CN106596211A - Stool sample preservation liquid, preparation method and application thereof - Google Patents
Stool sample preservation liquid, preparation method and application thereof Download PDFInfo
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- CN106596211A CN106596211A CN201510671258.9A CN201510671258A CN106596211A CN 106596211 A CN106596211 A CN 106596211A CN 201510671258 A CN201510671258 A CN 201510671258A CN 106596211 A CN106596211 A CN 106596211A
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Classifications
-
- G—PHYSICS
- 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
-
- G—PHYSICS
- 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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/305—Fixative compositions
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a stool sample preservation liquid, a preparation method and application thereof. Specifically, the stool sample preservation liquid contains: 45 vol%-60 vol% of a fixing agent; 0.1 mass%-15 mass% of a fixing agent adjuvant; 0.01 mass%-1.5 mass% of an anticoagulant; 10 vol%-15 vol% of a buffer solution; 0.01 mass%-1 mass% of an ionic strength maintenance agent; 0.01 vol%-0.5 vol% of a nonionic detergent; and the balance water. The stool sample preservation liquid provided by the invention has the characteristics of easy preparation and low cost, can preserve stool samples at room temperature, also can effectively inhibit DNA degradation and cell damage of stool samples, maintain cell shape, and can preserve samples for a long time, and the preserved samples have high qualification rate during reutilization.
Description
Technical Field
The invention relates to a fecal sample preserving fluid and a preparation method and application thereof.
Background
Non-invasive samples such as feces, hair, saliva, urine and the like are widely applied to forensic medicine, wild animal research and clinical research, wherein the feces sample is an effective means of clinical examination and the most common in general investigation, and has the characteristics of simplicity, convenience, practicability, low cost, no wound and suitability for large-scale crowd screening. With the development of molecular biology, the occurrence and development of tumors are attributed to related gene mutation, and exfoliated cells in feces contain mutant genes closely related to colorectal cancer, so that gene detection in feces is expected to become a new method for screening and diagnosing gastrointestinal tumors in early stage. It has been reported that a U.S. company developed a Stool-based DNA (sDNA) screening test for detecting DNA markers associated with colon cancer. 10000 cases of clinical trial data showed 92% sensitivity in colon cancer detection, 42% sensitivity in detection of precancerous polyps, 66% sensitivity in detection of polyps larger than 2 cm, and 87% specificity of all clinical data.
In addition, a large number of microbial colonies exist in feces, the composition of intestinal flora is a result of long-term co-evolution with a host and is closely related to the health and diseases of the host, such as obesity, diabetes, inflammatory bowel disease and the like, and the research of the total genome of the intestinal tract in feces (intestinal metagenomics) has become a hot research field at present.
However, fecal components are complex and contain large amounts of digestive residues and various nucleic acid degrading enzymes produced by bacteria such as proteases, DNases (DNases) and RNases (RNases), and various PCR inhibitors such as bile salts, bilirubin, humus, and pigments, making cells and nucleic acids in fecal samples extremely vulnerable to destruction and degradation. The conventional excrement sample storage method has short storage time, low sample reutilization qualification rate, harsh storage conditions and high cost.
Therefore, the current fecal sample solution and preservation method still need to be improved.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, an object of the present invention is to provide a feces sample storage means which has a long storage time, a high sample reuse yield, and is convenient for storage.
It should be noted that the present invention has been completed based on the following findings of the inventors:
researchers at home and abroad have developed various methods for preserving feces, and the principle comprises: 1) reducing the activity of various degrading enzymes at low temperature, and performing low-temperature treatment, namely freezing method, and generally storing at-20 ℃; 2) the dehydration drying can inhibit various biochemical reactions such as silica gel drying, silica dioxide drying, microwave drying and the like; 3) the preservation solution is usually used for inhibiting degrading enzyme and microbial activity, such as anhydrous ethanol, 70% ethanol, dimethyl sulfoxide (DMSO), dimethyl sulfoxide saturated salt Solution (DETs), etc. The preservation methods have simple components and single effect, and the preservation effect is not obvious along with the prolonging of time.
The inventor tries various preservation methods in the long-term gene research of the fecal sample, including the common methods, the effects are not ideal, the low-temperature preservation limits the collection of the sample in different places, the transportation cost of the sample is greatly increased, some samples have short-term effects after optimization (preservation solution transportation, preservation solution and low-temperature long-term preservation), but the sample is still obviously degraded after being placed for 1-3 months, the insufficient DNA amount brings great trouble to the subject research, and the inventor is also prompted to carry out various experimental exploration, so as to provide a normal-temperature, simple, low-cost and long-acting fecal sample preservation method.
