Rapid and non-destructive sampling method for Chinese pangolin

Abstract

The invention belongs to the field of molecular biology research of pangolins, and particularly relates to a rapid non-destructive sampling method of Chinese pangolins, which comprises the following steps: scraping oral saliva on the inner wall of the oral cavity of the Chinese pangolins by using a sterilized disposable swab with a cotton swab at the top; the method comprises the following steps: putting a disposable swab adhered with the oral saliva of the Chinese pangolin as a sample into a centrifugal tube filled with DNA (Deoxyribose Nucleic Acid) preserving fluid; and then extracting the DNA of the sample. The method disclosed by the invention has the technical advantages of saving resources, protecting wild animals and quickly and effectively extracting the DNA of the pangolin sinensis.

Description

Technical Field

[0001] The invention belongs to the field of pangolin molecular biology research, and particularly relates to a rapid and non-destructive sampling method for Chinese pangolins. Background Art

[0002] The Chinese pangolin (Manis pentadactyla) is a placental mammal of the order Scolopendra, one of the most unusual mammals due to its overlapping epidermal scales, ant-eating diet, lack of teeth, and unusually elongated tongue. They play important roles in their ecosystems, including being a predator of social insects, a burrow creator, a host for internal and external parasites, and prey for other predators.

[0003] On the other hand, because pangolins can be used as luxury delicacies and their scales can be used in traditional medicine, pangolins are hunted and used in large numbers, and the eight existing pangolin species have experienced serious population declines. Among them, the Chinese pangolin is one of the most threatened species. At the same time, it has been listed as a critically endangered species in the Red List of the International Union for Conservation of Nature (IUCN). Laboratory molecular research on Chinese pangolins must extract the genomic DNA of Chinese pangolins. Traditional pangolin sampling methods mostly extract pangolin blood for DNA extraction. Recently, some scholars have used skin / scales for improved experiments, but they still cause potential damage to pangolins. Extracting DNA through feces is also a non-invasive method, but its DNA extraction efficiency is low and is easily affected by environmental factors.

[0004] Currently available methods for extracting DNA from the oral cavity of animals (including humans) using mucus and mucous membranes include invention application number 201210554081.0 "DNA extraction reagent preparation method and extraction method suitable for a variety of samples" and invention application number 201610097433.2 "Rapid and non-destructive sampling method for spiny-chested frogs".

[0005] The invention of 201610097433.2, "Rapid and non-destructive sampling method for spiny-chested frogs", states: Use a homemade sterilized plastic film cotton swab to sample cells in the mouth of spiny-chested frogs, put the taken oral cells into a centrifuge tube, add PBS to allow the oral cells to fully integrate into the PBS, and then use the kit genomic DNA Mini Kit to extract the sample DNA.

[0006] However, the above method is not effective in extracting Chinese pangolin DNA. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a method for rapid and non-destructive sampling of Chinese pangolins, so as to realize rapid and non-destructive extraction of Chinese pangolin genomic DNA.

[0008] In order to solve the above technical problems, the present invention provides a rapid and non-destructive sampling method for Chinese pangolins (a simple method for rapidly and non-destructively extracting Chinese pangolin DNA): DNA is extracted based on the oral saliva on the inner wall of the Chinese pangolin's mouth.

[0009] As an improvement of the rapid non-destructive sampling method of Chinese pangolin of the present invention, the method comprises the following steps in sequence:

[0010] 1) Use a sterilized disposable swab with a cotton swab (cotton head) on the top to scrape the oral saliva (including oral epithelial cells) on the inner wall of the oral cavity of the Chinese pangolin;

[0011] 2) A disposable swab with saliva from the oral cavity of the Chinese pangolin was used as a sample and placed in a centrifuge tube (1.5 ml centrifuge tube) containing a DNA preservation solution, wherein the height of the DNA preservation solution at least covers the top cotton swab of the disposable swab;

[0012] 3) Using the product obtained in step 2), sample DNA is extracted using a kit TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0.

