Method for rapidly extracting turtle blood DNA from turtle blood and radix bupleuri decoction pieces and application

Abstract

The invention relates to the technical field of molecular biology, and particularly discloses a method for rapidly extracting turtle blood DNA from turtle blood and radix bupleuri decoction pieces and application thereof.The method comprises the steps that a turtle blood and radix bupleuri sample is soaked with a soaking solution, phenols and polysaccharide plant components in the sample are removed, and a turtle blood and radix bupleuri soaking solution is obtained; performing low-speed centrifugation on the obtained turtle blood and radix bupleuri soak solution to remove granular impurity components in the soak solution, and performing high-speed centrifugation to obtain precipitates containing turtle blood components; extracting DNA in the precipitate of the turtle blood component by adopting an improved blood DNA extracting solution to obtain turtle blood DNA; compared with a common blood genome kit method for extracting the turtle blood DNA in the turtle blood radix bupleuri in research, the method disclosed by the invention has the advantages that the interference of phenols, polysaccharides and other components in the radix bupleuri on DNA extraction can be effectively removed, the DNA extraction efficiency can be remarkably improved, and the concentration of the DNA extracted by the method is 6.91 times that of the kit method.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and particularly relates to a method for rapidly extracting turtle blood DNA from turtle blood Bupleuri Radix Praeparatus and its application. Background Art

[0002] Bupleuri Radix was first recorded in "Shennong Ben Cao Jing" in the Eastern Han Dynasty, with the correct name of "Zi Hu". "Zi" is the same as "Zi", which may refer to the purple color of its medicinal material properties. In the Tang Dynasty, it was also recorded in "Xin Xiu Ben Cao" that "Zi" is the same as the ancient character "Chai". "Zi is the ancient character of Chai... and this herb has a purple root. Now, the commonly used Zi Hu is like this. Also, using wood instead of a string, it has been called Zi Hu successively." There are also corresponding records in the Northern Song Dynasty, and the term "Bupleuri Radix" was first proposed in "Ben Cao Tu Jing". The famous medical scientist Li Shizhen in the Ming Dynasty also had some insights on the term "Bupleuri Radix". He believed that the young seedlings of Bupleuri Radix can be eaten, and its rhizomes become like firewood after getting old, so its root is named Bupleuri Radix. Bupleuri Radix tastes slightly bitter and has a slightly cold nature. It is a good medicine for relieving exterior syndrome and reducing fever, soothing the liver and relieving depression, and also has the effect of lifting yang qi. It is often used to treat exterior syndrome fever, qi stagnation and liver depression, etc. There have been phenomena of medical accidents caused by unclear variety confusion in past dynasties. The saying of "Bupleuri Radix robbing liver yin" formed in the late Ming Dynasty was continued by the febrile disease doctors in the Qing Dynasty, and there was once a phenomenon of fearing to use Bupleuri Radix. However, the unique medicinal properties of turtle blood exactly make up for this shortcoming. Turtle blood is formed by drying the blood of the turtle (Trionyx sinensis Wiegmann) of the Trionychidae family. It has a slightly cold nature and a salty taste, and can enter the liver meridian. It has a unique curative effect in clearing heat and nourishing yin, and removing stasis and detoxifying. The prepared turtle blood Bupleuri Radix obtained by processing turtle blood and Bupleuri Radix has better effects.

[0003] Turtle blood is the fresh blood of the soft-shelled turtle (Trionyx sinensis Wiegmann) of the Trionychidae family. The whole body of the turtle is rich in nutrients, so it has been a highly praised dual-purpose product of medicine and food among the common people for many years. According to "Chinese Herbal Medicine", turtle blood has the effects of nourishing yin and clearing heat, promoting blood circulation and dredging collaterals. It tastes sweet and salty, has a neutral nature, and enters the liver meridian, and is mainly used to treat consumptive fever, yin deficiency and low fever, hypochondriac pain, deviation of the mouth and eye slant, rectal prolapse, etc. The medicinal history of turtle blood was recorded as early as the Tang Dynasty, and its efficacy was further confirmed in the Yuan and Ming dynasties. In modern times, the application of turtle blood as a processing auxiliary material has experienced a prosperous period, and some varieties are still in use today. Especially for the turtle blood Bupleuri Radix, a characteristic prepared slice of Jiangxi Zhangbang, after being processed with turtle blood, its floating nature can be inhibited, and its effects of clearing the liver and reducing fever, and treating malaria can be increased, which fully reflects the medication characteristics of traditional Chinese medicine processing according to the prescription. In addition, turtle blood is a traditional processing auxiliary material for traditional Chinese medicine, and its processed products include turtle blood Bupleuri Radix, turtle blood Stellariae Radix, turtle blood Artemisiae Annuae Herba, turtle blood Moutan Cortex, turtle blood Salviae Miltiorrhizae Radix, turtle blood Paeoniae Radix Alba, turtle blood black soybeans, etc.