According to one aspect of the invention, a fecal specimen preservation fluid is provided. According to an embodiment of the invention, the stool sample preservation solution comprises: 45-60% by volume of a fixative; 0.1 to 15 mass% of a fixing agent auxiliary agent; 0.01 to 1.5 mass% of an anticoagulant; 10% to 15% by volume of a buffer; 0.01 to 1 mass% of an ionic strength-maintaining agent; 0.01% to 0.5% by volume of a non-ionic detergent; and the balance of water, wherein the fixing agent is at least one selected from methanol, ethanol, glycol and isopropanol, the fixing auxiliary agent is at least one selected from acetic acid and acetate, the anticoagulant agent is at least one selected from ethylenediamine tetraacetic acid and water-soluble salts of ethylenediamine tetraacetic acid, the buffer solution is at least one selected from phosphate buffer solution, acetate buffer solution, ACES buffer solution, ADA buffer solution, BIS-TRIS buffer solution, MES buffer solution and PIPES buffer solution, the ionic strength maintaining agent is chloride salt, and the non-ionic detergent is at least one selected from Tween 20, NP-40 and TritonX-100.
The inventor surprisingly finds that the excrement sample preservation solution can effectively inhibit DNA degradation and cell damage in the excrement sample, maintain cell morphology and ensure sample quality, so that nucleic acid and cells in the preserved excrement sample can meet the requirement of further research. In addition, according to the embodiment of the invention, the fecal sample preservation solution is easy to prepare, low in cost, capable of preserving the fecal sample at normal temperature, long in sample preservation time and high in sample reutilization qualification rate after preservation.
In addition, the fecal specimen preservation solution according to the above embodiment of the present invention may also have the following additional technical features:
according to an embodiment of the invention, the pH of the buffer is 7.4-8.0. Therefore, the pH stability of the fecal sample can be effectively maintained, and the cell morphology can be maintained.
According to some embodiments of the invention, the pH of the stool sample preservation solution is 7.4 to 8.0. Therefore, when the DNA is used for preserving the excrement sample, the DNA in the sample is not easy to degrade and break.
According to some specific examples of the present invention, the ionic strength maintaining agent is at least one selected from the group consisting of sodium chloride and potassium chloride.
According to some preferred embodiments of the invention, the anticoagulant is disodium edetate.
According to some preferred embodiments of the invention, the fixing agent is present in an amount of 50% to 60% by volume. Therefore, the effects of immobilizing protein and inhibiting DNA degradation are good.
According to some preferred embodiments of the present invention, the content of the anticoagulant is 0.1% by mass to 1.5% by mass. Therefore, the anticoagulation effect is good, and the cost is low.
According to some preferred embodiments of the present invention, the ionic strength maintaining agent is contained in an amount of 0.5% by mass to 1% by mass. This results in a good cell morphology maintenance effect.
According to some embodiments of the invention, the stool sample preservation solution of the invention comprises: 50% to 60% by volume of methanol; 0.1 to 15 mass% of acetic acid; 0.1 to 1.5 mass% of disodium ethylenediaminetetraacetate; 10% to 15% by volume of a phosphate buffer; 0.5 to 1 mass% of sodium chloride; tween-20 in an amount of 0.01% to 0.5% by volume; and the balance being water. Therefore, the preservation effect of the fecal specimen preservation solution is outstanding.
According to a specific example of the present invention, 1000ml of the stool specimen preservation solution contains:
500ml of methanol;
3.5g of acetic acid;
1g of disodium ethylene diamine tetraacetate;
100ml of phosphate buffer;
10g of sodium chloride;
0.5ml of Tween-20; and
399.5ml of water.
According to some embodiments of the invention, 1000ml of the stool sample preservation solution comprises:
450ml of ethanol;
1g of acetic acid;
3g of disodium ethylene diamine tetraacetate;
100ml acetate buffer;
1g of potassium chloride;
0.1ml of Tween-20; and
449.9ml of water.
According to further embodiments of the present invention, 1000ml of the stool sample preservation solution comprises:
550ml of ethylene glycol;
2.5g of acetic acid;
7g of disodium ethylene diamine tetraacetate;
150ml of PIPES buffer;
5g of sodium chloride;
2.5ml NP-40; and
297.5ml of deionized water.
According to another aspect of the present invention, the present invention also provides a method for preparing a preservation solution for a stool sample. According to an embodiment of the invention, the method comprises the steps of:
1) providing raw materials according to the formula of the excrement sample preservation solution;
2) adding an ionic strength maintaining agent, a buffer solution and an anticoagulant into deionized water, and dissolving and uniformly mixing the components to obtain a mixture;
3) adjusting the pH value of the mixture to 7.4-8.0;
4) and adding a fixing agent, a fixing agent auxiliary agent and a non-ionic detergent into the mixture subjected to pH value adjustment, uniformly mixing, and sterilizing to obtain the fecal sample preservation solution.
Therefore, the excrement sample preservation solution can be prepared and obtained efficiently, the cost is low, the obtained excrement sample preservation solution is good in quality, the excrement sample preservation solution can be effectively used for preserving excrement samples at normal temperature for a long time, the sample DNA degradation and cell damage after preservation are few, and the sample reutilization qualification rate is high.
According to a further aspect of the invention, there is also provided the use of a faecal sample preservation solution as hereinbefore described for preserving a faecal sample. According to the embodiment of the invention, the excrement sample preservation solution can be used for effectively preserving the excrement sample at normal temperature for a long time, and has the advantages of low preservation cost, good effect and high sample reutilization qualification rate.
