Method for extracting extracellular adsorption nucleic acid in water
By using sterilized beef paste solution and centrifugal separation and other technical means in the tap water system, extracellular adsorption of nucleic acids in the water were successfully isolated and extracted, and the nucleic acid confounding caused by the filter membrane intercepting suspended particles in the prior art was solved, and more accurate water quality monitoring and high-quality water supply were achieved.
Patent Information
- Application Number
- CN202510315505.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-18
- Publication Date
- 2025-06-06
AI Technical Summary
When extracting nucleic acids in tap water systems, the 0.22μm filter membrane will intercept suspended particles, resulting in the mixed nucleic acid and intracellular nucleic acid, affecting the extraction effect.
A new extraction method is adopted to elute the sample by sterilized beef paste solution, and the extracellular adsorption nucleic acids in water are isolated and extracted.
The separation of intracellular nucleic acid and extracellular adsorbed nucleic acid is achieved, pure intracellular nucleic acid samples are provided, and extracellular adsorbed nucleic acid is recovered, supporting more accurate water quality monitoring and risk assessment.
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Figure CN120098989A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of molecular biology, and in particular to a method for extracting extracellular adsorbed nucleic acids in water. Background Art
[0002] Nucleic acids in water systems are mainly divided into intracellular nucleic acids and extracellular nucleic acids, among which extracellular nucleic acids can be further divided into extracellular free nucleic acids and extracellular adsorbed nucleic acids. Intracellular nucleic acids exist in microorganisms in water, extracellular free nucleic acids exist in water bodies, and extracellular adsorbed nucleic acids exist on solid particles in water or in biofilms in water.
[0003] At present, the method for recovering nucleic acids in tap water systems mainly relies on filtration technology. Specifically, the water sample is first filtered through a glass sand core filter device equipped with a 0.22μm pore size filter membrane, the purpose of which is to intercept microorganisms in the water and separate extracellular free nucleic acids, so that intracellular nucleic acids can be extracted using a commercial nucleic acid extraction kit.
[0004] However, this method has a significant limitation: the 0.22μm filter membrane can not only retain microorganisms in the water, but also retain suspended particles in the water. These suspended particles usually adsorb a large amount of extracellular nucleic acids, and therefore will be extracted together in the subsequent nucleic acid extraction process, resulting in the final "intracellular nucleic acid" sample mixed with extracellular nucleic acids.
[0005] Therefore, the present invention further optimizes the method to separate the intracellular nucleic acid from the extracellular adsorbed nucleic acid to obtain pure intracellular nucleic acid and to recover the extracellular adsorbed nucleic acid. Summary of the invention
[0006] The purpose of the present invention is to provide a method for extracting extracellular adsorbed nucleic acids in water based on the above technical problems.
[0007] In order to achieve the above object, the first aspect of the present invention provides a method for extracting extracellular adsorbed nucleic acids in water, the method comprising the following steps:
[0008] (1) sterilizing the eluate to obtain a sterilized eluate;
[0009] (2) placing the sample to be tested in the sterilized eluent and stirring for 20 to 30 minutes to obtain a suspension;
[0010] (3) The suspension is divided into two centrifuge tubes for separation, the supernatant is taken, filtered, the filtrate is collected, isopropanol is added, shaken, and allowed to stand for 16 to 18 hours, shaken again, divided into centrifuge tubes for separation, the supernatant is discarded, ethanol solution is added to the precipitate, resuspended, divided into centrifuge tubes for separation, the supernatant is discarded, the supernatant is allowed to stand for 5 to 10 minutes, sterile double distilled water is added, and resuspended to obtain extracellular adsorbed nucleic acid.
[0011] Preferably, in step (1), the eluent is a beef extract solution, and the concentration of the eluent is 3%.
[0012] Further preferably, in step (1), the sterilization treatment conditions meet the following conditions: temperature is 121° C. and time is 20 min.
[0013] Preferably, in step (2), the sample to be tested is a filter membrane of a filtered water sample or a collected biofilm sample; and the volume of the filtered water sample is 3 to 10 L.
