WO2018138913A1 - Preservation solution and sample preservation method using said preservation solution, in particular preservation solution for sample dna and chemical substance such as organic acid or polyamine and preservation method using said preservation solution - Google Patents

Abstract

[Problem] To provide a preservation solution that can be safely stored and transported at room temperature, and that preserves samples in a condition that allows at least one of following to be carried out: microbial population analysis in which a molecular biological technique is used; and physical and chemical analysis of chemical substances such as organic acids and polyamine. In addition, to provide a method for preserving chemical substances using the preservation solution. [Solution] Provided is a preservation solution for DNA or a chemical substance, characterized by being formed by mixing at least one selected from the group consisting of 3 types of reagents: 4 mol/dm³ of guanidine thiocyanate; 0.1 mol/dm³ of Tris-HCl having a pH of 7.0-9.0; and 0.04 mol/dm³ of EDTA. Also provided is a method for preserving a sample DNA or chemical substance using the preservation solution.

Description

Preservation solution and method for preserving specimen using the preservation solution, particularly preservation solution for specimen DNA and chemical substances such as organic acids and polyamines, and preservation method using the preservation solution

In the present invention, at least one of chemical substances such as DNA, organic acids and polyamines present in living bodies such as humans and animals and in environments such as water systems, soils, and air is stable at room temperature (about 1 to 40 ° C.). Relates to a preservable storage solution. The present invention also relates to a specimen storage method using the storage solution.

Currently, microorganisms are considered to be able to separate only about 1% existing on the earth, and the remaining 99% are in a VNC (viable but non-culturable) state, which means that they are in a “living but unculturable” state. Such microorganisms that are incapable of culturing are understood as unculturable microorganisms and difficult-to-cultivate microorganisms. In other words, it means that there are many microorganisms that require unknown growth factors for culturing and microorganisms that are impossible or extremely difficult to purely culture. For the analysis of the microbial community structure, various molecular biological techniques using DNA that do not require culturing have been developed and used. However, there has been a problem that the microbial community structure differs from that at the time of collection depending on the temperature and method of storage from the collection of the specimen to analysis. It is an important issue to know the true microbial community structure to stably store and transport the DNA at room temperature without changing the DNA of the microbial community in the specimen at the time of collection.

Various molecular biological methods using this DNA include real-time PCR analysis, metagenomic analysis using next-generation sequencers, microbial community structure analysis such as T-RFLP analysis and DGGE analysis, DNA base sequence analysis of microorganism culture strains and strains There are RAPD analysis and microsatellite analysis, which are DNA polymorphism analysis methods for identifying the above.

In recent years, not only microbial community structure analysis, but also comprehensive analysis and analysis techniques of metabolites in living organisms and biological chemicals have been used for research to comprehensively elucidate metabolic activities in living organisms and the environment. Began to be. This comprehensive analysis and analysis method for metabolites is called metabolomic analysis and has been actively performed.

Quantitative analysis of chemical substances includes liquid chromatography, gas chromatography, capillary electrophoresis, and analysis combining these mass spectrometers.

Conventionally, when conducting analysis of microbial community structure and chemical substances such as organic acids and polyamines, sample collection is required to suppress the growth, death, and decomposition of microorganisms and the oxidation, decomposition, and volatilization of chemical substances. It was common to store frozen immediately. Therefore, it is virtually impossible to collect specimens in places where refrigeration equipment is difficult to install, and specimens can also be used in homes where it is difficult to freeze and store biological specimens such as feces due to sanitary problems. The collection was difficult.

In addition, when comparing frozen samples with samples immediately after collection, the presence of microbial communities that change easily, the amount of organic acid changes when frozen and stored, and during freezing and thawing during pretreatment There was a problem such as.

In the absence of refrigeration equipment, pretreatment, analysis and analysis should be started immediately after refrigerated storage in order to conduct microbial community structure analysis reflecting the state at the time of collection and physicochemical analysis of organic acids, polyamines, etc. In other words, preparation for analysis and analysis and handling of a large number of specimens become complicated, and when much time is required, it is difficult to immediately analyze and analyze.

