A nucleic acid preservative for giant panda fecal samples and a preparation method thereof

Abstract

The application provides a nucleic acid preservative for giant panda fecal samples and a preparation method thereof. The preservative is composed of the following components in the final concentration: buffer, 1-200 mM; metal ion chelator, 0.01-1 M; ion strength maintaining agent, 0.01-2 M; denaturant, 0.01-10 M; antioxidant, 1%-5% (V / V); antifreeze, 5%-30% (V / V); nucleic acid protective agent, 0.1%-5% (W / V); bacteriostatic agent, 0.5-500 ppm; and all the components are dissolved in deionized water. The preservative has the characteristics of convenient field operation and safe transportation. The DNA structure of the giant panda can be effectively prevented from being damaged by various fecal inhibitors by only soaking at room temperature, the preservative is suitable for the preservation of fresh or old fecal samples, and the DNA in the giant panda fecal samples can be stably preserved for a long time (6 months).

Description

Technical Field

[0001] This invention relates to the field of fecal preservation solution technology, specifically to a nucleic acid preservation solution for giant panda fecal samples and its preparation method. Background Technology

[0002] The primary condition for achieving substantial progress in genetic research on endangered wildlife conservation worldwide is obtaining sufficient quantity and quality of genetic resources. For endangered wild animals that are scarce and elusive, feces are an excellent source of their DNA. However, feces contain a large number of fecal inhibitors, including digestive metabolites, microorganisms, various DNA-degrading enzymes, and PCR inhibitors, making intestinal exfoliated cells and DNA in fecal samples highly susceptible to damage. Furthermore, as the time feces remain outside the body increases, external environmental factors such as microorganisms, high temperatures, precipitation, and ultraviolet radiation accelerate intestinal cell apoptosis and promote animal DNA degradation. Therefore, feces need to be fixed as quickly as possible after collection to maintain the quality of the wild animal's DNA at the time of collection. Early researchers conducted screening studies on optimal preservation methods using various tissue sample preservation techniques, such as freezing, drying, and immersion in preservation media, attempting to find the best fecal preservation method for specific wild animals. However, these tissue sample preservation methods do not consistently maintain superior preservation performance for fecal samples. Even the same research team may reach two different conclusions when recommending the optimal fecal preservation method for a specific species due to various variables. Therefore, some scholars advocate using various tissue sample preservation media and shortening the preservation time in the media to delay the loss of DNA in wild animal feces until a suitable method for preserving fecal samples is found.

[0003] Currently, in the field of giant panda research, scholars recommend drying, anhydrous ethanol immersion, or Chinese patent CN107177590A for preserving feces. The goal is to dehydrate exfoliated intestinal cells and prevent cell degradation. However, these preservation methods have significant drawbacks. First, these methods require a sufficient number of exfoliated intestinal cells, thus demanding a certain level of freshness from the fecal sample. However, in the wild, opportunities to collect fresh feces are limited, while readily available old feces contain fewer exfoliated intestinal cells and more degraded DNA fragments. Therefore, it is difficult to fully utilize old feces using these methods. Second, these preservation methods lack effective components for protecting nucleic acids. Feces are filled with various inhibitors, including microorganisms, DNA-degrading enzymes, and digestive metabolites, which can either bind to DNA to form complexes or directly degrade DNA. Simultaneously, feces containing bamboo residues contain large amounts of polyphenols and their secondary metabolites, which are easily oxidized after the feces are removed from the body, forming quinones and further damaging DNA structure. Simple drying or treatment with anhydrous ethanol cannot completely stop the effects of the aforementioned inhibitors on cells that have not been dehydrated in time and on DNA that has already been ruptured and released. Therefore, panda DNA is prone to continuous damage during preservation. Thirdly, the reagent properties or operational characteristics of these preservation methods are not easily adapted to various field application scenarios. For drying methods, the time required to dry the sample is long; various drying equipment is difficult to use in the field; silica gel desiccants have a limited sample capacity and need to be replaced regularly. Moreover, samples need to be left for a period of time to dry fully, and detached cells may suffer further loss during this period. For anhydrous ethanol immersion methods, although samples can be immediately immersed and used in the field, anhydrous ethanol is flammable, explosive, and volatile, generally requiring special packaging, and transportation is often subject to various restrictions. Chinese patent CN107177590A uses a method of drying first and then immersing in anhydrous ethanol, thus combining some of the disadvantages of the first two methods.

[0004] Therefore, there is an urgent need to develop a specialized nucleic acid preservation solution for giant panda fecal samples. This solution can conveniently and quickly fix fresh or old feces under various outdoor environmental temperatures, and can also provide continuous protection for DNA by targeting the characteristics of giant panda fecal inhibitors, thus achieving long-term stable preservation of giant panda DNA. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing preservation technologies and provide a nucleic acid preservation solution for giant panda fecal samples and its preparation method. This preservation solution is convenient for field operation and safe for transportation. It can effectively prevent various fecal inhibitors from damaging the DNA structure of giant pandas by simply soaking at room temperature. It is suitable for the preservation of fresh or old fecal samples.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows.

