High recovery rate of unwrapped DNA soil water tracer and preparation method thereof

Abstract

The application provides a high-recovery unwrapped DNA soil water tracer, and the composition of the tracer is as follows: 0.1-20 micromoles per liter of single-stranded DNA with a length of 80-120 base pairs, 0.01-0.2 moles per liter of Tris, 0.001-0.2 moles per liter of EDTA, 0.3-1 moles per liter of sodium monohydrogen phosphate or 0.05-0.1 moles per liter of sodium hexametaphosphate solution, 0.2-0.8 grams per liter of brilliant blue, and the pH is adjusted to 7.0-9.0 by using 5-6 moles per liter of NaOH or 5-6 moles per liter of HCl. The recovery rate experiment of the penetration of a saturated soil column shows that, compared with the existing DNA tracer, the unwrapped DNA soil water tracer has the advantages of not being easy to decompose, not being easy to adsorb and having a higher penetration rate in real soil.

Description

Technical Field

[0001] This invention belongs to the field of environmental protection, specifically, it relates to a high-recovery-rate uncoated DNA soil water tracer and its preparation method. Background Technology

[0002] DNA tracers can theoretically be any sequence, and artificially synthesized DNA can be synthesized with a zero background value in the environment, unaffected by other tests or other DNA in the environment. Therefore, artificially synthesized DNA tracers have become an emerging type of water and soil environmental tracer due to their advantages of coding ability, specificity, independence from environmental background values, and no environmental pollution.

[0003] Unencapsulated DNA is easily decomposed in the environment, with a half-life of only a few days in soil, and is easily adsorbed by soil particles, resulting in low recovery rates. In sand column experiments, the peak height / recovery rate of the DNA tracer penetration curve decreases with increasing DNA length, and the recovery rate of DNA tracers is typically 2-4 orders of magnitude lower than that of conservative tracers. In recent years, new technologies using materials such as PLGA, PLA, and SiO2 to encapsulate DNA have emerged. Although encapsulated DNA tracers are less prone to decomposition and have been well applied in tracing surface water and pollutant emissions, the larger particle size of encapsulated DNA tracers makes it difficult to penetrate soil pores, resulting in low tracer recovery rates and unsuitability for application in aquatic and soil environments. Currently, no researchers have improved the DNA recovery rate by refining the formulation of unencapsulated DNA tracers.

[0004] Currently, both uncoated and coated DNA tracers have limitations when applied to different soil and water environments. Uncoated DNA tracers are easily decomposed and adsorbed, resulting in low penetration in real soil. Coated DNA tracers, on the other hand, have large particle sizes, are easily filtered by soil, and have even lower penetration in soil. Therefore, there is an urgent need to develop a novel soil water tracer. Summary of the Invention

[0005] The purpose of this invention is to provide a soil water tracer with high recovery rate for uncoated DNA and its preparation method.

[0006] To achieve the objectives of this invention, in a first aspect, this invention provides a high-recovery-rate uncoated DNA soil water tracer (tracer I), the tracer comprising the following components: 0.1-20 μmol / L single-stranded DNA of 80-120 bp in length, 0.01-0.2 mol / L Tris, 0.001-0.2 mol / L EDTA, 0.3-1 mol / L sodium monohydrogen phosphate, 0.2-0.8 g / L brilliant blue, and pH adjusted to 7.0-9.0 with NaOH or HCl solution.

[0007] Preferably, the tracer comprises the following components: 0.2 μmol / L single-stranded DNA of 80-120 bp in length, 0.1 mol / L Tris, 0.1 mol / L EDTA, 0.5 mol / L sodium monohydrogen phosphate, 0.8 g / L brilliant blue, and adjusted to pH 7.0-9.0 with 5-6 mol / L NaOH or 5-6 mol / L HCl.

[0008] In one specific embodiment of the present invention, the single-stranded DNA is an 88bp single-stranded T12 DNA, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0009] The present invention also provides a method for preparing the tracer I, wherein the synthesized single-stranded DNA is purified by HPLC and then mixed with a pre-prepared solution containing Tris, EDTA, sodium hydrogen phosphate and brilliant blue in a certain proportion, and finally the pH is adjusted with NaOH or HCl solution.