From such a background, the inventors cannot proliferate the collected specimen by denaturing the biological cell wall protein in an aqueous solution containing a protein denaturant, a DNA degrading enzyme inactivator, a chelating agent, etc. Development of a storage solution that can stably store and transport DNA of biological communities and culture strains at room temperature without affecting the structure of biological communities at the time of collection by suppressing DNA resynthesis and degradation. I came.

As a result, for the purpose of stably storing and transporting DNA derived from microbial communities, the inventors have conventionally added 0.1 mol as an enzyme inactivator to 4 mol / dm ³ guanidine thiocynate, which has been known as a powerful protein denaturant. A method has been proposed in which the specimen is immersed in a storage solution to which 0.04 mol / dm ³ EDTA is added as a chelating agent and / dm ³ Tris-HCl (pH 9.0) (see Non-Patent Document 1).

However, since detailed studies on the concentration of guanidine thiothionate in the preservation solution were not carried out, as a result of earnest examination of this point, the inventors were able to reduce the concentration of guanidine thiothionate in the preservation solution. I found out. Reduced costs, increased efficiency of PCR amplification of DNA and reduced handling risks such as effects on the human body, and transport of infectious disease specimens falls under the prevention of infectious diseases and restrictions on transportation by medical laws for patients with infectious diseases Transportation methods that take safety into account are now possible. (See Patent Document 1).

Koji NAGASHIMA, Daisuke YASOKAWA, Kentaro ABE, Ryoji NAKAGAWA, Tooru KITAMURA, Toshiharu MIURA, Shu KOGAWA, Effect of a Lactobacillus species on incidence of diarrhea in calves micro , 97-110.

NAGASHIMA (K.), HISADA (T.), SATOU (M.), MOCHIZUKI (J.), Application of New Primer-Enzyme Combination to Terminal Restriction Fragment Length Polymorphism Profiling Environmental ., 2003, 69, 1251-1262.

NAGASHIMA (K.), MOCHIZUKI. (J.), HISADA (T.), SUZUKI (S.), SHIMOMURA (K.), Phylogenetic analysis of 16S ribosomal RNA gene sequences fromcal microbiotautility polymorphism profiling. Bioscience Microflora, 2006, 25, 99-107.

JP 2016-136911 A

On the other hand, there is no storage solution for storing specimens in a state where chemical substances such as organic acids and polyamines can be stably stored and transported at room temperature for physicochemical analysis, and microorganisms using molecular biological techniques There was no storage solution for storing a specimen in a state where at least one of a crowd analysis and a physicochemical analysis could be performed, and a storage method using the storage solution.

Because of this, it can be stored and transported stably at room temperature, and at least one of microbial community analysis using molecular biological techniques and physicochemical analysis of chemical substances such as organic acids and polyamines can be performed. It has been desired to develop a storage solution for storing the specimen in a stable state.

Furthermore, in the case of stool specimens, a larger amount of specimen is required in physicochemical analysis than in molecular biological techniques, while the size of the container must be kept small in terms of storage and transportation. In other words, the amount of the sample must be taken into consideration due to the effect on the preservation of DNA and chemical substances such as organic acids and polyamines due to the decrease in the concentration of guanidine thiothionate in the preservation solution by the specimen collection, and the mixing efficiency of the preservation solution and the specimen. It was necessary to set an optimal guanidine thiothionate concentration for the ratio of the storage solution amount to the ratio and the ratio.

The present invention can meet the above-mentioned demands, and has the following configuration. That is,
(1) From three types of reagent groups consisting of 4 mol / dm ³ guanidine thiocynate, 0.1 mol / dm ³ Tris-HCl having a pH of 7.0 to 9.0, and 0.04 mol / dm ³ EDTA, It is a preservation solution obtained by mixing at least one selected reagent. In addition, 0.1 mol / dm ³ Tris-HCl is more preferably set to pH 8.0.
(2) A storage solution characterized by storing at least one of DNA and a chemical substance such as an organic acid or polyamines in addition to the above-described configuration of the storage solution.
(3) A storage method comprising: adding a storage solution having the above-described configuration to a specimen, and storing the specimen at room temperature.
(4) In addition to the configuration of the storage method described above, the storage solution contains 4 mol / dm ³ guanidine thiocynate reagent, and after the storage solution is added to the specimen, the final concentration of guanidine thiocynate in the storage solution is 0.01 mol. / Dm ³ or more, and the volume of the specimen is 50% or less with respect to the volume of the storage solution. Note that the final concentration of guanidine thiocyanate in ^{0.15mol / dm 3 ~ 0.75mol / dm} 3, the capacity of the specimen is below 25% relative to volume of the stock solution, but it is more preferable.