[0004] The use of Bupleuri Radix Praeparata Cum Sanguine Trionycis was detailedly recorded in *Ben Cao Hai Li*: "When using Bupleuri Radix, adding a few spoons of Trionyx sinensis blood can prevent over-acting on the exterior." It was recorded in *Leng Lu Yi Hua* in the Qing Dynasty that "Bupleuri Radix Praeparata Cum Sanguine Trionycis" can soothe the liver without damaging liver yin; it was also recorded in *Chang Sha Fang Ge Kuo* in the Qing Dynasty about the processing method of "mixing with Trionyx sinensis blood and steaming"; it was also detailedly recorded in the 1963 edition of *Chinese Pharmacopoeia*: "For Bupleuri Radix Praeparata Cum Sanguine Trionycis, take Bupleuri Radix slices, place them in a large basin, pour in Trionyx sinensis blood diluted with a little warm water, mix well, moisten, stir-fry gently over low heat in an inner pot, take out and let cool." In local processing, there are slight differences in the processing methods of Bupleuri Radix Praeparata Cum Sanguine Trionycis. For example, in Zhejiang and Jiangsu, it is always processed by mixing with Trionyx sinensis blood and steaming. Take Bupleuri Radix Praeparata Cum Sanguine Trionycis slices, mix them with Trionyx sinensis blood, yellow rice wine or clear water, let it sit for a while, place in a pot, heat over low heat, stir-fry until dry, take out and let cool. For every 100 kg of Bupleuri Radix slices, use 12.5 kg of Trionyx sinensis blood and 12.5 kg of yellow rice wine.

[0005] Under the current increasingly strict requirements for drug quality and safety, the authenticity and content of animal-derived components in Bupleuri Radix Praeparata Cum Sanguine Trionycis tablets have become one of the key factors affecting its clinical application effect. Therefore, the research on the extraction method of animal-derived nucleic acids from Bupleuri Radix Praeparata Cum Sanguine Trionycis tablets is particularly important. The research on the technology of quickly and efficiently extracting Trionyx sinensis blood DNA from Bupleuri Radix Praeparata Cum Sanguine Trionycis tablets can not only effectively verify the existence and proportion of Trionyx sinensis blood components, but also ensure the quality of genuine tablets.

[0006] In response to this, the inventor proposes a method for quickly extracting Trionyx sinensis blood DNA from Bupleuri Radix Praeparata Cum Sanguine Trionycis tablets and its application to solve the above problems. Summary of the Invention

[0007] The aim of the present invention is to address the current situation in the market where there is a lack of technology specifically for extracting animal-derived (specifically Trionyx sinensis blood) genomic DNA from Bupleuri Radix Praeparata Cum Sanguine Trionycis. Existing research generally relies on commercial blood DNA extraction kits to extract Trionyx sinensis blood genomic DNA from Bupleuri Radix Praeparata Cum Sanguine Trionycis. However, this method is not only costly but also has limitations in extraction efficiency. In view of this, the present invention proposes an innovative method that can quickly and efficiently obtain high-quality Trionyx sinensis blood DNA from Bupleuri Radix Praeparata Cum Sanguine Trionycis, while reducing costs and improving the feasibility of operation. And during the extraction process, with the same amount of Bupleuri Radix Praeparata Cum Sanguine Trionycis used, the method of the present invention can obtain a higher concentration of DNA, which can save the consumption of Bupleuri Radix Praeparata Cum Sanguine Trionycis tablets in future research applications.

