CN117129687A - A kind of feces preservation liquid and preparation method thereof - Google Patents

Abstract

The application relates to a fecal preservation solution and a preparation method thereof in the technical field of fecal calprotectin preservation, wherein the protective solution comprises the following components in percentage by volume: 0.85% -0.9% of ionic strength stabilizer; 0.1% -2% of protein protectant; amino acid 0.1% -2%; 0.1% -1% of a surfactant; 0.1% -1% of preservative; the balance being buffer solution. The application can improve the stable existence time of fecal calprotectin in fecal samples.

Description

A kind of feces preservation liquid and preparation method thereof

Technical field

The present application relates to the field of fecal calprotectin preservation technology, and in particular to a fecal preservation solution and a preparation method thereof.

Background technique

Inflammatory bowel disease is a chronic intestinal disease. In recent years, the number of patients with inflammatory bowel disease has gradually increased and it has become one of the common diseases of the digestive system. Fecal calprotectin is used as a marker of inflammatory bowel disease.

Calprotectin is a calcium-zinc-binding protein in the S100 protein family. It exists in the cytoplasm of neutrophils. When intestinal inflammation permeability increases, it will promote the migration of neutrophils into the intestinal lumen, resulting in fecal calcium Calprotectin levels are increased, so measuring the concentration of fecal calprotectin can determine the presence of inflammation and reflect the severity of the disease to a certain extent.

Since calprotectin contains 14 potential cleavage sites that may be degraded by trypsin, although the trypsin activity in feces is very low and unstable, it may still be degraded if the feces is separated from the body for a long time. For example, the literature "Fecal Calcium "Calprotectin Detection and Its Experimental Influencing Factors" mentioned that "Acevedo et al. studied 287 fecal samples collected within 2 days from 18 patients with active UC and found that the fecal calprotectin level in the samples dropped by 28% after being left at room temperature for 7 days. "So generally, when patients with enteritis seek medical treatment in the hospital, they are often required to take stool samples on the spot to detect fecal calprotectin to assist diagnosis.

However, there are some situations, such as difficulty in patient sampling and difficulty in obtaining fecal samples while in the hospital; and situations where it is difficult to detect fecal samples in time for some reasons after sampling. Fecal samples are stored for a long time, which leads to the occurrence of fecal calprotectin. There is a certain degree of degradation, thus affecting the accuracy of the detection.

Contents of the invention

In order to increase the stable existence time of fecal calprotectin in fecal samples, this application provides a fecal preservation solution and a preparation method thereof.

The feces preservation solution and its preparation method provided by this application adopt the following technical solutions:

A feces preservation solution, the protective solution includes the following components in volume percentage: ionic strength stabilizer 0.85%-0.9%; protein protective agent 0.1%-2%; amino acid 0.1%-2%; surfactant 0.1%- 1%; preservative 0.1%-1%; the balance is buffer solution.

By adopting the above technical solution, both the protein protective agent and the amino acid can protect the protein structure of fecal calprotectin. The protein protective agent and the amino acid work synergistically to reduce the degree of decomposition of fecal calprotectin by trypsin; the preservative has the ability to inhibit The bacterial effect makes it difficult for fecal samples to breed bacteria and other microorganisms in the preservation solution, thereby reducing the possibility of fecal calprotectin being decomposed by microorganisms, making fecal calprotectin less prone to degradation during storage, and increasing the storage of fecal calprotectin. cycle. Surfactant makes the stool sample dissolve to a higher degree in the preservation solution, so that the protein protectant, amino acid and preservative can protect fecal calprotectin more comprehensively. The ionic strength stabilizer stabilizes the total ionic strength of the preservation solution to reduce the occurrence of some adverse reactions in the preservation solution that promote the decomposition of fecal calprotectin. The buffer solution can resist the pH change that occurs when the above components and stool samples are added to the preservation solution, so that the pH of the preservation solution can be maintained within the range where fecal calprotectin can stably exist, further extending the storage period of fecal calprotectin.

Optionally, the buffer solution of the buffer solution is selected from one of phosphate buffer and Tris-HCL buffer.

By adopting the above technical solution, the PB buffer has the advantages of being easy to prepare into various concentrations and having a wide range of pH values; the pH is less affected by temperature. Tris-HCL buffer has little interference with biochemical processes and does not precipitate with calcium, magnesium ions and heavy metal ions.

