Unencapsulated DNA contaminant tracer and tracing method

Abstract

The present invention provides an unencapsulated DNA contaminant tracer and a tracing method. The tracer comprises: 0.1-20 μmol / L double-stranded DNA, 10-50 mmol / L KBr or NaBr, 0.05-0.1 mol / L Tris, pH 7.0-9.0; the structure of the double-stranded DNA is: flanking sequence 1-core replication segment-flanking sequence 2, wherein the structure of the core replication segment is: forward primer-Linker1-probe-Linker2-reverse primer, and the double-stranded DNA has no homology with the DNA of known organisms. The present invention combines an unencapsulated DNA tracer with a pollutant migration and transformation model, and has the advantage of tracing soluble chain-like high-molecular pollutants with different adsorption rates and decomposition rates in porous media, and can realize multi-source tracing of pollutants in porous media.

Description

Technical Field

[0001] The present invention belongs to the field of environmental protection, and in particular relates to an unwrapped DNA pollutant tracer and a tracing method. Background Art

[0002] In theory, DNA tracers can be of any sequence, and synthetic DNA can be synthesized to ensure that its sequence has zero background value in the environment, free from the influence of other experiments or other DNA in the environment. Therefore, synthetic DNA tracers have become an emerging environmental tracer due to their codability, specificity, independence from environmental background values, and environmental safety.

[0003] Unencapsulated DNA readily decomposes in the environment, has a half-life of only a few days in soil, and is easily adsorbed by soil particles. In recent years, new technologies have emerged for encapsulating DNA using materials such as PLGA, PLA, and SiO2. Although these encapsulated DNA tracers are resistant to decomposition and have found success in tracing surface water and pollutant emissions, their larger particle size makes them difficult to penetrate soil pores, resulting in a low tracer recovery rate and making them unsuitable for use in water and soil environments.

[0004] Currently, both unencapsulated and encapsulated DNA tracers have their own shortcomings when applied to different soil and water environments. Unencapsulated DNA tracers are prone to decomposition and adsorption, resulting in low penetration rates in real soil. Encapsulated DNA tracers, on the other hand, have large particle sizes and are easily filtered by soil, resulting in even lower penetration rates. Therefore, there is an urgent need to develop a new type of DNA porous media pollutant tracer with controllable adsorption and predictable decomposition, so as to track pollutants with different adsorption and decomposition rates in porous media. Summary of the Invention

[0005] The purpose of the present invention is to provide a non-encapsulated DNA pollutant tracer and a tracing method, in particular a soluble chain polymer pollutant tracer and a tracing method with different adsorption rates and decomposition rates in a porous medium.

[0006] To achieve the purpose of the present invention, in a first aspect, the present invention provides an unencapsulated DNA contaminant tracer, comprising the following components: 0.1-20 μmol / L double-stranded DNA, 10-50 mmol / L KBr or NaBr (conservative tracer), 0.05-0.1 mol / L Tris, pH 7.0-9.0.

[0007] The double-stranded DNA has a structure of: flanking sequence 1-core replication segment-flanking sequence 2, with a total length of 65-2000 bp, and the double-stranded DNA has no homology with the DNA of known organisms.

[0008] The lengths of the flanking sequence 1 and the flanking sequence 2 are independently 1-1934 bp, and the sum of the lengths of the flanking sequence 1 and the flanking sequence 2 is 2-1935 bp.

[0009] The structure of the core replication segment is: forward primer-Linker1-probe-Linker2-reverse primer; the length of the core replication segment is 63-200 bp; the lengths of the forward primer and reverse primer are each independently 18-25 bp; the length of the probe segment is 25-32 bp; the lengths of Linker1 and Linker2 are each independently 1-138 bp, and the sum of the lengths of Linker1 and Linker2 is 2-139 bp.

[0010] The core replication segment should meet the following conditions: (1) the GC base content is 40%-60%; (2) the GC base content in the probe is 60%-65%; (3) the GC base content in the primer is 40%-60%; (4) the annealing temperature of the probe is 68-70°C, and the annealing temperature of the primer is 55-62°C.

[0011] For example, the total length of the double-stranded DNA is 82 bp, 100 bp, or 120 bp, and the length of the core replication segment is 80 bp.

[0012] For example, the total length of double-stranded DNA is 100 bp, and the length of the core replication segment is 98 bp.

[0013] Furthermore, the pH of the tracer can be adjusted to 7.0-9.0 using 5-6 mol / L NaOH or 5-6 mol / L HCl.

[0014] In a second aspect, the present invention provides the use of the tracer in multi-source tracing of pollutants in porous media;

[0015] In the present invention, the pollutant is a soluble chain polymer, preferably PAM (polyacrylamide), PAA (polyacrylic acid), PVA (polyvinyl alcohol) and the like.

[0016] Furthermore, porous media include but are not limited to underground aquifers and soil.

[0017] In a third aspect, the present invention provides a method for multi-source tracing of pollutants in a porous medium, comprising the following steps:

[0018] 1) Identify a certain soluble chain polymer present in porous media as the target pollutant;

[0019] 2) Conduct short-term column tests on the target pollutant and nucleic acid tracer to obtain the penetration curves of the target pollutant and nucleic acid tracer;

[0020] The nucleic acid tracer comprises the following components: a nucleic acid molecule, 10-50 mmol / L KBr or NaBr, 0.05-0.1 mol / L Tris, pH 7.0-9.0; the nucleic acid molecule comprises n different double-stranded DNAs, each having a concentration of 0.1-20 μmol / L; the structure of each double-stranded DNA is as described above, and each double-stranded DNA has a different total length and a different sequence of the core replication segment;

[0021] 3) Compare the penetration curves of the target pollutant and the nucleic acid tracer, and select the double-stranded DNA with the closest penetration curve to the target pollutant, which is recorded as DNA , which is used to simulate the diffusion and adsorption characteristics of target pollutants;