[0007] In a first aspect, the present invention provides a nucleic acid preservation solution for giant panda fecal samples, comprising, according to the final concentration: buffer solution, 1-200 mM; metal ion chelating agent, 0.01-1 M; ionic strength maintaining agent, 0.01-2 M; denaturing agent, 0.01-10 M; antioxidant, 1%-5% (V / V); antifreeze agent, 5%-30% (V / V); nucleic acid protectant, 0.1%-5% (W / V); antibacterial agent, 0.5-500 ppm; all the above components are dissolved in deionized water.

[0008] Preferably, the nucleic acid preservation solution comprises, according to the final concentration: buffer solution, 10-100 mM; metal ion chelating agent, 0.01-0.1 M; ionic strength maintaining agent, 0.01-1 M; denaturing agent, 0.01 M-5.5 M; antioxidant, 1%-3% (V / V); antifreeze, 5%-10% (V / V); nucleic acid protectant, 0.3%-3% (W / V); antibacterial agent, 1-300 ppm; all the above components are dissolved in deionized water.

[0009] Further, the buffer solution is tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) or phosphate buffer solution (PBS).

[0010] Further, the metal ion chelating agent is at least one selected from polyaluminum chloride, polyferric chloride, sodium aluminate, sodium aluminate, ethylenediaminetetraacetic acid, diethyltriaminepentaacetic acid, 1,2-cyclohexanediaminetetraacetic acid, sodium ethylenediaminedi-o-phenylacetate, and hydroxyethylethylenediaminetriacetic acid.

[0011] Furthermore, the ionic strength maintaining agent is sodium chloride or potassium chloride.

[0012] Further, the denaturing agent is at least one of hexadecyltrimethylammonium bromide, sodium dodecyl sulfate, dodecyl dimethyl betaine, ethylphenyl polyethylene glycol, polyethylene glycol octylphenyl ether, ammonium sulfate, lithium chloride, guanidine hydrochloride, guanidine isothiocyanate, and urea.

[0013] Furthermore, the antioxidant is at least one of tris(2-carbonylethyl)phosphohydrochloride, β-mercaptoethanol, dithiothreitol, and dithioerythritol.

[0014] Furthermore, the antifreeze agent is glycerin.

[0015] Furthermore, the nucleic acid protectant is at least one of polyaluminum chloride, polyferric chloride, activated carbon, diatomaceous earth, polyvinylpyrrolidone (PVP), polyvinylpyrrolidone (PVPP), polyethylene glycol PEG200-8000, type 4A molecular sieve, type 5A molecular sieve, sodium aluminate, and sodium metaaluminate.

[0016] Furthermore, the antibacterial agent is at least one of the Proclin series, KroVin series, and BND-10.

[0017] Furthermore, the preservation solution also includes a pH adjuster for adjusting the pH range of the preservation solution to 7.0-10.0.

[0018] Furthermore, the pH adjuster is sodium hydroxide or potassium hydroxide.

[0019] Secondly, the present invention also provides a method for preparing the above-mentioned nucleic acid preservation solution for giant panda fecal samples, comprising the following steps:

[0020] (1) According to the final concentration composition of the preservation solution, first mix the buffer solution, metal ion chelating agent, ionic strength maintaining agent, denaturant, antioxidant, antifreeze, antibacterial agent and deionized water evenly and dissolve them.

[0021] (2) After adjusting the pH value, filter out insoluble impurities;

[0022] (3) Add nucleic acid protectant to obtain the product.

[0023] Furthermore, in step (2), sodium hydroxide or potassium hydroxide is used to adjust the pH to 7.0-10.0.

[0024] Thirdly, the present invention provides a method for preserving both old and new feces of giant pandas, which uses the aforementioned preservation solution and includes the following steps:

[0025] Step 1, Sample Collection: Collect fresh (ex-vivo time ≤ 15 days) or old (ex-vivo time > 15 days) whole feces of giant pandas and place them in a sample bottle;

[0026] Step 2: Add preservation solution to the sample bottle at a mass-to-volume ratio of 1g:3mL for the collected feces, ensuring that the preservation solution completely submerges the feces;

[0027] Step 3: Store the sample vials away from light.

[0028] Fourthly, the present invention provides the application of the above-mentioned preservation solution in the extraction of DNA from giant panda feces.

[0029] Furthermore, the method of application is as follows: panda feces are preserved using the preservation solution, and then DNA is extracted from the panda feces.

[0030] Furthermore, the process of extracting DNA from giant panda feces includes:

[0031] 1) Collect the filtrate from giant panda fecal samples that have been filtered and preserved using two layers of sterile gauze;

[0032] 2) Add an equal volume of isopropanol or twice the volume of anhydrous ethanol to the filtrate and let it stand for 30-40 minutes;

[0033] 3) Centrifuge at 10000~12000g for 10~15min, and retain the precipitate;

[0034] 4) DNA extraction was performed using a DNA extraction kit.

