Uncoated dna soil water tracer and method of making

Abstract

The application provides an unwrapped DNA soil water tracer, and the composition of the tracer is as follows: 0.1-20 mu mol / L unwrapped single-stranded DNA with a length of 80-120 bp, 0.01-0.2 mol / L Tris, 0.001-0.2 mol / L EDTA, 0.2-0.8 g / L bright blue, and the pH is adjusted to 7.0-9.0 by using 5-6 mol / L NaOH or 5-6 mol / L HCl solution. The recovery rate experiment of penetrating 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 soil water tracer without encapsulated DNA 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 tracking 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 making them unsuitable for application in aquatic and soil environments. Currently, there are no research reports on improving 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 without encapsulated DNA and its preparation method.

[0006] To achieve the objective of this invention, this invention provides a soil water tracer without encapsulated DNA. The tracer comprises the following components: 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 LEDTA, 0.2-0.8 g / L Brilliant Blue, and adjusted to pH 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 g / L Brilliant Blue, and adjusted to pH 7.0-9.0 with 5-6 mol / L NaOH or 5-6 mol / L HCl solution.

[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, wherein 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, and finally the pH is adjusted with NaOH solution.

[0010] The design principle of the tracer of this invention 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 the 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 the 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+ (e.g.), inhibiting the activity of DNase (DNA enzyme) and reducing 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.

[0011] 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.

[0012] 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.

[0013] Preferably, the amount of tracer added is 10%-20% of the total pore volume of the soil.

[0014] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0015] (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 (such as silica microspheres with a minimum diameter of 60 nm, most of which are in the hundreds of nanometers or even micrometers) in the soil.

[0016] (ii) Compared with existing DNA tracers, the Tris buffer in the tracer of the present invention maintains a stable pH in soil water tracers without DNA encapsulation (which can be adjusted in the range of 7.0-9.0), making it suitable for most sandy soils in northern China. Furthermore, the pH of the tracer 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.

[0017] (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.

[0018] (iv) The recovery rate experiment of penetrating saturated soil column shows that the DNA-free 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

[0019] 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.

[0020] Figure 2 This is a particle size distribution curve of sand in Example 5 of the present invention.

[0021] Figure 3 This is the breakthrough curve of the unencapsulated DNA soil water tracer in Example 5 of the present invention.

[0022] Figure 4 This is the breakthrough curve of the DNA tracer containing low concentrations of Tris and EDTA in Example 5 of the present invention.

[0023] Figure 5 This is the penetration curve of the unencapsulated DNA soil water tracer in Example 3 of the present invention.

[0024] Figure 6This is the penetration curve of the DNA tracer encapsulated in polylactic acid (PLA) in Example 3 of the present invention.

[0025] Figure 7 This is a schematic diagram of the experimental setup for comparing uncoated DNA soil water tracer with polylactic acid (PLA)-coated DNA tracer in Example 3 of the present invention. Detailed Implementation

[0026] 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. Example 1: Preparation of DNA-free soil water tracer

[0027] 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 adjusted to pH 8.0 with 5 mol / L NaOH solution. 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.

[0028] 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 food-grade brilliant blue in a certain proportion. Finally, the pH is adjusted with NaOH solution.

[0029] Example 2: Preparation of DNA tracer encapsulated in polylactic acid (PLA)

[0030] First, 2.0017 g of polylactic acid (PLA) and 20.02 mL of dichloromethane solvent (CH2Cl2) were mixed thoroughly in a flask. Then, 200 μl of 1 μM HPLC-purified T12 DNA (SEQ ID NO:1) was mixed with 300 μL of nuclease-free water to prepare a DNA solution. While stirring, the DNA solution was added dropwise to the CH2Cl2-PLA solution; the mixture was then sonicated on ice at 95% energy for 15 s, with the sonicator tip held 2 mm above the bottom of the flask. The sonication step was repeated 3 times, and the mixture was shaken between each sonication to obtain a first polymer emulsion.

[0031] To harden the polymer microspheres, 40 mL of 1% polyvinyl alcohol (PVA) was added dropwise to the first emulsion while stirring. The PVA first emulsion mixture was then sonicated at 95% energy for 30 seconds. The sonication step was repeated three times, with the mixture shaken between each repetition to obtain the second emulsion. During the first and second sonications, the tip of the sonicator was kept approximately 2 mm from the bottom of the flask, and during the third repetition, it was kept approximately 2 cm from the bottom of the flask.