According to the first and second aspects of the present invention, it is possible to provide a novel storage solution capable of stably storing at least one of DNA and a chemical substance at room temperature. It is possible to provide a preservation solution capable of preserving DNA constituent ratios or chemical substances such as organic acids and polyamines, or DNA and DNA constituent ratios and chemical substances such as organic acids and polyamines. is there.

According to the invention described in claim 3, it is possible to newly provide a storage method for stably storing at least one of a sample DNA and an organic acid or a chemical compound of polyamines at room temperature.

According to the invention of claim 4, at least one of the sample DNA and the chemical substance can be stably stored and transported at room temperature, and the microbial community structure analysis, DNA and DNA using molecular biological techniques It is possible to newly provide a storage method that can use at least one of the specimens immersed in a solution for the physicochemical analysis of chemical substances such as composition ratios, organic acids, and polyamines.

"Comparison of fecal microbial community structure (fungal flora) after storage for 7 days at 30 ° C in a storage solution at the final concentration of each of guanidineine thiocynate by freezing and T-RFLP analysis" FIG. The vertical axis shows the abundance ratio of various bacterial species in feces. "Comparison of fecal microbial community structure (fungal flora) after storage for 7 days at 30 ° C in a storage solution at the final concentration of frozen storage and each guanidine thiocynate by molecular biological method T-RFLP analysis" FIG. The vertical axis shows the abundance ratio of various bacterial species in feces. It is the figure of the test subject A about "the organic acid of the stool after 30 days and 30 degreeC preservation | save with the preservation | save solution of 30 degreeC and the preservation | save solution of the final density | concentration of each guanidine thiocynate by the physical and chemical analysis. It is the figure of the test subject B about "comparison of the organic acid of the stool after a freezing preservation | save and the preservation | save solution of the final concentration of each guanidineinethiocynate by 30 days at 30 degreeC by a physicochemical analysis." "By physicochemical analysis, saliva polyamine after room temperature storage of with H 2 _O saved and the preservation solution" is a diagram for. The mix in the figure is a mixed solution of EDTA and Tris.

(Embodiment)
A preferred embodiment of the present invention will be described in detail.

The preservation solution in the present embodiment includes three types consisting of 4 mol / dm ³ guanidine thiocynate, pH 7.0 to 9.0 mol 0.1 mol / dm ³ Tris-HCl, and 0.04 mol / dm ³ EDTA. From the reagent group, at least one selected reagent is mixed. In addition, 0.1 mol / dm ³ Tris-HCl is more preferably set to pH 8.0.

The storage method in the present embodiment is characterized in that the above-described storage solution is added to a specimen, and at least one of the DNA of the specimen and a chemical substance such as an organic acid or a polyamine is stored at room temperature. .

Further, in the storage method described above, the storage solution contains 4 mol / dm ³ guanidine thiocynate reagent, and after the storage solution is added to the specimen, the final concentration of guanidine thiocynate in the storage solution is 0.01 mol / dm ³ or more. In addition, it is preferable that the volume of the specimen is 50% or less with respect to the volume of the storage solution. Note that the final concentration of guanidine thiocyanate in ^{0.15mol / dm 3 ~ 0.75mol / dm} 3, and more preferably be 25% or less with respect to the capacity of the storage of the sample solutions.

Samples include soils, sludges, liquids, etc. of environmental samples including various organisms, and feces, saliva, blood, etc. of biological samples.

Experiments (Experiment Method 1 and Experiment Method 2) to be described later were performed in order to demonstrate the operational effects of the preservation solution prepared based on the above-described embodiment and the preservation method using the preservation solution.

