table 17 repeatability data analysis
Example 4 sample detection method for sample amount requirement
Taking a fecal sample as a test sample, preparing 9 parts of test sample solutions (3 parts of samples with sampling amounts of about 0.01g, 0.03g and 0.09g respectively) respectively,
Sample amount was 0.01g of the test piece, and sample preparation method was as described in "1. Sample preparation method" in example 2, samples of sample amounts of 0.03g and 0.09g were homogenized, and then added with a sample dilution step so that the ratio was unchanged (0.01 g sample: 1.8mL acetonitrile solution), and the other was unchanged. The specific dilution and content results are shown in table 18:
TABLE 18 content of SCFA in samples measured at different sampling amounts
Note that "-" indicates that the short chain fatty acid CV value was not calculated in this project, because the material content in the sample was below the linear quantification limit.
As can be seen from the above table, the sample mainly contains AA (acetic acid) and PA (propionic acid), and the content of other short-chain fatty acids is lower than the quantitative limit, so that the variation coefficients of acetic acid and propionic acid are calculated only. As shown by the results, the content variation coefficients of the acetic acid and the propionic acid detected by the sampling amount of about 0.01 g-0.09 g are less than 15%, which indicates that the short chain fatty acid content has no obvious change in the value range, and the sampling amount range can be set to be 0.01 g-0.09 g.
Example 5 optimization of sample preparation method in sample detection
As is clear from 4.1 in example 3, in order to ensure the accuracy of the measurement of the sample content, the sample solution is preferably detected as soon as possible, the high-concentration sample solution can be placed for 6 hours at the temperature of less than or equal to 4 ℃, the medium-concentration sample solution can be placed for 3 hours at the temperature of less than or equal to 4 ℃, and the low-concentration sample is required to be detected and analyzed as soon as possible. This condition limits the detection efficiency, and to solve this problem, the inventors optimized the sample preparation method, added the steps of slowing down the derivatization reaction (low temperature centrifugation for 1min after derivatization) and stopping the derivatization reaction (50% acn with 0.1% formic acid was used for dilution of the on-press dilution).
1. The optimized sample detection method comprises the following steps:
(1) Sample collection (1 metal grinding bead in collection tube):
① A sample with preservation solution is prepared by taking a certain amount of fresh feces or intestinal contents and preserving in the preservation solution, wherein the preservation solution 1 is 50% ACN, and the preservation solution 2 is 75% (V/V) ethanol water solution, and the steps are reversed for tens of times.
② Control samples without preservation solution were collected directly from feces and immediately frozen for preservation at-80 ℃.
③ The fresh feces sample without preservation liquid is obtained by directly collecting feces and is used immediately.
(2) Sample pretreatment:
a) And (3) weighing a certain weight of sample (0.01 g-0.09 g) into a 2mL centrifuge tube, adding 1mL of 50% ACN, adding 1 particle of metal grinding beads, and directly homogenizing the sample containing preservation solution for the control sample without ① preservation solution and the fresh fecal sample without ② preservation solution.
B) Sample homogenization ①、②、③ samples were all handled identically by placing them in a tissue mill for 1min at 60Hz and then in a 4℃centrifuge at 13000rpm for 10min (4 ℃).
C) Sample dilution, namely sucking a certain amount of supernatant, diluting the sample proportionally until the sample contains 0.01g of the sample in every 1.8mL of 50% ACN solution, vortex oscillating for 30s and centrifuging for 30s. For example, for a sample of 0.03g stool, 0.0298g of the sample was weighed, 1ml of 50% ACN solution was initially dissolved, 100ul of supernatant was homogenized, 436ul of 50% ACN (0.0298g+5.364mL 50%ACN equivalent) was added, and finally 1.8ml of 50% ACN solution contained 0.01g of the sample.
