CN111826371A - Excrement nucleic acid preservation solution and preparation method and application thereof - Google Patents
Excrement nucleic acid preservation solution and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a fecal nucleic acid preserving fluid and a preparation method and application thereof. The fecal nucleic acid preserving fluid comprises: Tris-HCl buffer solution with the concentration of 0.01-1M, Ethylene Diamine Tetraacetic Acid (EDTA) with the concentration of 0.01-1M, sodium chloride with the concentration of 0.01-0.5M, sodium citrate with the concentration of 0.01-0.5M and absolute ethyl alcohol with the volume concentration of 10-30%; the pH value of the preservation solution is 7.0-10.0. The excrement nucleic acid preservation solution provided by the invention can effectively maintain the stability of nucleic acid in excrement, and is convenient for a user to sample at any time and transport at normal temperature. And does not affect the downstream detection of the intestinal flora.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a fecal nucleic acid sample preserving fluid and a preparation method and application thereof.
Background
The intestine is one of the most important organs of the human body. It has been reported that as many as 39 trillion bacterial cells are present in the human gut, 1.3 times the number of human cells, and that these bacteria encode genes 100 times more than human genes. The human intestinal flora is used as a microecosystem, and all members are mutually restricted and interdependent to form an ecological balance in quality and quantity. The stable and functional intestinal flora has important positive effects on the normal physiological metabolism, nutrient absorption, immune maintenance, hormone balance, central nervous system maturation and behavior characteristics of human bodies. The imbalance of the intestinal flora is related to the occurrence and development of various diseases, such as metabolic syndromes of obesity, diabetes and the like, rheumatoid arthritis, inflammatory bowel diseases, influenza virus infection, atherosclerosis and the like. Therefore, the method for detecting the composition structure and the change rule of the intestinal flora by taking the intestinal flora as a target point and using high-throughput sequencing, gene chips and other means becomes a new method for measuring the health condition of a human body and even for accompanying diagnosis of specific diseases.
The detection of fecal microbial composition is the most convenient and noninvasive method for studying intestinal flora structure and function. After the feces are collected, DNA information of almost all microorganisms in the feces is obtained through methods such as nucleic acid extraction, gene detection, bioinformatics analysis and the like, and composition information of all microorganism types in the feces can be obtained.
However, the collection and transportation of fecal samples are greatly restricted and still need to be improved.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, the invention aims to provide the excrement nucleic acid preservation solution and the preparation method and application thereof.
The inventor of the invention finds out in the research process that: during collection and transport, the composition of microorganisms and their nucleic acids in fecal samples is affected by the following factors: first, most of the feces are anaerobic bacteria, which may rapidly stop growing and die due to changes in the oxygen content of the environment, resulting in cell wall rupture and nucleic acid degradation; secondly, the change of temperature can also cause the change of growth rate of different bacteria, so that the composition of the fecal flora changes, certain temperature-sensitive microorganism species die and decompose, and nucleic acid degradation even disappears. The conventional storage mode, namely ultralow temperature (-80 ℃) storage is not suitable for conventional household sampling and transportation, so that part of users abandon detection or the detection result is not in accordance with the reality. At present, a stable and effective method is urgently needed to be developed, microorganisms and nucleic acid in the fecal sample are protected at normal temperature, and the reliability of the fecal flora detection result is ensured.
Therefore, the invention aims to provide the excrement nucleic acid preservation solution which can be used for normal-temperature preservation and transportation, the preservation solution is prepared according to the formula, and the collected fresh excrement sample is directly preserved in the preservation solution, so that the effect of stably and reliably preserving microorganisms and nucleic acid in the sample can be achieved. The excrement sample preserved by the preservation solution can achieve the effect of ultralow temperature cryopreservation immediately after sampling. The preserving fluid is nontoxic and harmless, and can be used for home sampling and normal-temperature long-distance transportation. The components of the preservation solution do not influence the extraction and detection of downstream nucleic acid.