[0013] As a further improvement of the rapid and non-destructive sampling method for Chinese pangolins of the present invention, the step 3) comprises the following steps in sequence:

[0014] 3.1) Add 180±10 μl of Buffer GL, 20±1 μl of Proteinase K and 10±0.5 μl of RNase A (10 mg / ml) to the centrifuge tube obtained in step 2) (i.e., the centrifuge tube in which the oral epithelial cells were collected), mix thoroughly (shake with a shaker for about 1 min to ensure that it is fully mixed with the collected oral epithelial cells), and discard the cotton swab;

[0015] Then, place in a constant temperature water bath at 56±1°C for 2-3h (to completely lyse the oral epithelial cells) to obtain a lysate;

[0016] 3.2) Add 20±1 μl RNase A (10 mg / mL) to the lysate obtained in step 3.1) and place at room temperature for 2-5 min;

[0017] 3.3) Add 200±10μl of Buffer GB and 200±10μl of ethanol (pure ethanol) to the product obtained in step 3.2) and mix (mix gently to avoid breaking of genomic DNA);

[0018] 3.4) Place the Spin Column on the Collection Tube to form a columnar collection tube;

[0019] Add all the products obtained in step 3.3) into the column collection tube and let it stand for 1.5 to 2.5 minutes; then centrifuge the column collection tube for 1 to 2 minutes and discard the filtrate;

[0020] 3.5) Add 500±10μl of Buffer WA to the column collection tube obtained in step 3.4) (make sure to completely empty the previous filtrate before adding Buffer WA); then centrifuge for 1-2 minutes and discard the filtrate;

[0021] 3.6) Add 700±10μl of Buffer WB ethanol solution to the column collection tube obtained in step 3.5) along the wall of the Spin Column tube; then centrifuge for 1-2 minutes and discard the filtrate;

[0022] The preparation method of the Buffer WB ethanol solution is as follows: add 56 ml of pure ethanol to every 24 ml of Buffer WB;

[0023] 3.7) Directly take the Spin Column in the column manifold obtained in step 3.6) and proceed to step 3.8) as follows;

[0024] Alternatively, the column collection tube obtained in step 3.6) is subjected to step 3.6) for 1 to 2 more times; and then the spin column in the column collection tube obtained in step 3.7) is taken to perform the following step 3.8);

[0025] Note: The purpose of repeated operation is to increase the amount of DNA obtained;

[0026] 3.8) Take out the Spin Column from the column collection tube and place it on a centrifuge tube (a new 1.5 ml centrifuge tube); add 50-200 μl (preferably 50 μl) of Elution Buffer to the center of the Spin Column membrane, then open the cover of the adsorption column and let it stand at room temperature for 4-6 minutes (the purpose is to completely dry the Spin Column to prevent residual ethanol from affecting subsequent steps);

[0027] 3.9) Centrifuge the column collection tube obtained in step 3.8) for 1 to 2 minutes; the liquid obtained by centrifugation is the extracted DNA.

[0028] Note: The DNA can be used for the next experiment immediately, or it can be stored at -4℃ for a short time or at -20℃ for a long time.

[0029] As a further improvement of the rapid and non-destructive sampling method for Chinese pangolins of the present invention: the DNA preservation solution is composed of trishydroxymethylaminomethane (Tris), ethylenediaminetetraacetic acid (EDTA), sodium azide (as a preservative) and deionized water; in the DNA preservation solution: the concentration of Tris is (10±1) mM, the concentration of EDTA is (1±0.1) mM, and the concentration of sodium azide is 0.025-0.035% (mass%, preferably 0.03%).

[0030] As a further improvement of the rapid and non-destructive sampling method of Chinese pangolin of the present invention:

[0031] In the step 3.8), the Elution Buffer is Elution Buffer that has been heated to 65±5° C. in advance.

[0032] That is, the Elution Buffer is heated to 65±5°C in advance and then added to the center of the Spin Column membrane in order to improve the elution efficiency.

[0033] As a further improvement of the rapid and non-destructive sampling method for Chinese pangolins of the present invention: the centrifugal speed is (12,000±1000) rpm.

[0034] In all the above steps of the present invention, if the temperature is not clearly stated, they are all carried out at room temperature.

[0035] The apparatuses contacting the Chinese pangolin DNA in the steps of the present invention are all sterilized at high temperature.

[0036] Proteinase K, ribonuclease A, and various washing buffers (Buffer GL, Buffer GB, Buffer WA, Buffer WB, Elution Buffer) used in the present invention can all be provided by the kit TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0.

