A method for extracting DNA from the surface of microplastics in water

Abstract

The present invention discloses a method for extracting DNA from the surface of microplastics in water. The method comprises separating microplastics from a water sample so that the microplastics are enriched on a filter membrane a. The intracellular DNA and extracellular DNA on the microplastics on the filter membrane a are then separated and processed to obtain a filter membrane b containing intracellular DNA and a filtrate containing extracellular DNA, respectively. The intracellular DNA is extracted from the filter membrane b containing intracellular DNA, and the extracellular DNA is extracted from the filtrate containing extracellular DNA. The present invention can provide a new research method for the study of microplastics. The microplastics are separated by flotation and then separated to obtain a filter membrane b containing intracellular DNA and a filtrate containing extracellular DNA, so that the intracellular DNA and extracellular DNA can be obtained separately in the future, thereby providing a basic sample for experimental analysis of the DNA on the surface of microplastics.

Description

Technical Field

[0001] The present invention relates to the technical field of DNA extraction on microplastics, and in particular to a method for extracting DNA from the surface of microplastics in water. Background Art

[0002] Plastic pollution is one of the most pressing environmental threats facing the world today. The larger plastic fragments released by plastic pollution can be further broken down into smaller fragments, known as microplastics (MPs), through physical, chemical, and biological degradation. The increasing plastic pollution in the oceans, soil, and atmosphere inevitably leads to direct and unconscious ingestion, inhalation, and contact with small plastic particles, causing microplastics to accumulate in the human body and posing a new threat to human health.

[0003] At the same time, microplastics can also serve as carriers of heavy metals, organic pollutants, harmful algae and pathogens. Under the influence of ocean currents or wind, microplastic particles carrying organic pollutants or pathogens can migrate over long distances, posing a threat to the ecological safety of the ocean and coast. It can be seen that exploring the DNA on the surface of microplastics is of great significance. Therefore, a method for extracting DNA from the surface of microplastics is needed to provide a basis for the exploration and analysis of DNA on the surface of microplastics. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a method for extracting DNA from the surface of microplastics in water.

[0005] The technical solution of the present invention is: a method for extracting DNA from the surface of microplastics in water, which comprises separating microplastics in a water sample so that the microplastics are enriched on a filter membrane a, and then separating and treating the intracellular DNA and extracellular DNA on the microplastics on the filter membrane a to obtain a filter membrane b containing intracellular DNA and a filtrate containing extracellular DNA, respectively; extracting intracellular DNA from the filter membrane b containing intracellular DNA, and extracting extracellular DNA from the filtrate containing extracellular DNA.

[0006] Furthermore, the method for separating microplastics in water samples and enriching the microplastics on filter membrane a is as follows:

[0007] S101, taking out the sampling net bag from the water sample and scraping the collected material on the sampling net bag into a beaker, and preparing a saturated sodium chloride solution;

[0008] S102. Then, flotation is performed using a saturated sodium chloride solution as the flotation liquid. After the flotation is completed, the flotation liquid in the culture dish is filtered through a 100 μm filter membrane a to enrich the microplastics on the filter membrane a.

[0009] Description: Flotation is the optimal method for separating microplastics. It has the advantages of high efficiency, flexible operation, and environmental protection and energy saving. By using saturated sodium chloride solution as a reagent for microplastic flotation separation, it can effectively separate microplastics. Because different substances respond differently to saturated sodium chloride solution, this allows the use of saturated sodium chloride solution to accurately separate microplastics during the flotation process.

[0010] And because the saturated sodium chloride solution regulates the wettability and adsorption of microplastics, microplastics are more likely to adhere to bubbles and float up, thereby improving flotation efficiency. In addition, the production cost of saturated sodium chloride solution is low, and the waste generated during the flotation process is relatively small, which can reduce the impact on the environment.

[0011] Furthermore, the separation method is:

[0012] S201, cut the filter membrane a obtained by flotation into multiple vertical strips, place them into a centrifuge tube with tweezers, add PBS buffer to the strips, and sonicate and vortex for a total of 10-15 minutes to desorb microplastics from the filter membrane a;

[0013] S202. Rinse filter membrane a with PBS buffer, discard filter membrane a, vortex again for 10-15 minutes to desorb DNA from the microplastics, and filter the solution through a 0.22 μm filter membrane b to obtain filter membrane b containing intracellular DNA and a filtrate containing extracellular DNA.