According to a first aspect of the present invention, there is provided a fecal nucleic acid preservation solution comprising: the Tris-HCl buffer solution is 0.01-1M in concentration; EDTA, wherein the concentration of the EDTA is 0.01-1M; sodium chloride, wherein the concentration of the sodium chloride is 0.01-0.5M; sodium citrate, wherein the concentration of the sodium citrate is 0.01-0.5M; absolute ethyl alcohol, wherein the volume concentration of the absolute ethyl alcohol is 10-30%; the pH value of the preservation solution is 7.0-10.0. The fecal nucleic acid preserving fluid can stably preserve microorganisms and nucleic acid in a sample. The Tris-HCl buffer solution in the fecal nucleic acid preservation solution can provide a stable buffer environment for a sample, and the EDTA can inhibit the action of nuclease or protease in the fecal sample, thereby reducing the degradation of nucleic acid; the absolute ethyl alcohol is equivalent to a preservative of the strains, and can provide a stable environment for the strains in the fecal samples. The components are matched with sodium chloride and sodium citrate, so that microorganisms and nucleic acid in the excrement sample can be stably stored, the excrement sample can be stored for 7 days at 40 ℃ after being mixed with the excrement sample, the excrement sample can still be stabilized, and the effect of ultralow-temperature cryopreservation immediately after sampling is achieved. And the method is non-toxic and harmless, is suitable for home sampling and normal-temperature long-distance transportation, and does not influence downstream detection.
According to an embodiment of the present invention, the fecal nucleic acid preservation solution described above may further include the following technical features:
in some embodiments of the invention, the concentration of the Tris-HCl buffer solution is 0.1-0.5M; the concentration of the EDTA is 0.1-0.5M; the concentration of the sodium chloride is 0.05-0.5M; the concentration of the sodium citrate is 0.05-0.3M; the volume concentration of the absolute ethyl alcohol is 15-30%, and the pH value of the preservation solution is 7.5-9.0. The fecal nucleic acid preservative fluid can stabilize nucleic acid samples, and compared with fresh samples, the fecal nucleic acid preservative fluid does not affect microorganisms and nucleic acids in the samples.
In some embodiments of the invention, the concentration of the Tris-HCl buffer is 0.5M; the concentration of the EDTA is 0.15M; the concentration of the sodium chloride is 0.5M; the concentration of the sodium citrate is 0.1M; the volume concentration of the absolute ethyl alcohol is 20 percent; the pH value of the preservation solution is 8.0. Thus, when mixed with a fecal sample, there is no effect on the microorganisms or nucleic acids in the sample during storage.
According to a second aspect of the present invention, there is provided a method for producing a fecal nucleic acid preservation solution, comprising: (1) weighing required amount of Tris, and adjusting the pH to 5.0-10.0 by hydrochloric acid to prepare Tris-HCl buffer solution; (2) dissolving EDTA with required amount in sterile deionized water, and adjusting the pH value to 5.0-10.0 to obtain EDTA solution; (3) dissolving required amounts of sodium chloride and sodium citrate in sterile deionized water, mixing with the Tris-HCl buffer solution and the EDTA solution, adjusting the pH value to 7.0-10.0, and performing autoclaving to obtain a sterilized solution; (4) and mixing the required amount of absolute ethyl alcohol with the sterilized solution, and sterilizing by using a microporous filter membrane to obtain the preservation solution.
According to an embodiment of the present invention, in the above preparation method, the autoclaving is autoclaving at 121 ℃ for 30 minutes.
According to a third aspect of the present invention, there is provided a method of preserving a stool sample, comprising: and mixing the fecal sample and the preservation solution in equal volume, sealing and shading, and preserving at the temperature below 40 ℃, wherein the preservation solution is the preservation solution in any embodiment of the first aspect of the invention.
In some embodiments of the invention, the fecal sample and the preservation solution are mixed in equal volumes, sealed in the dark, and stored at a temperature of-80 ℃.
According to a fourth aspect of the present invention, there is provided a kit comprising a fecal nucleic acid preservation solution according to any of the embodiments of the first aspect of the present invention. In some embodiments of the invention, the kit further comprises a sampling tube, wherein all of the fecal nucleic acid preservation solution is contained in the sampling tube, and the sampling tube is hermetically packaged.
According to a fifth aspect of the invention, there is provided a kit comprising: Tris-HCl buffer solution, EDTA, sodium chloride, sodium citrate and absolute ethyl alcohol; the Tris-HCl buffer solution, the EDTA, the sodium chloride, the sodium citrate and the absolute ethyl alcohol are respectively arranged in different containers.