[0008] The present invention first soaks the Bupleuri Radix Praeparata Cum Sanguine Trionycis tablet samples with an immersion buffer solution. On the one hand, it removes plant components such as phenols and polysaccharides in the samples to prevent their influence on DNA extraction later; on the other hand, it soaks out the Trionyx sinensis blood components in the Bupleuri Radix Praeparata Cum Sanguine Trionycis tablets and obtains a precipitate containing Trionyx sinensis blood cell components by centrifugation.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] A method for rapidly extracting turtle blood DNA from turtle blood Bupleuri Radix tablets, comprising the following steps:

[0011] Soak the turtle blood Bupleuri Radix sample with the soaking solution to wash the turtle blood from the turtle blood Bupleuri Radix and remove phenolic and polysaccharide plant components in the sample, obtaining the turtle blood Bupleuri Radix soaking solution;

[0012] Centrifuge the obtained turtle blood Bupleuri Radix soaking solution at a low speed to remove particulate impurities in the soaking solution, and then obtain a precipitate containing turtle blood components by high-speed centrifugation;

[0013] Extract DNA from the precipitate of the turtle blood components using a modified blood DNA extraction solution to obtain turtle blood DNA.

[0014] Reagents used in the extraction process:

[0015] Turtle blood Bupleuri Radix soaking solution: Ascorbic acid 0.09 g / L, Tris-HCl 0.1 mol / L, Polyvinylpyrrolidone 0.1 g / L.

[0016] Blood DNA extraction buffer: Chelex100 0.1 g / L, Polyvinylpyrrolidone 0.1 g / L, Proteinase K 400 μg / mL.

[0017] Chloroform: Isoamyl alcohol mixed solution: Prepared according to a volume ratio of 24:1.

[0018] 75% (v / v) ethanol solution: Mix absolute ethanol and double-distilled water according to a volume ratio of 3:1

[0019] Proteinase K solution: The stock solution contains Proteinase K at a concentration of 20 mg / ml, and the working concentration of Proteinase K is 400 μg / mL.

[0020] Specific extraction process:

[0021] Weigh 1 g of turtle blood Bupleuri Radix into a 5 mL sterilized centrifuge tube, add 3 mL of turtle blood Bupleuri Radix soaking buffer, manually shake for 20 min to wash out the turtle blood components in the turtle blood Bupleuri Radix.

[0022] The Bupleuri Radix granules will absorb part of the soaking solution. Use a pipette to transfer part of the soaking solution to a new sterilized 1.5 mL centrifuge tube, first centrifuge at 1000 rpm for 1 min to remove impurity precipitates and retain the supernatant.

[0023] Transfer the supernatant to a new centrifuge tube, and then centrifuge at a speed of 12000 rpm for 5 min, discard the supernatant, and retain the precipitate part.

[0024] Add 200 μL of extraction buffer (containing 100 mmol / L Tris-Hcl, 10 g / L polyvinylpyrrolidone, 400 μg / mL proteinase K) to the precipitate, and pipette to mix well to ensure that all the precipitate at the bottom of the tube is dissolved.

[0025] Transfer the above aqueous solution to a centrifuge tube containing 0.02 g of Chelex 100 beads, mix well, incubate in a water bath at 56 °C for 30 min, and shake vigorously for 5 - 10 s at intervals of 5 min during this period.

[0026] After the water bath, open the lid of the centrifuge tube, place it in a boiling water bath at 99 °C for 5 min, then cover the lid and shake for 5 - 10 s, and centrifuge at 12000 r / min for 5 min.

[0027] Transfer the supernatant to a new centrifuge tube, then add 500 μL of a mixture of chloroform and isoamyl alcohol (ratio 24:1), shake well to mix evenly, and then centrifuge at 12000 rpm for 5 min.

[0028] Aspirate the upper aqueous phase and transfer it to a new sterilized centrifuge tube, add an equal volume of isopropanol, shake well, and place it in a refrigerator at 4 °C for 15 min to precipitate nucleic acids.

