A sample storage reagent for tiger frog environmental detection and a preparation method thereof

Abstract

The present application relates to the field of bioengineering technology, and more particularly to a kind of tiger frog environment detection sample storage reagent and its preparation method, consisting of the following components by mass fraction: preservative 95-99 parts and microcapsule 1-5 parts;The present application is composed of the composite system of environmental protection type preservative matrix and temperature-sensitive microcapsule encapsulating glycerol aldehyde-3-phosphate dehydrogenase inhibitor by design, so that the reagent has the double-stage temperature change response function of "in situ slow release under normal temperature transportation to stabilize sample microbial metabolic state" and "incubation triggered rapid release in laboratory", can maximize the preservation of tiger frog environmental sample collection instant microbial activity and community structure information, thereby significantly improving the authenticity and reliability of subsequent molecular detection results, providing an efficient and convenient tool for tiger frog habitat health assessment, biodiversity monitoring and ecological environment research, and has broad application prospect.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a sample preservation reagent for environmental testing of tiger frogs and its preparation method. Background Technology

[0002] Tiger frog environmental monitoring is a method that analyzes biomarkers such as environmental DNA and microbial communities in samples from its habitat to assess the population's survival status, ecological health, and level of environmental pollution.

[0003] With the increasing demand for ecological monitoring and the popularization of citizen science projects, the development of a sample preservation reagent that is easy to use in the field, can stably preserve a variety of biological information, and is environmentally friendly has become a clear market demand in this field. At present, the sample preservation technology in this field mainly revolves around general-purpose stabilizers, which are limited in terms of specificity and functionality.

[0004] Existing technologies rely on traditional chemical preservatives, such as high-concentration ethanol or commercial nucleic acid preservation solutions. While ethanol can inhibit degradation, it is flammable and volatile, posing safety risks. It can also cause complete cell lysis and protein denaturation, completely destroying the activity status information of microorganisms. Although commercially available general-purpose preservation solutions are convenient to use, their formulations are optimized for human cells or blood, and their protective efficacy against complex microbial communities, eukaryotic DNA, and environmental enzyme systems in amphibian environmental samples is not optimized. Under normal temperature conditions, the preservation time and integrity of tiger frog-specific eDNA are often insufficient. At the same time, the above technologies all focus on the physical integrity of nucleic acid molecules and cannot "freeze" the immediate metabolic state of microorganisms during the preservation period. Metabolic activity is an important dimension reflecting the ecological function of habitat. In addition, existing solutions mostly rely on cold chain transportation and preservation, which is costly and logistically difficult in field monitoring practices in remote habitats.

[0005] Therefore, based on the relevant technologies mentioned above, there is an urgent need to develop a sample preservation reagent for environmental testing of tiger frogs and its preparation method. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a sample preservation reagent for environmental testing of tiger frogs and its preparation method, so as to solve the problems of poor safety, poor universality, insufficient stability and inconvenient transportation in the prior art.

[0007] To achieve the above objectives, this invention provides a sample preservation reagent for environmental testing of tiger frogs and its preparation method.

[0008] A sample preservation reagent for environmental testing of tiger frogs, comprising the following components in parts by weight: 95-99 parts of preservation solution and 1-5 parts of microcapsules; The preservation solution consists of trehalose, citrate-phosphate buffer, disodium EDTA, chitosan hydrochloride, cell membrane stabilizer, and reducing agent. The microcapsules are composed of an olealdehyde-3-phosphate dehydrogenase inhibitor, modified poly(N-isopropylacrylamide), and modified polylactic acid.

[0009] Preferably, the preparation steps of the preservation solution are as follows: Step A1: Add trehalose, citrate-phosphate buffer, disodium EDTA and chitosan hydrochloride to deionized water, heat to 20-30℃, stir at 300-400 rpm for 30-50 minutes, and the mixture is obtained after stirring. Step A2: Add the cell stabilizer polyethylene glycol 1000 vitamin E succinate and the reducing agent ascorbic acid to the mixture, cool to 15-25℃, stir at 150-250 rpm for 15-25 minutes, add 0.1 mol / L sodium hydroxide solution, adjust the pH to 7.3-7.5, stir until finished, and filter through a 0.22 μm filter membrane to obtain the preservation solution.

[0010] By using trehalose as the core stabilizer, excellent room-temperature glassy protection is provided to stabilize nucleic acid structure. Disodium EDTA efficiently chelates metal ions to inhibit environmental and microbial nucleases. Meanwhile, polysaccharide hydrochloride provides mild antibacterial effects, while cell stabilizers provide both cell membrane stability and antioxidant functions. In addition, ascorbic acid can prevent oxidative damage to cells. Therefore, the goal of room-temperature, long-lasting, and environmentally friendly preservation is achieved through the synergistic interaction of the components. By employing a "primary followed by secondary" dissolution sequence and controlling the cooling process, the activity of TPGS and ascorbic acid can be effectively protected. At the same time, precise pH adjustment (7.3-7.5) provides an optimal stable environment for nucleic acids and inhibitors. Therefore, the prepared preservation solution matrix is ​​not only stable and environmentally friendly, but also provides a compatible and stable carrier environment for the uniform dispersion and functional performance of subsequent smart microcapsules.