According to yet another aspect of the invention, a method of preserving a stool sample is also provided. According to an embodiment of the invention, the method comprises: mixing the above feces sample preservation solution and feces sample according to a volume ratio of 1-3: 1, and storing the mixture at room temperature in a sealed and dark place. The inventor finds that the method for preserving the fecal sample can effectively realize the long-term preservation of the fecal sample at normal temperature, and has the advantages of simple operation, low preservation cost, good effect, long preservation time and high sample reutilization qualification rate.
According to some preferred embodiments of the invention, the volume ratio of the stool sample preserving fluid to the stool sample is 1.5: 1. therefore, the storage cost is low, and the storage effect is outstanding.
In addition, the fecal sample preservation solution and the preservation method according to the embodiment of the present invention have at least one of the following advantages:
1) the excrement sample preservation solution can inhibit the degradation of DNA by a large amount of digestive enzymes generated by bacteria in excrement, can maintain the cell morphology, and is beneficial to morphological observation of cell separation.
2) The existing feces sample preservation solution has single component, and some feces sample preservation solutions are only suitable for short-time preservation, such as absolute ethyl alcohol preservation, feces dehydration, uneven dispersion and incomplete contact with the solution; some samples need low-temperature refrigeration, have strict requirements on instruments and transportation conditions, and have low quality degradation and short storage time although the sample degradation is slowed down. The excrement sample preservation liquid can be used for preserving samples at normal temperature, various components of the excrement sample preservation liquid are beneficial to homogenizing the excrement in the preservation liquid, so that the excrement can be fully contacted with the solution, the preservation time is long, and the components do not influence subsequent nucleic acid extraction and enzymatic reaction.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a comparison result of OD value method of the change of the DNA extraction amount of the fecal samples with the preservation time under different preservation conditions in example 4;
FIG. 2 is a comparison of the results of gel electrophoresis of the DNA extraction of the stool sample with the change of the storage time under different storage conditions in example 4;
FIG. 3 is a comparison of real-time fluorometry of human DNA extracted from stool samples of example 5 as a function of storage time; and
FIG. 4 is a comparison of the observation of epithelial cell morphology as a function of storage time in the stool samples of example 6 (40X).
Detailed Description
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.
According to one aspect of the invention, a fecal specimen preservation fluid is provided. According to an embodiment of the invention, the stool sample preservation solution comprises: 45-60% by volume of a fixative; 0.1 to 15 mass% of a fixing agent auxiliary agent; 0.01 to 1.5 mass% of an anticoagulant; 10% to 15% by volume of a buffer; 0.01 to 1 mass% of an ionic strength-maintaining agent; 0.01% to 0.5% by volume of a non-ionic detergent; and the balance of water, wherein the fixing agent is at least one selected from methanol, ethanol, glycol and isopropanol, the fixing auxiliary agent is at least one selected from acetic acid and acetate, the anticoagulant agent is at least one selected from ethylenediamine tetraacetic acid and water-soluble salts of ethylenediamine tetraacetic acid, the buffer solution is at least one selected from phosphate buffer solution, acetate buffer solution, ACES buffer solution, ADA buffer solution, BIS-TRIS buffer solution, MES buffer solution and PIPES buffer solution, the ionic strength maintaining agent is chloride salt, and the non-ionic detergent is at least one selected from Tween 20, NP-40 and TritonX-100.
The inventor surprisingly finds that the excrement sample preservation solution can effectively inhibit DNA degradation and cell damage in the excrement sample in a long time, maintain cell morphology and ensure sample quality, so that nucleic acid and cells in the preserved excrement sample can meet the requirement of further research. In addition, according to the embodiment of the invention, the fecal sample preservation solution is easy to prepare, low in cost, capable of preserving the fecal sample at normal temperature, long in sample preservation time and high in sample reutilization qualification rate after preservation.
In the fecal specimen preservation solution of the present invention, the fixative mainly serves to maintain the integrity of DNA and protein in the fecal specimen, denature protease, prevent degradation of protein by enzyme, and fix cellular structure. The inventor finds that the content of the fixative in the excrement sample storage solution is below 45 volume percent, although protein can be fixed, the effect is poor, and DNA degradation can be caused; and the content of the fixing agent is more than 60 percent by volume, which can cause the cells to be seriously deformed and condensed, influence the observation and interpretation of the cell morphology when the subsequent sample is used, and simultaneously increase the difficulty of DNA extraction. According to some preferred embodiments of the invention, the fixing agent is present in an amount of 50% to 60% by volume. Therefore, the effects of immobilizing protein and inhibiting DNA degradation are good.
The function of the fixing auxiliary agent in the excrement sample preservation solution is to expand cells and reduce the contraction effect of the fixing agent on the cells. However, if the content of the fixing aid exceeds 15% by mass, the cells may be excessively swollen, and the cell morphology may be affected.