[0010] Furthermore, the solution containing Tris, EDTA, sodium hydrogen phosphate, and brilliant blue in tracer I is prepared as follows: Weigh 1.2138g of Tris powder, 2.9371g of EDTA powder, 18.0879g of sodium hydrogen phosphate powder, and 0.5g of brilliant blue powder, stir to dissolve, adjust the pH to 8.0 with 5mol / L NaOH solution, and then bring the volume to 100mL.

[0011] The design principle of tracer I is as follows: (1) pH 8.0 is the optimal pH for DNA storage. Within the pH range of 3.0-9.0, the adsorption of DNA by the soil decreases significantly with the increase of tracer pH. Therefore, pH 8.0 is a compromise tracer pH that is suitable for DNA storage, has a low adsorption capacity, and is closest to the pH of soil in northern China (7.0-9.0); (2) Tris buffer solution has the function of stabilizing the pH of the tracer system, which can keep DNA at the optimal pH 8.0 for storage. EDTA can chelate divalent and high-valent metal cations (such as Mg). 2+ Mn 2+ Fe 2+ Ca 2+ Al 3+ (2) Inhibit the activity of DNase (DNA enzyme) and reduce DNA decomposition; (3) Since divalent metal cations can form cation bridges and promote DNA adsorption in the soil, EDTA can chelate divalent metal cations in the soil and solution without affecting the DNA tracing effect, thereby reducing DNA adsorption in the soil and improving the DNA recovery rate; (4) The phosphate groups in the tracer compete strongly with the DNA molecules on the surface of clay particles for adsorption sites. Phosphate can reduce DNA adsorption in the soil and improve the DNA tracer recovery rate by occupying the adsorption sites on the surface of soil particles.

[0012] Secondly, the present invention provides a high-recovery-rate uncoated DNA water and soil environmental pollutant tracer (tracer II), the tracer having the following composition: 0.1-20 μmol / L single-stranded DNA with a length of 80-120 bp, 0.01-0.2 mol / L Tris, 0.001-0.2 mol / L EDTA, 0.05-0.1 mol / L sodium hexametaphosphate solution, 0.2-0.8 g / L brilliant blue, and pH adjusted to 7.0-9.0 with NaOH or HCl solution.

[0013] Preferably, the tracer comprises the following components: 0.2 μmol / L single-stranded DNA of 80-120 bp in length, 0.1 mol / L Tris, 0.1 mol / L EDTA, 0.1 mol / L sodium hexametaphosphate solution, 0.8 g / L brilliant blue, and pH adjusted to 7.0-9.0 with 5-6 mol / L NaOH.

[0014] In one specific embodiment of the present invention, the single-stranded DNA is an 88bp single-stranded T12 DNA, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0015] The present invention also provides a method for preparing the tracer II, wherein the synthesized single-stranded DNA is purified by HPLC and then mixed with a pre-prepared solution containing Tris, EDTA, sodium hexametaphosphate and brilliant blue in a certain proportion, and finally the pH is adjusted with NaOH solution.

[0016] Furthermore, the solution containing Tris, EDTA, sodium hexametaphosphate, and brilliant blue in tracer II is prepared as follows: Weigh 1.2138g of Tris powder, 2.9371g of EDTA powder, 6.7974g of sodium hexametaphosphate powder, and 0.5g of brilliant blue powder, stir to dissolve, adjust the pH to 8.0 with 5mol / L NaOH solution, and then bring the volume to 100mL.

[0017] The design principle of tracer II is as follows: (1) pH 8.0 is the optimal pH for DNA storage. Within the pH range of 3.0-9.0, the adsorption of DNA by the soil decreases significantly with the increase of tracer pH. Therefore, pH 8.0 is a compromise tracer pH that is suitable for DNA storage, has a low adsorption capacity, and is closest to the pH of soil in northern China (7.0-9.0); (2) Tris buffer solution has the function of stabilizing the pH of the tracer system, which can keep DNA at the optimal pH 8.0 for storage. EDTA can chelate divalent and high-valent metal cations (such as Mg). 2+ Mn 2+ Fe 2+ Ca 2+Al 3+ (2) Inhibit the activity of DNase (DNA enzyme) and reduce DNA decomposition; (3) Since divalent metal cations can form cation bridges and promote DNA adsorption in the soil, EDTA can chelate divalent metal cations in the soil and solution without affecting the DNA tracing effect, thereby reducing DNA adsorption in the soil and improving the DNA recovery rate; (4) Sodium hexametaphosphate in the tracer is hydrolyzed into sodium orthophosphate and phosphite after dissolving in water, which competes strongly with the DNA molecules in the tracer for adsorption sites, occupying a large number of adsorption sites on the surface of soil particles, reducing DNA adsorption in the soil and improving the DNA tracer recovery rate.