[0011] Preferably, the mass ratio of trehalose, citrate-phosphate buffer, disodium EDTA, chitosan hydrochloride, and deionized water in step A1 is 0.1-0.12:0.01-0.015:0.005-0.006:0.002-0.003:1. The mass ratio of cell stabilizer, reducing agent and mixture in step A2 is 0.001-0.0015:0.0005-0.0008:1.

[0012] Preferably, the modified poly-N-isopropylacrylamide is prepared as follows: Under a nitrogen atmosphere, N-isopropylacrylamide and acrylic acid were added to deionized water, heated to 20-30°C, stirred at 200-300 rpm for 20-30 min, heated to 60-70°C, and ammonium persulfate initiator was added. The stirring speed was increased to 250-350 rpm, and the reaction was carried out for 4-6 h. After the reaction was completed, the temperature was lowered to 20-30°C, and the mixture was transferred to a 4000-7000 Da dialysis bag and dialyzed for 48-72 h. The mixture was then freeze-dried to obtain modified poly-N-isopropylacrylamide.

[0013] By introducing a specific ratio of acrylic acid to N-isopropylacrylamide copolymerization, the hydrophilic carboxyl groups can successfully raise the lower critical dissolution temperature of the polymer from approximately 32°C of homopolymer PNIPAM to the target range, achieving precise control of temperature response. In addition, by employing aqueous free radical polymerization, the reaction conditions are mild and the initiator ratio is appropriate, ensuring a high monomer conversion rate and a suitable molecular weight, with good reproducibility.

[0014] Preferably, the mass ratio of N-isopropylacrylamide, acrylic acid and initiator is 1:0.04-0.08:0.005-0.015.

[0015] Preferably, the preparation steps of the modified polylactic acid are as follows: Under a nitrogen atmosphere, lactide, glycolide, and a 0.01 mol / L stannous octoate toluene solution were added to 1-dodecaneol. The mixture was heated to 125-135℃, stirred at 200-300 rpm for 18-24 hours until the reaction was complete. The mixture was then cooled to 20-30℃, dissolved in anhydrous dichloromethane, precipitated with cold ethanol, washed, filtered, and dried under vacuum to obtain modified polylactic acid.

[0016] By controlling the mass ratio of lactide to glycolide, the degradation rate and mechanical properties of the copolymer can be precisely controlled. At the same time, the precise control of the target molecular weight can be achieved by using a trace amount of the initiator 1-dodecanool, which further ensures that the microcapsule wall material has suitable mechanical strength. In addition, ring-opening polymerization is carried out in a strictly anhydrous and oxygen-free environment at a precise temperature, which effectively avoids side reactions and hydrolysis, and ensures the regularity of the polymer chain structure and the stability of its performance.

[0017] Preferably, the mass ratio of lactide, glycolide, catalyst and 1-dodecaneol is 1:0.57-0.59:0.02-0.05:0.001-0.003.

[0018] Preferably, the preparation steps of the microcapsules are as follows: Step B1: Add sodium iodoacetate, the glyceraldehyde-3-phosphate dehydrogenase inhibitor, to 10 mmol / L phosphate buffer and stir for 1-3 min at 2500-3500 rpm to obtain the aqueous phase; Step B2: Add modified poly-N-isopropylacrylamide and modified polylactic acid to ethyl acetate solvent, heat to 20-30℃, stir at 300-400 rpm for 2-3 hours, cool to 0-4℃, add aqueous phase, increase the stirring speed to 8500-9500 rpm, stir for 2-4 minutes to obtain primary emulsion; Step B3: Add polyvinyl alcohol to deionized water, heat to 90-100℃, stir at 300-400 rpm for 60-90 min, cool to 20-30℃, add Tween-80 and the primary emulsion, increase the speed to 450-550 rpm, emulsify for 8-12 min, decrease the speed to 200-300 rpm, and solidify for 8-10 h. After solidification, centrifuge and wash to obtain microcapsules.

[0019] The double emulsification-solvent evaporation method can efficiently encapsulate water-soluble glyceraldehyde-3-phosphate dehydrogenase inhibitors and encapsulate them inside the polymer wall material. This can achieve physical isolation and protection of the active ingredient and create conditions for controlled release. In addition, high-speed emulsification and low-temperature operation can form a small-particle-size, uniformly distributed emulsion to improve the encapsulation efficiency.

[0020] Preferably, the mass ratio of sodium iodoacetate to phosphate buffer in step B1 is 1:8-12; The mass ratio of the modified poly(N-isopropylacrylamide), modified polylactic acid, and aqueous phase in step B2 is 0.7-0.9:1:1.6-1.7; The mass ratio of polyvinyl alcohol, deionized water, Tween-80 and the primary emulsion in step B3 is 0.07-0.09:7.8-8.0:0.007-0.009:1.

[0021] A method for preparing a sample preservation reagent for environmental testing of tiger frogs, the preparation method being as follows: Add the microcapsules to the preservation solution, cool to 2-8℃, stir at 100-140 rpm for 15-25 minutes, increase the speed to 120-180 rpm, stir for 20-40 minutes, and the sample preservation reagent is obtained after stirring is completed.

[0022] By controlling the stirring speed and time in stages, the microcapsules are ensured to be uniformly and stably dispersed in the viscous preservation liquid matrix, avoiding sedimentation or aggregation and ensuring the consistency of performance of each packaged product.