In the fecal sample preservation solution of the present invention, the anticoagulant mainly functions to remove complex heavy metal ions and prevent cells in the fecal sample from coagulating. According to some preferred embodiments of the invention, the anticoagulant is disodium edetate. In addition, the prior art contains more anticoagulant, which is usually 15-50% (w/w), however, the inventor finds that when the dosage of anticoagulant in the actual excrement sample storage liquid is reduced to 0.01-1.5% (w/w), preferably 0.1-1.5% (w/w), the same anticoagulant effect can be still ensured on the basis of cost reduction. This is because the number of cells in the fecal sample is small, and when the amount of free protein is small, the amount of heavy metal ions bound to the free protein is small. Thus, according to some preferred embodiments of the invention, the anticoagulant is present in an amount of 0.1% to 1.5% by mass.
In the fecal sample preservation solution of the present invention, the ionic strength maintaining agent has the main functions of maintaining the osmotic pressure of cells, maintaining the cell morphology and preventing DNA release into the preservation solution due to cell rupture. According to some specific examples of the present invention, the ionic strength maintaining agent is at least one selected from the group consisting of sodium chloride and potassium chloride. According to some preferred embodiments of the present invention, the ionic strength maintaining agent is contained in an amount of 0.5% by mass to 1% by mass.
In the fecal sample preserving fluid, the buffer solution can automatically adjust the pH change caused by autolysis byproducts released by cell bacteria in the fecal sample, maintain the pH stability and the cell morphology, and simultaneously reduce the natural degradation and breakage of DNA in the sample due to overhigh or overlow pH. According to an embodiment of the invention, the pH of the buffer is 7.4-8.0. According to some embodiments of the invention, the pH of the stool sample preservation solution is 7.4 to 8.0.
In the fecal sample preservation solution of the present invention, the main function of the non-ionic detergent is to dissociate mucus of the feces and increase the dispersion rate of the fecal sample.
According to some embodiments of the invention, the stool sample preservation solution of the invention comprises: 50% to 60% by volume of methanol; 0.1 to 15 mass% of acetic acid; 0.1 to 1.5 mass% of disodium ethylenediaminetetraacetate; 10% to 15% by volume of a phosphate buffer; 0.5 to 1 mass% of sodium chloride; tween-20 in an amount of 0.01% to 0.5% by volume; and the balance being water. Therefore, the preservation effect of the fecal specimen preservation solution is outstanding.
According to a specific example of the present invention, 1000ml of the stool specimen preservation solution contains:
500ml of methanol;
3.5g of acetic acid;
1g of disodium ethylene diamine tetraacetate;
100ml of phosphate buffer;
10g of sodium chloride;
0.5ml of Tween-20; and
399.5ml of water.
According to a specific example of the present invention, 1000ml of the stool specimen preservation solution contains:
450ml of ethanol;
1g of acetic acid;
3g of disodium ethylene diamine tetraacetate;
100ml acetate buffer;
1g of potassium chloride;
0.1ml of Tween-20; and
449.9ml of water.
According to a specific example of the present invention, 1000ml of the stool specimen preservation solution contains:
550ml of ethylene glycol;
2.5g of acetic acid;
7g of disodium ethylene diamine tetraacetate;
150ml of PIPES buffer;
5g of sodium chloride;
2.5ml NP-40; and
297.5ml of deionized water.
According to another aspect of the present invention, the present invention also provides a method for preparing a preservation solution for a stool sample. According to an embodiment of the invention, the method comprises the steps of:
1) providing raw materials according to the formula of the excrement sample preservation solution;
2) adding an ionic strength maintaining agent, a buffer solution and an anticoagulant into deionized water, and dissolving and uniformly mixing the components to obtain a mixture;
3) adjusting the pH value of the mixture to 7.4-8.0;
4) and adding a fixing agent, a fixing agent auxiliary agent and a non-ionic detergent into the mixture subjected to pH value adjustment, uniformly mixing, and sterilizing to obtain the fecal sample preservation solution.
Therefore, the excrement sample preservation solution can be prepared and obtained efficiently, the cost is low, the obtained excrement sample preservation solution is good in quality, the excrement sample preservation solution can be effectively used for preserving excrement samples at normal temperature for a long time, the sample DNA degradation and cell damage after preservation are few, and the sample reutilization qualification rate is high.
According to a further aspect of the invention, there is also provided the use of a faecal sample preservation solution as hereinbefore described for preserving a faecal sample. Therefore, the excrement sample preservation solution can be used for effectively preserving the excrement sample at normal temperature for a long time, and has the advantages of low preservation cost, good effect and high sample reutilization qualification rate.
According to yet another aspect of the invention, a method of preserving a stool sample is also provided. According to an embodiment of the invention, the method comprises: mixing the above feces sample preservation solution and feces sample according to a volume ratio of 1-3: 1, and storing the mixture at room temperature in a sealed and dark place. According to the embodiment of the invention, the method for preserving the fecal sample can effectively realize the long-term preservation of the fecal sample at normal temperature, and has the advantages of simple operation, low preservation cost, good effect, long preservation time and high sample reutilization qualification rate.