[0018] This invention also provides the application of the tracer in soil environmental tracing. The tracer of this invention is recommended for use in sandy soils.

[0019] In the aforementioned applications, the soil pH is tested beforehand, and the pH of the tracer is adjusted to the soil pH (e.g., pH 8.0) before use.

[0020] Preferably, the amount of tracer added is 10%-20% of the total pore volume of the soil. Through the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0021] (i) The tracer of this invention uses small-sized uncoated single-stranded DNA (straight length less than 30 nm) as the main component, which avoids the physical filtration of larger coated DNA (the smallest being silica microspheres with a diameter of 60 nm, and most being in the hundreds of nanometers or even micrometers) in the soil.

[0022] (ii) Compared with existing DNA tracers, the Tris buffer in the tracer of the present invention maintains a stable pH (adjustable in the range of 7.0-9.0) for soil water tracers without DNA coating, making it suitable for most sandy soils in northern China. Furthermore, the pH can be pre-adjusted according to the soil to be tested, which improves the stability of the tracer when it is put into use and minimizes the impact of pH changes on DNA adsorption, making it easier for quantitative analysis.

[0023] (III) The EDTA in the tracer of this invention chelates divalent and high-valent metal cations, such as Mg 2+ Mn 2+ Fe 2+ Ca 2+ Al 3+ On the one hand, it inhibits the action of DNase, reducing the decomposition of DNA; on the other hand, it inhibits the formation of cation bridges, reducing DNA adsorption, thereby improving the penetration recovery rate of DNA tracers.

[0024] (iv) Compared with uncoated DNA soil water tracers (containing 0.2 μmol / L single-stranded DNA of 88 bp, 0.1 mol / L Tris, 0.1 mol / L EDTA, 0.5 mg / L Brilliant Blue, and pH adjusted to 8.0 with 5 mol / L NaOH, without phosphate competing groups), the two tracers of the present invention further improve the recovery rate of the tracers. The recovery rate of tracer I containing sodium monohydrogen phosphate is increased to nearly 3 times, and the recovery rate of tracer II containing sodium hexametaphosphate is increased to more than 4 times, which greatly improves the recovery rate of existing uncoated DNA soil water tracers.

[0025] (v) The recovery rate experiment of penetrating saturated soil column shows that the high recovery rate of the unencapsulated DNA soil water tracer of the present invention has the advantages of being less prone to decomposition and adsorption in real soil and having a higher penetration rate compared with existing DNA tracers. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the experimental apparatus in Embodiment 5 of the present invention. In the figure, 1-Mastachian bottle; 2-lifting platform; 3-soil column (or plexiglass column); 4-peristaltic pump; 5-automatic partial collector.

[0027] Figure 2 This is a physical diagram of the experimental apparatus in Embodiment 5 of the present invention.

[0028] Figure 3 This is a particle size distribution curve of sand in Example 5 of the present invention.

[0029] Figure 4 This is the penetration curve of the soil water tracer without DNA encapsulation in Example 3 of the present invention.

[0030] Figure 5 This is the breakthrough curve of uncoated DNA soil water tracer I with high recovery rate in Example 1 of the present invention.

[0031] Figure 6 This is the breakthrough curve of uncoated DNA soil water tracer II with high recovery rate in Example 2 of the present invention.

[0032] Figure 7 This is the breakthrough curve of the uncoated DNA soil water tracer containing a low concentration of sodium monohydrogen phosphate in Example 4 of the present invention. Detailed Implementation

[0033] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the 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.

[0034] Example 1: Preparation of high-recovery-rate uncoated DNA soil water tracer I (tracer I)

[0035] This embodiment provides a high-recovery-rate uncoated DNA soil water tracer I, with the following composition: 0.2 μmol / L single-stranded T12 DNA (SEQ ID NO:1), 0.1 mol / L Tris, 0.1 mol / L EDTA, 0.5 mol / L sodium monohydrogen phosphate, and pH adjusted to 8.0 with 5 mol / L NaOH. Furthermore, 0.5 g / L food-grade brilliant blue dye is added to the tracer as a pigment, providing a visual indication of the experimental process without affecting the migration of the DNA tracer.