The beneficial effects obtained by the invention are as follows: the invention relates to a fecal nucleic acid preserving fluid, which is used for preparing and collecting fresh fecal samples, has the characteristics of protecting microorganisms and nucleic acids thereof in feces from degradation, protecting the diversity of the microorganisms, facilitating the collection and transportation of the fecal samples, facilitating downstream gene detection and the like, is beneficial to the research of the diversity of intestinal flora, and is applied to gene detection with the intestinal flora as a target spot.
Drawings
FIG. 1 is a comparison chart of the DNA extraction results of 5 volunteer fecal samples preserved by 5 preservation methods according to the embodiment of the present invention.
FIG. 2 is a comparison of microbial diversity index for 5 volunteer fecal samples stored by 5 storage regimes provided in accordance with an embodiment of the present invention.
FIG. 3 is a comparison graph of microbial principal component analysis of 5 volunteer fecal samples preserved by 5 preservation regimes provided in accordance with an embodiment of the present invention.
FIG. 4 is a comparison of the levels of classification of the microbiota of 5 volunteer faeces samples preserved by 5 preservation regimes provided in accordance with an embodiment of the present invention.
Wherein the reference number 1 in fig. 1-4 corresponds to the processing group No. 1, i.e. the fresh sample is directly extracted; the number 2 corresponds to the number 2 treatment group, namely, the fresh sample is stored for 7 days at the temperature of minus 80 ℃; the label 3 corresponds to the treatment group No. 3, namely, the fresh sample and the preservation solution are mixed, preserved for 3 days at 40 ℃ and then preserved for 4 days at-80 ℃; the number 4 corresponds to the number 4 treatment group, namely, the fresh sample and the preservation solution are mixed and preserved for 7 days at the temperature of 40 ℃; reference numeral 5 corresponds to treatment group 5, i.e., fresh samples were left at 40 ℃ for 7 days.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
The excrement nucleic acid preservation solution provided by the invention can effectively maintain the stability of nucleic acid in excrement, and is convenient for a user to sample at any time and transport at normal temperature. And does not affect the downstream detection process. The mixture of the preservation solution and the excrement can be directly used for downstream detection (nucleic acid extraction and related detection) without sample pretreatment, so that the flexibility of sample preservation and transportation is greatly facilitated, and the efficiency and the accuracy of gene detection with intestinal flora as a target point are improved.
Therefore, the invention provides a fecal nucleic acid preservation solution, which comprises: the Tris-HCl buffer solution is 0.01-1M in concentration; EDTA, wherein the concentration of the EDTA is 0.01-1M; sodium chloride, wherein the concentration of the sodium chloride is 0.01-0.5M; sodium citrate, wherein the concentration of the sodium citrate is 0.01-0.5M; absolute ethyl alcohol, wherein the volume concentration of the absolute ethyl alcohol is 10-30%; the pH value of the preservation solution is 7.0-10.0.
The invention also provides a preparation method of the fecal nucleic acid preserving fluid, which comprises the following steps:
(1) weighing required Tris, and regulating the pH value to 5.0-10.0 by using hydrochloric acid to prepare a Tris-HCl buffer solution;
(2) dissolving EDTA in required amount in sterile deionized water, and regulating pH to 5.0-10.0 with hydrochloric acid to obtain EDTA solution;
(3) dissolving required amount of sodium chloride and sodium citrate in sterile deionized water, adding Tris-HCl solution and EDTA solution, mixing, adjusting pH to 7.0-10.0 with NaOH, and autoclaving at 121 deg.C for 30 min;
(4) adding the required amount of absolute ethyl alcohol into the solution obtained in the step (3), and filtering and sterilizing through a microporous filter membrane.
(5) And (4) subpackaging the prepared preservation solution into sampling tubes, and strictly sealing and preserving at normal temperature.
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.