(Experiment Method 1)
In two subjects, the concentration of stool specimens immediately frozen after stool and guanidine thiocynate was 1 mol / dm ³ (final concentration after stool collection 0.75 mol / dm ³ ), 0.6 mol / dm ³ (after stool collection) Final concentration of 0.45 mol / dm ³ ), 0.4 mol / dm ³ (final concentration after stool collection 0.30 mol / dm ³ ), 0.2 mol / dm ³ (final concentration after stool collection 0.15 mol / dm ³ ) ³ ) and 4 kinds of guanidine thiocynate concentration storage solutions prepared by mixing 0.1 mol / dm ³ Tris-HCl (pH 8.0) and 0.04 mol / dm ³ EDTA. The stool was collected so that the stool concentration (specimen amount) was 250 g / dm ³ (25%). The collected stool samples are stored at 30 ° C. for 7 days, followed by comparison of microbial community structure (microflora) by T-RFLP analysis and comparison of organic acids by liquid chromatography (succinic acid, lactic acid, formic acid, acetic acid, propion) Acid, iso-butyric acid, n-butyric acid, iso-valeric acid, n-valeric acid). The examination conditions are shown in Table 1.

Microbial community structure (microflora) analysis by T-RFLP analysis is to extract the total bacterial DNA in feces, label the bacterial 16S rRNA gene region with forward and reverse primers, and either primer with fluorescent dye, After performing PCR amplification and cleaving with a restriction enzyme, the fluorescently labeled terminal DNA fragments cleaved using a capillary sequencer are fractionated, and the length and relative amount of each DNA fragment are analyzed to determine the bacterial community structure. It is a method of examining.

The examination of this case was based on the method of Nagashima et al. (See Non-Patent Documents 2 and 3). For fragment analysis, ABIＢPRISM 3130xl DNA Sequencer (Applied Biosystems, CA, USA) and GeneMapper (Applied Biosystems, CA, USA) were used. The classification of each fragment was judged by the operational taxonomic unit.

In the physicochemical analysis of organic acids, a certain amount of stool specimen was precisely weighed in a bead tube, suspended in an extraction solution, and then heat-treated (85 ° C., 20 minutes). The sample was crushed with a bead beater, centrifuged at 233.3 rps for 10 minutes, the supernatant was filtered through a membrane filter with a pore size of 0.45 μm, and this was analyzed as a sample solution by a high performance liquid chromatogram. The analysis conditions are shown in Table 2.

(Experimental result 1)
The results are shown in FIGS. In both the microbial community structure (bacteria flora) analysis and the physicochemical analysis of organic acids, in 2 subjects, which preservation of 0.2 mol / dm ³ or more (final concentration after stool collection 0.15 mol / dm ³ or more) The solution also preserved the microbial community structure (bacteria flora) and the amount of various organic acids, indicating that there was no difference from frozen storage. In the subject, iso-butyric acid, iso-valeric acid, and n-valeric acid were not detected. However, the same study using 0.2 mol / dm ³ stock solution in other subjects showed that iso-butyric acid, iso-valeric acid, and n-valeric acid were also stably retained (data) Not shown).

From the above results, guanidine thiocynate was added to a solution containing 0.1 mol / dm ³ Tris-HCl (pH 8.0), 0.04 mol / dm ³ EDTA and 0.2 mol / dm ³ to 1 mol / dm ³ of guanidine thiocynate. by final concentration after stool collection to collect feces to be ^{0.15mol / dm 3 ~ 0.75mol / dm} 3, microbial community structure (lawn) analysis and organic acids using molecular biology techniques It was confirmed that feces for physicochemical analysis can be maintained and stored at room temperature for 7 days in the same manner as frozen storage.

In addition, since the microbial community structure can be maintained and preserved, by dissolving the cell membranes of the microorganisms of infectious disease samples and clinical samples to make them unable to grow or infect, it becomes possible to handle safely without restrictions on transportation. It was also confirmed that it can be used for microbial community structure (bacteria flora) analysis and microbial gene analysis (identification, classification, strain identification) using various molecular biological techniques.