[0035] The beneficial effects of this invention are as follows:

[0036] (i) The nucleic acid preservation solution of the present invention is formulated with buffer, metal ion chelating agent, ionic strength maintainer, denaturant, antioxidant, antifreeze, nucleic acid protectant and antibacterial agent in appropriate proportions. It can lyse intestinal exfoliated cells and release mitochondrial and nucleal DNA, effectively inhibit the destructive effects of metabolic inhibitors and microorganisms in giant panda feces on DNA structure, protect the integrity of DNA structure, and keep giant panda DNA in a stable state for a long time.

[0037] (ii) The nucleic acid preservation solution of the present invention is applicable to both fresh and old giant panda fecal samples. It can stably preserve the DNA in giant panda fecal samples for a long time (6 months) simply by soaking at room temperature, and it is convenient for field collection operations.

[0038] (iii) The nucleic acid preservation solution of the present invention is simple to prepare, safe to transport, and can provide continuous protection for giant panda DNA, and is expected to be further extended to the preservation of DNA from other animal samples. Attached Figure Description

[0039] Figure 1 This is the electrophoretic detection pattern of the PCR amplification product in Example 4 of the present invention.

[0040] Figure 2 This is the electrophoretic detection pattern of the gpz-47 amplification product in Example 5 of the present invention.

[0041] Figure 3 This is the electrophoretic detection pattern of the gpz-20 amplification product in Example 5 of the present invention.

[0042] Figure 4 This is the electrophoretic detection pattern of the gpz-51 amplification product in Example 5 of the present invention.

[0043] Figure 5 This is the electrophoretic detection pattern of the gpz-29 amplification product in Example 5 of the present invention.

[0044] Figure 6 This is the electrophoretic detection pattern of the target fragment corresponding to the primers for mitochondrial DNA detection in 35 sample replicates in Example 6 of the present invention.

[0045] Figure 7This is the electrophoretic detection pattern of the gpz-47 amplification product in Example 6 of the present invention.

[0046] Figure 8 This is the electrophoretic detection pattern of the gpz-20 amplification product in Example 6 of the present invention.

[0047] Figure 9 This is the electrophoretic detection pattern of the gpz-51 amplification product in Example 6 of the present invention.

[0048] Figure 10 This is the electrophoretic detection pattern of the gpz-29 amplification product in Example 6 of the present invention.

[0049] Figure 11 This is the electrophoretic detection pattern of the nucleic acid extraction product in Example 7 of the present invention.

[0050] Figure 12 This is the electrophoretic detection pattern of the gpz-51 amplification product in Example 19 of the present invention.

[0051] Figure 13 This is the electrophoretic detection pattern of the gpl-60 amplification product in Example 19 of the present invention. Detailed Implementation

[0052] The DNA extraction kit used in this embodiment of the invention is the QIAamp DNA Stool Mini Kit.

[0053] In the description of this invention, it should be noted that unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0054] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0055] Example 1

[0056] This embodiment provides a nucleic acid preservation solution for giant panda fecal samples, comprising the following components at the final concentration: buffer solution, 20 mM; metal ion chelating agent, 0.06 M; ionic strength maintainer, 1 M; denaturant, 0.1 M; antioxidant, 3% (V / V); antifreeze, 5% (V / V); nucleic acid protectant, 3% (W / V); and antibacterial agent, 20 ppm. All components are dissolved in deionized water, and the pH of the preservation solution is adjusted to 8.50 using a pH adjuster.

[0057] The buffer solution was Tris-HCl, the metal ion chelating agent was 0.05M ethylenediaminetetraacetic acid + 0.01M polyaluminum chloride, the ionic strength maintainer was sodium chloride, the denaturing agent was guanidine hydrochloride (0.05M) + polyethylene glycol octylphenyl ether (0.05M), the antioxidant was tris(2-carbonylethyl)phosphohydrochloride, the antifreeze was glycerol, the nucleic acid protectant was polyethylene glycol PEG6000 0.5% (w / v) + PVP 2% (w / v), the antibacterial agent was Proclin 300, and the pH adjuster was sodium hydroxide.

[0058] The preparation method of the above-mentioned nucleic acid preservation solution includes the following steps:

[0059] (1) According to the final concentration composition of the preservation solution, first mix the buffer solution, metal ion chelating agent, ionic strength maintaining agent, denaturant, antioxidant, antifreeze, antibacterial agent and deionized water evenly and dissolve them.

[0060] (2) After adjusting the pH value, filter out insoluble impurities;

[0061] (3) Add nucleic acid protectant to obtain the product.

[0062] Example 2

[0063] This embodiment provides a nucleic acid preservation solution for giant panda fecal samples, which differs from Embodiment 1 in that the nucleic acid protectant is 0.5% (w / v) polyaluminum chloride + 0.5% (w / v) activated carbon + 1% (w / v) PVPP.

[0064] Example 3

[0065] This embodiment provides a nucleic acid preservation solution for giant panda fecal samples, which differs from Embodiment 1 in that the nucleic acid protectant is 0.5% (w / v) polyferric chloride + 0.5% (w / v) diatomaceous earth + 1% (w / v) PVP.