Example 1
Example 1 fresh stool samples from 5 volunteers were stored under the following 5 conditions, respectively, and the effect of each storage condition on the nucleic acid of the stool samples was verified by nucleic acid extraction. The process is as follows:
first, fresh fecal samples from 5 volunteers were collected and stored under the following 5 conditions:
treatment group No. 1 (extracted immediately after sampling): collecting about 0.2g of fresh excrement sample, and immediately extracting DNA;
treatment group No. 2 (direct cryopreservation for 7 days): collecting about 0.2g of fresh excrement sample, immediately freezing and storing at-80 ℃, and performing DNA extraction after 7 days of storage;
treatment group No. 3 (preservation solution at 40 ℃ for 3 days and ultra-low temperature for 4 days): collecting about 0.2g fresh feces sample, immediately adding equal volume of preservation solution, reversing and mixing, preserving at 40 deg.C for 3 days, preserving at-80 deg.C for 4 days, and extracting DNA;
treatment group No. 4 (preservation solution combined with preservation at 40 ℃ for 7 days): collecting about 0.2g of fresh excrement sample, immediately putting into an equal volume of preservation solution, reversing and uniformly mixing, preserving at 40 ℃ for 7 days, and then extracting DNA;
treatment group No. 5 (one week at 40 ℃): about 0.2g of fresh fecal sample was collected, and DNA extraction was performed after standing at 40 ℃ for 7 days without addition of a stock solution.
Wherein, the preservation solution that No. 3 handled group and No. 4 handled group and used includes Tris-HCl, EDTA, NaCl, sodium citrate and absolute ethyl alcohol, wherein: the concentration of the Tris-HCl buffer solution is 0.5M; the concentration of the EDTA is 0.15M; the concentration of the sodium chloride is 0.5M; the concentration of the sodium citrate is 0.1M; the volume concentration of the absolute ethyl alcohol is 20%, and the pH value of the preservation solution is 8.0.
Specifically, the processes of fecal sample treatment and nucleic acid extraction are as follows:
(1) processing of preserved fecal samples
Treatment group No. 1: about 0.2g of solid fecal sample was placed in a centrifuge tube and 1.4mL Buffer ASL (R) was added
DNA pool Kit, Qiagen, Germany), vortexed on a vortexer to be sufficiently uniform;
treatment group No. 2: about 0.2g of solid fecal sample was placed in a centrifuge tube and 1.4mL Buffer ASL (R) was added
DNA pool Kit, Qiagen, Germany), vortexed on a vortexer to be sufficiently uniform;
treatment group No. 3 and treatment group No. 4: the whole storage tube was shaken well and mixed on a vortex apparatus, and 500. mu.L of 300-
DNA pool Kit, Qiagen, Germany), vortexed on a vortexer to be sufficiently uniform;
treatment group No. 5: about 0.2g of solid fecal sample was placed in a centrifuge tube and 1.4mL Buffer ASL (R) was added
DNA pool Kit, Qiagen, Germany), vortexed on a vortexer to be sufficiently uniform;
(2) incubating for 5min at a constant temperature of 70 ℃ on a metal bath;
(3) fully whirling for 15 seconds, and centrifuging at a high speed for 1 minute to precipitate impurities;
(4) sucking 1.2mL of supernatant, and discarding the precipitate;
(5) to each sample tube (supernatant) was added 1 inhibiex Tablet (b.c.)
DNA StoolKit, Qiagen, Germany), immediately vortexed thoroughly for 1 minute to dissolve the tablets thoroughly. Incubating for 1 minute at normal temperature;
(6) centrifuging at high speed for 3min, sucking all supernatant, and removing precipitate;
(7) to each new empty tube, 200. mu.L of the above supernatant solution and 15. mu.L of proteinase K (
DNAStool Kit, Qiagen, Germany), mixed well; then 200 mu L of Buffer AL is added, and the mixture is evenly mixed by swirling again;
(8) incubating in a constant temperature metal bath or water bath at 70 deg.C for 10 min;
(9) adding 500 mu L of absolute ethyl alcohol, shaking, uniformly mixing and carrying out microcentrifugation;
(10) the mixture was slowly added to a QIAamp spin column (
DNA pool Kit, Qiagen, Germany), centrifuging at 20000rcf for 1min, and discarding the residual solution;
(11) open QIAamp spin column, add 500. mu.L Buffer AW1 (b)
DNA pool Kit, Qiagen, Germany), centrifuging at 20000rcf for 1min, and discarding the residual solution;
(12) open QIAamp spin column, add 500. mu.L buffereAW 2 (b)
DNA pool Kit, Qiagen, Germany), centrifuging at 20000rcf for 1min, and discarding the residual solution; centrifuging repeatedly for one time;
(13) putting the QIAamp centrifugal column into a new 2mL EPP tube, and drying at room temperature for 2 min;
(14) add 100. mu.L Buffer ATE: (
DNA pool Kit, Qiagen, Germany), incubated at room temperature for 3min, centrifuged at 20000rcf for 1min, DNA eluted, and DNA solution collected.