In addition, since organic acids can be maintained and preserved, by dissolving the cell membranes of microorganisms in infectious disease samples and clinical samples so that they cannot grow or infect, it becomes possible to handle them safely without restrictions on transportation. It was also confirmed that it can be used for physicochemical analysis by various methods.

This enables analysis and analysis that can be stably stored and transported at room temperature, and can perform at least one of microbial community structure (bacteria flora) analysis using molecular biological techniques and physicochemical analysis such as organic acids. It became possible to use it.

And since microbial community structure can be maintained and preserved, real-time PCR analysis, metagenomic analysis using next-generation sequencers, microbial community structure analysis such as T-RFLP analysis and DGGE analysis, DNA base sequence analysis of biological culture strains and strain identification Specimens that can be used for various molecular biology techniques using DNA such as RAPD analysis and microsatellite analysis, which are DNA polymorphism analysis techniques to be performed, and that enable specimens to be collected at various locations A combined product such as a collection container, a specimen transport container that enables the specimen to be transported at room temperature, and a specimen storage container that enables the specimen to be stored at room temperature can be expected. In addition, products that can handle infectious disease samples and clinical samples can be expected.

(Experiment Method 2)
Next, a saliva specimen was prepared by adding 1.5 μmol / dm ³ polyamines standard 30 mm ³ to saliva 30 mm ³ and H ₂ O as a control, and each storage solution 30 mm ³ as a control, and immediately after addition and at room temperature. Changes in polyamines after storage for ˜2 weeks were measured. Table 3 shows the conditions for studying the stock solution.

The physicochemical analysis of polyamines was carried out by adding acetonitrile (MeCN) 120 mm ³ and 70 nmol / dm ³ diaminohexane to each saliva sample, deproteinizing it, centrifuging at 3000 g for 5 minutes, and using the supernatant as the sample solution, 40 mmol / dm ³ DBD-F in acetonitrile (90 mm ³⁾ and 100 mmol / dm ³ triethylamine aqueous solution (60 mm ³⁾ were added, and after derivatizing the polyamine at 60 ° C. for 30 minutes, 12 types of polyamines were analyzed by a high performance liquid chromatogram mass spectrometer. The analysis conditions are shown in Table 4.

(Experimental result 2)
The results are shown in FIG. With control H ₂ O and Tris alone, data of 12 types of polyamines immediately after addition and after 2 weeks changed, but EDTA alone or a mixed storage solution (mix) of EDTA and Tris can be stably stored It was confirmed that it can be used as a storage solution that can withstand actual analysis.

Therefore, EDTA alone can be stably stored and transported at room temperature, and can be used as a storage solution that can be used for physicochemical analysis of chemical substances such as polyamines.

Sample collection containers that allow samples to be collected at various locations, sample transport containers that allow samples to be transported at room temperature, and sample storage that enables samples to be stored at room temperature We can expect products that combine containers.

Since chemical substances such as organic acids and polyamines can be maintained and stored, it can be confirmed that they can be used for physicochemical analysis in combination with liquid chromatography, gas chromatography, capillary electrophoresis and their mass spectrometers, and samples can be used in various places. A product that combines a sample collection container that can be collected at room temperature, a sample transport container that enables the sample to be transported at room temperature, and a sample storage container that enables the sample to be stored at room temperature. I can expect.

Claims (4)

At least one kind from three kinds of reagent groups consisting of 4 mol / dm ³ guanidine thiocynate, pH 7.0 or more and 9.0 or less 0.1 mol / dm ³ Tris-HCl, and 0.04 mol / dm ³ EDTA A preservation solution obtained by mixing the above selected reagents. 2. The preservation solution according to claim 1, wherein at least one of DNA and a chemical substance such as an organic acid or a polyamine is preserved. A storage method comprising: adding the storage solution according to claim 1 or 2 to a specimen, and storing the specimen at room temperature. The preservation solution contains a reagent of 4 mol / dm ³ guanidine thiocynate, and after the preservation solution is added to the specimen, the final concentration of guanidine thiocynate in the preservation solution is 0.01 mol / dm ³ or more. The storage method according to claim 3, wherein the volume of the specimen is 50% or less with respect to the volume.

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