[0023] The beneficial effects of this invention are: This invention provides a sample preservation reagent for tiger frog environmental testing and its preparation method. The invention utilizes a composite system composed of an environmentally friendly preservation solution matrix and temperature-sensitive microcapsules encapsulating a glyceraldehyde-3-phosphate dehydrogenase inhibitor. This allows the reagent to exhibit a two-stage temperature-sensitive response function: "slow release in situ during room temperature transportation to stabilize the microbial metabolic state of the sample" and "rapid release triggered by incubation in the laboratory." Compared to existing technologies, this invention, without relying on a cold chain, can maximize the preservation of microbial activity and community structure information of tiger frog environmental samples (such as water samples and sediments) at the moment of collection, thereby significantly improving the accuracy and reliability of subsequent molecular detection results. It also boasts advantages such as environmental friendliness, operational safety, and broad downstream compatibility. This reagent provides an efficient and convenient tool for tiger frog habitat health assessment, biodiversity monitoring, and ecological environment research, and has broad application prospects. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only for this invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a bar chart showing the results of the preservation efficiency test of tiger frog-specific environmental DNA (eDNA) on day 7 and day 30 in this invention. Figure 2 This is a bar chart showing the test results on day 3 and day 7 in the microbial metabolic activity stabilization effect test of this invention; Figure 3 This is a bar chart showing the release function verification test in this invention; Figure 4 This is a line graph showing the biocompatibility test results in this invention. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0027] Example 1: The preparation steps of a preservation solution are as follows: S1: Add 10g of trehalose, 1g of citrate-phosphate buffer, 0.5g of disodium EDTA and 0.2g of chitosan hydrochloride to 100g of deionized water, heat to 20℃, stir at 400rpm for 30min, and after stirring is complete, a mixture is obtained. S2: Add 0.1g of the cell stabilizer polyethylene glycol 1000 vitamin E succinate and 0.05g of the reducing agent ascorbic acid to 100g of the mixture, cool to 15℃, stir at 250rpm for 15min, add 0.1mol / L sodium hydroxide solution, adjust the pH to 7.3-7.5, stir until finished, and filter through a 0.22μm filter membrane to obtain the preservation solution.

[0028] Example 2: The preparation steps of a preservation solution are as follows: S1: Add 11g of trehalose, 1.3g of citrate-phosphate buffer, 0.55g of disodium EDTA and 0.25g of chitosan hydrochloride to 100g of deionized water, heat to 25℃, stir at 350rpm for 40min, and after stirring is complete, a mixture is obtained. S2: Add 0.13g of the cell stabilizer polyethylene glycol 1000 vitamin E succinate and 0.065g of the reducing agent ascorbic acid to 100g of the mixture, cool to 20℃, stir at 200rpm for 20min, add 0.1mol / L sodium hydroxide solution, adjust the pH to 7.3-7.5, stir until finished, and filter through a 0.22μm filter membrane to obtain the preservation solution.

[0029] Example 3: The preparation steps of a preservation solution are as follows: S1: Add 12g of trehalose, 1.5g of citrate-phosphate buffer, 0.6g of disodium EDTA and 0.3g of chitosan hydrochloride to 100g of deionized water, heat to 30℃, stir at 300rpm for 50min, and after stirring is complete, a mixture is obtained. S2: Add 0.15g of the cell stabilizer polyethylene glycol 1000 vitamin E succinate and 0.08g of the reducing agent ascorbic acid to 100g of the mixture, cool to 25℃, stir at 150rpm for 25min, add 0.1mol / L sodium hydroxide solution, adjust the pH to 7.3-7.5, stir until finished, and filter through a 0.22μm filter membrane to obtain the preservation solution.

[0030] Example 4: The preparation steps of a preservation solution are as follows: S1: Add 10g of trehalose, 1g of citrate-phosphate buffer, 0.5g of disodium EDTA, 0.2g of chitosan hydrochloride and 1g of mannitol to 100g of deionized water, heat to 20℃, stir at 400rpm for 30min, and after stirring is complete, a mixture is obtained. S2: Add 0.1g of the cell stabilizer polyethylene glycol 1000 vitamin E succinate and 0.05g of the reducing agent ascorbic acid to 100g of the mixture, cool to 15℃, stir at 250rpm for 15min, add 0.1mol / L sodium hydroxide solution, adjust the pH to 7.3-7.5, add 5mg of lysozyme, stir until finished, filter through a 0.22μm filter membrane to obtain the preservation solution.

[0031] Example 5: The preparation steps of a preservation solution are as follows: S1: Add 10g of trehalose, 1g of citrate-phosphate buffer, 0.5g of disodium EDTA, 0.2g of chitosan hydrochloride, 0.2g of sodium citrate and 0.1g of sodium chloride to 100g of deionized water, heat to 20℃, stir at 400rpm for 30min, and after stirring is complete, a mixture is obtained; S2: Add 0.1g of the cell stabilizer polyethylene glycol 1000 vitamin E succinate and 0.05g of the reducing agent ascorbic acid to 100g of the mixture, cool to 15℃, stir at 250rpm for 15min, add 0.1mol / L sodium hydroxide solution, adjust the pH to 7.3-7.5, stir until finished, and filter through a 0.22μm filter membrane to obtain the preservation solution.