[0022] 4) Prepare the following DNA Tracer: 0.1-20 μmol / L DNA , 10-50 mmol / L KBr or NaBr, 0.05-0.1 mol / L Tris, pH 7.0-9.0;

[0023] 5) In the water environment (such as groundwater, soil water) where porous media exist, the target pollutants and DNA are detected. The tracer was used for decomposition test to obtain the target pollutants and DNA. The decomposition rate;

[0024] 6) In the pollutant migration and transformation model, the Br penetration curve is used to inversely solve the saturated hydraulic conductivity of porous media and the diffusion coefficient in the convection-diffusion equation;

[0025] 7) Input the DNA obtained in step 5) into the pollutant transport and transformation model. the rate of decomposition of the tracer;

[0026] 8) Use the DNA obtained in step 2) The tracer breakthrough curve is used to inversely analyze the adsorption and desorption parameters in the pollutant migration and transformation model;

[0027] 9) The saturated hydraulic conductivity of the porous medium and the diffusion coefficient in the convection-diffusion equation obtained by inversely solving the Br penetration curve in step 6), the decomposition rate of the target pollutant measured in step 5), and the adsorption and desorption parameters obtained by inversely solving in step 8) are introduced into the pollutant migration and transformation model to obtain a simulated penetration curve of the target pollutant; then, the simulated penetration curve of the target pollutant is compared with the penetration curve of the target pollutant obtained by the column test in step 2), and the selected DNA is verified by referring to formulas (1) and (2). Whether the tracer can be used to trace the target pollutant; when the determination coefficient R 2If the value is above 0.8 and the Nash coefficient NSE is above 0.6, it indicates that the DNA screened The tracer and the established pollutant migration and transformation model can be used to trace the migration and decomposition process of target pollutants in porous media;

[0028] Among them, the coefficient of determination R 2 The Nash coefficient NSE is an evaluation index used to describe the similarity between the simulated breakthrough curve and the breakthrough curve of the target pollutant obtained by the column test;

[0029] (1)

[0030] (2)

[0031] in, t At different time points, T is the total time; Y t The time point in the penetration curve of the target pollutant calculated by the inverse solution parameters t The corresponding concentration value; X t This is the time point in the breakthrough curve of the target pollutant obtained by column testing in step 2). t The corresponding concentration value; yes X t The average value of .

[0032] Furthermore, when there are m pollution sources of the target pollutant in the target porous medium, m DNA The core replication segments of the double-stranded DNA are identical in length but different in sequence and identical in total length, respectively, and are denoted as DNA 1. DNA 2. DNA m, and prepare DNA according to step 4) 1. DNA 2. DNA mtracer;

[0033] Add DNA to contamination source 1 1 tracer, put DNA at the source of contamination 2 2 tracers, and so on, by injecting DNA into the contamination source m mtracer;

[0034] Through DNA 1. DNA 2. DNA The penetration curve of m is inversely solved using the pollutant migration and transformation model to obtain the adsorption and desorption parameters; a decomposition experiment of the target pollutant is carried out in the water environment where the porous medium exists to obtain the decomposition rate of the target pollutant; the inversely solved adsorption and desorption parameters and the decomposition rate measured in the decomposition experiment are substituted into the established pollutant migration and transformation model to achieve multi-source tracing of the target pollutant.

[0035] Preferably, the column 4 used in the column test in step 2) has an inner diameter of 1 cm and a height of 50 cm, and is filled with 50 cm of a porous medium sterilized by high temperature.

[0036] Furthermore, a column filled with a porous medium is pre-saturated with a buffer solution before the column test is performed.

[0037] Preferably, the buffer solution comprises TE buffer at pH 8.0, containing 10 mM Tris-HCl and 1 mM EDTA.

[0038] Preferably, the pollutant migration and transformation model used in step 6) is the software HYDRUS-1D (see https: / / www.pc-progress.com / en / Default.aspx?hydrus). For "inverse solution", please refer to the software help and manual.

[0039] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0040] This invention combines unwrapped DNA tracers of varying total lengths and core replication segment lengths with a pollutant migration and transformation model. Adsorption is controlled by total length, while decomposition rates are predicted by core replication segment length. This approach offers the advantage of tracing soluble, chain-like polymer pollutants with varying adsorption characteristics and decomposition rates in porous media, enabling multi-source tracing of pollutants in porous media. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a decomposition curve of the unencapsulated DNA tracer in Example 2 of the present invention.

[0042] Figure 2 This is a graph showing the decomposition rate of the unencapsulated DNA tracer in Example 2 of the present invention.

[0043] Figure 3 This is a diagram of the experimental device in Example 3 of the present invention. In the figure, 1 is a conical flask; 2 is a peristaltic pump; 3 is a syringe; 4 is a column (or a plexiglass column); 5 is an automatic fraction collector.

[0044] Figure 4 Graph showing the particle size distribution of Ottawa quartz sand and fine quartz sand in Example 3 of the present invention.

[0045] Figure 5 This is the penetration curve of the unencapsulated DNA soil water tracer in Example 3 of the present invention in Ottawa quartz sand.

[0046] Figure 6 This is the penetration curve of the unencapsulated DNA soil water tracer in fine quartz sand in Example 3 of the present invention.

[0047] Figure 7 is the decomposition rate of the unencapsulated DNA soil water tracer in Example 3 of the present invention in two porous media.

[0048] Figure 8 is the adsorption amount of the unencapsulated DNA soil water tracer in Example 3 of the present invention in the two porous media.

[0049] Figure 9 This is the method for tracing water flow in porous media using an unencapsulated DNA series with controllable adsorption and predictable decomposition in Example 4 of the present invention. DETAILED DESCRIPTION

[0050] The present invention provides a method for tracing pollutants in porous media using unencapsulated DNA with predictable adsorption, controllable decomposition, and predictable properties. Specifically, based on a series of unencapsulated double-stranded DNA tracers, this method utilizes decomposition experiments in the water environment of the target porous medium, as well as column experiments using the target porous medium. Combined with a pollutant migration and transformation model, this method provides a method for tracing the migration and decomposition of pollutants in porous media.