[0037] The present invention has the following beneficial effects:

[0038] 1) The simple method for extracting Chinese pangolin DNA quickly and without damage of the present invention replaces the traditional sampling method (such as blood or tissue sampling) by extracting oral saliva (containing oral epithelial cells) on the inner wall of the oral cavity of the Chinese pangolin, which reduces the physical and psychological damage to the pangolin and can be carried out without interfering with the normal life of the animal. Since the sampling process is harmless to the pangolin, multiple sampling can be performed on the same individual, which is helpful for long-term detection of its physiological condition and genetic changes. By regularly collecting saliva samples, the number, distribution and health status of wild populations can be better understood, providing a scientific basis for formulating effective protection strategies.

[0039] 2) Collecting oral saliva (including oral epithelial cells) from the oral cavity of Chinese pangolins is relatively simple and does not require complex equipment and professional technicians, which makes it possible to efficiently carry out sampling work in the field. Moreover, according to the experimental results, the extracted DNA can be used for further experimental research, and a large amount of scientific research funds can also be saved. The use of non-destructive sampling technology can also reduce the additional hunting pressure caused by sampling needs for endangered species such as Chinese pangolins.

[0040] In summary, the present invention is a rapid and non-destructive sampling method for Chinese pangolins. The present invention uses the characteristics of saliva secretion by the salivary glands in the oral cavity of Chinese pangolins, uses a disposable swab to sample the oral saliva on the inner wall of the oral cavity of Chinese pangolins, adds a DNA preservation solution to the extracted saliva, makes the top cotton swab part of the swab fully blend into the DNA preservation solution, and then uses the kit TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 to extract the sample DNA. The method of the present invention has the technical advantages of saving resources, protecting wild animals, and extracting DNA from Chinese pangolins quickly and effectively. The method is applied to pangolin sampling, which can protect pangolin resources, and can also save a lot of scientific research funds, so as to achieve the purpose of repeated sampling. The present invention provides new possibilities for conducting scientific research under the premise of respecting life, and also provides new ideas and methods for non-invasive sampling technology of other wild animals. The rapid and non-destructive sampling method for Chinese pangolins of the present invention has the characteristics of reducing interference and potential damage to the pangolin body, fast sampling speed, good DNA extraction effect, simple method and easy operation, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0042] Figure 1 This is a schematic flow chart of a simple method for rapidly and non-destructively extracting Chinese pangolin DNA according to the present invention;

[0043] Figure 2 It is an electrophoresis diagram. DETAILED DESCRIPTION

[0044] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0045] The preparation method of the sterilized disposable swab is as follows:

[0046] Take a disposable swab with a cotton swab (cotton head) on the top, and then sterilize it with high-pressure moist heat steam (0.1 MPa, 110° C., sterilization for 40 minutes) to obtain a sterilized disposable swab.

[0047] Example 1: A rapid and non-destructive sampling method for Chinese pangolins, comprising the following steps:

[0048] 1) Use a sterilized disposable swab to collect (repeatedly, generally at least 10 seconds) oral saliva (including oral epithelial cells) from the inner wall of the oral cavity of the Chinese pangolin;

[0049] 2) The collected oral saliva (including oral epithelial cells) was placed in a 1.5 ml centrifuge tube (i.e., the top cotton swab of the disposable swab with the oral saliva of the Chinese pangolin was placed in a 1.5 ml centrifuge tube), and 1 ml of DNA preservation solution was added; at this time, the height of the DNA preservation solution was higher than the top cotton swab of the disposable swab.

[0050] The DNA preservation solution is composed of tris(hydroxymethyl)aminomethane (Tris), ethylenediaminetetraacetic acid (EDTA), sodium azide (as a preservative) and deionized water; in the DNA preservation solution: the concentration of Tris is 10mM, the concentration of EDTA is 1mM, and the concentration of sodium azide is 0.03% (mass %).

[0051] 3) Extract the sample DNA from the product obtained in step 2) using the kit TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0;

[0052] The details are as follows:

[0053] 3.1) Heat the water bath or constant temperature oscillator to 56°C in advance;

[0054] Add 180 μl of Buffer GL, 20 μl of Proteinase K and 10 μl of RNase A (10 mg / ml) to the centrifuge tube where the oral epithelial cells were collected (i.e., the centrifuge tube obtained in step 2), and shake with a shaker for about 1 min to ensure that the collected oral epithelial cells are fully mixed, and discard the cotton swab;

[0055] Then, the cells were placed in a constant temperature water bath at 56°C for 2 h to completely lyse the oral epithelial cells to obtain lysate products.