[0014] More preferably, in step (2), the amount of the sterilized eluent used is 40 to 100 mL.
[0015] Preferably, in step (3), the separation treatment conditions meet the following requirements: room temperature, rotation speed of 12000 rpm, and time of 10 min.
[0016] More preferably, in step (3), the amount of isopropanol used is 40-100 mL.
[0017] Preferably, in step (3), the concentration of the ethanol solution is 70% to 75%.
[0018] Preferably, in step (3), the sterile double distilled water is specifically sterilized ultrapure water or PCR-grade sterile enzyme-free water, and the amount of the sterile double distilled water is 1 to 2 mL.
[0019] The method provided by the present invention has at least the following beneficial effects:
[0020] (1) The method provided by the present invention realizes the separation of intracellular nucleic acids and extracellular adsorbed nucleic acids, which is of great significance for accurately evaluating the distribution and content of nucleic acids in water systems. Extracellular adsorbed nucleic acids usually come from the lysis and secretion of microorganisms or free nucleic acids in the external environment. Their content and composition can reflect the microbial activity in the water body, the degree of environmental pollution, and the trend of water quality changes. By separating and quantifying extracellular adsorbed nucleic acids, more accurate data support can be provided for water quality monitoring and risk assessment, which will help to carry out subsequent in-depth research, achieve high-quality water supply, and ensure the safety of residents' water use.
[0021] (2) The method for extracting extracellular adsorbed nucleic acids in the prior art is the magnetic bead method, that is, preparing a separation solution to separate intracellular nucleic acids from extracellular adsorbed nucleic acids, then adding nanomagnetic beads to the solution containing extracellular adsorbed nucleic acids, and using the adsorption of magnetic beads to nucleic acids to recover extracellular nucleic acids in the solution. The method provided by the present invention changes the composition of the separation solution (i.e., the eluent) and uses isopropanol to precipitate nucleic acids, thereby reducing costs to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 is a Venn diagram of the microbial community structure of intracellular nucleic acid, extracellular free nucleic acid and extracellular adsorbed nucleic acid of the water sample in Example 1;
[0023] Figure 2 This is an ASV abundance difference analysis diagram of the intracellular nucleic acid and the extracellular adsorbed nucleic acid of the water sample in Example 1, wherein red in the figure represents intracellular nucleic acid and green represents extracellular adsorbed nucleic acid. DETAILED DESCRIPTION
[0024] The endpoints and any values of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and the individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.
[0025] The present invention will be described in detail below by way of examples. In the following examples, unless otherwise specified, the raw materials are all commercially available.
[0026] In the following examples, unless otherwise specified, the room temperature is 20-25°C.
[0027] Example 1
[0028] This example is used to provide a method for extracting extracellular adsorbed nucleic acids in water from a tap water system, the method comprising the following steps:
[0029] (1) placing 70 mL of a 3% (w / v) beef extract solution in an autoclave for sterilization to obtain a sterilized eluate;
[0030] The beef extract solution was prepared as follows: 3 g of beef extract was fully dissolved in 100 mL of ultrapure water;
[0031] The sterilization treatment conditions are as follows: temperature is 121° C. and time is 20 min.
[0032] (2) 10 L of tap water was filtered through a glass sand core filter device, the filter membrane was removed, and placed in 70 mL of the sterilized eluent, a magnetic stirrer was added, and stirred at room temperature for 25 min to obtain a suspension;
[0033] The glass sand core filter device is equipped with a filter membrane with a pore size of 0.22 μm;
[0034] The amount of the magnetic stirrer added is 1.