According to some preferred embodiments of the invention, the volume ratio of the stool sample preserving fluid to the stool sample is 1.5: 1. therefore, the storage cost is low, and the storage effect is outstanding.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
The general method comprises the following steps:
the preparation method of the fecal sample preservation solution referred to in the following examples generally comprises the following steps:
1) providing raw materials according to the formula of the excrement sample preservation solution;
2) adding an ionic strength maintaining agent, a buffer solution and an anticoagulant into deionized water, and dissolving and uniformly mixing the components to obtain a mixture;
3) adjusting the pH value of the mixture to 7.4-8.0;
4) and adding a fixing agent, a fixing agent auxiliary agent and a non-ionic detergent into the mixture subjected to pH value adjustment, uniformly mixing, and sterilizing to obtain the fecal sample preservation solution.
Example 1
Faecal specimen preservative solutions were prepared according to the general method described previously, according to the formulations indicated in the table below. The method comprises the following specific steps:
adding deionized water (Milli-Q ultrapure water), adding ionic strength maintaining agent, buffer solution and anticoagulant, adjusting pH to 8.0, adding fixative, fixative adjuvant and nonionic detergent, mixing, and sterilizing.
TABLE 1
| Component name | Amount of addition |
| Methanol | 500ml |
| Acetic acid | 3.5g |
| Ethylenediaminetetraacetic acid disodium salt | 1g |
| Phosphate buffer | 100ml |
| Sodium chloride | 10g |
| Tween-20 | 0.5ml |
| Deionized water | 399.5ml |
Wherein, the formula of the phosphate buffer solution is as follows: 100ml of deionized water was added with sodium dihydrogen phosphate (NaH)2PO4·H20.9g of O), disodium hydrogen phosphate (Na)2HPO4·7H2O)24.97g。
Example 2
A stool specimen preservative solution was prepared according to the formulation shown in table 2 below, with reference to the method described in example 1. Wherein, the formula of the acetate buffer solution is as follows: to 100ml of deionized water was added 12.89g of acetic acid (CH)3COOH)0.32g, sodium acetate (CH)3COONa)。
TABLE 2
| Component name | Amount of addition |
| Ethanol | 450ml |
| Acetic acid | 1g |
| Ethylenediaminetetraacetic acid disodium salt | 3g |
| Acetate buffer solution | 100ml |
| Potassium chloride | 1g |
| Tween-20 | 0.1ml |
| Deionized water | 449.9ml |
Example 3
A stool specimen preservative solution was prepared according to the formulation shown in table 3 below, with reference to the method described in example 1. Wherein, the formula of the PIPES buffer solution is as follows: to 150ml of deionized water was added 3.02g of piperazine-1, 4-diethylsulfonic acid (PIPES).
TABLE 3
| Component name | Amount of addition |
| Ethylene glycol | 550ml |
| Acetic acid | 2.5g |
| Ethylenediaminetetraacetic acid disodium salt | 7g |
| PIPES buffer | 150ml |
| Sodium chloride | 5g |
| NP-40 | 2.5ml |
| Deionized water | 297.5ml |
Example 4
A healthy person who had about 10g of feces per pass was put into a 60ml sterile feces collection cup (about 1/4 collection cup), and evenly divided into 8 parallel specimens, and then subjected to feces preservation verification test as follows:
the experiment was set up with 5 treatment groups: anhydrous alcohol group, preservation solution 1 group, preservation solution 2 group, refrigeration group and room temperature group. Wherein, the absolute ethanol group is stored at room temperature by adopting absolute ethanol with the volume of 1.5 times of that of the feces sample; the preservation solution 1 group was prepared by using DETs buffer (20% DMSO, 0.25M EDTA-Na) 1.5 times the volume of the fecal specimen2) Storing at room temperature; preservation solution 2 group, the fecal sample preservation solution of the present invention prepared in example 1 with 1.5 times volume of the fecal sample was used for room temperature preservation; a refrigeration group, wherein the excrement sample is refrigerated and stored at the temperature of-20 ℃; room temperature group, only stool samples were stored at room temperature. And, absolute alcohol group, preservation solution2 replicates (i.e., 2 samples) were set for both group 1 and group 2 of the preservation solution.
For each treatment group, fecal DNA was extracted at different time points of 0h, 24h, and 72h of storage, and then the DNA quality of each treatment sample was compared by OD and gel electrophoresis.