Optionally, the pH value of the buffer solution ranges from 7.4 to 8.0.

By adopting the above technical solution, within the above pH range, the stability of calprotectin is better, further reducing the degree of decomposition of fecal calprotectin by trypsin.

Optionally, the buffer concentration in the buffer solution is 10mM-200mM.

By adopting the above technical solution, within the concentration range of the buffer solution, the buffer solution has a better buffering capacity and can better stabilize the pH of the preservation solution.

Optionally, the surfactant is selected from Tween 20 and Tween 80.

By adopting the above technical solution, Tween 20 and Tween 80 have properties such as emulsification, diffusion, solubilization, and stability, and can increase the solubility of stool samples and various components in the preservation solution.

Optionally, the ionic strength stabilizer is selected from one of sodium chloride and potassium chloride.

By adopting the above technical solution, sodium chloride and potassium chloride, as inert electrolytes, can maintain the stability of the total ionic strength to maintain and balance the ion concentration in the protective solution, prevent the reaction rate and characteristics from changing, and prevent the promotion of fecal calprotectin Decompose the occurrence of adverse reactions.

Optionally, the protein protective agent is one of bovine serum albumin and casein sodium salt.

By adopting the above technical solution, bovine serum albumin has high solubility and can be dissolved and stably existed in the preservation solution; bovine serum albumin has high affinity, and after being dissolved in the protective solution, bovine serum albumin can interact with fecal calcium. The proteins bind together to form stable complexes that protect fecal calprotectin. Casein sodium salt can protect fecal calprotectin by inhibiting proteases, thereby reducing the degree of decomposition of fecal calprotectin by proteases.

Optionally, the amino acid is glycine.

By adopting the above technical solution, glycine can be used as a protein stabilizer, which can prevent the aggregation, crystallization or denaturation of fecal calprotectin and maintain the activity and stability of fecal calprotectin, making it relatively easier to detect fecal calprotectin during detection. , to improve the detection accuracy of fecal calprotectin in fecal samples.

Optionally, the preservative is selected from proclin300 and Krovin500.

By adopting the above technical solution, both proclin300 and Krovin500 have broad-spectrum antibacterial capabilities, are effective against bacteria, fungi and yeasts, and can quickly eliminate the activity of microorganisms, making the antibacterial effect on the preservation solution better.

In the second aspect, the preparation method of a stool preservation solution provided by this application adopts the following technical solution.

A method for preparing a feces preservation solution, including the following steps:

At room temperature, prepare a buffer solution and adjust the pH, then add the ionic strength stabilizer, protein protectant, amino acid, surfactant and preservative, stir to dissolve and then dilute to volume.

By adopting the above technical solution, the preservation solution can be prepared conveniently and quickly through the above steps; preparing the buffer solution and preparing the preservation solution at room temperature can enable the preservation solution to adapt to and simulate the temperature conditions of normal temperature storage of stool samples, and more Facilitates preservation of stool samples

To sum up, this application includes at least one of the following beneficial technical effects:

1. Both protein protectants and amino acids can protect the protein structure of fecal calprotectin. Preservatives reduce the possibility of fecal calprotectin being decomposed by microorganisms, making fecal calprotectin less prone to degradation during storage and increasing the number of fecal calprotectin. The storage period of calprotectin; surfactant makes the fecal sample dissolve to a higher degree in the preservation solution, so that protein protectants, amino acids and preservatives can protect fecal calprotectin more comprehensively;

2. The ionic strength stabilizer stabilizes the total ionic strength of the preservation solution to reduce the occurrence of some adverse reactions in the preservation solution that promote the decomposition of fecal calprotectin;

3. The buffer solution keeps the pH of the preservation solution within the range where fecal calprotectin can stably exist, further extending the storage period of fecal calprotectin.

Detailed ways

1. Embodiments

At room temperature, add NaOH solution to PB (phosphate buffer) with a concentration of 50mmol/L to adjust the pH to 7.6 to obtain a PB buffer solution; add 90mL PB buffer solution to the Erlenmeyer flask, and then add 0.85g sodium chloride, Stir and dissolve 0.1g bovine serum albumin, 0.1g glycine, 0.1mL Tween 20 and 0.1mL proclin300, then transfer to a 100ML volumetric flask, and adjust the volume to the mark to obtain a preservation solution.