The results of preserving stool samples and extracting DNA in the above 5 ways are shown in FIG. 1. The extracted target DNA has a main band of about 1.5Kb, and if a bright band is obvious at the position and the band is clear and has no dragging, the microbial DNA in the fecal sample is successfully extracted, and the integrity of the DNA molecule is not damaged. As can be seen from fig. 1, each of treatment groups No. 1 to 4 obtained a relatively clear band compared to treatment group No. 5.
The results show that complete DNA can be extracted from samples which are immediately extracted after fresh excrement samples are obtained and immediately frozen at-80 ℃ after sampling; complete DNA can be extracted from samples respectively stored under two conditions (3 days at 40 ℃, 4 days at ultralow temperature or 7 days at 40 ℃) by using the storage solution provided by the invention; and the sample is directly placed at 40 ℃ for 7 days without adding a preservation solution, the DNA of the sample is seriously degraded, no obvious main strip exists, and only a small fragment is arranged below the gel picture. The result shows that the preservation solution provided by the invention can effectively protect the microbial DNA under the tested two temperature preservation conditions, and has good extraction effect.
Example 2
1. Ribosomal 16S rRNA gene V3-V4 region fragment amplification and sequencing
(1) Reagent: high fidelity PCR polymerase system (Kapa Biosystems)
(2) The primer sequence is as follows:
upstream primer (341F): 5' -ACTCCTACGGGAGGCAGCAG (SEQ ID NO:1)
Downstream primer (806R): 5' -GGACTACHVGGGTWTCTAAT (SEQ ID NO:2), wherein V represents base A or base G or base C; w represents the base A or T; h represents base A or base C or base T.
(3) The method comprises the following operation steps: set up PCR total 30. mu.L, including 15. mu.L of 2 XKAPA HiFi HotStartStreadMix (Kapa Biosystems), 0.2. mu.M each of upstream and downstream primers, 30-50ng of DNA template. The reaction was carried out according to the following PCR reaction conditions:
TABLE 1 PCR reaction conditions
(4) And (3) carrying out agarose gel electrophoresis gel cutting purification on the amplified fragment, and sequencing by using an Illumina MiSeq sequencing platform in a PE300 mode.
2. Sequencing results analysis and comparison
(1) Through the above experiments, we obtained the sequencing result of the diversity of the flora measured after 5 volunteers' stool samples were stored under 5 conditions. Through bioinformatics analysis, we obtained the results of the microorganism Alpha-Diversity analysis of each sample, which were represented by species abundance (i.e., number of species, in terms of Operational TaxonomicUnits, number of OTUs) and Diversity Index (Shannon Diversity Index).
As shown in fig. 2, the results indicated that treatment groups 1-4 of each volunteer, namely: samples which are frozen at-80 ℃ immediately after being extracted and sampled and samples which are stored in a storage solution and stored under two conditions respectively have consistent flora abundance and diversity; and treatment group No. 5 of each volunteer, namely: after sampling, the samples are directly placed for 7 days at 40 ℃, the flora abundance and diversity of the samples are obviously lower than those of the No. 1-4 treatment groups, and the fact that the variety of the detected species is obviously reduced due to DNA degradation is suggested. The results show that the preservation solution provided by the invention can effectively reflect the species diversity information of the flora under the two temperature preservation conditions tested.
(2) By bioinformatics Analysis, weighted UniFrac distances between different samples were calculated and compared for Principal component Analysis (PCoA). PCoA based on weighted UniFrac distance shows the correlation between samples as shown in figure 3. Each point on the graph in fig. 3 represents the information on the composition of the fecal flora of 1 volunteer obtained from 1 storage method. The closer the two points are, the more similar the constitutive structures of the two flora and vice versa.
The results in FIG. 3 show that treatment groups 1-4 of each volunteer, namely: samples frozen at-80 ℃ immediately after extraction and sampling, samples stored in a preservation solution and stored under two conditions, respectively, were more similar in the species composition of the flora, while sample No. 5 from each volunteer, i.e.: the samples were directly placed at 40 ℃ for 7 days after sampling, and the species composition of the flora was greatly different from that of the treatment groups No. 1-4. In particular, in volunteers No. 1-4, it was observed that the flora species composition was more similar to that of the immediately extracted sample under both conditions of preservation with preservation solution.
