A normal-temperature soil preserving solution and a method for preparing the same

Abstract

This invention relates to a soil preservation solution for room temperature preservation and its preparation method, belonging to the field of soil microbial preservation technology. The soil preservation solution for room temperature preservation of this invention contains the following components: a dissociation agent, a chelating agent, a salt ion balancer, an inhibitor, a nucleic acid protectant, and water; the pH of the preservation solution is 7.5-9.5; the dissociation agent includes thiocyanate, guanidine salt, and a surfactant; the thiocyanate is selected from any one or a combination of sodium thiocyanate, ammonium thiocyanate, potassium thiocyanate, and lithium thiocyanate; the concentration of the thiocyanate in the preservation solution is 0.1-0.5M; the guanidine salt is selected from any one or a combination of guanidine sulfate and guanidine isosulfate; the concentration of the guanidine salt in the preservation solution is 0.1-0.5M; the surfactant is selected from any one or a combination of SDS, Tween-20, and PVP. Treating soil samples with the preservation solution of this invention can effectively maintain the stability of microbial genomic DNA nucleic acids in the soil samples and is beneficial for the extraction of soil microbial nucleic acids.

Description

Technical Field

[0001] This invention relates to a soil preservation solution for preserving soil at room temperature and its preparation method, belonging to the field of soil microbial preservation technology. Background Technology

[0002] In recent years, microbiome research has received increasing attention and importance. Among them, the study of soil microorganisms has gradually become favored by researchers. Sequencing analysis of soil microorganisms is crucial for microbial research, and the extraction of soil microorganisms is necessary before sequencing analysis. The ability to obtain high-quality nucleic acids is the foundation for sequencing and other research.

[0003] Nucleic acid extraction using fresh materials or cryopreserved samples is often an important method for obtaining high-quality nucleic acids. However, there are many inconveniences in collecting and extracting soil samples or performing cryopreservation in remote areas. Therefore, if freshly collected soil samples could be placed in a preservation solution and stored at room temperature, and then nucleic acid extraction and other operations could be performed in the laboratory, the efficiency of sample preservation and extraction would be greatly improved.

[0004] To date, there are no soil microbial preservation solutions on the market, and there are no reported patents related to soil preservation solutions.

[0005] Chinese patent CN110004212A discloses a fecal preservation solution, its preparation method, and a method for preserving feces. The fecal preservation solution comprises: 0.10-0.20M Tris-based; 0.25-0.75M EDTA; 10mM sodium chloride (NaCl), saturated at room temperature; 5-15% dimethyl sulfoxide (DMSO); 2.5-10% ethanol; and 5-20% glycerol. The combination of dimethyl sulfoxide and glycerol is more suitable for preserving bacterial cultures with fewer inhibitors, but not for preserving sample types rich in inhibitors, such as soil and feces.

[0006] Regarding other existing technologies, the inventors have found that the current technologies mainly have the following technical problems: When preserving samples with high levels of inhibitors such as feces and soil, the liquid salt used is mainly guanidine salt. A high guanidine salt content is detrimental to the purity of extracted nucleic acids, reducing their purity and hindering PCR amplification and sequencing. Furthermore, the lack of nucleic acid protective agents may be detrimental to long-term sample preservation; additionally, some inhibitors in the soil may competitively bind to the binding solution during microbial DNA extraction, which is unfavorable for subsequent microbial nucleic acid extraction.

[0007] Based on the above background, there is a need for a preservation solution suitable for storing soil at room temperature and its preparation method. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a soil preservation solution and its preparation method that can effectively preserve soil nucleic acids for a long time at room temperature.

[0009] Based on the technical problems existing in the prior art, the inventors made the following improvements: reducing the amount of guanidine salt and using thiocyanate; adding a nucleic acid protectant; adding an inhibitor, thus providing a soil preservation solution and its preparation method that can effectively preserve soil nucleic acids for a long time at room temperature.

[0010] The technical solution of this invention to solve the technical problem is as follows:

[0011] In a first aspect of the invention, a preservation solution for preserving soil at room temperature is provided.

[0012] The preservation solution contains the following components: a liquid release agent, a chelating agent, a salt ion balancer, an inhibitor, a nucleic acid protectant, and water; the pH of the preservation solution is 7.5-9.5; the preferred pH of the preservation solution is 8.0.