Example 2-3:

A feces preservation solution and its preparation method are different from Example 1 in that: as shown in Table 1.

Table 1:

Example 4:

A fecal preservation solution and a preparation method thereof are different from Example 2 in that the concentration of the phosphate buffer solution is 10 mmol/L.

Example 5:

A fecal preservation solution and a preparation method thereof are different from Example 2 in that the concentration of the phosphate buffer solution is 200 mmol/L.

2. Comparative ratio

Comparative example 1:

A stool preservation solution and its preparation method are different from Example 2 in that equal volumes of sodium chloride are replaced with PB buffer solution.

Comparative example 2:

A feces preservation solution and its preparation method are different from Example 2 in that equal volumes of bovine serum albumin are replaced with PB buffer solution.

Comparative example 3:

A stool preservation solution and its preparation method are different from Example 2 in that equal volumes of glycine are replaced with PB buffer solution.

Comparative example 4:

A feces preservation solution and a preparation method thereof are different from Example 2 in that: the pH value of the PB buffer solution is 7.2.

Comparative example 5:

A feces preservation solution and its preparation method are different from Example 2 in that equal volumes of sodium chloride, bovine serum albumin, glycine, Tween 20, and proclin300 are replaced with a PB buffer solution with a pH value of 7.6.

3. Performance testing test

1) Label stool samples from different sources as sample 1, sample 2, and sample 3. Take multiple groups of 50 mg samples 1-3 respectively, add them to 2.5 mL of conventional diluent, and mix well to obtain multiple groups of sample diluents. Instantly Take a quantitative sample diluent and put it into the assay kit, and use fluorescence immunochromatography to detect the fecal calprotectin concentration in the sample diluent.

2) Take multiple groups of 100 mg sample 1 and add them to the preservation solution prepared in Examples 1-5 and Comparative Examples 1-5 respectively, mix well for storage, and then take out 50 μL from the preservation solution on the 7th day and the 15th day respectively. After adding to 625 μL of diluent for dilution and mixing, multiple sets of sample diluents are obtained. A quantitative amount of the sample diluents is put into the assay kit, and fluorescence immunochromatography is used to detect the concentration of fecal calprotectin in the sample diluents. .

3) Test the fecal calprotectin concentration in both Sample 2 and Sample 3 as described above.

The above performance test results are shown in Table 2:

Table 2:

4. Result Analysis and Summary

Combining Examples 1-5 and Comparative Examples 1-5 and Table 2, it can be seen that since the critical value of fecal calprotectin concentration to determine whether inflammatory bowel disease exists is 60ug/g, in Example 1, Samples 1-3 The fecal calprotectin concentrations measured in the fresh samples were 29.36ug/g, 63.05ug/g and 574.73ug/g respectively. The fecal calprotectin concentrations measured in samples 1-3 were below 60ug/g and 60ug/g respectively. g and above 60ug/g. Correspondingly, the fecal calprotectin concentration measured in samples 1-3 on the 7th and 15th days did not change much compared with the fresh samples. Therefore, it can be concluded from the above that the preservation solution prepared in this application can be used to store various types of sources. Effective preservation of fecal calprotectin in fecal samples from (without inflammatory bowel disease, on the verge of having inflammatory bowel disease, and already with inflammatory bowel disease).

Compared with Examples 1-5, Comparative Example 5 does not contain sodium chloride, bovine serum albumin, glycine, Tween 20, proclin300 and other components, which is equivalent to using only PB buffer solution (diluent) as a preservation solution for stool samples. save. As can be seen from Table 2, the fecal calprotectin concentrations measured in Examples 1-3 on the 7th and 15th days were much higher than those in Comparative Example 5; and the fecal calprotectin concentrations measured on the 7th and 15th days of Comparative Example 5 were The calprotectin concentration was much lower than the fecal calprotectin concentration measured in the fresh sample of Comparative Example 5, and the fecal calprotectin concentration measured on the 15th day was originally lower than the fresh sample. It can be seen from the above that the preservation effect of stool samples using only PB buffer solution (diluent) is very poor.