[0051] In short, the unwrapped double-stranded DNA tracer series provides the migration and adsorption parameters of the target pollutant by conducting a migration experiment in the target porous medium. The target pollutant provides its own decomposition rate by conducting a decomposition experiment in the water environment where the target porous medium exists. Finally, through the pollutant migration and transformation model, the migration and adsorption parameters provided by the unwrapped double-stranded DNA tracer series are combined with the decomposition rate of the target pollutant itself to achieve the tracing of the migration and transformation process of the target pollutant in the target porous medium.

[0052] The present invention adopts the following technical solutions:

[0053] The present invention provides an unencapsulated DNA contaminant tracer comprising the following components: 0.1-20 μmol / L double-stranded DNA, 10-50 mmol / L KBr or NaBr, 0.05-0.1 mol / L Tris, and the pH adjusted to 7.0-9.0 using 5-6 mol / L NaOH or 5-6 mol / L HCl. This tracer, when combined with a contaminant migration and transformation model, offers the advantage of tracing soluble, chain-like, high-molecular-weight contaminants with varying adsorption and decomposition rates in porous media.

[0054] The sequence of each DNA tracer is randomly generated by a DNA random generator (https: / / molbiotools.com / randomsequencegenerator.php) and meets the following conditions:

[0055] 1) Core replication segment structure: forward primer-Linker1-probe-Linker2-reverse primer; the forward and reverse primers are 18-25 bp long; the probe segment is 25-32 bp long; the Linker1 and Linker2 are 1-138 bp long, respectively, and the combined length of Linker1 and Linker2 is 2-139 bp; the core replication segment is 63-200 bp long.

[0056] 2) Requirements for the core replication segment: The GC content of the synthetic DNA sequence should be 40%-60% of all bases, the GC content of the probe sequence should be 60%-65% of the probe bases, and the GC content of the primer sequence should be 40%-60% of the primer bases. The annealing temperature of the probe should be 68-70°C, and the annealing temperature of the primer should be 55-62°C. Following these principles, each single-stranded DNA and its corresponding primer and probe were designed using the Primer-BLAST tool (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) to confirm the uniqueness of the primers and ensure that they do not correspond to the DNA of any documented existing organisms.

[0057] 3) The structure of the DNA sequence is: flanking sequence 1-core replication segment-flanking sequence 2; the length of flanking sequence 1 and flanking sequence 2 is 1-1934 bp, and the sum of the lengths of flanking sequence 1 and flanking sequence 2 is 2-1935 bp; the total length is 65-2000 bp.

[0058] 4) Preparation of single-stranded DNA consisting of "Flanking Sequence 1-Core Replicating Segment-Flanking Sequence 2": For DNA sequences with a total length of 65-200 bp, we recommend synthesis. For DNA sequences with a total length of 201-2000 bp, we recommend cloning (available from Integrated DNA Technologies (https: / / sg.idtdna.com / pages / products / genes-and-gene-fragments / megamers-single-stranded-dna-fragments)).

[0059] 5) Preparation of the double-stranded DNA tracer for "Flanking Sequence 1-Core Replicating Segment-Flanking Sequence 2": Add 20 μL of DNA annealing buffer, 20 μL of single-stranded DNA (50 μM), 20 μL of complementary single-stranded DNA (50 μM), and 40 μL of ultrapure water to a 0.2 mL centrifuge tube and mix thoroughly. Place the 0.2 mL centrifuge tube in a qPCR instrument and denature the DNA at 95°C for 2 minutes. Then, anneal the DNA from 95°C to 25°C for approximately 90 minutes. Finally, remove the sample and freeze it at -20°C.

[0060] The double-stranded DNA of the present invention has no homology with DNA of known organisms.

[0061] The present invention also provides a method for predicting the decomposition rate of a soluble chain-like high-molecular pollutant tracer that is not wrapped with DNA.

[0062] In one embodiment of the present invention, the following decomposition experiments were conducted in groundwater:

[0063] Before the experiment began, 100 mL of groundwater was thoroughly mixed with 0.8 mL of unencapsulated DNA tracer with known concentrations for each DNA sequence. A 0.1 mL initial sample was frozen at -20°C to determine the initial concentration. The remaining sample was aliquoted into 10 tubes, labeled with the sampling time, and then the experiment was performed. Each tube was opened only once, at the designated sampling time, and then frozen at -20°C. The concentration of each DNA sequence in the sample was measured, and a decomposition curve was plotted to calculate the decomposition rate of each DNA sequence.

[0064] The present invention also provides a method for controlling adsorption of unwrapped DNA series soluble chain-shaped high-molecular pollutant tracers.

[0065] In one embodiment of the present invention, column 4 has an inner diameter of 1 cm and a height of 50 cm. It is filled with 50 cm of the target porous medium, which has been sterilized at high temperatures. Both the tracer and the water flow from bottom to top. A conical flask 1 is used to provide a water supply. Water in the flask is slowly pumped out from above along a flexible tube via a peristaltic pump 2 at a constant flow rate. After passing through the porous medium in the column from below, the water flows out from above. The filtrate is collected regularly and quantitatively by an automatic fraction collector 5 into a centrifuge tube, where the tracer concentration is measured.

[0066] Before the experiment begins, start the peristaltic pump 2 and the automatic fraction collector 5, and flush the porous medium in the column from bottom to top with 3 pore volumes of groundwater with a pH of 8.8 to expel air, so that the entire column system is saturated and the flow rate is stable. Figure 4As shown in Figure 1, 1 mL of the prepared tracer was injected into the bottom of the saturated column 4, and the filtrate sample was collected from above the column 4. Samples were taken every 2 minutes from 0 to 60 min, with each sample volume being approximately 1 mL. After sampling, the concentrations of different DNA sequences and Br ions in the samples were measured, and penetration curves were plotted to calculate the tracer recovery rate.