[0014] Description: A secondary separation treatment is used to desorb DNA from microplastics. First, PBS buffer is used as a separation reagent. The filter membrane a is sheared multiple times using the above method. This is combined with an ultrasonic and vortex treatment method to further desorb the microplastics from the filter membrane a.

[0015] Moreover, on this basis, by continuing to use PBS buffer and the vortex time used, the extracellular DNA can be effectively desorbed from the microplastics, and then the filter membrane b containing intracellular DNA and the filtrate containing extracellular DNA can be accurately obtained through a 0.22μm filter membrane b.

[0016] Furthermore, before the ultrasound, the temperature of the PBS buffer solution is adjusted to room temperature, and temperature-control microbeads accounting for 15% to 25% of the volume of the PBS buffer solution are added to the centrifuge tube, the temperature of the temperature-control microbeads is controlled at 8 to 12° C., and the ultrasound treatment is performed at 40 to 50° C. with continuous stirring.

[0017] Note: Generally, higher temperatures and longer times help to improve desorption efficiency, but this may also lead to DNA degradation. Therefore, based on this, the present invention introduces an appropriate amount of temperature-control microbeads into the PBS buffer during the ultrasonic treatment process, and utilizes the slow-release low-temperature characteristics of the temperature-control microbeads to achieve autonomous temperature control of the PBS buffer subjected to heated ultrasonic treatment. During this period, as stirring proceeds, it can quickly act on the PBS buffer desorption system, while ensuring the desorption efficiency, avoiding DNA degradation and affecting the DNA extraction amount; at the same time, by improving the efficiency of DNA desorption from microplastics, the amount of extracted DNA can be greatly increased, thereby improving the extraction effect of the extraction method of the present invention and providing more accurate sample data for exploring DNA on the surface of microplastics.

[0018] Furthermore, within the first 3 s of ultrasonic treatment, the PBS buffer was rapidly heated from room temperature to 40-50 °C, maintained for 2 s, and then temperature-controlled microbeads were added in multiple times until the ultrasonic treatment was completed;

[0019] The temperature-controlled microbeads were added to the PBS buffer in stages according to the mass percentage, specifically:

[0020] 1) Within the first 3 seconds of ultrasonic treatment, the amount of temperature-controlled microbeads added was 60-70%;

[0021] 2) After ultrasonic treatment for 2 seconds, the amount of temperature-controlled microbeads added at a single time is 5-10%.

[0022] Description: By introducing an appropriate amount of temperature-control microbeads into the PBS buffer during the ultrasonic treatment process, we precisely control the addition stage of the temperature-control microbeads. We use 60-70% by mass of temperature-control microbeads mainly as protection for the rapid temperature rise 3s before ultrasonic treatment. Then, we use the amount of temperature-control microbeads added in a single time to gradually lower the temperature of the PBS buffer to enhance the desorption effect of DNA from microplastics, thereby greatly increasing the amount of extracted DNA and improving the extraction effect of the extraction method.

[0023] Furthermore, in the ultrasonic and vortexing, the duration of the ultrasonic treatment accounts for 1 / 60 to 3 / 100 of the total treatment time; and the intensity of the ultrasonic treatment is 2 to 3 W / cm 2 , the frequency of ultrasound is 20kHz.

[0024] Note: Traditional ultrasonic treatment will generate shear force under its high-frequency sound waves, which can easily lead to DNA breakage. With the continuous development of ultrasonic technology, short time, low power and high frequency can reduce or avoid DNA breakage and degradation. By introducing temperature-controlled microbeads, we have optimized and adjusted the ultrasonic treatment to avoid affecting DNA breakage and degradation, and enhance the effect of DNA desorption from microplastics. The above-mentioned ultrasonic treatment time can well cooperate with the DNA separation treatment. If the ultrasonic time is too short, the microplastics cannot be effectively and quickly desorbed from the filter membrane a, and if the ultrasonic time is too long, it is easy for the DNA separation treatment to affect the subsequent determination of the intracellular and extracellular DNA content.