In Examples 1-3, the concentrations of fecal calprotectin measured in Example 2 on days 7 and 15 were both higher than those in Example 1 and Example 3. Therefore, it can be concluded from the above that each raw material of Example 2 Compared with Example 1 and Example 3, its volume percentage has a better preservation effect on fecal calprotectin in fecal samples and can better extend the storage period of fecal calprotectin.

The phosphate buffer concentrations of Examples 4-5 are different from those of Example 2. As can be seen from Table 2, the fecal calprotectin concentrations measured on the 7th and 15th days of Example 2 are both higher than those of Example 4, and The concentration of fecal calprotectin measured in Example 5 on the 7th and 15th days increased very little compared to Example 2. Therefore, the phosphate buffer concentration in the protective solution at 50mM can preserve the fecal samples very well. At the same time, it will not cause any waste of phosphate groups in the phosphate buffer solution.

Compared with Example 2, Comparative Example 1 lacks the sodium chloride component. From Table 2, it can be seen that the fecal calprotectin concentration measured on the 7th day and the 15th day of Example 2 was higher than that of Comparative Example 1. It can be seen from this that As an ionic strength stabilizer, sodium chloride can improve the preservation effect of fecal calprotectin in the preservation solution.

Compared with Example 2, Comparative Example 2 lacks the bovine serum albumin component. From Table 2, it can be seen that the fecal calprotectin concentration measured on the 7th day and the 15th day of Example 2 is higher than that of Comparative Example 2. Therefore, It can be seen that bovine serum albumin, as a protein protectant, can improve the preservation effect of fecal calprotectin in the preservation solution.

Compared with Example 2, Comparative Example 3 lacks the glycine component. It can be seen from Table 2 that the fecal calprotectin concentration measured on the 7th and 15th days of Example 2 was higher than that of Comparative Example 3. It can be seen that glycine can Improve the preservation effect of fecal calprotectin in the preservation solution.

The pH value of the PB buffer solution in Comparative Example 4 is outside the pH value range of the PB buffer solution in this application. As can be seen from Table 2, the fecal calprotectin concentrations measured on the 7th and 15th days of Example 2 were both high. From Comparative Example 4, it can be seen that the pH value of the PB buffer solution is between 7.4 and 8.0, which can keep the pH of the preservation solution within the range where fecal calprotectin can stably exist, so as to extend the storage period of fecal calprotectin. .

The above are all preferred embodiments of the present application, and are not intended to limit the scope of protection of the present application. Therefore, any equivalent changes made based on the structure, shape, and principle of the present application shall be covered by the scope of protection of the present application. Inside.

Claims (10)

1. A feces preservation solution, characterized in that the protective solution includes the following components in volume percentage: ionic strength stabilizer 0.85%-0.9%; protein protective agent 0.1%-2%; amino acid 0.1%-2%; Surfactant 0.1%-1%; preservative 0.1%-1%; the balance is buffer solution. 2. A stool preservation solution according to claim 1, characterized in that the buffer solution of the buffer solution is selected from one of phosphate buffer and Tris-HCL buffer. 3. A stool preservation solution according to claim 1, characterized in that: the pH value of the buffer solution ranges from 7.4 to 8.0. 4. A feces preservation solution according to claim 1, characterized in that: the buffer concentration in the buffer solution is 10mM-200mM. 5. A stool preservation solution according to claim 1, characterized in that the surfactant is selected from one of Tween 20 and Tween 80. 6. A stool preservation solution according to claim 1, characterized in that the ionic strength stabilizer is selected from the group consisting of sodium chloride and potassium chloride. 7. A feces preservation solution according to claim 1, characterized in that the protein protective agent is selected from the group consisting of bovine serum albumin and casein sodium salt. 8. A feces preservation solution according to claim 1, characterized in that: the amino acid is glycine. 9. A stool preservation solution according to claim 1, characterized in that the preservative is selected from the group consisting of proclin300 and Krovin500. 10. A method for preparing a feces preservation solution according to any one of claims 1 to 9, characterized in that it includes the following steps: at room temperature, prepare a buffer solution and adjust the pH, and then add the ionic strength to stabilize the solution. agents, protein protectants, amino acids, surfactants and preservatives, stir to dissolve and then dilute to volume.