[0066] Example 4

[0067] This embodiment provides a nucleic acid preservation solution for giant panda fecal samples, which differs from Embodiment 1 in that the nucleic acid protectant is 1% (w / v) polyaluminum chloride + 0.5% (w / v) diatomaceous earth + 0.5% (w / v) polyethylene glycol PEG8000.

[0068] Example 5

[0069] This embodiment provides a nucleic acid preservation solution for giant panda fecal samples, which differs from Example 1 in that the nucleic acid protectant is polyethylene glycol PEG6000 1% (W / V) + PVP 1.5% (W / V).

[0070] Example 6

[0071] This embodiment provides a nucleic acid preservation solution for giant panda fecal samples, which differs from Example 1 in that the nucleic acid protectant is polyethylene glycol PEG6000 0.5% (W / V) + PVP 1% (W / V).

[0072] Example 7

[0073] This embodiment provides a nucleic acid preservation solution for giant panda fecal samples, which differs from Example 1 in that the nucleic acid protectant is polyethylene glycol PEG6000 0.5% (W / V) + PVPP 1% (W / V).

[0074] Example 8

[0075] This embodiment provides a nucleic acid preservation solution for giant panda fecal samples, which differs from Embodiment 1 in that the nucleic acid protectant is 0.5% (w / v) of type 4A molecular sieve + 1% (w / v) of PVPP.

[0076] Example 9

[0077] This embodiment provides a nucleic acid preservation solution for giant panda fecal samples, which differs from Embodiment 1 in that the nucleic acid protectant is 0.5% (w / v) of 5A molecular sieve + 1% (w / v) of PVPP.

[0078] Example 10

[0079] This embodiment provides a nucleic acid preservation solution for giant panda fecal samples, which differs from Example 1 in that the nucleic acid protectant is 0.1% sodium aluminate (W / V) + 0.5% polyethylene glycol PEG6000 (W / V).

[0080] Example 11

[0081] This embodiment provides a nucleic acid preservation solution for giant panda fecal samples, which differs from Example 1 in that the nucleic acid protectant is 0.1% (w / v) sodium aluminate + 0.5% (w / v) polyethylene glycol PEG8000.

[0082] Example 12

[0083] This embodiment provides a nucleic acid preservation solution for giant panda fecal samples, comprising the following components at the final concentration: buffer solution, 200 mM; metal ion chelating agent, 0.2 M; ionic strength maintainer, 2 M; denaturant, 6 M; antioxidant, 5% (V / V); antifreeze, 15% (V / V); nucleic acid protectant, 0.2% (W / V); and antibacterial agent, 0.5 ppm. All components are dissolved in deionized water, and the pH of the preservation solution is adjusted to 7.50 using a pH adjuster.

[0084] The buffer solution was Tris-HCl, the metal ion chelating agent was ethylenediaminetetraacetic acid (0.15M) + polyferric chloride (0.05M), the ionic strength maintainer was potassium chloride, the denaturing agent was guanidine isothiocyanate (3.95M) + urea (2M) + polyethylene glycol octylphenyl ether (0.05M), the antioxidant was dithiothreitol, the antifreeze was glycerol, the nucleic acid protectant was polyaluminum chloride 0.01% (w / v) + activated carbon 0.09% (w / v) + PVP 0.1% (w / v), the antibacterial agent was KroVin500, and the pH adjuster was sodium hydroxide.

[0085] The preparation method of the above-mentioned fecal sample nucleic acid preservation solution is the same as that in Example 1.

[0086] Example 13

[0087] This embodiment provides a nucleic acid preservation solution for giant panda fecal samples, which differs from Example 12 in that: the buffer is PBS, the metal ion chelating agent is hydroxyethyl ethylenediamine triacetic acid (0.15M) + polyferric chloride (0.05M), the ionic strength maintainer is potassium chloride, the denaturing agent is hexadecyltrimethylammonium bromide (3.9M) + polyethylene glycol octylphenyl ether (0.1M) + sodium dodecyl sulfate (0.3M), the antioxidant is β-mercaptoethanol, the antifreeze is glycerol, the nucleic acid protectant is polyethylene glycol PEG6000 0.1% (w / v) + activated carbon 0.1% (w / v), and the antibacterial agent is KroVin500.

[0088] The preparation method of the above-mentioned fecal sample nucleic acid preservation solution is the same as that in Example 1.

[0089] Example 14

[0090] This embodiment provides a nucleic acid preservation solution for giant panda fecal samples, which differs from Example 12 in that: the buffer solution is PBS, the metal ion chelating agent is sodium aluminate (0.15M) + polyaluminum chloride (0.05M), the ionic strength maintainer is potassium chloride, the denaturing agent is dodecyl dimethyl betaine (1M) + lithium chloride (1M) + ethyl phenyl polyethylene glycol (1M) + guanidine hydrochloride (3M), the antioxidant is dithioerythritol, the antifreeze agent is glycerol, the nucleic acid protectant is polyferric chloride, and the antibacterial agent is BND-10.