[0032] Example 6: Preparation of a modified poly(N-isopropylacrylamide) Under a nitrogen atmosphere, 100 g of N-isopropylacrylamide and 4 g of acrylic acid were added to 150 mL of deionized water. The mixture was heated to 20 °C, stirred at 300 rpm for 20 min, heated to 70 °C, and 0.5 g of ammonium persulfate initiator was added. The stirring speed was increased to 250 rpm, and the reaction was carried out for 6 h. After the reaction was completed, the mixture was cooled to 20 °C and transferred to a 4000-7000 Da dialysis bag. Dialysis was performed for 72 h, and the mixture was freeze-dried to obtain modified poly(N-isopropylacrylamide).

[0033] Example 7: Preparation of a modified poly(N-isopropylacrylamide) Under a nitrogen atmosphere, 100 g of N-isopropylacrylamide and 6 g of acrylic acid were added to 150 mL of deionized water. The mixture was heated to 25 °C, stirred at 250 rpm for 25 min, heated to 65 °C, and 1 g of ammonium persulfate initiator was added. The stirring speed was increased to 300 rpm, and the reaction was carried out for 5 h. After the reaction was completed, the mixture was cooled to 25 °C and transferred to a 4000-7000 Da dialysis bag. Dialysis was performed for 60 h, and the mixture was freeze-dried to obtain modified poly-N-isopropylacrylamide.

[0034] Example 8: Preparation of a modified poly(N-isopropylacrylamide) Under a nitrogen atmosphere, 100 g of N-isopropylacrylamide and 8 g of acrylic acid were added to 150 mL of deionized water. The mixture was heated to 30 °C, stirred at 200 rpm for 30 min, heated to 60 °C, and 1.5 g of ammonium persulfate initiator was added. The stirring speed was increased to 350 rpm, and the reaction was carried out for 4 h. After the reaction was completed, the mixture was cooled to 30 °C and transferred to a 4000-7000 Da dialysis bag. Dialysis was performed for 48 h, and the mixture was freeze-dried to obtain modified poly(N-isopropylacrylamide).

[0035] Example 9: Preparation of a modified polylactic acid Under a nitrogen atmosphere, 100g of lactide, 57g of glycolide, and 2g of catalyst in a 0.01mol / L stannous octoate toluene solution were added to 0.1g of 1-dodecaneol. The mixture was heated to 125℃, stirred at 300rpm for 18h until the reaction was complete. The mixture was then cooled to 30℃, dissolved in anhydrous dichloromethane, precipitated with cold ethanol, washed, filtered, and dried under vacuum to obtain modified polylactic acid.

[0036] Example 10: Preparation of a modified polylactic acid Under a nitrogen atmosphere, 100g of lactide, 58g of glycolide, and 3g of catalyst in a 0.01mol / L stannous octoate toluene solution were added to 0.2g of 1-dodecaneol. The mixture was heated to 130℃, stirred at 250rpm for 20h until the reaction was complete. The mixture was then cooled to 25℃, dissolved in anhydrous dichloromethane, precipitated with cold ethanol, washed, filtered, and dried under vacuum to obtain modified polylactic acid.

[0037] Example 11: Preparation of a modified polylactic acid Under a nitrogen atmosphere, 100 g of lactide, 59 g of glycolide, and 5 g of catalyst in a 0.01 mol / L stannous octoate toluene solution were added to 0.3 g of 1-dodecaneol. The mixture was heated to 135 °C, stirred at 200 rpm for 24 h until the reaction was complete. The mixture was then cooled to 20 °C, dissolved in anhydrous dichloromethane, precipitated with cold ethanol, washed, filtered, and dried under vacuum to obtain modified polylactic acid.

[0038] Example 12: Preparation of a microcapsule: S1: Add 10g of glyceraldehyde-3-phosphate dehydrogenase inhibitor sodium iodoacetate to 80g of 10mmol / L phosphate buffer, stir at 2500rpm for 3min to obtain the aqueous phase; S2: 70g of modified poly-N-isopropylacrylamide (Example 6) and 100g of modified polylactic acid (Example 9) were added to 200mL of ethyl acetate solvent, heated to 20°C, stirred at 400rpm for 2h, cooled to 4°C, added 160g of aqueous phase, increased the stirring speed to 8500rpm, and stirred for 4min to obtain the primary emulsion; S3: Add 70g of polyvinyl alcohol to 780g of deionized water, heat to 90℃, stir at 400rpm for 60min, cool to 30℃, add 0.7g of Tween-80 and 100g of primary emulsion, increase the speed to 450rpm, emulsify for 12min, reduce the speed to 200rpm, and solidify for 10h. After solidification, centrifuge and wash to obtain microcapsules.