[0029] Take out the sample from the refrigerator and centrifuge at 12000 rpm for 5 min. The white precipitate obtained is DNA.

[0030] Use a pipette to aspirate and discard the supernatant, add 1 mL of 75% ethanol to the precipitate, gently pipette to wash the precipitate, and then centrifuge at 4 °C and 12000 rpm for 5 min.

[0031] Use a pipette to aspirate and discard the 75% ethanol, remove as much liquid as possible, and then open the lid of the centrifuge tube and place it in a laminar flow hood for about 15 min to dry the DNA precipitate.

[0032] Finally, add 50 μL of ultrapure water to the obtained DNA sample to dissolve the DNA. Thus, the DNA solution of turtle blood in Bupleurum chinense DC. var. marginatum W. T. Wang et Hsiao is obtained, and this DNA can be used for subsequent gene amplification.

[0033] Compared with the existing methods for extracting animal-derived DNA from Bupleurum chinense DC. var. marginatum W. T. Wang et Hsiao, the present invention has the following beneficial effects:

[0034] 1. Turtle blood Bupleurum is prepared by stir-frying Bupleurum, turtle blood and yellow rice wine. When extracting the DNA of turtle blood, liquid reagents are needed to soak out the components of turtle blood. However, phenols and polysaccharides in Bupleurum will inevitably enter the soaking solution, both of which will interfere with DNA extraction. In the soaking step of turtle blood Bupleurum in the present invention, ascorbic acid and polyvinylpyrrolidone are added to the soaking solution. The former can effectively prevent the oxidation of polyphenolic substances, and the latter can form complexes with polyphenolic and polysaccharide substances, which can be removed during centrifugation or chloroform extraction, reducing the interference of phenols and polysaccharides in Bupleurum on subsequent DNA extraction and improving the quality of DNA extraction.

[0035] 2. The leaching solution of turtle blood Bupleurum contains a large amount of granular impurities. In the present invention, the impurities are first removed by short-term low-speed centrifugation (1000 rpm, 1 min), and then a precipitate containing turtle blood components is obtained by high-speed centrifugation (12000 rpm, 1 min). The present invention effectively separates and obtains turtle blood components from turtle blood Bupleurum by combining low-speed and high-speed centrifugation methods.

[0036] 3. Due to the special material of turtle blood Bupleurum, which contains both animal and plant components, it is difficult to remove the components that interfere with DNA extraction, such as phenols and polysaccharides in Bupleurum, by using conventional blood DNA extraction methods to obtain turtle blood DNA. In the DNA extraction step of the present invention, Chelex100 is added to the DNA extraction buffer as the main component for lysing turtle blood cells. Chelex100 is a high-molecular-weight anion exchange resin, mainly composed of copolymerization of polystyrene and divinylbenzene. It contains paired iminodiacetate ions, which can selectively bind polyvalent cations, especially heavy metal ions. Under heating conditions, Chelex100 can destroy the cell structure and release the DNA inside the cells. Chelex-100 can also inactivate nucleases and stabilize DNA to prevent its degradation. In addition, polyvinylpyrrolidone is added to the extraction buffer to further remove phenolic and polysaccharide compounds in the sample. Such a setting in the present invention can effectively improve the DNA extraction efficiency and quality, providing a guarantee for subsequent DNA barcode identification. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a comparison chart of the extraction efficiency of obtaining turtle blood DNA in the embodiment of the present invention and extracting turtle blood DNA in turtle blood Bupleurum with a blood genome kit.

[0038] Figure 2 It is a comparison chart of A260 / A280 of obtaining turtle blood DNA in the embodiment of the present invention and extracting turtle blood DNA in turtle blood Bupleurum with a blood genome kit.

[0039] Figure 3This is a comparison graph of the turtle blood DNA obtained in the embodiments of the present invention and the turtle blood DNA extracted from turtle blood Bupleurum in the kit at A260 / A230.

[0040] Figure 4 This is the PCR electrophoresis result graph of the turtle blood DNA amplified by the mitochondrial gene BX of the turtle after extracting the turtle blood DNA from turtle blood Bupleurum by the method in the embodiments of the present invention.