[0091] The preparation method of the above-mentioned fecal sample nucleic acid preservation solution is the same as that in Example 1.

[0092] Example 15

[0093] Preservation period of giant panda mitochondrial DNA at room temperature:

[0094] Experimental materials: whole feces of 3 giant pandas, anhydrous ethanol, and fecal sample nucleic acid preservation solution of Example 1 of this invention;

[0095] Experimental procedure: (1) Homogenize fecal samples from three giant pandas to ensure uniform distribution of exfoliated intestinal cells. (2) Divide the samples from each individual into 10 tubes at 5g / tube. Label 5 tubes as the preservation solution group and the other 5 tubes as the control group (preserved with anhydrous ethanol). (3) Add fecal sample nucleic acid preservation solution and anhydrous ethanol to the fecal samples according to their labels to soak them. Refer to the description in "4.4 Sample Collection Method" of the "Technical Specification for Establishing Genetic Archives of Giant Panda Population" (LY / T2896-2017) for the volume of solution added. (4) When the samples in (3) reached the soaking time points of 0 days, 2 weeks, 4 weeks, 12 weeks and 24 weeks at room temperature (15-25℃), the anhydrous ethanol preservation group collected the precipitate according to the method of Zhang Baowei et al. [Zhang Baowei, Wei Fuwen, Li Ming, et al. Simple method for DNA extraction from feces of giant panda and red panda [J]. Acta Zoologica Sinica, 2004, 50(3): 452-458.], and the preservation solution preservation group collected the precipitate according to the method provided in this invention. All precipitates were subjected to DNA extraction according to the instructions of the QIAamp DNA Stool Mini Kit. The elution volume was 100µL. (5) According to the requirements of "7.1 DNA quality and mitochondrial marker PCR amplification" and "8.2 PCR amplification system and procedure" in the "Technical Specification for Establishing Genetic Archives of Giant Panda Population" (LY / T2896-2017), the extracted products were PCR amplified using the mitochondrial marker primers specified in the technical specification. The primer sequences were P-tp (5'-CTCCCTAAGACTCAAGGAAG-3') and BEDH (5'-GGGTGATCTATAGTGTTATGTCC-3'), with an amplified sequence length of 750 bp. The template volume in the 20 µL reaction system was 6 µL. If no target band was amplified, it indicated that the solution had poor preservation ability for mitochondrial DNA. (8) Electrophoretic detection of PCR amplification products: A 1.5% agarose gel was prepared for electrophoresis detection.

[0096] Experimental results: such as Figure 1 As shown, the target band was amplified in all three individual replicates of the preservation solution group, and the DNA quality of all individual replicates met the requirements of the "Technical Specifications for Establishing Genetic Archives of Giant Panda Populations" (LY / T2896-2017). In the anhydrous ethanol preservation group, only two individuals amplified the target band, but the DNA quality of individual replicate 1 did not meet the requirements. Among all individual replicates, the target band obtained in the anhydrous ethanol preservation group was significantly weaker than that obtained in the preservation solution group. These results indicate that for 5g fecal samples, immersion in the preservation solution is more effective in maintaining the stability of mitochondrial DNA quality over the preservation period (24 weeks).

[0097] Example 16

[0098] Preservation period of panda cell nuclear genomic DNA at room temperature:

[0099] Experimental materials: whole feces of 3 giant pandas, anhydrous ethanol, and fecal sample nucleic acid preservation solution of Example 1 of this invention;

[0100] Experimental procedure: (1) Homogenize fecal samples from three giant pandas to ensure uniform distribution of exfoliated intestinal cells. (2) Divide the samples from each individual into 10 tubes at 5g / tube. Label 5 tubes as the preservation solution group and the other 5 tubes as the control group (anhydrous ethanol preservation group). (3) Add fecal preservation solution and anhydrous ethanol to the feces according to their labels to soak them. Refer to the description in "4.4 Sample Collection Method" of the "Technical Specification for Establishing Genetic Archives of Giant Panda Population" (LY / T2896-2017) for the volume of solution added. (4) When the samples in (3) reached the soaking time points of 0 days, 2 weeks, 4 weeks, 12 weeks and 24 weeks at room temperature (15-25℃), the precipitate of the anhydrous ethanol preservation group was collected according to the method of Zhang Baowei et al. [Zhang Baowei, Wei Fuwen, Li Ming, et al. A simple method for extracting DNA from feces of giant panda and red panda [J]. Acta Zoologica Sinica, 2004, 50(3): 452-458.], and the precipitate of the preservation solution group was collected according to the method provided in this invention. All precipitates were subjected to DNA extraction according to the instructions of the QIAamp DNA Stool Mini Kit. The elution volume was 100µL. (5) In accordance with the requirements of "7.2 Individual Identification" and "8.2 PCR Amplification System and Procedure" in the "Technical Specification for Establishing Genetic Archives of Giant Panda Populations" (LY / T2896-2017), the extracted products were amplified by PCR using primers 2-5 (gpz-47, gpz-20, gpz-51 and GPL-29) in Appendix B of the technical specification. The template added to the 20µL reaction system was 6µL. If the amplification was unsuccessful, it indicates that the solution had poor preservation ability for genomic DNA in the cell nucleus. (6) Electrophoretic detection of PCR amplification products: 1.5% agarose gel was prepared for electrophoresis detection.