[0013] The liquid release agent includes thiocyanate, guanidine salt, and surfactant;

[0014] The thiocyanate is selected from any one or a combination of several of sodium thiocyanate, ammonium thiocyanate, potassium thiocyanate, and lithium thiocyanate; the concentration of the thiocyanate in the storage solution is 0.1-0.5M;

[0015] The guanidine salt is selected from any one or a combination of several of guanidine sulfate and guanidine isosulfate; the concentration of the guanidine salt in the preservation solution is 0.1-0.5M;

[0016] The surfactant is selected from any one or a combination of several of SDS, Tween-20, and PVP; the concentration of the surfactant in the preservation solution is 1-5%.

[0017] The functions of each component in this invention are explained below:

[0018] Dissociation agent: Allows cells carrying nucleic acid molecules to separate from impurities;

[0019] Chelating agents: Chelating agents are used to bind with metal ions to form large molecular substances that precipitate.

[0020] Salt ion balancer: ensures the balance of salt ions in the solution;

[0021] Blocking agents: These agents block or reduce the interaction between contaminants in a sample and nucleic acids, thereby promoting the separation of nucleic acid molecules.

[0022] Nucleic acid protectants: ensure the integrity of nucleic acid molecules during the separation process.

[0023] Preferably, the specific components of the liquid release agent and its concentration in the preservation solution are as follows: 0.3M sodium thiocyanate, 0.2M guanidine sulfate and 2% PVP.

[0024] Further, the chelating agent is selected from any one or a combination of several of ethylenediaminetetraacetic acid (EDTA), citric acid, ethylene glycol diethyl ether diaminetetraacetic acid (EGTA), and trisodium hypotriacetate monohydrate (NTA). Preferably, the specific components of the chelating agent and its concentration in the preservation solution are as follows: 0.1 M EDTA.

[0025] Further, the salt ion balancing agent is an inorganic salt ion buffer solution, which is selected from any one or a combination of several of sodium chloride, potassium chloride, lithium chloride, sodium fluoride, and Tris-HCl. Preferably, the specific components of the salt ion balancing agent and its concentration in the preservation solution are as follows: 150 mM sodium chloride, 50 mM potassium chloride, 50 mM sodium fluoride, and 0.1 M Tris-HCl.

[0026] Further, the blocking agent is a short-chain fatty acid salt, which is selected from any one or a combination of several of sodium polystyrene sulfonate, sodium polyacrylate, sodium acetate, ammonium acetate, and ammonium glycolate. Preferably, the specific components of the blocking agent and its concentration in the preservation solution are as follows: 1% sodium polystyrene sulfonate.

[0027] Furthermore, the nucleic acid protectant is any one or a combination of several of trehalose, glycine, and mannitol. More preferably, the specific components of the nucleic acid protectant and its concentration in the preservation solution are as follows: 20 mM trehalose, 0.5 M glycine, and 80 mM mannitol.

[0028] In a preferred embodiment of the present invention, the specific components of the liquid preparation solution for preserving soil at room temperature, including the liquid separation agent, chelating agent, salt ion balancer, blocking agent, and nucleic acid protectant, and the concentrations of each component in the liquid preparation solution, are as follows:

[0029] Liquid release agents: 0.3M sodium thiocyanate, 0.2M guanidine sulfate, and 2% PVP;

[0030] Chelating agent: 0.1M EDTA;

[0031] Salt ion balancer: 150mM sodium chloride, 50mM potassium chloride, 50mM sodium fluoride and 0.1M Tris-HCl;

[0032] Blocking agent: 1% sodium polystyrene sulfonate;

[0033] Nucleic acid protective agents: 20mM trehalose, 0.5M glycine, and 80mM mannitol;

[0034] The pH of the preservation solution is 8.0.

[0035] In this invention, all percentages (%) refer to weight / volume percentages (w / v%), expressed in g / mL.

[0036] In a second aspect of the invention, a method for preparing a preservation solution for preserving soil at room temperature as described in the first aspect is provided.

[0037] The preparation method is as follows: Weigh each component according to the above formula, dissolve them in sterile deionized water, adjust the pH, and store at room temperature.