[0067] Furthermore, the present invention provides a method for tracing pollutants in porous media using a series of unwrapped DNA soluble chain polymer pollutant tracers, which specifically includes the following steps:

[0068] 1) Identify target pollutants present in porous media: chain-like water-soluble high-molecular-weight pollutants.

[0069] 2) Conduct a short-term column experiment to obtain penetration curves for the target contaminant and a series of unencapsulated double-stranded DNA tracers. The nucleic acid tracer comprises the following components: a nucleic acid molecule, 10-50 mmol / L KBr or NaBr, 0.05-0.1 mol / L Tris, pH 7.0-9.0; the nucleic acid molecule contains n different double-stranded DNAs, each with a concentration of 0.1-20 μmol / L; and each double-stranded DNA has a different total length and core replication sequence. The experimental water should be carefully selected to ignore the target contaminant and implement the decomposition of the double-stranded DNA series in the short-term column experiment. For example, TE buffer (containing 10 mM Tris-HCl and 1 mM EDTA) at pH 8.0, which is used to store the DNA tracer, can be used.

[0070] 3) Compare the penetration curves of the target pollutant and the unwrapped double-stranded DNA tracer series, and select the unwrapped double-stranded DNA tracer (denoted as DNA) with the penetration curve closest to the target pollutant. ) to simulate the diffusion and adsorption characteristics of target pollutants.

[0071] 4) Prepare the following DNA Tracer: 0.3 μmol / L DNA , 10 mmol / L KBr or NaBr, 0.05 mol / LTris, pH 8.0.

[0072] 5) Conduct decomposition tests in a water environment (such as groundwater, soil water) where porous media exist to obtain target pollutants and DNA decomposition rate.

[0073] 6) In the pollutant migration and transformation model, the saturated hydraulic conductivity of the porous medium and the diffusion coefficient in the convection-diffusion equation are inversely solved using the Br penetration curve.

[0074] 7) Input the target pollutant and DNA obtained in step 5) into the pollutant migration and transformation model. decomposition rate.

[0075] 8) Use the DNA obtained in step 2) The penetration curve is used to inversely analyze the adsorption and desorption parameters in the pollutant migration and transformation model.

[0076] 9) The diffusion coefficient in the convection diffusion equation obtained by back-solving Br in step 6), the decomposition rate of the target pollutant measured in step 5), and the adsorption and desorption parameters obtained by back-solving in step 8) are introduced into the pollutant migration and transformation model to obtain the simulated penetration curve of the target pollutant; then, the penetration curve of the target pollutant obtained by the column test in step 2) is compared with the penetration curve of the target pollutant obtained by the column test in step 2), and the selected DNA is verified by referring to formulas (1) and (2). Whether the tracer can be used to trace the target pollutant; when the determination coefficient R 2 If the value is above 0.8 and the Nash coefficient NSE is above 0.6, it indicates that the DNA screened The tracer and the established pollutant migration and transformation model can be used to trace the migration and decomposition process of target pollutants in porous media;

[0077] Among them, the coefficient of determination R 2 The Nash coefficient NSE is an evaluation index used to describe the similarity between the simulated breakthrough curve and the breakthrough curve of the target pollutant obtained by the column test;

[0078] (1)

[0079] (2)

[0080] in, t At different time points, T is the total time; Y t The time point in the penetration curve of the target pollutant calculated by the inverse solution parameters t The corresponding concentration value; X t This is the time point in the breakthrough curve of the target pollutant obtained by column testing in step 2). t The corresponding concentration value; yes X t The average value of .

[0081] 10) Through the above steps, you can use DNA Tracers and the established pollutant migration and transformation model are used to trace the migration and decomposition of pollutants in porous media.

[0082] 11) When there are m pollution sources of target pollutants in the target porous medium, design m DNA The core replication segments of the double-stranded DNA are identical in length but different in sequence and identical in total length, respectively, and are denoted as DNA 1. DNA 2. DNA m, and prepare DNA according to step 4) 1. DNA 2. DNA m tracer.

[0083] 12) Add DNA to contamination source 1 1 tracer, put DNA at the source of contamination 2 2 tracers, and so on, by injecting DNA into the contamination source m m tracer.

[0084] 13) Through DNA 1. DNA 2. DNA The penetration curve of m is inversely solved using the pollutant migration and transformation model to obtain the adsorption and desorption parameters; a decomposition experiment of the target pollutant is carried out in the water environment where the porous medium exists to obtain the decomposition rate of the target pollutant; the inversely solved adsorption and desorption parameters and the decomposition rate measured in the decomposition experiment are substituted into the established pollutant migration and transformation model to achieve multi-source tracing of the target pollutant.

[0085] In the present invention,

[0086] Target porous media: underground aquifers / soils.

[0087] Water environment where the target porous media exists: groundwater / soil water.

[0088] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0089] Single-stranded DNA and its complementary sequence were purchased from Integrated DNA Technologies (https: / / sg.idtdna.com / pages / products / genes-and-gene-fragments / megamers-single-stranded-dna-fragments).

[0090] DNA annealing buffer was purchased from Shanghai Beyotime Biotechnology Co., Ltd. (https: / / www.beyotime.com / product / D0251.htm).

[0091] Ottawa quartz sand was purchased from LAGUNA CLAY COMPANY (https: / / www.axner.com / sand-silica-sand.aspx).

[0092] Fine quartz sand was purchased from Qingmiaoyuan (https: / / item.taobao.com / item.htm?_u=32n3nuh4f6cd&id=685104918034&spm=a1z09.2.0.0.be712e8da2EZno).

[0093] The device used for column test was self-assembled.