[0025] Further, the intracellular DNA was extracted from the filter membrane b containing the intracellular DNA using Fast DNA TM Spin Kit for Soil was used to extract intracellular DNA.

[0026] Instructions: Using Fast DNA TM The Spin Kit for Soil has the advantages of high efficiency, simplicity, high purity, high quality and high safety. It can quickly and effectively extract intracellular DNA. The kit is easier to operate, reduces tedious steps and time consumption, and improves experimental efficiency. Fast DNA TM The reagents in the Spin Kit for Soil can effectively remove impurities and inhibitors to obtain highly pure intracellular DNA. They can also reduce or avoid DNA breakage and degradation, ensuring the quality of the extracted intracellular DNA.

[0027] Furthermore, the extracellular DNA is extracted from the filtrate containing the extracellular DNA using a magnetic bead method.

[0028] Description: The magnetic bead method has the advantages of high efficiency, high purity, simple operation, wide application range, controllability and quantification, as well as economy and environmental protection. By utilizing the affinity molecules on the surface of hydroxyl magnetic beads to bind to the affinity of DNA, efficient separation of DNA is achieved. The magnetic bead method can remove inhibitory substances (organic solvents, detergents, metal ions, etc.) in the sample, thereby obtaining high-purity extracellular DNA; and the number of functional groups on the surface of hydroxyl magnetic beads can be controlled, thereby realizing the control of the concentration information of the extractable DNA solution, enabling the magnetic bead method to meet the quantitative requirements. In addition, the magnetic bead method does not require complex equipment and technology and is simple and easy to operate.

[0029] The beneficial effects of the present invention are:

[0030] (1) The method for extracting DNA from the surface of microplastics of the present invention can provide a new research approach for the study of microplastics. By flotation separation of microplastics and then separation treatment to obtain a filter membrane b containing intracellular DNA and a filtrate containing extracellular DNA, the intracellular DNA and extracellular DNA can be obtained separately in the subsequent study, thereby providing a basic sample for experimental analysis of the DNA on the surface of microplastics.

[0031] (2) The present invention desorbs the DNA on the microplastics by adopting a secondary separation treatment method, first desorbing the microplastics on the filter membrane a; on this basis, by continuing to use PBS buffer, etc., the extracellular DNA is effectively desorbed from the microplastics, thereby accurately obtaining the filter membrane b containing the intracellular DNA and the filtrate containing the extracellular DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a flow chart of the method for extracting DNA from the surface of microplastics in water according to the present invention.

[0033] Figure 2 It is a schematic diagram of the simulation structure of the temperature-control microbeads of the present invention. DETAILED DESCRIPTION

[0034] The present invention will be further described in detail below in conjunction with specific implementation methods to better demonstrate the advantages of the present invention.

[0035] Example 1: A method for extracting DNA from the surface of microplastics in water, such as Figure 1 As shown, the following steps are included:

[0036] S1. Separate the microplastics from the water sample and enrich them on the filter membrane a:

[0037] S101, take out the sampling net bag from the water sample and scrape the collected material on the sampling net bag into a beaker, and prepare a saturated sodium chloride solution. The solubility of sodium chloride at room temperature is 360g per liter of water;

[0038] S102, flotation is then performed using a saturated sodium chloride solution as a flotation liquid. After the flotation is completed, the flotation liquid in the culture dish is filtered through a 100 μm filter membrane a to enrich the microplastics on the filter membrane a;

[0039] Among them, the flotation method is:

[0040] 1) Clean the outer wall and bottom of the beaker, place it in an 180 mm crystallizing dish, add saturated sodium chloride solution until the beaker is almost full (95% of the beaker capacity), stir slowly with a glass rod for 5 minutes, and let it stand for 30 minutes;

[0041] 2) After standing for 30 minutes, use a 50 mL graduated cylinder to add a certain amount of saturated sodium chloride solution to the beaker until it is about to overflow but not overflowing (100% of the beaker capacity);

[0042] 3) Slowly add 50 mL of saturated sodium chloride solution to the beaker several times (blow gently to allow visible material on the surface to enter the crystallizing dish. If no material is visible on the surface after several additions, wait a while before adding again) and record the amount used;

[0043] 4) Use a 10 mL pipette to slowly pipette the solution in the beaker to the halfway mark several times and place the pipetted liquid in a crystallizing dish.