[0038] The present invention has the following technical effects:

[0039] 1) This invention provides a preservation solution for preserving soil at room temperature. The preservation solution contains a liquid release agent, a chelating agent, an ionic strength maintainer, an inhibitor, a nucleic acid protectant, and water. Treating soil samples with the preservation solution of this invention can effectively maintain the stability of microbial genomic DNA nucleic acids in the soil samples and facilitates the extraction of soil microbial nucleic acids.

[0040] 2) As can be seen from Example 2, the preservation solution of the present invention can effectively maintain the stability of DNA nucleic acids at room temperature, and at least the nucleic acids can still be effectively detected in the preservation solution after 30 days. As can be seen from Example 3, the soil preservation solution of the present invention is as effective as soil preservation at -80℃, and even slightly better than -80℃ freezing, with unexpected technical effects.

[0041] 3) As can be seen from Example 4, the concentration and purity of soil microbial nucleic acid extracted using the soil preservation solution of the present invention are superior to those of the prior art, indicating that the preservation solution can effectively maintain the stability of host nucleic acid at room temperature. Attached Figure Description

[0042] Figure 1 To test the preservation effect of soil preservation solution on nucleic acids.

[0043] Figure 2 Geloscouring images of nucleic acids extracted from soil using different methods for preservation.

[0044] Figure 3 The image shows the 16S amplification of nucleic acid extracted from soil after 30 days of preservation using different methods. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0046] Example 1

[0047] The soil preservation solution formulation of Example 1 consists of the following components: 0.3M sodium thiocyanate, 0.2M guanidine sulfate, 2% PVP, 0.1M EDTA, 150mM sodium chloride, 50mM potassium chloride, 50mM sodium fluoride, 0.1M Tris-HCl, 1% sodium polystyrene sulfonate, 20mM trehalose, 0.5M glycine, and 80mM mannitol.

[0048] The soil preservation solution of Example 1 is prepared as follows: Weigh each component according to the above formula, dissolve it in sterile deionized water, adjust the pH, and store at room temperature.

[0049] Example 2: Detection of the nucleic acid preservation ability of the soil preservation solution from Example 1

[0050] The soil preservation solution from Example 1 was used to test its ability to preserve nucleic acids. The specific experimental steps are as follows:

[0051] 1. Sample preparation: Three types of surface soil of the same type were randomly selected within the area of ​​119°54'35"E and 32°23'59"N.

[0052] 2. Experimental Methods: Add 5 mL of the preservation solution from Example 1 to the soil sample. Take an equal amount of 100 μL of Helicobacter pylori nucleic acid, vortex to mix, and divide each sample into two equal parts. Place them at room temperature for 0 days and 30 days, respectively. Take 600 μL of the preservation solution from each part and use the Soil Genomic DNA Extraction Kit (CW2091) to extract DNA for comparison. For detailed operation steps, please refer to the product instructions. Elute with 100 μL. Take 5 μL of each sample as a template for real-time quantitative qPCR to detect the amplification Ct value of Helicobacter pylori.

[0053] According to patent CN115044688A, fluorescent probe detection was performed on the extracted genomic DNA, and the primers used were also referenced in patent CN115044688A. Control 1 used Helicobacter pylori extraction and amplification in sterile water with added bacteria as control 2, and control 2 used soil extraction and amplification in sterile water with added bacteria as control 3. Specific nucleic acid extraction procedures are described in the instructions.

[0054] All tests were repeated, and the average cycle threshold was used for calculation.

[0055] The experimental results are shown in Table 1:

[0056] Table 1. Preservation effect of soil preservation solution on DNA and nucleic acid by qPCR.

[0057]

[0058] As can be seen from Table 1, compared with the internal control Ct of the 0-day control group, the Ct difference of nucleic acids stored in the preservation solution for 0 days and 30 days after extraction and recovery is basically within 1 Ct range, and the Ct value of the corresponding samples is not significantly different. This indicates that the preservation solution of the present invention can effectively maintain the stability of DNA nucleic acid under room temperature conditions, and at least the nucleic acid can still be effectively detected after 30 days in the preservation solution.

[0059] Figure 1 These are the detection values ​​in the "Ct values ​​saved for 0 days" and "Ct values ​​saved for 30 days" columns corresponding to Table 1.