[0041] Figure 5 This is the PCR electrophoresis result graph of the pig blood DNA amplified by the nuclear gene ZH of the pig after extracting the pig blood DNA from pig blood Bupleurum by the method in the embodiments of the present invention.

[0042] Figure 6 This is the PCR electrophoresis result graph of the bovine blood DNA amplified by the mitochondrial gene NX of the bovine after extracting the bovine blood DNA from rabbit blood Bupleurum by the method in the embodiments of the present invention.

[0043] Figure 7 This is the PCR electrophoresis result graph of the sheep blood DNA amplified by the mitochondrial gene YX of the sheep after extracting the sheep blood DNA from sheep blood Bupleurum by the method in the embodiments of the present invention.

[0044] Figure 8 This is the PCR electrophoresis result graph of the chicken blood DNA amplified by the nuclear gene JH of the chicken after extracting the chicken blood DNA from chicken blood Bupleurum by the method in the embodiments of the present invention.

[0045] Figure 9 This is the PCR electrophoresis result graph of the duck blood DNA amplified by the nuclear gene YaH of the duck after extracting the duck blood DNA from duck blood Bupleurum by the method in the embodiments of the present invention.

[0046] Figure 10 This is the PCR electrophoresis result graph of the donkey blood DNA amplified by the mitochondrial gene LvX of the donkey after extracting the donkey blood DNA from donkey blood Bupleurum by the method in the embodiments of the present invention;

[0047] Figure 11 This is a flow chart of a method for rapidly extracting turtle blood DNA from turtle blood Bupleurum tablets in the present invention. Detailed implementation mode

[0048] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0049] Example 1:

[0050] Use the method of the present invention to extract the turtle blood DNA from turtle blood Bupleurum tablets. The specific steps are as follows:

[0051] 1. Take 1 g of Biexue Chaihu Granules and add 3 mL of the soaking solution. Manually shake and mix well for 30 min. Then transfer the soaked solution to a new sterilized 2 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, transfer the supernatant to a new tube, and then centrifuge at 12000 r / min for 10 min. Discard the supernatant and retain the precipitate.

[0052] 2. Add 200 μL of extraction buffer to the precipitate, and then add 10 μL of Proteinase K (20 mg / mL) to ensure that all the precipitate at the bottom of the tube is dissolved.

[0053] 3. Transfer the above aqueous solution to a centrifuge tube containing 0.02 g of Chelex 100 particles, mix well, and incubate in a water bath at 56 °C for 30 min, with vigorous shaking for 5 - 10 s at 5 min intervals.

[0054] 4. After the water bath, open the lid of the centrifuge tube, incubate in a boiling water bath at 99 °C for 5 min, then cover the lid and shake for 5 - 10 s, and centrifuge at 12000 r / min for 5 min.

[0055] 5. Transfer the supernatant to a new centrifuge tube, then add 500 μL of a chloroform and isoamyl alcohol mixture (24:1), shake vigorously, and then centrifuge at 12000 rpmin for 5 min.

[0056] 6. Pipette the upper aqueous phase and transfer it to a new sterilized centrifuge tube, add an equal volume of isopropanol, shake and mix well, and place it in a 4 °C refrigerator for 15 min to precipitate nucleic acids.

[0057] 7. Take out the sample from the refrigerator and centrifuge at 12000 rpm for 5 min. The white precipitate obtained is DNA.

[0058] 8. Use a pipette to aspirate and discard the supernatant, add 1 mL of 75% ethanol to the precipitate, gently pipette and wash the precipitate, and then centrifuge at 4 °C and 12000 rpm for 5 min.

[0059] 9. Use a pipette to aspirate and discard the 75% ethanol, remove as much liquid as possible, and then open the lid of the centrifuge tube and place it in a laminar flow hood for about 15 min to dry the DNA precipitate.

[0060] 10 Finally, add 50 μL of ultrapure water to the obtained DNA sample to dissolve the DNA. The Biexue DNA solution in Biexue Chaihu is obtained, and this DNA can be used for subsequent gene amplification.

[0061] 9. Use a nucleic acid quantifier to measure the concentration and purity of the obtained DNA (A260 / A280, A260 / A230).