[0044] 5) Add saturated sodium chloride solution to the beaker until full scale, stir, let it stand for 30 minutes, and pipette to the 50 mL mark on the sediment;

[0045] 6) After flotation, remove the beaker and rinse the outer wall and bottom with saturated sodium chloride solution (if the sample contains a lot of sediment, a second flotation can be performed);

[0046] S2, then separating the intracellular DNA and extracellular DNA on the microplastics of filter membrane a to obtain filter membrane b containing intracellular DNA and filtrate containing extracellular DNA;

[0047] S201. Cut the filter membrane a obtained by flotation into 4 vertical strips with scissors and place them in a 15 mL centrifuge tube with tweezers. Add 5 mL of PBS buffer and sonicate for 25 seconds and vortex for a total of 15 minutes. The intensity of the ultrasound is 2.5 W / cm 2 The frequency of ultrasound is 20 kHz, so that the microplastics are desorbed from the filter membrane a. The preparation method of PBS buffer is as follows: per 1 L, it contains 8.23 ​​g of disodium hydrogen phosphate, 1.2 g of sodium dihydrogen phosphate, 5.0 g of sodium chloride, 1.5 g of No. 3 bile salt, and 20 g of sorbitol, and the pH is 7.6;

[0048] S202, rinse filter membrane a with 4 mL of PBS buffer, discard filter membrane a, vortex again for 15 minutes to desorb DNA from the microplastics, and filter the solution through a 0.22 μm filter membrane b to obtain filter membrane b containing intracellular DNA and a filtrate containing extracellular DNA;

[0049] S3. Extraction of intracellular DNA from filter membrane b containing intracellular DNA using Fast DNA TM The Spin Kit for Soil kit was used to extract intracellular DNA.

[0050] S301. Cut the filter membrane b into pieces and place them in a 15 mL centrifuge tube. Add PBS buffer to immerse the membrane, sonicate for 25 seconds and vortex for 15 minutes. The intensity of the ultrasound is 2.5 W / cm 2 , the frequency of ultrasound is 20kHz, so that the material on the filter membrane b is dispersed, the filter membrane b is taken out, and the filter membrane b and tweezers are rinsed with PBS buffer, the filter membrane b is discarded, and the centrifuge tube is centrifuged at 10000rpm for 10min, all the supernatant is poured out, and the precipitate is retained;

[0051] S302, according to Fast DNA TM The Spin Kit for Soil kit process extracts intracellular DNA from the precipitate;

[0052] S4. Extract the extracellular DNA from the filtrate containing extracellular DNA using the magnetic bead method.

[0053] S401. Prepare Buffer CW1 and Buffer CW2: Buffer CW1: isopropanol: guanidine hydrochloride = 1:1 [e.g., 100 mL Buffer CW1: 50 mL isopropanol + 50 mL sterile water + 66.871 g guanidine hydrochloride]; Buffer CW2: 75% ethanol.

[0054] S402, take 5 mL of the filtrate and 4 mL of Buffer CL into a 50 mL centrifuge tube. It is understood that Buffer CL is purchased from Biomag Biotechnology;

[0055] S403, oscillate each centrifuge tube for 1 minute to mix;

[0056] S404, resuspend the hydroxy magnetic beads (shake to mix the hydroxy magnetic beads thoroughly), and shake thoroughly to make the hydroxy magnetic beads in the centrifuge tube completely and evenly suspended;

[0057] S405. Take 30 μL of hydroxyl magnetic beads into a centrifuge tube, invert and mix, shake for 4 minutes, tie the centrifuge tube to a large magnet, unscrew the lid, wait until the hydroxyl magnetic beads are completely adsorbed, and discard the liquid with a pipette;

[0058] S406. After removing the centrifuge tube from the large magnet, add 1 mL of Buffer CW1 (to wash away the hydroxy magnetic beads), shake for 1 minute, and tie it to the large magnet again. After the hydroxy magnetic beads are completely adsorbed, discard the liquid with a pipette to avoid touching the magnetic bead cluster;

[0059] S407. After removing the centrifuge tube from the large magnet, add 1 mL of Buffer CW2 (75% ethanol), shake for 1 minute, and tie it to the large magnet again. Repeat the above steps (wait until the hydroxy magnetic beads are completely adsorbed, and discard the liquid with a pipette to avoid touching the magnetic bead cluster) [At this time, the TE buffer can be preheated to 55°C];

[0060] S408. After removing the centrifuge tube from the large magnet, add 1 mL of Buffer CW2 and mix thoroughly with a pipette. Pipette the liquid into a 2 mL centrifuge tube and shake for 1 minute. Place the tube on a magnetic stand for adsorption and discard the liquid with a pipette.