[0032] The second emulsion was added dropwise to 1 L of 0.3% PVA. After continuously stirring the final solution at room temperature for 3 hours, the final solution was concentrated by repeated centrifugation, removal of the supernatant, and washing with Milli-Q water. In the final wash, no more Milli-Q water was added, and the concentrated, washed tracer was stored in a 4°C refrigerator for later use.

[0033] The average particle size of the prepared polylactic acid (PLA)-encapsulated DNA tracer was determined to be 850 nm using a Malvern particle size analyzer.

[0034] Example 3: Comparative test of the unencapsulated DNA soil water tracer of the present invention and the polylactic acid (PLA) encapsulated DNA tracer.

[0035] Fill the tilted earthen box with loamy sand measuring 1.9m long, 0.5m wide, and 1m high, and gravel measuring 0.1m long, 0.5m wide, and 1m high, such as... Figure 7 As shown. The experiment lasted for 10 days, with artificial rainfall occurring three times a day for up to 2 hours. Uncoated DNA soil water tracers from Example 1 and polylactic acid (PLA)-coated DNA tracers from Example 2 (with an average particle size of 850 nm after coating) were applied to the soil surface. The penetration curves and recovery rates of the two were compared. The recovery rate of the uncoated DNA tracer was 1.05% (…). Figure 5 The recovery rate of polylactic acid (PLA)-encapsulated DNA tracers was 0.01%. Figure 6 Therefore, the unencapsulated DNA tracer of the present invention, due to its small size, easily penetrates soil and exhibits superior penetration recovery rate compared to existing encapsulated DNA tracers. Furthermore, the preparation process of this tracer is relatively simple, reducing the complex preparation steps of encapsulated DNA, saving tracer manufacturing costs, and eliminating the need for the extraction of DNA from PLA microspheres before qPCR detection with PLA-encapsulated DNA tracers.

[0036] Example 4: Preparation of uncoated DNA tracers containing low concentrations of Tris and EDTA

[0037] The DNA tracer containing low concentrations of Tris and EDTA consisted of: 0.2 μmol / L HPLC-purified 88 bp uncoated single-stranded T12 DNA, 0.01 mol / L Tris, 0.001 mol / L EDTA, and 0.5 g / L Brilliant Blue. The pH of the tracer was adjusted to 8.0 with 5 mol / L NaOH. The preparation method was the same as in Example 1.

[0038] Example 5: Comparison Test between DNA Tracers Containing Low Concentrations of Tris and EDTA and the DNA Tracer of the Present Invention

[0039] like Figure 1 As shown, the soil column (or plexiglass 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. The Marshall bottle 1 is used to maintain the water head; water in the Marshall bottle 1 flows out from below along a flexible tube. According to the principle of communicating vessels, the water head above the soil column 3 is level with the air inlet of the Marshall bottle 1. The 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 the peristaltic pump 4. The filtrate is collected in centrifuge tubes at regular intervals and in quantitative quantities by the automatic partial collector 5, and the tracer concentration is to be measured.

[0040] 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 2 As shown. 5 mL of the tracer prepared in Example 1 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 bottle 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.

[0041] The above saturated soil column penetration test was performed in two groups, with each group having replicates. The first group used the DNA tracer prepared in Example 1, and the second group used the DNA tracer containing low concentrations of Tris and EDTA prepared in Example 4.

[0042] The experimental results showed that the recovery rate of the DNA tracer containing low concentrations of Tris and EDTA was 6.7% ± 1.9%. Figure 4 The recovery rate of the DNA tracer in this invention is 13.4% ± 1.9%. Figure 3 Therefore, the Tris and EDTA content of the present invention is superior.

[0043] 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> Uncoated DNA Soil Water Tracer and Preparation Method <130> KHP221114525.3 <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. A soil water tracer without DNA encapsulation, characterized in that, The tracer consists of the following components: 0.2 μmol / L single-stranded DNA with the nucleotide sequence shown in SEQ ID NO:1, 0.1 mol / L Tris, 0.1 mol / L EDTA, 0.5 g / L Brilliant Blue, and pH adjusted to 7.0-9.0 with 5-6 mol / L NaOH or 5-6 mol / L HCl solution.

2. The method for preparing the tracer 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, and brilliant blue in a specific ratio. Finally, the pH was adjusted with NaOH solution.

3. The application of the tracer of claim 1 in soil environmental tracing.

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

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

0.

6. The application according to any one of claims 3-5, characterized in that, The amount of tracer added is 10%-20% of the total pore volume of the soil.

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