[0060] Example 3: Detection of the ability of soil preservation solution to preserve microbial nucleic acids in soil samples

[0061] The soil preservation solution was prepared using the preservation solution from Example 1. Three soil samples were collected from the area described in Example 2 as experimental samples, and four different treatments were performed, with three replicates for each treatment. The specific treatments were as follows:

[0062] (1) Direct extraction group: Weigh 3 soil samples, 0.3g each, and immediately extract genomic DNA. Store the extracted nucleic acid at -80 degrees Celsius for later use.

[0063] (2) -80 degrees Celsius for 30 days: Weigh 3 soil samples, 0.3g each, and store them in a sealed container at -80 degrees Celsius for 30 days. Then, extract genomic DNA and use the extracted nucleic acid for later use.

[0064] (3) Soil preservation solution for 30 days: Weigh 3 soil samples, 3g each, add 15mL of soil preservation solution to each sample, mix well, seal and preserve at room temperature (18-25 degrees Celsius) for 30 days, take 1.5mL of soil preservation solution (containing about 0.3g of soil sample) for genomic DNA extraction, and use the extracted nucleic acid for later use.

[0065] (4) Sealed storage at room temperature for 30 days: Weigh 3 soil samples, 0.3g each, and seal and store at room temperature (18-25 degrees Celsius) for 30 days. Perform genomic DNA extraction, and use the extracted nucleic acid for later use.

[0066] (5) Sterile water storage for 30 days: Weigh 3 soil samples, 3g each, add 15mL of sterile water to each sample, mix well, and seal and store at room temperature (18-25 degrees Celsius) for 30 days. Take 1.5mL of the solution (containing about 0.3g of soil sample) for genomic DNA extraction. The extracted nucleic acid is then ready for use.

[0067] Genomic DNA was extracted using a soil genomic DNA extraction kit (Kangwei Century, catalog number CW2091). For detailed operating procedures, please refer to the product instructions. Finally, DNA and nucleic acid were eluted using 100 μL of elution buffer.

[0068] Nucleic acid concentration and purity detection: The nucleic acid concentration of the extracted genomic DNA was determined using Qubit, and the nucleic acid purity was determined using Nanodrop. The results are shown in Table 2-4.

[0069] Table 2

[0070] c(ng / uL) Sample 1 Sample 2 Sample 3 Direct extraction group 21.69 61.18 38.19 Store at -80 degrees Celsius for 30 days. 26.33 36.92 17.24 Soil preservation solution can be stored for 30 days. 29.79 83.49 34.38 Store in a sealed container at room temperature for 30 days. 101.38 101.90 80.35 Sterile water can be stored for 30 days. 11.44 40.91 9.76

[0071] Table 3

[0072] A260 / 280 Sample 1 Sample 2 Sample 3 Direct extraction group 1.82 1.87 1.78 Store at -80 degrees Celsius for 30 days. 1.83 1.90 1.75 Soil preservation solution can be stored for 30 days. 1.88 1.88 1.58 Store in a sealed container at room temperature for 30 days. 1.93 1.91 1.85 Sterile water can be stored for 30 days. 1.49 1.41 1.53

[0073] Table 4

[0074] A260 / 230 Sample 1 Sample 2 Sample 3 Direct extraction group 0.64 1.19 0.91 Store at -80 degrees Celsius for 30 days. 0.96 1.21 0.57 Soil preservation solution can be stored for 30 days. 0.62 1.19 0.57 Store in a sealed container at room temperature for 30 days. 1.05 1.30 1.04 Sterile water can be stored for 30 days. 0.37 0.58 0.33

[0075] As can be seen from Table 2-4:

[0076] Nucleic acid concentration and purity obtained by preserving soil using the soil preservation solution of this invention for 30 days are consistent with, or even slightly better than, nucleic acid extracted from samples preserved at ultra-low temperatures for 30 days. However, samples directly preserved in water for 30 days cannot yield high-quality nucleic acid.

[0077] The extracted genomic DNA from each group was compared for nucleic acid integrity using agarose gel electrophoresis. For example... Figure 2 As shown.

[0078] from Figure 2 It can be seen that the nucleic acid extracted after 30 days of preservation in soil preservation solution has the same integrity as that extracted after 30 days of preservation in ultra-low temperature freezer, and is better than that extracted after 30 days of preservation in water.