[0061] S409, centrifuge at 6000 rpm for 1 min, place on a magnetic rack for adsorption, remove waste liquid, and leave with the lid open at room temperature for 15 min;

[0062] S410, remove the centrifuge tubes from the magnetic rack, add 30 μL of TE buffer preheated at 55°C, and shake each centrifuge tube for 5 minutes;

[0063] Centrifuge at S411 and 6000r for 1 min. Collect the liquid with a magnet into a clean 2 mL centrifuge tube. Measure the nucleic acid concentration and store in a -80°C refrigerator.

[0064] The sewage from a municipal sewage treatment plant in this city was tested. The DNA extraction method on the surface of microplastics in water in Example 1 was used, and the nucleic acid concentration was detected using Qubit4.0. The operation steps are as follows:

[0065] 1) Standard Solution S1: Add 190 μL Qubit™ 1X dsDNA HS Working Solution and 10 μL Qubit™ 1X dsDNA HS Standard #1 to a test tube and mix thoroughly.

[0066] 2) Standard Solution S2: Add 190 μL Qubit™ 1X dsDNA HS Working Solution and 10 μL Qubit™ 1X dsDNA HS Standard #2 to a test tube and mix thoroughly.

[0067] 3) Sample tube: Add 199 μL Qubit™ 1X dsDNA HS Working Solution and 1 μL sample DNA to the sample tube and mix thoroughly.

[0068] 4) Place standard solution S1, standard solution S2, and the sample to be tested in a centrifuge and centrifuge briefly (to allow the liquid on the tube wall to centrifuge to the bottom) and let it stand at room temperature for 2 minutes (protect from light);

[0069] 5) Turn on the Qubit instrument and select dsDNA mode;

[0070] 6) Select dsDNA High Sensitivity mode;

[0071] 7) Click read standards and measure standard solution S1 and standard solution S2 respectively for calibration;

[0072] 8) Measure sample concentration: Set Enter original sample volume to "1μL" and Output sample units to "ng / μL", click Read tubes and record the measured value. The result is as follows:

[0073] The concentrations of eDNA and iDNA on the surface of microplastics in the sewage treatment unit of the municipal sewage treatment plant are shown in Table 1 below:

[0074] Table 1 Concentrations of eDNA and iDNA on microplastic surfaces

[0075]

[0076] As can be seen from Table 1 above, the method for extracting DNA from the surface of microplastics in water of the present invention can effectively obtain eDNA and iDNA from the influent of sewage treatment plants, aeration sedimentation tanks, high-efficiency sedimentation tanks, rotary disc filter beds, and effluent from contact disinfection tanks.

[0077] At the same time, in order to verify the comparison between the DNA extraction method of the present invention and the common extraction methods on the market, we conducted subsequent experiments using the indicator parameter of "wastewater treatment plant influent".

[0078] Set up a control, using the "Test Microplastics and Sample Preparation" mentioned in the journal "Comparison and Optimization of DNA Extraction Methods for Biofilms on Two Types of Soft Microplastics in Coastal Environments" and combined with the methods of steps S3 and S4 of this embodiment 1.

[0079] The concentrations of eDNA and iDNA on the surface of microplastics in the sewage treatment plant influent of the municipal sewage treatment plant are shown in Table 2 below:

[0080] Table 2 Concentrations of eDNA and iDNA on microplastic surfaces

[0081]

[0082] From the comparison in Table 2 above, it can be seen that when the control method was used to extract and separate DNA from microplastics, the concentrations of eDNA and iDNA on the surface of microplastics from the same sewage treatment plant influent were reduced to a certain extent. This may be due to DNA breakage and degradation, or due to incomplete extraction and separation, resulting in a decrease in the concentration of eDNA and iDNA. This has a certain impact on the types and quantities of eDNA and iDNA extracted from microplastics. It can be seen that the extraction method of the present invention provides a better experimental analysis basis sample for the exploration of DNA on the surface of microplastics.