[0094] The DNA sequences involved in the following examples are shown in Table 1 (SEQ ID NOs: 1-8).

[0095] Table 1 DNA sequences

[0096]

[0097] Example 1 Preparation of Unencapsulated DNA Series Soluble Chain Polymer Pollutant Tracers

[0098] The tracer composition of this example is as follows: a double-stranded DNA series with a total length of 82-120 bp at a concentration of approximately 0.3 μmol / L, 0.05 mol / L Tris, and 10 mmol / L KBr, and the pH is adjusted to 8.0 with 5 mol / L NaOH or 5 mol / L HCl.

[0099] The double-stranded DNAs are 82 bp, 100 bp and 120 bp double-stranded DNAs, and their nucleotide sequences are shown in Table 1.

[0100] The core replication segment is 80 bp long, and the total length is 82 bp, 100 bp and 120 bp of double-stranded DNA.

[0101] The core replication segment is 98 bp long, and the total length is 100 bp of double-stranded DNA.

[0102] The DNA sequences used are shown in Table 1. It should be noted that A80F1_1 and A80F1_2 in Table 1 represent a pair of tracers. They are not complementary strands, but rather two DNA sequences with equal core replication base number and total length, but different sequences. This is used to eliminate errors caused by specific sequences. Their core replication segments are randomly generated with different sequences, and their flanking segments are identical. This demonstrates that the rate of double-stranded DNA degradation is determined by the core replication base number, independent of the specific sequence, and that the adsorption of double-stranded DNA is determined by the total length, independent of the specific sequence. Similarly, A80F10_1 and A80F10_2, A98F1_1 and A98F1_2, and A80F20_1 and A80F20_2 also have equal core replication base number and total length, but their core replication segments are randomly generated with different sequences, and their flanking segments are identical.

[0103] The sequence of each DNA tracer is randomly generated by a DNA random generator (https: / / molbiotools.com / randomsequencegenerator.php) and meets the following conditions:

[0104] 1) Core replication segment structure: forward primer-Linker1-probe-Linker2-reverse primer; the forward and reverse primers are 18-25 bp long; the probe segment is 25-32 bp long; the Linker1 and Linker2 are 1-138 bp long, and the sum of the Linker1 and Linker2 lengths is 2-139 bp; the core replication segment is 63-200 bp long.

[0105] 2) Requirements for the core replication segment: The GC content of the synthetic DNA sequence should be 40%-60% of all bases, the GC content of the probe sequence should be 60%-65% of the probe bases, and the GC content of the primer sequence should be 40%-60% of the primer bases. The annealing temperature of the probe should be 68-70°C, and the annealing temperature of the primer should be 55-62°C. Based on the above principles, each single-stranded DNA and its corresponding primer and probe were designed using the Primer-BLAST tool (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) to confirm the uniqueness of the primers and ensure that the primers do not correspond to the DNA of any documented existing organisms.

[0106] 3) The structure of the DNA sequence is: flanking sequence 1-core replication segment-flanking sequence 2; the length of flanking sequence 1 and flanking sequence 2 is 1-1934 bp, and the sum of the lengths of flanking sequence 1 and flanking sequence 2 is 2-1935 bp; the total length is 65-2000 bp.

[0107] 4) Preparation of single-stranded DNA consisting of Flanking Sequence 1-Core Replication Segment-Flanking Sequence 2: For DNA sequences with a total length of 65-200 bp, synthesis is recommended. For DNA sequences with a total length of 201-2000 bp, cloning is recommended (available from Integrated DNA Technologies (https: / / sg.idtdna.com / pages / products / genes-and-gene-fragments / megamers-single-stranded-dna-fragments)).

[0108] 5) Preparation of double-stranded DNA tracer (Flanking Sequence 1-Core Replication Segment-Flanking Sequence 2): Add 20 μL of DNA annealing buffer, 20 μL of single-stranded DNA (50 μM), 20 μL of complementary single-stranded DNA (50 μM), and 40 μL of ultrapure water to a 0.2 mL centrifuge tube and mix thoroughly. Place the 0.2 mL centrifuge tube in a qPCR instrument and denature the DNA at 95°C for 2 min. Then, anneal the DNA from 95°C to 25°C for approximately 90 min. Finally, remove the sample and freeze it at -20°C.

[0109] Example 2: Method for Predicting the Decomposition Rate of Unwrapped DNA Series Soluble Chain Polymolecular Pollutant Tracers

[0110] A decomposition experiment was conducted on the unencapsulated DNA series tracer in Example 1 in the water environment where the target porous medium exists. Before the experiment began, 100 mL of groundwater and 0.8 mL of unencapsulated DNA series tracer with known concentrations of each DNA sequence were thoroughly mixed, and 0.1 mL of the initial sample was taken and frozen at -20°C to measure the initial concentration. The remaining sample was divided into 10 tubes, the sampling time was marked, and the experiment was carried out. Each tube of sample was opened only once at the specified sampling time, and the sample was frozen at -20°C. The concentration of each DNA sequence in the sample was measured and plotted as shown below. Figure 1 The degradation curves shown are used to calculate the degradation rates of different DNA sequences.

[0111] Preferably, the target porous medium is located in groundwater, and the decomposition experiment is conducted at room temperature in the dark to simulate decomposition in an underground aquifer. The experiment lasts for three days and is repeated three times to eliminate experimental errors.

[0112] like Figure 2 As shown, by comparison, it can be seen that during the 3-day decomposition experiment, the decomposition rate of the DNA tracer with an unwrapped core replication segment of 80 bp in Example 1 is similar and less than that of the DNA tracer with an unwrapped core replication segment of 98 bp in Example 1, which proves that the decomposition rate of the DNA chain is determined by the length of the core replication segment and is not related to the total length.

[0113] Based on the existing DNA tracers, the present invention proposes that the decomposition rate of the DNA tracer can be predicted by the length of the core replication segment of the DNA tracer.