[0036] The above tracer is prepared as follows: the synthesized single-stranded DNA is purified by HPLC and then mixed with a pre-prepared solution containing Tris, EDTA, sodium hydrogen phosphate and brilliant blue in a certain proportion. Finally, the pH is adjusted with NaOH solution.

[0037] Example 2: Preparation of high-recovery-rate uncoated DNA soil water tracer II (tracer II)

[0038] This embodiment provides a high-recovery-rate uncoated DNA soil water tracer II, with the following composition: 0.2 μmol / L single-stranded T12 DNA (SEQ ID NO:1), 0.1 mol / L Tris, 0.1 mol / L EDTA, 0.1 mol / L sodium hexametaphosphate solution, and pH adjusted to 8.0 with 5 mol / L NaOH. Furthermore, 0.5 g / L food-grade brilliant blue dye is added to the tracer as a pigment, providing a visual indication of the experimental process without affecting the migration of the DNA tracer.

[0039] The above tracer is prepared as follows: the synthesized single-stranded DNA is purified by HPLC and then mixed with a pre-prepared solution containing Tris, EDTA, sodium hexametaphosphate and brilliant blue in a certain proportion. Finally, the pH is adjusted with NaOH solution.

[0040] Example 3: Preparation of DNA-free soil water tracer

[0041] This embodiment provides a soil water tracer without encapsulated DNA, comprising: 0.2 μmol / L single-stranded T12 DNA (SEQ ID NO:1), 0.1 mol / L Tris, 0.1 mol / L EDTA, and pH adjusted to 8.0 with 5 mol / L NaOH. Furthermore, 0.5 g / L food-grade brilliant blue dye is added as a pigment to the tracer, providing a visual indication of the experimental process without affecting the migration of the DNA tracer.

[0042] The above tracer is prepared as follows: the synthesized single-stranded DNA is purified by HPLC and then mixed with a pre-prepared solution containing Tris, EDTA and brilliant blue in a certain proportion. Finally, the pH is adjusted with NaOH solution.

[0043] Example 4: Preparation of a soil water tracer containing low concentrations of sodium phosphate and no DNA coating

[0044] This embodiment provides a soil water tracer containing low concentrations of sodium phosphate for uncoated DNA, with the following composition: 0.2 μmol / L single-stranded T12 DNA (SEQ ID NO:1), 0.1 mol / L Tris, 0.1 mol / L EDTA, 0.1 mol / L sodium monohydrogen phosphate, and pH adjusted to 8.0 with 5 mol / L NaOH. Furthermore, 0.5 g / L food-grade brilliant blue dye is added to the tracer as a pigment, providing a visual indication of the experimental process without affecting the migration of the DNA tracer.

[0045] The above tracer is prepared as follows: the synthesized single-stranded DNA is purified by HPLC and then mixed with a pre-prepared solution containing Tris, EDTA, sodium hydrogen phosphate and brilliant blue in a certain proportion. Finally, the pH is adjusted with NaOH solution.

[0046] Example 5: Experiment comparing the recovery rates of different DNA soil water tracers

[0047] like Figure 1 and Figure 2 As shown, soil column 3 has an inner diameter of 3cm and a height of 25cm, filled with 10cm of dried sandy soil. Both the tracer and water flow vertically downwards along the direction of gravity. Marshall bottle 1 is used to maintain the water head; water in Marshall bottle 1 flows out from below along a flexible tube. According to the principle of communicating vessels, the water head above soil column 3 is level with the air inlet of Marshall bottle 1. Lifting platform 2 is used to fine-tune the height of the Marshall bottle, thereby accurately controlling the water head position. After passing through the soil column, the water flows out from below and is slowly extracted at a constant flow rate by peristaltic pump 4. The filtrate is collected in centrifuge tubes at regular intervals and in quantitative quantities by automatic partial collector 5, and the tracer concentration is to be measured.