[0079] After the extracted genomic DNA groups were mixed, the bacterial 16S gene was amplified by real-time quantitative PCR, and the nucleic acid stability of the host gene was compared and detected. The fluorescent dye used for amplifying the 16S gene was Ultra SYBR Mixture (Catalog No.: CW0957) from Kangwei Century.

[0080] Specific primers for the 16S gene include:

[0081] F: CCTACGGGAGGCAGCAG (SEQ ID NO: 1)

[0082] R: ATTACCGCGGCTGCTGG (SEQ ID NO: 2)

[0083] The amplification system is configured as follows:

[0084] name Volume / uL UltraSYBR Mixture (2×) 12.5 Upstream primer (10 μM) 0.5 Downstream primer (10uM) 0.5 template 2 Water (to bring the total to 25uL) 9.5 total 25

[0085] The quantitative PCR amplification reaction program is configured as follows:

[0086] Quantitative fluorescence amplification of 16S (using ABI 7500)

[0087]

[0088] Nucleic acid product PCR detection results as follows Figure 3 As shown. From Figure 3 It can be seen that the 16S gene of bacteria in the soil could not be amplified by real-time PCR in soil that was sealed and stored at room temperature for 30 days. This indicates that the nucleic acid extracted after 30 days of sealed storage at room temperature is no longer the nucleic acid of the original microorganisms in the soil. The types of microorganisms in the soil have changed. Therefore, direct storage at room temperature cannot meet the conditions for preserving microorganisms in the soil.

[0089] Amplification of 16S:

[0090]

[0091]

[0092] Experimental results: The results of quantitative fluorescence are shown in the table above (Table and...). Figure 3 Correspondingly, during the 16S amplification, except for the 30 groups sealed at room temperature, the other 4 groups all showed amplification, indicating that the bacterial community structure in the 30-day sealed at room temperature group may have changed. Therefore, it can be concluded that sealed at room temperature cannot preserve soil microorganisms. In addition, directly preserving soil with sterile water may lead to the degradation of nucleic acids in soil microorganisms. Preserving soil using the soil preservation solution of this invention is as effective as preserving soil at -80℃, and even slightly better than freezing at -80℃.

[0093] Example 4

[0094] The soil preservation solution was prepared using the preferred method of the preservation solution in the embodiments of the present invention, and a fecal preservation solution was prepared according to patent CN113637723A as a comparative example.

[0095] Three soil samples were collected from the area in Example 2 as experimental samples. Each soil sample was divided into two parts. One part was added to the soil preservation solution of Example 1 of this invention, and the other part was added to the comparative sample. After being sealed and stored at room temperature for 30 days, 1.5 mL of the soil preservation mixture (containing about 0.3 g of soil sample) was taken for genomic DNA extraction. The extracted nucleic acid was then used for future reference.

[0096] The results are shown in the table below:

[0097] c(ng / uL) This invention Comparative Invention Methods Sample 1 34.56 16.81 Sample 2 24.92 16.24 Sample 3 7.41 5.79 A260 / 280 This invention Comparative Invention Methods Sample 1 1.85 1.91 Sample 2 1.92 1.83 Sample 3 193 185 A260 / 230 This invention Comparative Invention Methods Sample 1 1.06 0.49 Sample 2 0.57 0.66 Sample 3 0.35 0.24

[0098] Conclusion: The concentration and purity of soil microbial nucleic acids extracted using the soil preservation solution of the present invention are superior to those of the comparative example, indicating that the preservation solution can effectively maintain the stability of host nucleic acids at room temperature.

[0099] The above are merely embodiments of the present invention and do not limit the scope of the patent. Any equivalent modifications made based on the content of this specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A preservation solution for soil at room temperature, characterized in that, The preservation solution is formulated as follows: 0.3M sodium thiocyanate, 0.2M guanidine sulfate, 2% PVP, 0.1M EDTA, 150mM sodium chloride, 50mM potassium chloride, 50mM sodium fluoride, 0.1M Tris-HCl, 1% sodium polystyrene sulfonate, 20mM trehalose, 0.5M glycine and 80mM mannitol, and the pH of the preservation solution is 7.5-9.5.

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