(3) The compositional differences of different samples at the phylum of bacteria classification level were compared by bioinformatics analysis and comparison. Relative abundance of bacteria based on phylum levels is shown in FIG. 4, where each bar represents fecal flora information from 1 volunteer from 1 storage format and the vertical axis represents the relative abundance of each bacterial phylum in each sample.
The results in FIG. 4 show that treatment groups 1-4 of each volunteer, namely: samples which are immediately extracted and frozen at the temperature of minus 80 ℃ after sampling, samples which are respectively preserved in the preservation solution under two conditions, and the species compositions of the floras are similar; in particular, samples taken immediately and stored in the present preservation solution under both conditions were significantly more similar in terms of the species composition of the flora. And treatment group No. 5 of each volunteer, namely: the samples were directly placed at 40 ℃ for 7 days after sampling, and the species composition of the flora was greatly different from that of the treatment groups No. 1-4. In particular, the results of the tests on the 5 volunteer samples all showed that Bacteroidetes (Bacteroidetes) decreases in abundance under storage conditions at 40 ℃ for 7 days, whereas Proteobacteria (Proteobacteria) increases in abundance under these conditions. Under the condition of preservation, the relative abundance of the bacteria with low relative abundance also increases remarkably.
Based on the DNA extraction effect of fecal microorganisms, the abundance of detected bacteria, diversity analysis, the detected flora structure, the composition of detected phyla levels of bacteria, it can be concluded: the fecal nucleic acid preserving fluid is used for preserving fecal samples under different temperature conditions, the nucleic acid extraction effect is consistent with the effect of extracting nucleic acid immediately after collecting fresh fecal samples or immediately after ultralow temperature cryopreservation, and the nucleic acid extraction effect is superior to the effect of extracting nucleic acid after direct placement at 40 ℃.
Meanwhile, the preservation effect of the fecal nucleic acid preservation solution with different components on the fecal samples is researched.
Experiments are carried out by designing different gradients of reagent of the fecal nucleic acid preservative solution formula, 5 experimental groups are carried out by using the same method for the preservative solution in the formula disclosed in the patent, the effect of the preservative solution in different formulas is verified by using the same method disclosed in the patent, and the result shows that the formula of the preservative solution disclosed in the patent has the best effect. Meanwhile, a formula with similar reagent composition is designed, 5 experimental groups are carried out by the same method with the preservation solution in the formula in the patent, the effect of the preservation solution in different formulas is verified by the same method in the patent, and the result shows that the preservation solution formula in the patent has the best effect.
The result shows that the excrement nucleic acid preservation solution provided by the invention can maintain the stability of nucleic acid in excrement, is convenient for a user to sample at any time and transport at normal temperature, can be directly used for downstream detection without pretreatment after being mixed with excrement, facilitates the flexibility of sample preservation and transport, and is beneficial to improving the efficiency and the accuracy of gene detection taking intestinal flora as a target.
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 are not necessarily intended to 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. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
<110> Shanghai Sharp next Biotech Co., Ltd
<120> excrement nucleic acid preservation solution and preparation method and application thereof
<130>PIDC4180303
<160>2
<170>PatentIn version 3.5
<210>1
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<213> Artificial sequence
<220>
<223> primer
<400>1
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<211>20
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<220>
<223> primer
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Claims (10)
1. A fecal nucleic acid preserving fluid, which is characterized in that: the method comprises the following steps:
the Tris-HCl buffer solution is 0.01-1M in concentration;
EDTA, wherein the concentration of the EDTA is 0.01-1M;
sodium chloride, wherein the concentration of the sodium chloride is 0.01-0.5M;
sodium citrate, wherein the concentration of the sodium citrate is 0.01-0.5M;
absolute ethyl alcohol, wherein the volume concentration of the absolute ethyl alcohol is 10-30%;
the pH value of the preservation solution is 7.0-10.0.