[0083] Example 2: This example differs from Example 1 in that, in step S201 and step 301, ultrasound is applied for 12 seconds and vortex treatment is performed, the total treatment time is 12 minutes, and the ultrasound intensity is 2W / cm 2 , the frequency of ultrasound is 20 kHz; in step S202, vortex again for 12 minutes.

[0084] Example 3: This example differs from Example 1 in that, in steps S201 and 301, ultrasound is applied for 18 seconds and vortex treatment is performed, the total treatment time is 10 minutes, and the ultrasound intensity is 3W / cm 2 , the frequency of ultrasound is 20 kHz; in step S202, vortex again for 10 minutes.

[0085] In order to verify the comparison of the DNA extraction method of the present invention on the surface of microplastics in water with the common extraction methods on the market, we conducted subsequent experimental exploration using the indicator parameter "wastewater treatment plant influent". The concentrations of eDNA and iDNA on the surface of microplastics in the influent of municipal sewage treatment plants are shown in Table 3 below:

[0086] Table 3 Concentrations of eDNA and iDNA on microplastic surfaces

[0087]

[0088] From the comparison in Table 3 above, it can be seen that by reducing or extending the ultrasonic treatment time and adjusting the ultrasonic intensity, it was found that when extracting and testing the eDNA and iDNA on the surface of microplastics in the same sewage treatment plant influent, the eDNA and iDNA concentrations of Examples 2 and 3 decreased to a certain extent. This may be due to the breakage and degradation of DNA, or due to incomplete extraction and separation, resulting in a decrease in the concentration of eDNA and iDNA. Among them, the ultrasonic treatment time and adjustment of the ultrasonic intensity in Example 1 are relatively optimal.

[0089] Example 4: This example differs from Example 1 in that, before the ultrasound treatment, the temperature of the PBS buffer solution is adjusted to room temperature, i.e., 25°C, and temperature-control microbeads accounting for 21% of the volume of the PBS buffer solution are added to the centrifuge tube. The temperature of the temperature-control microbeads is controlled at 10°C, and the ultrasound treatment is performed at 45°C with continuous stirring.

[0090] Among them, such as Figure 2 As shown, the temperature-control microbeads are 1 cm diameter microspheres consisting of a ceramic shell (1 mm thick) and a phase change cold storage core. The phase change cold storage material can be commercially available paraffin wax, etc., and is not limited to the use of this phase change cold storage material. The phase change cold storage material is encapsulated in a thin plastic shell. The diameter of the phase change cold storage core is about 8 mm, and a copper sheet with an area of ​​1 / 3 of the spherical surface is embedded on one side of the plastic shell (0.5 mm thick) for cooling, and a counterweight is provided on the other side of the spherical surface for eccentric rotation of the phase change cold storage core in the ceramic shell. A copper sheet with an area of ​​1 / 3 of the spherical surface is embedded on one side of the ceramic shell for heat conduction. The phase change cold storage core rotates irregularly in the ceramic shell, so that the two copper sheets are in contact for cooling and heat conduction.

[0091] It is understandable that both ceramic shells and plastic thin shells can be used to prepare and assemble temperature-controlled microspheres using the hemisphere splicing technology, but the preparation and assembly method is not limited to this one.

[0092] Example 5: This example differs from Example 4 in that temperature-controlled microbeads accounting for 15% of the volume of PBS buffer are added to the centrifuge tube.

[0093] Example 6: This example differs from Example 4 in that temperature-controlled microbeads accounting for 25% of the volume of PBS buffer are added to the centrifuge tube.

[0094] Example 7: This example differs from Example 4 in that the temperature of the temperature-controlling microbeads is controlled at 8°C.

[0095] Example 8: This example differs from Example 4 in that the temperature of the temperature-controlling microbeads is controlled at 12°C.

[0096] Example 9: This example differs from Example 4 in that ultrasonic treatment at 40° C. is performed with continuous stirring.

[0097] Example 10: This example differs from Example 4 in that ultrasonic treatment at 50° C. is performed with continuous stirring.