[0101] Experimental results: such as Figure 2-5As shown, in the preservation solution group, all three individual replicates showed target bands corresponding to the four STR primer pairs at all soaking time points, with a genotyping success rate of 100%. In contrast, the anhydrous ethanol preservation group only detected target fragments of the three STR primer pairs gpz-47, gpz-51, and GPL-29, and individual repeatability was poor. Among the individual replicates that showed target bands in both soaking preservation media, the target band brightness in the preservation solution group was significantly higher than that in the anhydrous ethanol group. In the preservation solution group, there was no significant difference in the brightness of all target bands within the preservation period from 0 days to 24 weeks. These results indicate that for a 5g fecal sample, the preservation solution group significantly outperformed anhydrous ethanol in preserving the diversity of giant panda DNA fragments, maintaining the stability of nuclear genomic DNA quality within the preservation period (24 weeks).

[0102] Example 17

[0103] Performance study of fecal preservation solution:

[0104] Experimental materials: whole feces from 35 giant pandas, anhydrous ethanol, and fecal sample nucleic acid preservation solution from Example 1 of this invention.

[0105] Experimental protocol: (1) Collect whole fecal samples from 35 giant pandas, homogenize them, and divide them into two tubes of 5g each. (2) Randomly select one tube and label it as the preservation solution group, and the other tube as the control group (anhydrous ethanol preservation group). (3) Add fecal preservation solution and anhydrous ethanol to soak the feces according to their respective labels. Refer to the description of “4.4 Sample collection method” in the “Technical Specification for Establishing Genetic Archives of Giant Panda Population” (LY / T2896-2017) for the volume of solution added. (4) Soak the feces for 24 hours. Collect the precipitate in the anhydrous ethanol preservation group according to the method of Zhang Baowei et al. [Zhang Baowei, Wei Fuwen, Li Ming, et al. Simple method for DNA extraction from feces of giant panda and red panda [J]. Acta Zoologica Sinica, 2004, 50(3): 452-458.] Collect the precipitate in the preservation solution group according to the method provided in this invention. All precipitates were subjected to DNA extraction according to the instructions of the QIAamp DNA Stool Mini Kit. The elution volume was 100 µL. (5) According to the requirements of "7.1 DNA quality and mitochondrial marker PCR amplification" and "8.2 PCR amplification system and procedure" in the "Technical Specification for Establishing Genetic Archives of Giant Panda Population" (LY / T2896-2017), the extracted products were amplified by PCR using the mitochondrial marker primers specified in the technical specification. The primer sequences were P-tp (5'-CTCCCTAAGACTCAAGGAAG-3') and BEDH (5'-GGGTGATCTATAGTGTTATGTCC-3'), and the amplified sequence length was 750 bp. The template added to the 20 µL reaction system was 6 µL. If no target band was amplified, it indicates that the solution had poor preservation ability for mitochondrial DNA. (6) In accordance with the requirements of "7.2 Individual Identification" and "8.2 PCR Amplification System and Procedure" in the "Technical Specification for Establishing Genetic Archives of Giant Panda Populations" (LY / T2896-2017), the extracted products were amplified by PCR using primers 2-5 (gpz-47, gpz-20, gpz-51 and GPL-29) in Appendix B of the technical specification. The template added to the 20µL reaction system was 6µL. If the amplification was unsuccessful, it indicates that the solution had poor preservation ability for genomic DNA in the cell nucleus. (7) Electrophoretic detection of PCR amplification products: 1.5% agarose gel was prepared for electrophoresis detection.

[0106] Experimental results: such as Figure 6 As shown, the detection rate of the target fragment corresponding to the primers for mitochondrial DNA detection in the preservation solution group was significantly higher than that in the control group preserved in anhydrous ethanol in 35 sample replicates. Figure 7-10As shown, the detection rate of the target fragments corresponding to the primers for nuclear genomic DNA detection in the preservation solution group was significantly higher than that in the control group preserved with anhydrous ethanol in 35 sample replicates. These results indicate that for 5g fecal samples, the preservation solution has a relatively higher preservation efficiency for mitochondrial DNA and nuclear genomic DNA, and its overall performance is superior to anhydrous ethanol.

[0107] Example 18

[0108] Study on protective efficacy:

[0109] Experimental materials: whole feces of 2 giant pandas, fecal sample nucleic acid preservation solution with nucleic acid protectant removed (others are the same as in Example 1), and fecal sample nucleic acid preservation solution of Example 1 of this invention.