[0114] Example 3 Method for Controlling the Adsorption Rate of Unwrapped DNA Series Soluble Chain Polymer Pollutant Tracers

[0115] Conduct short-term column tests, e.g. Figure 3 As shown, column 4 has an inner diameter of 1 cm and a height of 50 cm. It is filled with 50 cm of the target porous medium, which has been sterilized at high temperatures. Both the tracer and the water flow from bottom to top. Conical flask 1 is used to provide a water supply. Water in flask 1 is slowly pumped out from above along a flexible tube via peristaltic pump 2 at a constant flow rate. After passing through the porous medium in the column from below, the water flows out from above. The filtrate is collected regularly and quantitatively by an automatic fraction collector 5 into a centrifuge tube, where the tracer concentration is measured.

[0116] Before the experiment begins, start the peristaltic pump 2 and the automatic fraction collector 5, and flush the porous medium in the column from bottom to top with 3 pore volumes of groundwater with a pH of 8.8 to expel air, so that the entire column system is saturated and the flow rate is stable. Figure 4 As shown. 1 mL of the tracer prepared in Example 1 was simultaneously injected into the lower portion of the saturated column 4, and filtrate samples were collected from above the column 4. Samples were taken every 2 minutes from 0 to 60 min, with each sample volume being approximately 1 mL. After sampling, the concentrations of DNA and Br ions in the samples were measured, and penetration curves were plotted to calculate the tracer recovery rate.

[0117] Preferably, the above saturated column penetration test is performed in 2 groups, with 3 repetitions in each group. 50 =0.288 mm Ottawa quartz sand, the second group is d 50 = 0.110 mm fine quartz sand to verify the universal applicability of the method of the present invention.

[0118] Figure 5 is the penetration curve of the unencapsulated DNA tracer in Ottawa quartz sand in Example 1, Figure 6 This is the penetration curve of the unencapsulated DNA tracer in fine quartz sand of Example 1. Figure 7 is the decomposition amount of the unencapsulated DNA tracer in two types of quartz sand in Example 1, Figure 8 The adsorption amount of the unencapsulated DNA tracer in Example 1 in the two types of quartz sand.

[0119] By comparison, it can be seen that the average DNA recovery rates of the unencapsulated DNA soil water tracers with a total length of 82 bp (A80F1-1 and A80F1-2), 100 bp (A80F10-1, A80F10-2, A98F1-1 and A98F1-2), and 120 bp (A80F20-1 and A80F20-2) in Ottawa quartz sand in Example 1 were 52.3%±0.35%, 50.9%±0.87%, and 49.5%±0.31%, respectively. Example 1 The average DNA recovery rates of unencapsulated DNA soil water tracers with a total length of 82 bp (A80F1-1 and A80F1-2), 100 bp (A80F10-1, A80F10-2, A98F1-1 and A98F1-2), and 120 bp (A80F20-1 and A80F20-2) in fine quartz sand were 26.1% ± 0.79%, 24.8% ± 0.65%, and 24.0% ± 0.85%, respectively.

[0120] The experimental results were simulated using a contaminant transport and transformation model, preferably using HYDRUS-1D. The decomposition conditions in the experiment were consistent with the decomposition conditions in groundwater under dark conditions in Example 2. By combining the decomposition rates in Example 2 with the HYDRUS-1D model, the average DNA degradation rates of the unencapsulated DNA soil water tracers in Example 1 with core copy segment lengths of 80 bp (A80F1-1, A80F1-2, A80F10-1, A80F10-2, A80F20-1, and A80F20-2) and 98 bp (A98F1-1 and A98F1-2) were calculated to be 1.33% ± 0.04% and 1.39% ± 0.87%, respectively, in Ottawa quartz sand. Example 1 The average DNA degradation amounts of unencapsulated DNA soil water tracers with core replica segment lengths of 80 bp (A80F1-1, A80F1-2, A80F10-1, A80F10-2, A80F20-1, and A80F20-2) and 98 bp (A98F1-1 and A98F1-2) in Ottawa quartz sand were 1.75% ± 0.04% and 1.86% ± 0.05%, respectively.

[0121] Calculations using the HYDRUS-1D model show that the average DNA adsorption amounts of the unencapsulated DNA soil water tracers in Example 1 with total lengths of 82 bp (A80F1-1 and A80F1-2), 100 bp (A80F10-1, A80F10-2, A98F1-1 and A98F1-2), and 120 bp (A80F20-1 and A80F20-2) in Ottawa quartz sand were 46.5% ± 0.31%, 47.7% ± 0.88%, and 49.2% ± 0.32%, respectively. There was a linear relationship between total length and adsorption amount: y = 0.0007x + 0.407 (y is the adsorption amount, x is the total length of the unencapsulated DNA tracer). Example 1 The average DNA adsorption amounts of unencapsulated DNA soil water tracers with total lengths of 82 bp (A80F1-1 and A80F1-2), 100 bp (A80F10-1, A80F10-2, A98F1-1 and A98F1-2), and 120 bp (A80F20-1 and A80F20-2) in fine quartz sand were 72.2% ± 0.81%, 73.4% ± 0.68%, and 74.3% ± 0.87%, respectively. There was a linear relationship between the total length and the adsorption amount, y = 0.0005x + 0.678 (y is the adsorption amount, x is the total length of the unencapsulated DNA tracer).

[0122] The present invention proves that under the premise that the adsorption sites in the porous medium are not fully occupied, there is a linear relationship between the total length and the adsorption amount, and proposes that the total length of the DNA tracer can be used to control the adsorption of DNA in the porous medium.