[0039] Example 13: Preparation of a microcapsule: S1: Add 10g of glyceraldehyde-3-phosphate dehydrogenase inhibitor sodium iodoacetate to 100g of 10mmol / L phosphate buffer, stir at 3000rpm for 2min to obtain the aqueous phase; S2: Add 80g of modified poly-N-isopropylacrylamide (Example 7) and 100g of modified polylactic acid (Example 10) to 200mL of ethyl acetate solvent, heat to 25°C, stir at 350rpm for 2.5h, cool to 2°C, add 165g of aqueous phase, increase the stirring speed to 9000rpm, stir for 3min to obtain the primary emulsion; S3: Add 80g of polyvinyl alcohol to 790g of deionized water, heat to 95℃, stir at 350rpm for 70min, cool to 25℃, add 0.8g of Tween-80 and 100g of primary emulsion, increase the speed to 500rpm, emulsify for 10min, reduce the speed to 250rpm, and solidify for 9h. After solidification, centrifuge and wash to obtain microcapsules.

[0040] Example 14: Preparation of a microcapsule: S1: Add 10g of glyceraldehyde-3-phosphate dehydrogenase inhibitor sodium iodoacetate to 120g of 10mmol / L phosphate buffer, stir at 3500rpm for 1min to obtain the aqueous phase; S2: 90g of modified poly-N-isopropylacrylamide (Example 8) and 100g of modified polylactic acid (Example 11) were added to 200mL of ethyl acetate solvent, heated to 30°C, stirred at 300rpm for 3h, cooled to 0°C, 170g of aqueous phase was added, the stirring speed was increased to 9500rpm, and stirred for 2min to obtain the primary emulsion; S3: Add 9g of polyvinyl alcohol to 800g of deionized water, heat to 100℃, stir at 300rpm for 90min, cool to 20℃, add 0.9g of Tween-80 and 100g of primary emulsion, increase the speed to 550rpm, emulsify for 8min, reduce the speed to 300rpm, and solidify for 8h. After solidification, centrifuge and wash to obtain microcapsules.

[0041] Example 15: Preparation method of a sample preservation reagent for environmental monitoring of tiger frogs 1g of microcapsules (Example 12) were added to 99g of preservation solution (Example 1), cooled to 2°C, stirred at 140 rpm for 15 min, increased to 180 rpm and stirred for 20 min. Once stirring was complete, the sample preservation reagent was obtained.

[0042] Example 16: A method for preparing a sample preservation reagent for environmental monitoring of tiger frogs. Add 3g of microcapsules (Example 13) to 97g of preservation solution (Example 2), cool to 6°C, stir at 120 rpm for 20 min, increase the speed to 160 rpm and stir for 30 min. Once stirring is complete, the sample preservation reagent is obtained.

[0043] Example 17: Preparation method of a sample preservation reagent for environmental monitoring of tiger frogs Add 5g of microcapsules (Example 14) to 95g of preservation solution (Example 3), cool to 8°C, stir at 100 rpm for 25 min, increase the speed to 120 rpm, stir for 40 min, and the stirring is complete to obtain the sample preservation reagent.

[0044] Example 18: A method for preparing a sample preservation reagent for environmental monitoring of tiger frogs. 1g of microcapsules (Example 12) were added to 99g of preservation solution (Example 4), cooled to 2°C, stirred at 140 rpm for 15 min, increased to 180 rpm and stirred for 20 min. Once stirring was complete, the sample preservation reagent was obtained.

[0045] Example 19: A method for preparing a sample preservation reagent for environmental monitoring of tiger frogs. 1g of microcapsules (Example 12) were added to 99g of preservation solution (Example 5), cooled to 2°C, stirred at 140 rpm for 15 min, increased to 180 rpm and stirred for 20 min. Once stirring was complete, the sample preservation reagent was obtained.

[0046] Comparative Example 1: Compared with Example 15, this comparative example did not add microcapsules in the preparation process of a sample preservation reagent for environmental testing of tiger frogs. Sodium iodoacetate was added directly, and the remaining steps and parameters were the same. This comparative example will not be repeated here. The final sample preservation reagent was obtained.

[0047] Comparative Example 2: This comparative example differs from Example 15 only in that "sodium iodoacetate" is replaced with "proteinase K". All other steps and parameters are the same, and will not be repeated here. The final sample preservation reagent is obtained.

[0048] Comparative Example 3: Compared with Example 15, this comparative example did not add modified poly-N-isopropylacrylamide in the preparation process of a microcapsule. All other steps and parameters were the same, and will not be repeated here. The final sample preservation reagent was obtained.

[0049] Comparative Example 4: Compared with Example 15, in the preparation process of a sample preservation reagent for environmental testing of tiger frog, modified poly-N-isopropylacrylamide, sodium iodoacetate and modified polylactic acid were directly added to the preservation solution. The remaining steps and parameters were the same, and will not be repeated in this comparative example. The final sample preservation reagent was obtained.

[0050] Performance testing: Tiger frog-specific environmental DNA (eDNA) preservation efficiency test (reference) Figure 1 ) The Applied Biosystems QuantStudio 5 real-time quantitative PCR instrument was used. 1. Take 5.0 mL of the sample preservation solution of Examples 15-19 and Comparative Examples 1-4 respectively, add 50 mL of freshly collected water sample from the pond in the habitat of Tiger Frog, and vortex for 10 s. 2. Positive control: Take 3 tubes of 50mL water sample, add 5.0mL of sterile phosphate buffer (pH 7.4) to each tube, and mix well; Negative control: Take 3 tubes of 50mL water samples and do not perform any treatment; 3. Place the sample in a constant temperature incubator at 25℃±1℃ and store for 30 days. Extract total DNA using the DNA extraction kit instructions and test it using a real-time fluorescence quantitative PCR instrument. 4. Formula for calculating eDNA retention rate: ; C0: Average eDNA copy number of the positive control group on day 0; Ct: The average eDNA copy number of the test group at a certain time point (t).