[0035] (3) The suspension was divided into multiple centrifuge tubes, centrifuged at room temperature and a speed of 12000 rpm for 10 min, all the supernatant was taken with a syringe (to avoid inhaling the precipitate), filtered through a 0.22 μm syringe filter, the filtrate was collected and placed in a 250 mL conical flask, and 70 mL of isopropanol was added, shaken, and allowed to stand for 18 h. The suspension was divided into multiple 50 mL centrifuge tubes, centrifuged at room temperature and a speed of 12000 rpm for 10 min, the supernatant was discarded, 1 mL of 75% ethanol solution was added to the precipitate, and the suspension was resuspended. The suspension was divided into 50 mL centrifuge tubes, centrifuged at room temperature and a speed of 12000 rpm for 10 min, the supernatant was discarded, and the suspension was allowed to stand at room temperature for 10 min. 1 mL of sterile double distilled water was added and the suspension was resuspended to obtain extracellular adsorbed nucleic acid.
[0036] Example 2
[0037] This example is used to provide a method for extracting extracellular adsorbed nucleic acids in water of a natural water system, the method comprising the following steps:
[0038] (1) placing 40 mL of a 3% (w / v) beef extract solution in an autoclave for sterilization to obtain a sterilized eluate;
[0039] The beef extract solution was prepared as follows: 3 g of beef extract was fully dissolved in 100 mL of ultrapure water;
[0040] The sterilization treatment conditions are as follows: temperature is 121° C. and time is 20 min.
[0041] (2) 3 L of river water was filtered through a glass sand core filter device, the filter membrane was removed, and placed in 40 mL of the sterilized eluent, a magnetic stirrer was added, and stirred at room temperature for 25 min to obtain a suspension;
[0042] The glass sand core filter device is equipped with a filter membrane with a pore size of 0.22 μm;
[0043] The amount of the magnetic stirrer added is 1.
[0044] (3) The suspension was divided into multiple centrifuge tubes, centrifuged at room temperature and a speed of 12000 rpm for 10 min, all the supernatant was taken with a syringe (to avoid inhaling the precipitate), filtered through a 0.22 μm syringe filter, the filtrate was collected and placed in a 250 mL conical flask, and 40 mL of isopropanol was added, shaken, and allowed to stand for 16 h. The suspension was divided into multiple 50 mL centrifuge tubes, centrifuged at room temperature and a speed of 12000 rpm for 10 min, the supernatant was discarded, 1 mL of 70% ethanol solution was added to the precipitate, and the suspension was resuspended. The suspension was divided into 50 mL centrifuge tubes, centrifuged at room temperature and a speed of 12000 rpm for 10 min, the supernatant was discarded, and the suspension was allowed to stand at room temperature for 5 min. 2 mL of sterile double distilled water was added and the suspension was resuspended to obtain extracellular adsorbed nucleic acid.
[0045] Example 3
[0046] This example is used to provide a method for extracting nucleic acids adsorbed outside of biofilms in a water system, the method comprising the following steps:
[0047] (1) placing 100 mL of 3% (w / v) beef extract solution in an autoclave for sterilization to obtain a sterilized eluate;
[0048] The beef extract solution was prepared as follows: 3 g of beef extract was fully dissolved in 100 mL of ultrapure water;
[0049] The sterilization treatment conditions are as follows: temperature is 121° C. and time is 20 min.
[0050] (2) placing the cotton swab collected from the biofilm sample of the water system pipe network into 100 mL of the sterilized eluent, adding a magnetic stirrer, and stirring at room temperature for 30 minutes to obtain a suspension;
[0051] The amount of the magnetic stirrer added is 1.
[0052] (3) The suspension was divided into multiple centrifuge tubes, centrifuged at room temperature and a speed of 12000 rpm for 10 min, all the supernatant was taken with a syringe (to avoid inhaling the precipitate), filtered through a 0.22 μm syringe filter, the filtrate was collected and placed in a 250 mL conical flask, and 100 mL of isopropanol was added, shaken, and allowed to stand for 17 h. The suspension was divided into multiple 50 mL centrifuge tubes, centrifuged at room temperature and a speed of 12000 rpm for 10 min, the supernatant was discarded, 1 mL of 70% ethanol solution was added to the precipitate, and the precipitate was resuspended. The suspension was divided into 50 mL centrifuge tubes, centrifuged at room temperature and a speed of 12000 rpm for 10 min, the supernatant was discarded, and the precipitate was allowed to stand at room temperature for 8 min. 2 mL of sterile double distilled water was added and resuspended to obtain extracellular adsorbed nucleic acid.