[0110] Experimental protocol: (1) Collect whole fecal samples from two giant pandas, homogenize them, and dispense them into two tubes at 40g / tube. (2) Randomly select one tube and label it as group A (without nucleic acid protectant) and the other tube as group B (with nucleic acid protectant). (3) Add fecal preservation solution containing or without nucleic acid protectant according to the label. (4) After the feces are fully soaked, collect the precipitate according to the method provided in this invention. All precipitates are subjected to DNA extraction according to the instructions of the QIAamp DNA Stool Mini Kit. The elution volume is 200µL. (5) Electrophoretic detection of nucleic acid extraction products: Prepare 1.5% agarose gel for electrophoretic detection.

[0111] Experimental results: such as Figure 11 As shown, the band brightness of the extracted genomic DNA products was consistent in both sample replicates, with the band brightness of fecal preservation solution B being significantly higher than that of fecal preservation solution A. These results indicate that the nucleic acid preservative component can significantly enhance the DNA preservation ability of the fecal preservation solution.

[0112] Example 19

[0113] Performance comparison study of Examples 1-14:

[0114] Experimental materials: whole feces of one giant panda, and fecal sample nucleic acid preservation solution from Examples 1 to 14 of this invention;

[0115] Experimental procedure: (1) Homogenize the fecal sample from one giant panda to ensure that the exfoliated intestinal cells are evenly distributed in the fecal sample. (2) Dispense 14 tubes of the above-mentioned individual samples at a rate of 5g / tube. (3) Soak the fecal samples in the nucleic acid preservation solution described in Examples 1-14. The volume of solution added is as described in "4.4 Sample Collection Method" of the Technical Specification for Establishing Genetic Archives of Giant Panda Population (LY / T2896-2017). (4) After soaking the samples in (3) at room temperature (15-25℃) for 7 days, collect the precipitate according to the method provided in this invention, and perform DNA extraction on all precipitates according to the instructions of the QIAamp DNA Stool MiniKit. The elution volume is 100µL. (5) In accordance with the requirements of "7.2 Individual Identification" and "8.2 PCR Amplification System and Procedure" in the "Technical Specification for Establishing Genetic Archives of Giant Panda Populations" (LY / T2896-2017), the extracted products were amplified by PCR using primers gpz-51 and gpl-60 in Appendix B of the technical specification. The amount of template added to the 20µL reaction system was 6µL, and the amount of positive control DNA added was 5ng. If the target band is relatively bright, it indicates that the fecal sample nucleic acid preservation solution of this embodiment has relatively strong nucleic acid preservation performance. (6) Electrophoretic detection of PCR amplification products: 1.5% agarose gel was prepared for electrophoretic detection.

[0116] Experimental results: such as Figure 12-13 As shown, all examples detected the target bands corresponding to the two pairs of STR primers (gpz-51 and gpl-60), with the target band in Example 1 being slightly brighter than that in the other examples. These results indicate that the fecal sample nucleic acid preservation solutions in Examples 1-14 all possess good nucleic acid preservation performance.

[0117] Comparative Example 1

[0118] Drying method: According to the scheme of Wan et al. [Wan QH, Zhu L, Wu H, et al. Majorhistocompatibility complex class II variation in the giant panda (Ailuropodamelanoleuca) [J]. Mol Ecol, 2006, 15(9):2441-2450.], giant panda feces (defecation time <24h) need to be dried overnight at 65°C in a laboratory environment, then transferred to paper bags, and finally sealed in a self-sealing bag containing silica gel desiccant.

[0119] Disadvantages: Since fecal collection does not occur in a laboratory, samples need to be left to stand for a period of time before being brought to the lab for drying. However, this standing period can easily lead to further loss of exfoliated intestinal cells. Furthermore, wild animals generally live in remote, inaccessible natural environments; shortening sample standing time would inevitably increase the transportation burden on collection personnel. In summary, the disadvantages of this technical solution are: A) It requires a high degree of fecal freshness; B) Thorough drying requires time, and fecal samples cannot be immediately fixed at the time of collection, as microorganisms may continue to act, leading to DNA loss; C) The drying process requires overnight standing, resulting in a long operation time; D) The silica gel desiccant needs to be replaced regularly.

[0120] Comparative Example 2

[0121] Anhydrous ethanol soaking method: According to the scheme of Zhang Baowei et al. [Zhang Baowei, Wei Fuwen, Li Ming, et al. Simple method for DNA extraction from feces of giant panda and red panda [J]. Acta Zoologica Sinica, 2004, 50(3): 452-458.], the mucus-rich part of the surface of fresh giant panda feces was collected and preserved by soaking in 2 times the volume of anhydrous ethanol.

[0122] The disadvantages of this technical solution are: A. It requires the freshness of the feces; B. It requires the collection personnel to peel off the mucous membrane layer of the fresh feces on site; C. Anhydrous ethanol poses a transportation safety risk.

[0123] Comparative Example 3

[0124] Two-step method: According to Chinese patent CN107177590A, giant panda feces (defecation time <12h) need to be soaked in anhydrous ethanol for 22-26h, then dried thoroughly in an oven at 60-70℃ (1.5-2h), and finally 15-20g of dried sample is transferred to a 50mL centrifuge tube containing 15mL silica gel desiccant, and stored for a long time (up to 24 months) after being isolated from air by degreased cotton.