[0051]

[0052] Microbial metabolic activity stabilization effect test (refer to) Figure 2 ) The BioTek Synergy H1 multi-functional microplate reader was used. 1. Take 5.0 mL of the sample preservation solution of Examples 15-19 and Comparative Examples 1-4 respectively, add 50 mL of freshly collected pond water sample from the habitat of Tiger Frog, and vortex for 30 s at a speed of 2500 rpm. Positive control group: Take 3 tubes of 50mL water sample, add 5.0mL of sterile phosphate buffer (pH 7.4) to each tube, and mix well; 2. Place the sample in a constant temperature incubator at 25℃±1℃ and store for 7 days. Then, according to the ATP assay kit instructions, reconstitute the lyophilized enzyme-substrate in the specified buffer solution 30 minutes in advance to prepare the working solution and equilibrate to room temperature. 3. Take 100 μL of sample suspension and add it to a 96-well detection plate. Add 100 μL of ATP detection working solution, shake at 800 rpm for 2 min, add to the microplate reader, let stand for 5 min, and record the measured relative luminescence units (RLU). 4. Formula for calculating the rate of change in metabolic activity (ΔA%): ; Tn: Represents a point in time; 5. Results: A ΔA% value close to 0 indicates that the preservation reagent can effectively "freeze" the metabolic activity of microorganisms at the moment of sample collection, inhibiting their subsequent growth or decay.

[0053] A significantly negative ΔA% indicates that the reagent has a strong metabolic inhibitory effect, which may lead to distortion of activity information.

[0054] A significantly positive ΔA% indicates that the reagent failed to effectively inhibit microbial growth and that metabolic activity changed during storage.

[0055]

[0056] Release function verification test: (refer to) Figure 3 ) The BioTek Synergy H1 multi-functional microplate reader was used. 1. Take 5.0 mL of the sample preservation solution of Examples 15-19 and Comparative Examples 1-4 respectively, add 50 mL of freshly collected pond water sample from the habitat of Tiger Frog, invert 10 times to mix thoroughly, and place in a constant temperature incubator at 25℃ for 72 h. 2. Batch A (trigger group): Place 3 tubes in a 37℃ constant temperature shaker at 100 rpm for 15 min; Batch B (non-trigger control group): Place 3 tubes in a 25℃ constant temperature incubator and let stand for 15 minutes; 3. Take 100 μL of sample supernatant from the branch tubes of batch A and batch B respectively, add it to the well of the microplate, add 100 μL of ATP detection working solution, shake at 800 rpm for 30 s, let stand for 5 min, and use a microplate reader to detect the sample. Record the relative luminescent unit (RLU) value of each well. 4. Calculate the trigger release effect value: ; RLU A The average RLU of three parallel samples in batch A; RLU B The average RLU of three parallel samples in batch B; 5. Result determination: The effect value was significantly less than 1, indicating that incubation at 37°C effectively triggered the accelerated release of the inhibitor from the microcapsule, which significantly enhanced the inhibitory effect on microbial metabolism, thus verifying the temperature-sensitive triggering function. An effect value close to 1 indicates that temperature changes did not cause a significant change in the release behavior, meaning it does not have a temperature-sensitive triggering function.

[0057]

[0058] Stability test of the preservation solution The BioTek Synergy H1 multi-functional microplate reader was used in accordance with the GB / T 16886.5-2017 testing standard. 1. Take the preservation solutions prepared in Examples 1-5 and dispense them into 3 sterile centrifuge tubes, with each tube containing 4 mL; 2. Group A (room temperature group): Store in a constant temperature incubator at 25±1℃, protected from light; Group B (Refrigerated Group): Store in a refrigerator at 4±1℃, away from light, for 90 days; 3. After storage, observe whether the sample shows any color change, turbidity, precipitation or suspension, and use a pH meter to measure the pH value of the storage solution; Biocompatibility testing (refer to) Figure 4 ) 1. Take 0.1 mL of each of the preservation solutions prepared in Examples 1-5, add 0.9 mL of complete culture medium, mix well, filter through a 0.22 μm sterile membrane to obtain the extract; 2. Add 0.25% trypsin to each complete culture medium containing mouse fibroblasts, adjusting the cell density to 5 × 10⁻⁶ cells / cell. 4 The cells / mL were then seeded into 96-well plates at 100 μL per well and incubated at 37°C in a 5% CO2 incubator for 24 h. 3. Discard the old culture medium from the original culture plate. Experimental group: Add 100 μL of extraction solution; Negative control group: Add 100 μL of complete culture medium; Positive control group: Add 100 μL of complete culture medium containing 1% Triton X-100. After the sample collection was completed, the 96-well plate was returned to the incubator and cultured for another 24 hours. At the end of the culture, 10 μL of CCK-8 solution was added, and the plate was cultured for another 2 hours. The absorbance (OD) was then measured using a microplate reader at 450 nm. 450 ); 4. Calculate the relative cell viability (%): ; 5. Evaluation criteria: A relative cell viability of ≥80% indicates that the sample is not cytotoxic. A relative cell viability of 60%-79% suggests mild toxicity. Relative cell viability <60%: The sample is considered to have significant cytotoxicity.