[0056] illustrate:

[0057] To accelerate cell lysis, the sample can be shaken (e.g., inverted and mixed once every 30±1 min) or pipetted in a constant temperature water bath to accelerate lysis;

[0058] Before discarding the cotton swab, try to squeeze out the remaining liquid in the cotton swab. If the squeezing is incomplete, leave the cotton swab in the centrifuge tube and place it in a constant temperature water bath.

[0059] 3.2) Add 20 μl RNase A (10 mg / mL) to the lysate obtained in step 3.1) and place at room temperature for 2 to 5 minutes;

[0060] 3.3) Add 200 μl Buffer GB and 200 μl 100% ethanol to the product obtained in step 3.2) and mix gently (to avoid genomic DNA breakage);

[0061] 3.4) Place the Spin Column on the Collection Tube to form a columnar collection tube;

[0062] Use a pipette to add all the liquid in the centrifuge tube in step 3.3) to the column collection tube and let it stand for 2 minutes; then centrifuge the column collection tube at 12,000 rpm for 2 minutes and discard the filtrate (make sure to completely empty the filtrate before the subsequent steps);

[0063] 3.5) Add 500 μl of Buffer WA to the column collection tube obtained in step 3.4); then centrifuge at 12,000 rpm for 1 min and discard the filtrate;

[0064] 3.6) Add 700 μl of Buffer WB ethanol solution to the column collection tube obtained in step 3.5) along the wall of the Spin Column tube; then centrifuge at 12,000 rpm for 1 min and discard the filtrate;

[0065] The preparation method of the Buffer WB ethanol solution is as follows: add 56 ml of pure ethanol to every 24 ml of Buffer WB;

[0066] 3.7) Repeat the process of the previous step (i.e., add Buffer WB ethanol solution and subsequent centrifugation according to step 3.6) to the column collection tube obtained in step 3.6);

[0067] 3.8) Take out the Spin Column in the column collection tube obtained in step 3.7) and place the adsorption column in a new 1.5 ml centrifuge tube;

[0068] Heat the Elution Buffer to 65°C in advance;

[0069] Add 50 μl of pre-heated Elution Buffer to the center of the Spin Column membrane in the column collection tube, then open the cover of the adsorption column and let it stand at room temperature for 5 minutes (the purpose is to completely dry the Spin Column to prevent residual ethanol from affecting the subsequent steps);

[0070] 3.9) Centrifuge the column collection tube obtained in step 3.8) at 12,000 rpm for 2 min; the liquid obtained by centrifugation is the extracted DNA.

[0071] The DNA can be used for the next experiment immediately, or it can be stored at -4℃ for a short time or at -20℃ for a long time.

[0072] Experiment 1: According to the rapid non-destructive sampling method for Chinese pangolins described in Example 1, 10 different Chinese pangolins were randomly selected for DNA extraction. The obtained DNA performance data are shown in Table 1.

[0073] At the same time, the above 10 samples were amplified by PCR and electrophoresed. The gel image is as follows: Figure 2 As shown:

[0074] Lane 1 is 2000K Mark, and lanes 2-11 are amplified DNA.

[0075] According to Table 1 and Figure 2 It can be seen that the DNA extraction rate is high, the fragments are long, complete and not degraded, and the sample extraction quality is stable.

[0076] Table 1. DNA data obtained by rapid and non-destructive sampling of Chinese pangolins

[0077]

[0078] Note: When the OD value (A260 / A280) is close to 1.8, it indicates that the purity of the DNA sample is higher.

[0079] Comparative Example 1: Using 201210554081.0 "DNA extraction reagent preparation method and extraction method suitable for various samples", the results are shown in Table 2, which shows that the method is not effective in extracting Chinese pangolin DNA.

[0080] Table 2. DNA data obtained by sampling according to the method of 201210554081.0

[0081]

[0082]

[0083] Comparative Example 2: Using 201610097433.2 "Rapid and non-destructive sampling method for Rana spinosa", the results are shown in Table 3, which shows that this method is not effective in extracting Chinese pangolin DNA.