Wherein, the excrement DNA sample is extracted by adopting a QIAamp DNA pool Mini Kit of QIAGEN company, 200mg excrement is taken each time, and the extraction steps are as follows:
1) sampling: a tip-removing gun head sucks 1.5ml of homogeneous sample, the supernatant is removed by centrifugation at 1200rpm, 180 mg of excrement is conveniently taken out to be placed on ice in a 2ml centrifuge tube;
2) digestion: adding 1.6ml Buffer ASL, and shaking until the sample is homogeneous; centrifuging at 12000rpm for 1min, and collecting 1.4ml of supernatant to a new 2ml centrifuge tube; adding 1 inhibitor EX Tablet into each tube, shaking for 1min to dissolve completely, standing for 1min, centrifuging at 12000rpm for 3min, transferring the supernatant to a new 1.5ml centrifuge tube, and centrifuging at 12000rpm for 3 min; taking 600 mu l of supernatant to a 2ml centrifuge tube, adding 25 mu l of protease K, mixing uniformly, adding 600 mu l of Buffer AL, and shaking for 15 s; water bath at 70 deg.C for 10min, separating, adding 600 μ l anhydrous ethanol, and mixing;
3) column passing adsorption: adding 600 μ l of the extract into a labeled QIAamp spincolumn tube, centrifuging at 12000rpm for 1min, removing liquid, separating the liquid in step 5) twice, passing through a column, and removing the centrifuged liquid;
4) rinsing: adding 500 μ l Buffer AW1, centrifuging at 12000rpm for 1min, and removing the centrifugal liquid; adding 500 μ l Buffer AW2, centrifuging at 12000rpm for 3min, removing the centrifugal liquid, and then throwing at 12000rpm for 1 min;
5) and (3) elution: placing a QIAamp spincolumn centrifuge tube into a new 1.5ml centrifuge tube, carefully opening the cover and airing for 3min, adding 50. mu.l of Buffer AE into the center of the column, standing at room temperature for 3min, and centrifuging at 12000rpm for 1 min; then, 50. mu.l of Elution Buffer was added repeatedly for elution, the column was removed, and the resulting liquid was centrifuged to obtain the extracted DNA, which was stored at-20 ℃ for a labeling time and sample name.
The OD value method comprises the following steps: taking 1. mu.l of DNA solution to adoptAnd (4) carrying out ultraviolet absorption detection by an ultraviolet spectrophotometer, and taking an Elution Buffer as a blank control, wherein the reading unit is ng/mu l. The results of OD values at different time points for the 8 samples are shown in Table 4 and FIG. 1.
TABLE 4
The gel electrophoresis method comprises the following steps: after DNA extraction, RNAase enzyme digestion for 1h, 1% agarose gel, 160V electrophoresis for 45min, staining observation, 8 samples of different time electrophoresis results are shown in figure 2. In FIG. 2, panel A shows the electrophoresis pattern of DNA extracted from 8 parallel samples at 0h, panel B shows the electrophoresis pattern of DNA extracted from each sample after storage for 24h, and panel C shows the electrophoresis pattern of DNA extracted from each sample after storage for 72 h. And as shown in fig. 2, the bands in each figure are respectively as follows from left to right: the method comprises the following steps of preparing an anhydrous ethanol group sample 1, an anhydrous ethanol group sample 2, a preservation solution 1 group sample 3, a preservation solution 1 group sample 4, a preservation solution 2 group sample 5, a preservation solution 2 group sample 6, a refrigeration group sample 7 and a room temperature group sample 8; in each figure, M represents Mark, maximum segment 12K.
The results of fig. 1 and 2 show that: storing for 0h (immediately after sampling and storing) to extract DNA, wherein the measured concentration of the OD value of each processing group and the electrophoretic band display are not greatly different, the band is single, the main band is more than 12K, human genome DNA has incomplete removal of protein and RNA, and the measured OD value is larger; after the sample is placed for 24 hours, DNA bands of samples in a refrigerating group and a room temperature group are obviously weakened and seriously degraded, while DNA bands in other treatment groups (namely under other conditions) are weakened in brightness and reduced in concentration, which indicates that degradation exists; after 72 hours of storage, the sample degradation speed in each treatment group is relatively slowed, DNA bands of the samples are weak when the samples are placed at room temperature, RNA and protein impurity bands are reduced, OD value detection and electrophoresis detection results show that the degradation speed is fastest within 24 hours of the storage solution 2 groups, the degradation is slow, the DNA concentration tends to be stable, the DNA in the storage solution 2 groups (namely the excrement sample storage solution prepared in the embodiment 1) is not changed greatly all the time, and the storage effect is obvious.
In addition, in the experiment, after the sample is placed under different storage conditions (different treatment groups) after being collected, in the absolute ethyl alcohol group, the feces sample stored by the absolute ethyl alcohol is dehydrated, compacted and difficult to sample, and the sampling error is larger; in the refrigeration group, samples are refrigerated, the surface temperature is quickly reduced, and the interior of the samples is usually frozen within 2-3 h; in the room temperature group, a sample placed at room temperature extremely generates biochemical reaction, the color becomes dark, pungent and foul smell is generated, and the operation is seriously influenced; the samples in the group of preservative solution 2 were easily dispersed to be homogeneous, and the color change was small without any special odor.
Further, the above-described experimental verification was carried out on the feces sample storage solutions prepared in examples 2 to 3, and the results similarly showed that the feces sample storage solution of the present invention stored the sample DNA with the least change in quality and the best storage effect with respect to the other treatment groups.
Example 5
The method comprises the following steps of putting about 10g of excrement of a healthy person into a 60ml sterile excrement collecting cup (about 1/4 collecting cup), equally dividing the excrement into 4 parallel samples, and carrying out excrement storage verification experiments to further detect the timeliness of the excrement sample storage solution, wherein the excrement is specifically as follows:
experiment set 2 treatments: treatment 1, samples 1-3 the fecal sample preservation solution of the present invention prepared in example 2, which is 1.5 times the volume of the fecal sample, is stored at room temperature; treatment 2, sample 4 was performed using a feces sample of 1.5 volumes DETs buffer (20% DMSO, 0.25M EDTA-Na)2) Then, for each treated sample, DNA in the sample was extracted on days 1, 3, 7, 15 and 60 of storage, and changes in the content of human DNA in the sample were compared by fluorescent quantitative PCR with reference to human conserved gene β -action.