[0059]

[0060] Data Analysis: As can be seen from Tables 1-4, the sample preservation reagent for environmental detection of tiger frog prepared by the present invention has a higher eDNA preservation rate at room temperature, a better metabolic activity stability effect, a faster temperature effect triggering function, and good biocompatibility and stability. In contrast, Comparative Example 1, by directly adding the inhibitor sodium iodoacetate to the preservation solution, resulted in a sharp decline in its eDNA preservation capacity at room temperature and poor metabolic activity stability. This was because the free inhibitor molecules rapidly diffused and reached the effective concentration in the early stages of sample addition, leading to their large consumption in the early stages of preservation. At the same time, the lack of physical barrier protection allowed these active molecules to be directly exposed to the complex environment matrix, making them prone to non-specific binding or redox reactions with organic matter and metal ions in the sample, resulting in rapid inactivation. Furthermore, this simple solution system completely lacked a controlled release mechanism and responsive material basis, and could not regulate the release behavior according to time or external stimuli (such as temperature). Comparative Example 2, by replacing sodium iodoacetate with proteinase K, showed excellent eDNA extraction efficiency but completely destroyed the metabolic activity information of the microorganism. This is because proteinase K, as a potent and non-selective proteolytic enzyme, works by completely cleaving cell structures to release nucleic acids. This violent cleavage process irreversibly destroys the integrity of the cell and all its immediate biochemical reactions, resulting in the complete erasure of the microbial metabolic activity information. In addition, the enzyme activity of proteinase K itself increases with increasing temperature, which makes it exhibit a "pseudo-trigger" effect, but this is not based on the programmed release response designed by the material. Comparative Example 3, lacking the addition of modified poly(N-isopropylacrylamide), resulted in the microcapsules losing their temperature-responsive release function. This is because microcapsules using only modified polylactic acid as the wall material primarily rely on the slow hydrolytic diffusion of the polymer for drug release. This release mode is insensitive to temperature changes and lacks a rapid on / off mechanism. Furthermore, the key role of the modified poly(N-isopropylacrylamide) copolymer lies in its precisely controlled low critical dissolution temperature. This characteristic allows the wall material to undergo a rapid hydrophilic-hydrophobic phase transition under specific temperature conditions, thereby actively opening the release channel. In addition, the absence of this component means the microcapsules only possess basic sustained-release functionality and cannot translate external temperature stimuli into significant changes in release behavior. The consequence is that the microcapsules completely lose their temperature-responsive release capability, although their basic protective functions are partially retained. Comparative Example 4, due to the simple blending of the components, resulted in the worst long-term preservation effect of its eDNA, and neither metabolic stability nor temperature-responsive triggering function was observed. This is because the modified poly(N-isopropylacrylamide), modified polylactic acid, and active inhibitor were directly dispersed in the solution, which destroyed the core-shell structure necessary for achieving protection, controlled release, and responsiveness. At the same time, in this random mixture system, the inhibitor lost the isolation and protection of the microcapsule wall material, resulting in poorer stability. The polymer could not form an interface with mechanical strength and responsiveness and may exist in the form of unhelpful aggregates. In addition, unpredictable mutual interference may occur between the components and between them and the preservation solution matrix, rather than the designed functional synergy.

[0061] In Example 18, mannitol and lysozyme were added, resulting in excellent eDNA preservation rate, stable metabolic activity, and complete temperature response triggering function. This is because mannitol, as a polyol protective agent, can synergistically work with trehalose to further stabilize protein and cell membrane structures and enhance the system's protective ability under repeated freeze-thaw or drying stress. At the same time, the introduction of lysozyme is intended to specifically lyse the cell walls of some Gram-positive bacteria in the sample. This can gently promote the release of nucleic acids from these bacteria without disrupting the overall metabolic state, which may help improve the comprehensiveness of nucleic acid extraction. In addition, these added components are all compatible with the original buffer system and chelating agent and do not change the pH and basic physicochemical properties of the preservation solution. Example 19 used a preservation solution containing sodium citrate and sodium chloride, resulting in excellent eDNA preservation rate, stable metabolic activity, and significant temperature-responsive triggering function. This is because sodium citrate, as another effective metal ion chelating agent, can complement ethylenediaminetetraacetic acid (EDTA) to more comprehensively block the activity of metalloenzymes that may trigger nucleic acid degradation. At the same time, the addition of sodium chloride aims to regulate the ionic strength of the system, making it closer to the physiological environment, which helps maintain the relative stability of the morphology of microbial cells when they first come into contact with the preservation solution and reduces osmotic pressure shock. In addition, the addition of these two salts further consolidates the buffer capacity of the preservation solution and enhances its resistance to pH fluctuations from environmental samples of different sources.

[0062] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

[0063] This invention is intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A sample preservation reagent for environmental testing of tiger frogs, characterized in that, It consists of the following components in parts by weight: 95-99 parts of preservation solution and 1-5 parts of microcapsules; The preservation solution consists of trehalose, citrate-phosphate buffer, disodium EDTA, chitosan hydrochloride, cell membrane stabilizer, and reducing agent. The microcapsules are composed of an olealdehyde-3-phosphate dehydrogenase inhibitor, modified poly(N-isopropylacrylamide), and modified polylactic acid.