2. The preservation solution according to claim 1,
the concentration of the Tris-HCl buffer solution is 0.1-0.5M;
the concentration of the EDTA is 0.1-0.5M;
the concentration of the sodium chloride is 0.05-0.5M;
the concentration of the sodium citrate is 0.05-0.3M;
the volume concentration of the absolute ethyl alcohol is 15-30%;
the pH value of the preservation solution is 7.5-9.0.
3. The preservation solution according to claim 1,
the concentration of the Tris-HCl buffer solution is 0.5M;
the concentration of the EDTA is 0.15M;
the concentration of the sodium chloride is 0.5M;
the concentration of the sodium citrate is 0.1M;
the volume concentration of the absolute ethyl alcohol is 20 percent;
the pH value of the preservation solution is 8.0.
4. A method for producing a preserving fluid as claimed in any one of claims 1 to 3, which comprises: the method comprises the following steps:
(1) weighing required amount of Tris, and adjusting the pH to 5.0-10.0 by hydrochloric acid to prepare Tris-HCl buffer solution;
(2) dissolving EDTA with required amount in sterile deionized water, and adjusting the pH value to 5.0-10.0 to obtain EDTA solution;
(3) dissolving required amounts of sodium chloride and sodium citrate in sterile deionized water, mixing with the Tris-HCl buffer solution and the EDTA solution, adjusting the pH value to 7.0-10.0, and performing autoclaving to obtain a sterilized solution;
(4) and mixing the required amount of absolute ethyl alcohol with the sterilized solution, and sterilizing by using a microporous filter membrane to obtain the preservation solution.
5. The method of claim 4, wherein the autoclaving is autoclaving at 121 ℃ for 30 minutes.
6. A method of preserving a stool sample, comprising:
mixing the fecal sample and the preservation solution in equal volume, sealing and shading, preserving at the temperature below 40 ℃,
wherein the preservation solution is the preservation solution according to any one of claims 1 to 3.
7. The preservation method according to claim 6, wherein the fecal specimen and the preservation solution are mixed in equal volumes, sealed and shielded from light, and preserved at a temperature of-20 ℃.
8. A kit comprising the fecal nucleic acid preservation solution according to any one of claims 1 to 3.
9. The kit of claim 8, further comprising: and the sampling tube is filled with the excrement nucleic acid preservation solution and is packaged in a sealing way.
10. A kit, comprising: Tris-HCl buffer solution, EDTA, sodium chloride, sodium citrate and absolute ethyl alcohol; the Tris-HCl buffer solution, the EDTA, the sodium chloride, the sodium citrate and the absolute ethyl alcohol are respectively arranged in different containers.
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| CN201910298925.1A CN111826371A (en) | 2019-04-15 | 2019-04-15 | Excrement nucleic acid preservation solution and preparation method and application thereof |
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| CN109735600A (en) * | 2019-02-27 | 2019-05-10 | 深圳市金锐生物科技有限公司 | A kind of genital tract flora nucleic acid preservation formula of liquid, method of preparation and use |
| CN112522362A (en) * | 2020-12-25 | 2021-03-19 | 南京申友生物技术有限公司 | Preservation solution for preserving bacterial DNA in fecal sample at normal temperature |
| CN113729008A (en) * | 2021-09-30 | 2021-12-03 | 苏州中科先进技术研究院有限公司 | Excrement storage liquid, preparation method and application thereof |
| CN114323850A (en) * | 2021-12-22 | 2022-04-12 | 上海锐翌生物科技有限公司 | Sample preprocessing device and nucleic acid and stool sample preprocessing method |
| CN114657069A (en) * | 2022-04-24 | 2022-06-24 | 深圳净拓生物科技有限公司 | Excrement storage liquid and preparation method of excrement bacterial suspension |
| CN115486439A (en) * | 2022-09-23 | 2022-12-20 | 深圳微辰生命科技有限公司 | Feces preservation method and application of ethanol in preparation of feces preservation solution and short-chain fatty acid detection reagent or kit |
| CN117063920A (en) * | 2023-10-16 | 2023-11-17 | 苏州帕诺米克生物医药科技有限公司 | Preservation method of fecal sample and detection method of short chain fatty acid of fecal sample |
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| CN114657069A (en) * | 2022-04-24 | 2022-06-24 | 深圳净拓生物科技有限公司 | Excrement storage liquid and preparation method of excrement bacterial suspension |
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Application publication date: 20201027 |