[0123] Example 4 Method for tracing pollutants in porous media using unencapsulated DNA series soluble chain polymer pollutant tracers

[0124] Based on the unwrapped DNA series of soluble chain polymer pollutant tracers in Example 1, a method for tracing the migration of pollutants in porous media was proposed by conducting a decomposition experiment in a water environment with a target porous medium as shown in Example 2 and a migration experiment in a target porous medium as shown in Example 3, combined with a pollutant migration and transformation model. In short, Figure 9 As shown, the unwrapped DNA series of soluble chain polymer pollutant tracers provide the migration and adsorption parameters of the target pollutants by conducting migration experiments in the target porous medium. The target pollutants provide their own decomposition rates by conducting decomposition experiments in the water environment where the target porous medium exists. Finally, through the pollutant migration and transformation model, the migration and adsorption parameters provided by the unwrapped double-stranded DNA series tracers are combined with the decomposition rate provided by the target pollutant itself to achieve the tracing of the migration and transformation process of the target pollutant in the target porous medium.

[0125] Specifically,

[0126] 1) Identify target soluble chain polymer pollutants, such as PAM (polyacrylamide), PAA (polyacrylic acid), PVA (polyvinyl alcohol), etc.

[0127] 2) Conduct a short-term column experiment to obtain penetration curves for the target contaminant and a series of unencapsulated double-stranded DNA tracers. The nucleic acid tracer comprises the following components: a nucleic acid molecule, 10-50 mmol / L KBr or NaBr, 0.05-0.1 mol / L Tris, pH 7.0-9.0; the nucleic acid molecule contains n different double-stranded DNAs, each with a concentration of 0.1-20 μmol / L; and each double-stranded DNA has a different total length and core replication sequence. The experimental water should be carefully selected to minimize the target contaminant and allow for the degradation of the double-stranded DNA series during the short-term column experiment. For example, TE buffer (10 mM Tris-HCl and 1 mM EDTA) at pH 8.0, which is used to store the DNA tracer, can be used.

[0128] 3) Compare the penetration curves of the target pollutant and the unwrapped double-stranded DNA tracer series, and select the unwrapped double-stranded DNA tracer (denoted as DNA) with the penetration curve closest to the target pollutant. ) to simulate the diffusion and adsorption characteristics of target pollutants;

[0129] 4) Prepare the following DNA Tracer: 0.3 μmol / L DNA , 10 mmol / L KBr or NaBr, 0.05 mol / LTris, pH 8.0;

[0130] 5) Conduct decomposition tests in a water environment (such as groundwater, soil water) where porous media exist to obtain target pollutants and DNA The decomposition rate; for example, the decomposition rate of A80F1_1 is 1.08E-03 min -1 ;

[0131] 6) In the pollutant migration and transformation model, the saturated hydraulic conductivity of the porous medium and the diffusion coefficient in the convection-diffusion equation are inversely solved using the Br penetration curve. For example, the Ottawa sand column in Example 2 has a saturated hydraulic conductivity of 0.62 cm / min and a diffusion coefficient of 0.153 cm.

[0132] 7) Input the target pollutant and DNA obtained in step 5) into the pollutant migration and transformation model. The decomposition rate; for example, the decomposition rate of A80F1_1 is 1.08E-03 min -1 ;

[0133] 8) Use the DNA obtained in step 2) The penetration curve is used to inversely analyze the adsorption and desorption parameters in the pollutant migration and transformation model; for example, the adsorption and desorption parameters of A80F1_1 in the Ottawa sand column in Example 2 are: adsorption rate of the first adsorption site = 0.068 min -1 , the desorption rate of the first adsorption site = 3.98 min -1 , the adsorption rate of the second adsorption site = 0.021 min -1 , desorption rate of the second adsorption site = 3E-04 min -1 ;

[0134] 9) The diffusion coefficient in the convection diffusion equation obtained by back-solving Br in step 6), the decomposition rate of the target pollutant measured in step 5), and the adsorption and desorption parameters obtained by back-solving in step 8) are introduced into the pollutant migration and transformation model to obtain the simulated penetration curve of the target pollutant; then, the penetration curve of the target pollutant obtained by the column test in step 2) is compared with the penetration curve of the target pollutant obtained by the column test in step 2), and the selected DNA is verified by referring to formulas (1) and (2). Whether the tracer can be used to trace the target pollutant; when the determination coefficient R 2 If the value is above 0.8 and the Nash coefficient NSE is above 0.6, it indicates that the DNA screened The tracer and the established pollutant migration and transformation model can be used to trace the migration and decomposition process of target pollutants in porous media;

[0135] Among them, the coefficient of determination R 2 The Nash coefficient NSE is an evaluation index used to describe the similarity between the simulated breakthrough curve and the breakthrough curve of the target pollutant obtained by the column test;

[0136] (1)

[0137] (2)

[0138] in, t At different time points, T is the total time; Y t The time point in the penetration curve of the target pollutant calculated by the inverse solution parameters t The corresponding concentration value; X t This is the time point in the breakthrough curve of the target pollutant obtained by column testing in step 2). t The corresponding concentration value; yes X t The average value of .

[0139] 10) Through the above steps, you can use DNA Tracers and the established pollutant migration and transformation model are used to trace the migration and decomposition of pollutants in porous media.

[0140] 11) When there are m pollution sources of target pollutants in the target porous medium, design m DNA The core replication segments of the double-stranded DNA are identical in length but different in sequence and identical in total length, respectively, and are denoted as DNA 1. DNA 2. DNA m, and prepare DNA according to step 4) 1. DNA 2. DNA mtracer;

[0141] 12) Add DNA to contamination source 1 1 tracer, put DNA at the source of contamination 2 2 tracers, and so on, by injecting DNA into the contamination source m mtracer;

[0142] 13) Through DNA 1. DNA 2. DNA The penetration curve of m is inversely solved using the pollutant migration and transformation model to obtain the adsorption and desorption parameters; a decomposition experiment of the target pollutant is carried out in the water environment where the porous medium exists to obtain the decomposition rate of the target pollutant; the inversely solved adsorption and desorption parameters and the decomposition rate measured in the decomposition experiment are substituted into the established pollutant migration and transformation model to achieve multi-source tracing of the target pollutant.