2. The sample preservation reagent for environmental testing of tiger frogs according to claim 1, characterized in that, The preparation steps of the preservation solution are as follows: Step A1: Add trehalose, citrate-phosphate buffer, disodium EDTA and chitosan hydrochloride to deionized water, heat to 20-30℃, stir at 300-400 rpm for 30-50 minutes, and the mixture is obtained after stirring. Step A2: Add the cell stabilizer polyethylene glycol 1000 vitamin E succinate and the reducing agent ascorbic acid to the mixture, cool to 15-25℃, stir at 150-250 rpm for 15-25 minutes, add 0.1 mol / L sodium hydroxide solution, adjust the pH to 7.3-7.5, stir until finished, and filter through a 0.22 μm filter membrane to obtain the preservation solution.

3. The sample preservation reagent for environmental testing of tiger frogs according to claim 2, characterized in that, The mass ratio of trehalose, citrate-phosphate buffer, disodium EDTA, chitosan hydrochloride, and deionized water in step A1 is 0.1-0.12:0.01-0.015:0.005-0.006:0.002-0.003:

1. The mass ratio of cell stabilizer, reducing agent and mixture in step A2 is 0.001-0.0015:0.0005-0.0008:

1.

4. The sample preservation reagent for environmental testing of tiger frogs according to claim 1, characterized in that, The modified poly-N-isopropylacrylamide is prepared as follows: Under a nitrogen atmosphere, N-isopropylacrylamide and acrylic acid were added to deionized water, heated to 20-30°C, stirred at 200-300 rpm for 20-30 min, heated to 60-70°C, and ammonium persulfate initiator was added. The stirring speed was increased to 250-350 rpm, and the reaction was carried out for 4-6 h. After the reaction was completed, the temperature was lowered to 20-30°C, and the mixture was transferred to a 4000-7000 Da dialysis bag and dialyzed for 48-72 h. The mixture was then freeze-dried to obtain modified poly-N-isopropylacrylamide.

5. The sample preservation reagent for environmental testing of tiger frogs according to claim 4, characterized in that, The mass ratio of N-isopropylacrylamide, acrylic acid, and initiator is 1:0.04-0.08:0.005-0.

015.

6. The sample preservation reagent for environmental testing of tiger frogs according to claim 1, characterized in that, The preparation steps of the modified polylactic acid are as follows: Under a nitrogen atmosphere, lactide, glycolide, and a 0.01 mol / L stannous octoate toluene solution were added to 1-dodecaneol. The mixture was heated to 125-135℃, stirred at 200-300 rpm for 18-24 hours until the reaction was complete. The mixture was then cooled to 20-30℃, dissolved in anhydrous dichloromethane, precipitated with cold ethanol, washed, filtered, and dried under vacuum to obtain modified polylactic acid.

7. The sample preservation reagent for environmental testing of tiger frogs according to claim 6, characterized in that, The mass ratio of lactide, glycolide, catalyst and 1-dodecaneol is 1:0.57-0.59:0.02-0.05:0.001-0.

003.

8. The sample preservation reagent for environmental testing of tiger frogs according to claim 1, characterized in that, The preparation steps of the microcapsules are as follows: Step B1: Add sodium iodoacetate, the glyceraldehyde-3-phosphate dehydrogenase inhibitor, to 10 mmol / L phosphate buffer and stir for 1-3 min at 2500-3500 rpm to obtain the aqueous phase; Step B2: Add modified poly-N-isopropylacrylamide and modified polylactic acid to ethyl acetate solvent, heat to 20-30℃, stir at 300-400 rpm for 2-3 hours, cool to 0-4℃, add aqueous phase, increase the stirring speed to 8500-9500 rpm, stir for 2-4 minutes to obtain primary emulsion; Step B3: Add polyvinyl alcohol to deionized water, heat to 90-100℃, stir at 300-400 rpm for 60-90 min, cool to 20-30℃, add Tween-80 and the primary emulsion, increase the speed to 450-550 rpm, emulsify for 8-12 min, decrease the speed to 200-300 rpm, and solidify for 8-10 h. After solidification, centrifuge and wash to obtain microcapsules.

9. The sample preservation reagent for environmental testing of tiger frogs according to claim 8, characterized in that, The mass ratio of sodium iodoacetate to phosphate buffer in step B1 is 1:8-12; The mass ratio of the modified poly(N-isopropylacrylamide), modified polylactic acid, and aqueous phase in step B2 is 0.7-0.9:1:1.6-1.7; The mass ratio of polyvinyl alcohol, deionized water, Tween-80 and the primary emulsion in step B3 is 0.07-0.09:7.8-8.0:0.007-0.009:

1.

10. A method for preparing a sample preservation reagent for environmental testing of tiger frogs according to any one of claims 1-9, characterized in that, The preparation method is as follows: Add the microcapsules to the preservation solution, cool to 2-8℃, stir at 100-140 rpm for 15-25 minutes, increase the speed to 120-180 rpm, stir for 20-40 minutes, and the sample preservation reagent is obtained after stirring is completed.

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