Wherein, the extraction of the fecal DNA sample is performed by using QIAamp DNA pool Mini Kit of QIAGEN company, 200mg of feces are taken each time, and the extraction procedure is as described in example 3.
The fluorescent quantitative PCR adopts the conservative gene β -action for quantification, and utilizes primers β -action-F and β -action-R (target product 175bp), SYBR fluorescent quantitative kit (Takara company), 25 mul of reaction system, 0.5 mul of upstream and downstream primer (concentration 10 mul/L), 64 ℃ of annealing temperature, 45 cycles of annealing, H as negative control2O, positive control human AGS cell line DNA, and the result is taken as the CT value of the amplification curve, and the specific result is shown in figure 3.
Wherein the primer sequences are as follows:
β-actin-F：
5’-TGGTGATGGAGGAGGCTCAGCAAGT-3’(SEQ ID NO：1)；
β-actin-R：
5’-AGCCAATGGGACCTGCTCCTCCCTTGA-3’(SEQ ID NO：2)。
the fluorescence quantitative result in fig. 3 confirms that the preservation time is 60 days, the concentration of human DNA in the fecal samples 1-3 preserved by the fecal sample preservation solution of the invention has little change, the long-time effect is far stronger than the normal temperature preservation (sample 4) effect of DETs buffer solution, and the convenience degree is better than that of absolute ethyl alcohol and low temperature refrigeration.
Further, the above experiments were conducted to verify that the feces storage solutions prepared in examples 1 and 3 were excellent in storage effect with minimal change in the concentration of the sample DNA stored in the feces storage solution of the present invention compared to the other treatment groups when the storage time was up to 60 days.
Example 6 cell morphology preservation validation experiment
A feces amount of about 20g per excretion of a healthy person was put into a 60ml sterile feces collection cup (about 1/3 collection cup), 1.5 times by volume of the feces specimen preservation solution of the present invention prepared in example 3 was added, and about 5.0X10 was added5Individual oral epithelial cells (mixed to make a composition containing a quantity of exfoliated cellsTest stool samples), mix until the stool is homogenized, and stand in the shade at room temperature. Then, the exfoliated cells in the sample were collected and observed as follows:
1) cell separation
SCSR was used on days 1, 7 and 15 of storageTMThe method for separating the cells in the Fecal sample by using the Fecal Cell Isolation Kit comprises the following specific steps:
rough classification at the early stage: sucking with a tip-removing gun head for 4 times, taking about 3ml (about 1g of original excrement is contained, and the ratio of 20g of excrement to 30ml of preservation solution is converted) of a homogenized sample into a plastic bag of a 330-micron nylon net, adding 10ml of precooled PBS, extruding the bag by external force to further refine the sample, transferring the suspension passing through the nylon net into a prepared 50-ml centrifuge tube (the upper end of the centrifuge tube is provided with a removable 40-micron fine sieve), removing the fine sieve, and supplementing the filtrate in the centrifuge tube to 25ml by using the precooled PBS;
centrifugal subdivision: slowly adding 10ml of separation solution (put to room temperature in advance) at the bottom of the centrifugal tube to avoid air bubbles and mixing with the filtrate in the centrifugal tube, so that the interface of the two solutions is clear; centrifuging at room temperature of 200g for 10min, slowly sucking off the supernatant, and leaving about 3ml of residual liquid to suspend the bottom cells;
washing cells: precooled PBS (pH 7.2) was added to the cell suspension to 40ml, mixed well, centrifuged at 900g for 10min at 4 ℃ and the supernatant aspirated, repeated once with 15ml PBS (pH 7.2), carefully removed, 2.5ml of residual solution was left to resuspend the bottom cells and the suspension was put on ice.
2) Observation of cell staining
Cell staining observation was performed according to the following procedure:
smearing, namely preparing a smear (smear method) by taking 1 mu l of cell suspension, and quickly drying;
fixing, fixing the specimen with methanol for 5-10min (3-5 min), and rinsing with distilled water (or naturally drying);
dyeing, diluting the dye solution (9 parts of phosphate buffer solution and 1 part of Giemsa dye solution are fully mixed), dripping the dye solution on tissues or cells, dripping for 10-15min, rinsing with distilled water, and performing 20X and 40X microscopic examination on a cover plate when the cover plate is wet in emergency, wherein the result is shown in figure 4.
As shown in FIG. 4, the cell morphology on day 1 of storage is shown in Panel A, the cell morphology on day 7 of storage is shown in Panel B, the cell morphology on day 15 of storage is shown in Panel C, and most of the peripheral black particles are Escherichia coli and minute impurities in feces; panels D and E show the initial morphology of oral epithelial cells with the surrounding black particles as impurities.