[0048] A soil column was saturated from bottom to top with a 0.05 mol / L Tris-HCl solution (pH 8.0), air was expelled, and the column was allowed to stand for 8 hours until saturation. The soil particle size distribution is as follows: Figure 3As shown. 5 mL of the tracer prepared in Examples 1-4 was injected into the top of the saturated soil column 3. Simultaneously, the peristaltic pump 4 and the automatic partial collector 5 were started to collect the filtrate sample from below. After the tracer infiltration was complete, a 10 cm water head was added, and the water head and supply were maintained using a Maslow flask 1. Samples were taken every 1 minute from 0-30 min, with each sample volume approximately 5 mL. From 30-60 min, samples were taken every 3 minutes, with each sample volume approximately 15 mL. After sampling, the concentrations of DNA and brilliant blue tracer in the samples were measured, a breakthrough curve was plotted, and the tracer recovery rate was calculated.

[0049] The above saturated soil column penetration test was performed in 4 groups, with 3 replicates per group. The first group was the high-recovery-rate uncoated DNA soil water tracer I prepared in Example 1 of this invention, the second group was the high-recovery-rate uncoated DNA soil water tracer II prepared in Example 2 of this invention, the third group was the uncoated DNA soil water tracer prepared in Example 3 of this invention, and the fourth group was the uncoated DNA soil water tracer containing low concentration of sodium phosphate prepared in Example 4 of this invention.

[0050] Figure 4 The breakthrough curve of the soil water tracer without DNA encapsulation in Example 3 is shown. Figure 5 This is the breakthrough curve of tracer I in Example 1. Figure 6 This is the breakthrough curve of tracer II in Example 2. Figure 7 This is the breakthrough curve of the uncoated DNA soil water tracer containing a low concentration of sodium phosphate in Example 4.

[0051] The comparison shows that the DNA recovery rate of the unencapsulated DNA soil water tracer in Example 3 was 13.4% ± 1.9%. Tracer I in Example 1 and Tracer II in Example 2 further improved upon the tracer in Example 3. Penetration tests showed that the DNA recovery rate of Tracer I was 36.0% ± 1.7%, nearly three times that of the tracer in Example 3, and the DNA recovery rate of Tracer II was 55.3% ± 2.9%, more than four times that of the tracer in Example 3. However, the recovery rate of the unencapsulated DNA soil water tracer in Example 4, containing a low concentration of sodium monohydrogen phosphate (0.1 mol / L), was only 21.82%, which was only 60.6% of that of Tracer I in Example 1 (containing a low concentration of sodium monohydrogen phosphate 0.5 mol / L). Therefore, Tracer I containing 0.5 mol / L sodium monohydrogen phosphate is superior in this invention.

[0052] This invention adds sodium phosphate buffer solution or sodium hexametaphosphate solution to existing DNA tracers, providing soluble phosphate. The phosphate ions generated after the phosphate dissolves in water can participate in competitive adsorption, competing with the phosphate groups in DNA molecules for soil adsorption sites, thereby reducing DNA adsorption in the soil. All experiments were conducted at 22℃, and the results show that the two tracers of this invention have good tracking effects at this temperature.

[0053] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. sequence list <110> China Agricultural University <120> High-recovery-rate uncoated DNA soil water tracer and its preparation method <130> KHP221116051.9 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 88 <212> DNA <213> Artificial Sequence <400> 1 ccgtagagat ctcccatctg tcctttgctg aaggttaaaa ccccggaccg cctagaatat 60 tctttcttta gctccaaaat ggcctctc 88

Claims

1. Application of high-recovery-rate uncoated DNA tracers in soil environmental tracing; The composition of the high-recovery-rate uncoated DNA soil water tracer is as follows: 0.2 μmol / L single-stranded DNA with a length of 80-120 bp, 0.1 mol / L Tris, 0.1 mol / L EDTA, 0.1 mol / L sodium hexametaphosphate solution, 0.8 g / L brilliant blue, and pH adjusted to 7.0-9.0 with 5-6 mol / L NaOH or 5-6 mol / L HCl; The nucleotide sequence of the single-stranded DNA is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The synthesized single-stranded DNA was purified by HPLC and then mixed with a pre-prepared solution containing Tris, EDTA, sodium monohydrogen phosphate or sodium hexametaphosphate, and brilliant blue in a specific ratio. Finally, the pH was adjusted with NaOH solution.

3. The application according to claim 1, characterized in that, The soil pH was tested beforehand, and the tracer was adjusted to the soil pH before use.

4. The application according to claim 3, characterized in that, The soil pH is 8.

0.

5. The application according to any one of claims 1-4, characterized in that, The amount of tracer added is 10%-20% of the total pore volume of the soil.

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