[0143] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A multi-source tracing method for pollutants in porous media, characterized in that: The following steps are involved: 1) Identify soluble chain polymers present in porous media as target pollutants; 2) Conducting short-term column tests on the target pollutant and nucleic acid tracer to obtain the penetration curves of the target pollutant and nucleic acid tracer; The nucleic acid tracer comprises the following components: 0.1-20 μmol / L nucleic acid molecules, 10-50 mmol / L KBr or NaBr, 0.05-0.1 mol / L Tris, pH 7.0-9.0; the nucleic acid molecules contain n different double-stranded DNAs, each of which has a different total length and a different core replication segment sequence; 3) Compare the penetration curves of the target pollutant and the nucleic acid tracer, and select the double-stranded DNA with the closest penetration curve to the target pollutant, denoted as DNA*, to simulate the diffusion and adsorption characteristics of the target pollutant; 4) Prepare DNA* tracer: 0.1-20 μmol / L DNA*, 10-50 mmol / L KBr or NaBr, 0.05-0.1 mol / L Tris, pH 7.0-9.0; 5) Conduct decomposition tests on the target pollutant and the DNA* tracer in a water environment containing porous media, and measure the decomposition rates of the target pollutant and the DNA*; 6) In the pollutant migration and transformation model, the Br penetration curve is used to inversely solve the saturated hydraulic conductivity of porous media and the diffusion coefficient in the convection-diffusion equation; 7) Input the decomposition rate of the DNA* tracer obtained in step 5) into the pollutant transport and transformation model; 8) using the DNA* tracer breakthrough curve obtained in step 2) to inversely solve the adsorption and desorption parameters in the pollutant migration and transformation model; 9) The saturated hydraulic conductivity and diffusion coefficient in step 6), the decomposition rate of the target pollutant measured in step 5), and the adsorption and desorption parameters obtained by reverse analysis in step 8) are introduced into the pollutant migration and transformation model to obtain a simulated penetration curve of the target pollutant; then, the simulated penetration curve of the target pollutant is compared with the penetration curve of the target pollutant obtained by the column test in step 2). Referring to formulas (1) and (2), when the determination coefficient R 2 If the NSE is above 0.8 and the Nash coefficient NSE is above 0.6, it indicates that the screened DNA* tracer and the established pollutant migration and transformation model can be used to trace the migration and decomposition process of target pollutants in porous media; Among them, t is different time points, T is the total time; Y t is the concentration value corresponding to time point t in the penetration curve of the target pollutant calculated by the inverse solution parameters; X t is the concentration value corresponding to time point t in the breakthrough curve of the target pollutant obtained by the column test in step 2); It's X t The average value of The double-stranded DNA has a structure of: flanking sequence 1-core replication segment-flanking sequence 2, with a total length of 82-120 bp, and the double-stranded DNA has no homology with the DNA of known organisms; wherein the lengths of flanking sequence 1 and flanking sequence 2 are each independently 1-20 bp, and the sum of the lengths of flanking sequence 1 and flanking sequence 2 is 2-21 bp; The core replication segment has the following structure: forward primer-Linker1-probe-Linker2-reverse primer; the core replication segment is 80-100 bp long; the forward and reverse primers are each 18-25 bp long; the probe segment is 25-32 bp long; Linker1 and Linker2 are each 1-26 bp long, and the sum of Linker1 and Linker2 is 2-27 bp long; The core replication segment meets the following conditions: (1) the GC base content is 40%-60%; (2) the GC base content in the probe is 60%-65%; (3) the GC base content in the primer is 40%-60%; (4) the annealing temperature of the probe is 68-70°C, and the annealing temperature of the primer is 55-62°C.

2. The method according to claim 1, characterized in that When there are m sources of target pollutants in the target porous medium, m double-stranded DNAs are designed with the same length as the core replication segment of DNA* but different sequences and the same total length, and are respectively denoted as DNA*1, DNA*2, ...DNA*m. DNA*1, DNA*2, ...DNA*m tracers are prepared according to step 4). Add DNA*1 tracer to pollution source 1, add DNA*2 tracer to pollution source 2, and so on, add DNA*m tracer to pollution source m; The adsorption and desorption parameters were inversely solved using the pollutant migration and transformation model through the breakthrough curves of DNA*1, DNA*2, ..., DNA*m; Decomposition experiments of target pollutants are carried out in a water environment where porous media exist to obtain the decomposition rate of the target pollutants; the adsorption and desorption parameters obtained by the reverse decomposition and the decomposition rate measured by the decomposition experiment are substituted into the established pollutant migration and transformation model to achieve multi-source tracing of the target pollutants.

3. The method according to claim 1, characterized in that The column 4 used in the column test in step 2) has an inner diameter of 1 cm and a height of 50 cm, and is filled with 50 cm of porous medium that has been sterilized at high temperature; The column filled with the porous medium was pre-saturated with buffer before the column test.

4. The method according to claim 1, wherein The composition of the buffer is TE buffer at pH 8.0, containing 10 mM Tris-HCl and 1 mM EDTA.

5. The method according to claim 1, wherein The total length of the double-stranded DNA is 82 bp, 100 bp or 120 bp, and the length of the core replication segment is 80 bp; or, The total length of the double-stranded DNA is 100 bp, and the length of the core replication segment is 98 bp.

6. The method according to claim 1, characterized in that Adjust the pH to 7.0-9.0 with 5-6 mol / L NaOH or 5-6 mol / L HCl.

7. The method according to any one of claims 1 to 6, characterized in that The water environment in step 5) includes groundwater and soil water.

8. The method according to any one of claims 1 to 6, characterized in that The pollutant migration and transformation model used in step 6) is HYDRUS-1D.

9. The method according to claim 8, characterized in that The target pollutants are selected from PAM, PAA, and PVA.

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