[0084] Table 3. DNA data obtained by sampling according to the method of 201610097433.2

[0085]

[0086] Comparative Example 3: Step 3.2) of Example 1 is eliminated; that is, the cleavage product obtained in Step 3.1) is directly subjected to Step 3.3). The rest is the same as in Example 1.

[0087] The results are shown in Table 4, which shows that the method used to extract Chinese pangolin DNA is not effective.

[0088] Table 4

[0089]

[0090]

[0091] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A rapid and non-destructive sampling method for Chinese pangolins, characterized in that: DNA was extracted from the oral saliva on the inner wall of the Chinese pangolin's mouth.

2. The rapid and non-destructive sampling method for Chinese pangolin according to claim 1, characterized in that The steps are as follows: 1) Use a sterilized disposable swab with a cotton swab on the top to scrape the oral saliva on the inner wall of the oral cavity of the Chinese pangolin; 2) A disposable swab with saliva from the oral cavity of the Chinese pangolin is used as a sample and placed in a centrifuge tube containing a DNA preservation solution, wherein the height of the DNA preservation solution at least covers the top cotton swab of the disposable swab; 3) Extracting sample DNA using the product obtained in step 2).

3. The rapid and non-destructive sampling method for Chinese pangolin according to claim 2, characterized in that The step 3) comprises the following steps in sequence: 3.1) Add 180±10μl of Buffer GL, 20±1μl of Proteinase K and 10±0.5μl of RNase A to the centrifuge tube obtained in step 2), mix thoroughly and discard the cotton swab; Then, place in a constant temperature water bath at 56±1℃ for 2-3h to obtain the cleavage product; 3.2) Add 20±1 μl RNase A to the lysate obtained in step 3.1) and place at room temperature for 2-5 min; 3.3) Add 200±10μl of Buffer GB and 200±10μl of ethanol to the product obtained in step 3.2) and mix; 3.4) Place the Spin Column on the Collection Tube to form a columnar collection tube; Add all the products obtained in step 3.3) into the column collection tube and let it stand for 1.5 to 2.5 minutes; then centrifuge the column collection tube for 1 to 2 minutes and discard the filtrate; 3.5) Add 500±10μl of Buffer WA to the column collection tube obtained in step 3.4); then centrifuge for 1-2 minutes and discard the filtrate; 3.6) Add 700±10μl of Buffer WB ethanol solution to the column collection tube obtained in step 3.5) along the wall of the Spin Column tube; then centrifuge for 1-2 minutes and discard the filtrate; The preparation method of the Buffer WB ethanol solution is as follows: add 56 ml of pure ethanol to every 24 ml of Buffer WB; 3.7) Directly take the Spin Column in the column manifold obtained in step 3.6) and proceed to step 3.8) as follows; Alternatively, the column collection tube obtained in step 3.6) is subjected to step 3.6) for 1 to 2 more times; and then the spin column in the column collection tube obtained in step 3.7) is taken to perform the following step 3.8); 3.8) Take out the Spin Column from the column collection tube and place it on the centrifuge tube; add 50-200 μl of Elution Buffer to the center of the Spin Column membrane, then open the cover of the adsorption column and let it stand at room temperature for 4-6 minutes; 3.9) Centrifuge the column collection tube obtained in step 3.8) for 1 to 2 minutes; the liquid obtained by centrifugation is the extracted DNA.

4. The rapid and non-destructive sampling method of Chinese pangolin according to claim 3, characterized in that: The DNA preservation solution is composed of tris(hydroxymethyl)aminomethane (Tris), ethylenediaminetetraacetic acid (EDTA), sodium azide and deionized water; in the DNA preservation solution: the concentration of Tris is (10±1) mM, the concentration of EDTA is (1±0.1) mM, and the concentration of sodium azide is 0.025-0.035%.

5. The rapid and non-destructive sampling method of Chinese pangolin according to claim 4, characterized in that: In the step 3.8), the Elution Buffer is the Elution Buffer that has been heated to 65±5° C. in advance.

6. The rapid non-destructive sampling method for Chinese pangolin according to any one of claims 2 to 5, characterized in that: The centrifugal speed is (12,000±1000) rpm.

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