D) Derivatization by taking 20. Mu.L of supernatant, adding 10. Mu.L of 200mM 3-NPH. HCl, 10. Mu.L of 200mM EDC. HCl, 80. Mu.L of 50% ACN, 50. Mu.L of 7% Pyridine, 1. Mu.L of internal standard solution (ACETIC ACID-13C2 concentration 50. Mu.g/ml, isobutyric acid-d6 concentration 12. Mu.g/ml, propionic acid-d5, butanonic acid-d7, isovaleric acid-d9, VALERIC ACID-d3, caproic acid-d5 concentration 15. Mu.g/ml) respectively, vortex shaking for 1min, centrifuging for 30min at 40℃and centrifuging for 1min at 4℃and 13000 rpm.
(3) Dilution upper machine
First, an acetonitrile solution containing 0.1% formic acid was prepared, and 1mL of formic acid was dissolved in 999mL of an aqueous acetonitrile solution having a volume concentration of 50%. 280. Mu.L of the solution is taken, 20. Mu.L of the derivatized reaction solution is added, vortex oscillation is carried out for 30s, centrifugation is carried out at 13000rpm for 10min (4 ℃) and then the supernatant is sucked into a sample bottle for LC-MS/MS analysis.
2. Comparing the optimized sample preparation method with the sample preparation method shown in example 2
Four stool samples were prepared according to the "1. Sample preparation method" (hereinafter referred to as "original method") and the optimization method (hereinafter) respectively in example 2, and the results are shown in the data comparison table 18 of the original method and the optimization method. As can be seen from the comparison of the data of the original method and the optimized method in Table 19, the IBA, 2-MBA, IVA, VA and 3-MVA, ICA, CA contents in the four fecal samples are lower, the concentration does not reach the quantitative lower limit, the data result is only used as a reference, the difference between the original method and the optimized method is not calculated, the AA, PA and BA contents in the samples are higher, and the relative deviation range between the AA, PA and BA contents detected by the optimized method and the AA, PA and BA detected by the original method is 0.7% -6.5% (15%), namely, the short-chain fatty acid results detected by the two methods are not significantly different. As shown in a stability result table 20 of the sample prepared by the optimization method under the condition of not more than 4 ℃, the variation coefficient of the sample prepared by the optimization method under the condition of not more than 4 ℃ is in the range of 0.2% -1.8%, and according to the stability-resistant acceptable standard, the variation coefficient is not more than 15%, and the sample solution prepared by the optimization method can be stably placed under the condition of not more than 4 ℃ for 15 days.
Table 19 comparison of example 2 method and post-optimization method data
Note that a is the original method, B is the optimization method, ① is 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, CA did not reach the lower limit of quantification, the data results were only used as reference, and the difference between the two was not calculated.
Table 20 optimized method preparation samples stability of each short chain fatty acid at 4 ℃ or less.
Note that ① is Calculated Concentration (ug/ml), ② is the coefficient of variation of short chain fatty acid content detected on day 1, day 2, day 3, day 4 and day 15 of the optimization method, and the coefficient of variation is not calculated because the concentration results of IBA, 2-MBA, IVA, VA and 3-MVA, ICA, CA do not reach the lower limit of quantification, the data result is only used as a reference, and the coefficient of variation is not calculated.
Linear evaluation of the optimized method.
Based on the short chain fatty acid content in the actual samples and the results in table 13, the linear standard solution (specifically, table 21 below) was reconfigured, and 3 samples were prepared for each concentration level, each 1 needle was introduced. And (3) carrying out linear regression by taking the sample concentration as an abscissa and the peak area ratio y of the standard substance and the internal standard substance as an ordinate to obtain a linear determination coefficient R 2, and examining the linearity of the optimization method. As can be seen from the results in Table 22, AA, PA, IBA, BA, 2-MBA, IVA, VA, 3-MVA, ICA and CA have good linear relationships in the corresponding concentration ranges, the correlation coefficients are all >0.997, and the linear examination meets the acceptable standards.
Table 21 Standard sample concentration (μg/mL)
| 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 |
Table 22 Linear equation, correlation coefficient and quantitative Range of short chain fatty acids (optimization method)
Note that Eq μ ation is a linear equation, R 2 is a determination coefficient, WEIGHTING is a weight, LLOQ (μg/mL) and ULOQ (μg/mL) are a lower limit and an upper limit of quantification, respectively, and accurate quantification is possible in the range of both.