[0098] In order to verify the comparison between the DNA extraction method of the present invention and the common extraction methods on the market, we conducted subsequent experiments using the indicator parameter of “wastewater treatment plant influent”.

[0099] A control was set up, which was treated with ultrasonic treatment at 45°C with constant stirring, without adding temperature-controlled microbeads, and the rest of the extraction method was the same;

[0100] The concentrations of eDNA and iDNA on the surface of microplastics in the sewage treatment plant influent of the municipal sewage treatment plant are shown in Table 4 below:

[0101] Table 4 Concentrations of eDNA and iDNA on microplastic surfaces

[0102]

[0103] As can be seen from the comparison in Table 4 above, in Example 4, after the temperature-controlled microbeads were introduced for ultrasonic treatment at elevated temperatures, we found that when extracting and testing eDNA and iDNA from the surface of microplastics in the same sewage plant influent, the concentrations of eDNA and iDNA increased to a certain extent. This may be because while avoiding DNA breakage and degradation, the desorption effect was improved by increasing the temperature, thereby improving the extraction and separation effect, thereby obtaining a larger total amount of eDNA and iDNA;

[0104] At the same time, by comparing and analyzing different amounts of temperature-controlled microbeads added and different operating temperatures of the temperature-controlled microbeads, the concentrations of both eDNA and iDNA decreased to a certain extent. This may be due to the breakage and degradation of a small amount of DNA, or due to reduced extraction and separation efficiency. Among them, the amount of temperature-controlled microbeads added and the operating temperature of the temperature-controlled microbeads in Example 4 were relatively optimal.

[0105] Furthermore, by comparing and analyzing different ultrasonic treatment temperatures, the concentrations of eDNA and iDNA decreased to a certain extent. Lowering the temperature may have affected the extraction and separation efficiency, while raising the temperature may have caused a small amount of DNA to break and degrade, ultimately affecting the total amount of eDNA and iDNA extracted.

[0106] And compared with the control, it can be seen that when no temperature-controlled microbeads are added, only the temperature-raising ultrasonic treatment shows a significant decrease. Although there is still a certain improvement compared with Example 1, this improvement is not obvious. Therefore, the optimization method of Example 4 can significantly increase the extraction amount of eDNA and iDNA.

[0107] Example 11: This example differs from Example 4 in that, within the first 3 seconds of ultrasonic treatment, the PBS buffer solution is rapidly heated from room temperature to 45°C, maintained for 2 seconds, and then temperature-controlled microbeads are added seven times within 20 seconds until the ultrasonic treatment is completed; wherein, the temperature-controlled microbeads are added to the PBS buffer solution in stages according to the mass percentage, specifically:

[0108] 1) Within the first 3 seconds of ultrasonic treatment, the amount of temperature-controlled microbeads added was 65%;

[0109] 2) After ultrasonic treatment for 2 seconds, the amount of temperature-controlled microbeads added at a single dose was 5%.

[0110] Example 12: This example differs from Example 4 in that, within the first 3 seconds of ultrasonic treatment, the PBS buffer solution is rapidly heated from room temperature to 45°C, maintained for 2 seconds, and then temperature-controlled microbeads are added in 8 portions over 20 seconds until the ultrasonic treatment is completed; wherein, the temperature-controlled microbeads are added to the PBS buffer solution in stages according to the mass percentage, specifically:

[0111] 1) Within the first 3 seconds of ultrasonic treatment, the amount of temperature-controlled microbeads added was 60%;

[0112] 2) After ultrasonic treatment for 2 seconds, the amount of temperature-controlled microbeads added at a single dose was 5%.

[0113] Example 13: This example differs from Example 4 in that, within the first 3 seconds of ultrasonic treatment, the PBS buffer solution is rapidly heated from room temperature to 45°C, maintained for 2 seconds, and then temperature-controlled microbeads are added three times within 20 seconds until the ultrasonic treatment is completed; wherein, the temperature-controlled microbeads are added to the PBS buffer solution in stages according to the mass percentage, specifically:

[0114] 1) Within the first 3 seconds of ultrasonic treatment, the amount of temperature-controlled microbeads added was 70%;

[0115] 2) After ultrasonic treatment for 2 seconds, the amount of temperature-controlled microbeads added at a single time was 10%.