[0053] Comparative Example 1
[0054] The method of Example 1 is followed, except that in step (2), the volume of the tap water is 20 L, and the remaining steps and parameters are the same as those of Example 1.
[0055] Comparative Example 2
[0056] The method of Example 1 was followed, except that in step (2), the volume of the eluent used was 20 mL, and the remaining steps and parameters were the same as in Example 1.
[0057] Comparative Example 3
[0058] The method of Example 2 was followed, except that in step (2), the concentration of the eluent used was 6% (w / v). The remaining steps and parameters were the same as those of Example 2.
[0059] Comparative Example 4
[0060] The method of Example 3 was followed, except that in step (3), the standing time was 10 h, and the remaining steps and parameters were the same as those of Example 3.
[0061] In order to express the present invention more clearly, Figure 1 The Venn diagram of the microbial community structure of intracellular nucleic acid, extracellular free nucleic acid and extracellular adsorbed nucleic acid of the water sample in Example 1 is shown. Figure 2 The figure shows the difference analysis of ASV abundance between intracellular nucleic acid (red) and extracellular adsorbed nucleic acid (green) of the water sample in Example 1.
[0062] Depend on Figure 1 It can be seen that there are significant differences in the community structures of intracellular nucleic acids, extracellular free nucleic acids, and extracellular adsorbed nucleic acids; Figure 2 It can be seen that there is a significant difference in the ASV level between extracellular adsorbed nucleic acids and intracellular nucleic acids, which proves that the community components of the extracellular adsorbed nucleic acids and intracellular nucleic acids extracted by this method are different (that is, the sources of nucleic acids are different). Therefore, the nucleic acids extracted by the method in the embodiment of the present invention are not intracellular nucleic acids, but extracellular adsorbed nucleic acids.
[0063] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.
Claims
1. A method for extracting extracellular adsorbed nucleic acids in water, characterized in that: The method comprises the following steps: (1) sterilizing the eluate to obtain a sterilized eluate; (2) placing the sample to be tested in the sterilized eluent and stirring for 20 to 30 minutes to obtain a suspension; (3) The suspension is divided into two centrifuge tubes for separation, the supernatant is taken, filtered, the filtrate is collected, isopropanol is added, shaken, and allowed to stand for 16 to 18 hours, shaken again, divided into centrifuge tubes for separation, the supernatant is discarded, ethanol solution is added to the precipitate, resuspended, divided into centrifuge tubes for separation, the supernatant is discarded, the supernatant is allowed to stand for 5 to 10 minutes, sterile double distilled water is added, and resuspended to obtain extracellular adsorbed nucleic acid.
2. The method according to claim 1, characterized in that: In step (1), the eluent is a beef extract solution, and the concentration of the eluent is 3%.
3. The method according to claim 1 or 2, characterized in that: In step (1), the sterilization treatment conditions are as follows: temperature is 121° C. and time is 20 min.
4. The method according to claim 1 or 2, characterized in that: In step (2), the sample to be tested is a filter membrane of a filtered water sample or a collected biofilm sample; and the volume of the filtered water sample is 3 to 10 L; And / or, in step (2), the amount of the sterilized eluent used is 40 to 100 mL.
5. The method according to claim 1 or 2, characterized in that: In step (3), the separation treatment conditions are as follows: room temperature, rotation speed of 12000 rpm, and time of 10 min.
6. The method according to claim 1 or 2, characterized in that: In step (3), the amount of isopropanol used is 40 to 100 mL.
7. The method according to claim 1 or 2, characterized in that: In step (3), the concentration of the ethanol solution is 70% to 75%.
8. The method according to claim 1 or 2, characterized in that: In step (3), the sterile double distilled water is specifically sterilized ultrapure water or PCR-grade sterile enzyme-free water, and the amount of the sterile double distilled water is 1 to 2 mL.