In summary, the present invention tests two preservation solutions that allow stool samples to be collected and stored at Room Temperature (RT). The use of such preservation solutions allows participants to send samples by conventional mailing without the need for reservation, refrigeration or cold chain transport. The invention also evaluates the reliability and efficiency of storage fluid collection at ambient temperature without significant differences in SCFA content as compared to fresh feces and fresh feces immediately frozen at-80 ℃. The invention provides a method for preparing the sample, which highlights the potential of collecting and storing the sample at the ambient temperature by using the preservation solution, is convenient for the research aiming at collecting a large sample by the possibility that a participant can send the sample through mailing, further reduces the difficulty of accurately quantifying the short chain fatty acid, provides scientific basis for the short chain fatty acid detection to become clinical applications such as metabolic disease screening, early diagnosis, treatment monitoring, prognosis evaluation and the like, and can promote the clinical application of the short chain fatty acid spectrum.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (15)
1. Use of ethanol for the preparation of a faecal management solution, characterised in that the faecal management solution comprises only an aqueous ethanol solution having a concentration of 75% by volume, the faecal management solution being used to maintain a stable short chain fatty acid content in a faecal sample.
2. The application of the excrement preservation solution in preparing the short-chain fatty acid detection reagent or the kit is characterized in that the excrement preservation solution only contains an ethanol water solution with the volume concentration of 75%, and the excrement preservation solution is used for maintaining the stability of the short-chain fatty acid content in an excrement sample.
3. A method for preserving feces, characterized in that it comprises the steps of mixing a feces sample to be preserved with a feces preserving fluid, wherein the feces preserving fluid only contains an ethanol aqueous solution with a volume concentration of 75%, and the feces preserving fluid is used for maintaining the stability of the short chain fatty acid content in the feces sample.
4. A method of preserving faeces according to claim 3 characterised in that each 0.01-0.6g faeces sample is preserved in 1.8ml faeces preserving fluid.
5. A method of preserving faeces according to claim 3 characterised in that preserving faeces samples is carried out at-4 ℃ to 50 ℃.
6. The method of preserving feces according to claim 5, wherein preserving the feces sample is performed at 4 ℃, 25 ℃, 30 ℃, or 50 ℃.
7. A method for sample pretreatment for short chain fatty acid detection, comprising the steps of:
(a) Homogenizing the fecal sample to be treated;
(b) Centrifuging, sucking supernatant, diluting with acetonitrile solution, mixing, and centrifuging;
(c) Derivatizing the supernatant and centrifuging;
(d) Diluting the reaction solution obtained by centrifuging in the step (c) by using an acetonitrile solution containing formic acid;
The fecal sample is a fecal sample stored in a fecal preservation solution or a fecal sample without a fecal preservation solution, and the fecal preservation solution contains only an aqueous ethanol solution with a volume concentration of 75%.
8. The method for sample pretreatment for short chain fatty acid detection according to claim 7, wherein the acetonitrile solution in steps (b) and (d) is an acetonitrile aqueous solution, and the volume concentration of the acetonitrile aqueous solution is 50% -70%.
9. The method for sample pretreatment for short chain fatty acid detection according to claim 8, wherein 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, wherein in the step (b), the sample is diluted with acetonitrile solution to 0.005-0.05 g of stool sample per 1.8mL of acetonitrile solution.
11. The method for sample pretreatment for short chain fatty acid detection according to claim 7, wherein the derivatizing reagent employed in the derivatizing in step (c) is 3-nitrophenylhydrazine hydrochloride.
12. The method for sample pretreatment for short chain fatty acid detection according to claim 7, wherein the centrifugation in step (C) is centrifugation at 0 to 10 ℃.
13. The method for sample pretreatment for short chain fatty acid detection according to claim 12, characterized in that centrifugation is performed at 4 ℃ for 30-60s.
14. The method for sample pretreatment for short chain fatty acid detection according to claim 7, wherein the formic acid concentration by volume in the step (d) is 0.1% to 0.5%.
15. A method for detecting short chain fatty acids, comprising pretreating a sample to be detected by the method of any one of claims 7 to 14, and then detecting short chain fatty acids in the pretreated sample solution.
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