[0116] In order to verify the comparison of the DNA extraction method of the present invention on the surface of microplastics in water with the common extraction methods on the market, we conducted subsequent experimental exploration using the indicator parameter "wastewater treatment plant influent". The concentrations of eDNA and iDNA on the surface of microplastics in the influent of municipal sewage treatment plants are shown in Table 5 below:

[0117] Table 5 Concentrations of eDNA and iDNA on microplastic surfaces

[0118]

[0119] From the comparison in Table 5 above, it can be seen that by optimizing the addition method of the temperature-controlled microbeads, it was found that when extracting and testing the eDNA and iDNA on the surface of microplastics in the same sewage treatment plant influent, the eDNA and iDNA concentrations of Examples 11 to 13 were all increased to a certain extent, among which the addition method of the temperature-controlled microbeads in Example 11 was relatively optimal.

Claims

1. A method for extracting DNA from the surface of microplastics in water, characterized in that: The microplastics in the water sample are separated and enriched on the filter membrane a, and then the intracellular DNA and extracellular DNA on the microplastics on the filter membrane a are separated and processed to obtain the filter membrane b containing the intracellular DNA and the filtrate containing the extracellular DNA, respectively; the intracellular DNA is extracted from the filter membrane b containing the intracellular DNA, and the extracellular DNA is extracted from the filtrate containing the extracellular DNA; The separation method is as follows: S201, cutting the filter membrane a obtained by flotation into multiple vertical strips, placing them in a centrifuge tube, adding PBS buffer thereto, and sonicating and vortexing for a total of 15 minutes to desorb microplastics from the filter membrane a; S202, rinse filter membrane a with PBS buffer, discard filter membrane a, vortex again for 15 minutes to desorb DNA from the microplastics, and filter the solution through a 0.22 μm filter membrane b to obtain filter membrane b containing intracellular DNA and a filtrate containing extracellular DNA; Before the ultrasound, the temperature of the PBS buffer solution was adjusted to room temperature, and temperature-control microbeads accounting for 21% of the volume of the PBS buffer solution were added to the centrifuge tube, the temperature of the temperature-control microbeads was controlled at 10° C., and the tube was ultrasonically treated at 45° C. with continuous stirring; The temperature-control microspheres are 1cm in diameter and consist of a 1mm-thick ceramic shell and a phase-change thermal storage core. The phase-change thermal storage material is encapsulated in a thin plastic shell. The core has a diameter of 8mm, and a copper sheet covering 1 / 3 of the spherical surface is embedded on one side of the plastic shell for cooling. A counterweight is installed on the other side of the sphere to ensure eccentric rotation of the core within the ceramic shell. Within the first 3 s of ultrasonic treatment, the PBS buffer was rapidly heated from room temperature to 45 °C, maintained for 2 s, and then temperature-controlled microbeads were added in multiple doses until the ultrasonic treatment was completed; The temperature-controlled microbeads were added to the PBS buffer in stages according to the mass percentage, specifically: 1) Within the first 3 seconds of ultrasonic treatment, the amount of temperature-controlled microbeads added was 65%; 2) After ultrasonic treatment for 2 seconds, the single addition amount of temperature-controlled microbeads was 5%; During the ultrasonication and vortexing, the ultrasonication was performed for 25 seconds, and the total treatment time was 15 minutes; and the intensity of the ultrasonication was 2.5 W / cm 2 , the frequency of ultrasound is 20kHz; The method for separating microplastics from water samples and enriching them on filter membrane a is: S101, taking out the sampling net bag from the water sample and scraping the collected material on the sampling net bag into a beaker, and preparing a saturated sodium chloride solution; S102. Then, flotation is performed using a saturated sodium chloride solution as the flotation liquid. After the flotation is completed, the flotation liquid in the culture dish is filtered through a 100 μm filter membrane a to enrich the microplastics on the filter membrane a.

2. The method for extracting DNA from the surface of microplastics in water according to claim 1, wherein: Fast DNA was used to extract intracellular DNA from the filter membrane b. TM The intracellular DNA was extracted using the Spin Kit for Soil.

3. The method for extracting DNA from the surface of microplastics in water according to claim 1, wherein: The extracellular DNA was extracted from the filtrate containing the extracellular DNA using the magnetic bead method.

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