As can be seen from FIG. 4, the stained nuclei of the oral epithelial cells are initially purple red, the cytoplasm is light red, and some cell membranes are irregularly folded, and from FIGS. A-C, it can be seen that the cells in the fecal sample gradually deepen the color of the nuclei observed under the microscope with the increase of the preservation time, the cell morphology is not greatly influenced by the external action, the cell membranes are intact and can be easily identified, in addition, the number of the cells observed by the 1. mu.l cell suspension smear is irregular, and 10 cells are observed in the best result, which is consistent with the theoretical value (5.0 × 10) (5.0530ml) 3ml to 10 were the same, and there was no tendency for the 3 results to decrease, probably because the number of cells in the sample floated very much and the loss of the procedure differed, or the number of experiments was too small.
Further, the above-described experimental verification was carried out on the stool preservation solution prepared in example 1-2, and similarly, the results showed that the cells in the stool sample preserved by the stool preservation solution of the present invention did not change much in cell morphology due to external influences with the increase of preservation time, and the cell membrane was intact.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (10)
1. A stool specimen preservation fluid comprising:
45-60% by volume of a fixative;
0.1 to 15 mass% of a fixing agent auxiliary agent;
0.01 to 1.5 mass% of an anticoagulant;
10% to 15% by volume of a buffer;
0.01 to 1 mass% of an ionic strength-maintaining agent;
0.01% to 0.5% by volume of a non-ionic detergent; and
the balance of water,
wherein,
the fixing agent is at least one selected from methanol, ethanol, glycol and isopropanol,
the fixing auxiliary is at least one selected from acetic acid and acetate,
the anticoagulant is at least one of ethylenediamine tetraacetic acid and water-soluble salts of ethylenediamine tetraacetic acid,
the buffer solution is at least one selected from phosphate buffer solution, acetate buffer solution, ACES buffer solution, ADA buffer solution, BIS-TRIS buffer solution, MES buffer solution and PIPES buffer solution,
the ionic strength maintaining agent is a chloride salt,
the non-ionic detergent is at least one selected from Tween 20, NP-40 and TritonX-100.
2. The preservation solution for fecal samples according to claim 1 characterized in that the pH value of said buffer is 7.4-8.0.
3. The stool sample preservation solution according to claim 1, wherein the pH value of the stool sample preservation solution is 7.4 to 8.0.
4. The stool specimen preservation solution according to claim 1, wherein said ionic strength maintaining agent is at least one selected from the group consisting of sodium chloride and potassium chloride,
optionally, the anticoagulant is disodium edetate.
5. The stool sample preservative solution according to claim 1, wherein the fixative is present in an amount of 50 vol% to 60 vol%,
optionally, the content of the anticoagulant is 0.1-1.5% by mass,
optionally, the ionic strength maintaining agent is contained in an amount of 0.5% by mass to 1% by mass.
6. The stool specimen preservation solution according to claim 5, comprising:
50% to 60% by volume of methanol;
0.1 to 15 mass% of acetic acid;
0.1 to 1.5 mass% of disodium ethylenediaminetetraacetate;
10% to 15% by volume of a phosphate buffer;
0.5 to 1 mass% of sodium chloride;
tween-20 in an amount of 0.01% to 0.5% by volume; and
the balance of water,
optionally, 1000ml of the stool sample preservation solution comprises:
500ml of methanol;
3.5g of acetic acid;
1g of disodium ethylene diamine tetraacetate;
100ml of phosphate buffer;
10g of sodium chloride;
0.5ml of Tween-20; and
399.5ml of water.
7. The stool sample preservation solution according to claim 1, wherein 1000ml of the stool sample preservation solution comprises:
450ml of ethanol;
1g of acetic acid;
3g of disodium ethylene diamine tetraacetate;
100ml acetate buffer;
1g of potassium chloride;
0.1ml of Tween-20; and
449.9ml of water are added to the mixture,
optionally, 1000ml of the stool sample preservation solution comprises:
550ml of ethylene glycol;
2.5g of acetic acid;
7g of disodium ethylene diamine tetraacetate;
150ml of PIPES buffer solution;
5g of sodium chloride;
2.5ml NP-40; and
297.5ml of deionized water.
8. A method for preparing a stool specimen preservation solution, comprising the steps of:
1) providing a starting material according to the formulation of a stool specimen preservative solution according to any one of claims 1 to 7;
2) adding an ionic strength maintaining agent, a buffer solution and an anticoagulant into deionized water, and dissolving and uniformly mixing the components to obtain a mixture;
3) adjusting the pH value of the mixture to 7.4-8.0;
4) and adding a fixing agent, a fixing agent auxiliary agent and a non-ionic detergent into the mixture subjected to pH value adjustment, uniformly mixing, and sterilizing to obtain the fecal sample preservation solution.
9. Use of a stool specimen preservation solution according to any one of claims 1 to 7 for preserving a stool specimen.
10. A method of preserving a stool sample comprising:
a fecal sample preservation solution according to any of claims 1-7 and a fecal sample in a volume ratio of 1-3: 1, sealing and storing in dark at room temperature,
optionally, the volume ratio of the stool sample preservation fluid to the stool sample is 1.5: 1.
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