[0125] Disadvantages: This technical solution not only has a long total operation time (23.5-28 hours), but also involves numerous procedures. After using the drying instrument, multiple steps are required in a laboratory environment, including sample aliquoting, adding desiccant, and degreasing cotton. Field fecal sample collection conditions are often less than ideal; it is highly possible to collect fecal samples that have been soaked in anhydrous ethanol for more than 26 hours or have been outside the body for more than 12 hours. The patent document does not provide detection performance results for these two situations. In summary, the disadvantages of this technical solution are: A) It requires a high degree of fecal freshness; B) The total operation time is long; C) The drying procedure is cumbersome, and the fecal sample cannot be immediately fixed at the time of collection, potentially leading to DNA loss due to continued microbial action; D) There are time requirements for the anhydrous ethanol soaking step.

[0126] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the invention. Those skilled in the art will understand that implementing all or part of the above embodiments and making equivalent changes in accordance with the claims of the present invention are still within the scope of the invention.

[0127] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A nucleic acid preservative for a giant panda fecal sample, characterized by: The final concentration composition includes: buffer solution, 20 mM; metal ion chelating agent, 0.06 M; ionic strength maintainer, 1 M; denaturant, 0.1 M; antioxidant, 3% (V / V); cryoprotectant, 5% (V / V); nucleic acid protectant; and antibacterial agent, 20 ppm. All components are dissolved in deionized water. The buffer solution is tris(hydroxymethyl)aminomethane hydrochloride. The final concentration composition of the metal ion chelating agent is: ethylenediaminetetraacetic acid 0.05 M and polyaluminum chloride 0.01 M. The ionic strength maintainer is sodium chloride. The final concentration composition of the denaturant is: guanidine hydrochloride 0.05 M and polyethylene glycol octylphenyl ether 0.05 M. The antioxidant is tris(2-carbonylethyl)phosphohydrochloride. The cryoprotectant is glycerol. The nucleic acid protectant is selected from any of the following components: ​ (1) The nucleic acid protectant is composed of 0.5% (W / V) polyethylene glycol 6000 and 2% (W / V) polyvinylpyrrolidone; (2) The nucleic acid protectant is composed of 0.5% (W / V) polyaluminum chloride, 0.5% (W / V) activated carbon and 1% (W / V) polyvinylpyrrolidone; (3) The nucleic acid protectant is composed of 0.5% (W / V) polyferric chloride, 0.5% (W / V) diatomaceous earth and 1% (W / V) polyvinylpyrrolidone; (4) The nucleic acid protectant is composed of 1% (W / V) polyaluminum chloride, 0.5% (W / V) diatomaceous earth and 0.5% (W / V) polyethylene glycol 8000; (5) The nucleic acid protectant is composed of 1% (w / v) polyethylene glycol 6000 and 1.5% (w / v) polyvinylpyrrolidone; (6) The nucleic acid protectant is composed of 0.5% (w / v) polyethylene glycol 6000 and 1% (w / v) polyvinylpyrrolidone; (7) The nucleic acid protectant is composed of 0.5% (w / v) polyethylene glycol 6000 and 1% (w / v) polyvinylpyrrolidone; (8) The nucleic acid protectant is composed of 0.5% (W / V) of type 4A molecular sieve and 1% (W / V) of polyvinylpyrrolidone; (9) The nucleic acid protectant is composed of 0.5% (W / V) of type 5A molecular sieve and 1% (W / V) of polyvinylpyrrolidone; (10) The nucleic acid protectant is composed of 0.1% (w / v) sodium aluminate and 0.5% (w / v) polyethylene glycol 6000; (11) The nucleic acid protectant is composed of 0.1% (W / V) sodium aluminate and 0.5% (W / V) polyethylene glycol 8000.

2. The method for preparing the nucleic acid preservative of the fecal sample of Ailuropoda melanoleuca according to claim 1, characterized in that: Includes the following steps: (1) According to the final concentration composition of the preservation solution, first mix the buffer solution, metal ion chelating agent, ionic strength maintaining agent, denaturant, antioxidant, antifreeze, antibacterial agent and deionized water evenly and dissolve them. (2) After adjusting the pH value, filter out insoluble impurities; (3) Add nucleic acid protectant to obtain the product.

3. A method for preserving old and new feces of a giant panda, characterized by: The method uses the preservation solution described in claim 1, and includes the following steps: Step 1, sample collection: collect fresh feces or old feces of giant panda with ex situ time ≤ 15 days or ex situ time > 15 days, and put into a sample bottle; Step 2, in the sample bottle, add the preservative liquid according to the mass-volume ratio of 1g:3mL of the collected feces and the preservative liquid, and ensure that the preservative liquid completely immerses the feces; Step 3, store the sample bottle in the dark.

4. The application of the preservative liquid of claim 1 in the preservation of DNA of giant panda feces.

Images