[0062] Comparative Example 1

[0063] In this comparative example, the method for extracting turtle blood DNA from Bupleuri Radix Praeparata Cum Sanguine Pini was to use a blood genomic DNA extraction kit (Tiangen).

[0064] 1. Add 1 g of Bupleuri Radix Praeparata Cum Sanguine Pini granules to 4 mL of cell lysate CL, shake for 30 minutes, transfer the leachate to a 2 mL centrifuge tube, then centrifuge at 12000 rpm for 3 minutes, and discard the supernatant.

[0065] 2. Add 200 μL of GS to the precipitate, mix well, add it to the solution previously mixed with 200 μL of buffer GB and 20 μL of Proteinase K, then let it stand at 56 °C for 10 minutes, and invert it regularly to mix evenly. It can be observed that the solution becomes clear.

[0066] 3. After standing at room temperature for 3 minutes, add 350 μL of buffer BD, then continuously invert the centrifuge tube to mix the solution evenly. After standing for a while, flocculent precipitates can be observed.

[0067] 4. Add the solution obtained in step 3 and the flocculent precipitates therein to the adsorption column (the adsorption column is sleeved in the collection tube), centrifuge at 12000 rpm for 30 seconds, then pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0068] 5. Add 500 μL of buffer GDB to the adsorption column, centrifuge at 12000 rpm for 30 seconds, then pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0069] 6. Add 600 μL of washing solution PWB to the adsorption column, centrifuge at 12000 rpm for 30 seconds, then pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0070] 7. Repeat the previous step by adding 600 μL of washing solution PWB to the adsorption column, centrifuge at 12000 rpm for 2 minutes, and then pour out the waste liquid.

[0071] 8. Place the adsorption column at room temperature and let it stand still for 2 - 5 minutes to completely dry the washing solution on the surface layer of the adsorption column.

[0072] 9. Transfer the adsorption column to a new 1.5 mL centrifuge tube, and drop 30 μL of elution buffer TB into the middle part of the adsorption membrane in the air. Place it at room temperature for 2 minutes to dissolve as much DNA as possible in the adsorption column. Then centrifuge at 12000 rpm for 2 minutes, and collect the TB buffer into the centrifuge tube.

[0073] 10. Use a nucleic acid quantifier to measure the concentration and purity of the obtained DNA (A260 / A280, A260 / A230).

[0074] Example 2

[0075] Using the method in Example 1 of the present invention, animal-derived DNA in Bupleuri Radix processed with pig blood, rabbit blood, sheep blood, chicken blood, duck blood, and donkey blood was extracted respectively to obtain animal-derived DNA in Bupleuri Radix processed with pig blood, rabbit blood, sheep blood, chicken blood, duck blood, and donkey blood. Bupleuri Radix processed with different animal blood was provided by Jiangsu Chengkai Traditional Chinese Medicine Co., Ltd., and the Bupleuri Radix slices processed with pig blood, rabbit blood, sheep blood, chicken blood, duck blood, and donkey blood according to the processing method of Bupleuri Radix processed with soft-shelled turtle blood were used to simulate the adulteration of soft-shelled turtle blood in Bupleuri Radix processed with soft-shelled turtle blood.

[0076] PCR amplification was performed on the animal blood DNA in Bupleuri Radix processed with various animal blood obtained in Example 1 and Example 2. The PCR reaction system was 20 μL, including 2 μL of DNA, 0.5 μL of each upstream and downstream primer, 10 μL of 2×Es Taq Master Mix (Tiangen), and 7 μL of ultrapure water.

[0077] PCR was performed using the soft-shelled turtle blood DNA obtained in Example 1. The PCR program was: pre-denaturation at 94 °C for 3 minutes; denaturation at 94 °C for 30 seconds, annealing at 53.5 °C for 30 seconds, extension at 72 °C for 30 seconds, for 40 cycles. To amplify the soft-shelled turtle mitochondrial gene fragment, the primers were BX-F: 5’-TACTACCTTTTTTGATCCTTCT-3’; BX-R: 5,-ATTAGCATAGTACGTTACTACA-3,, and the product size was 147 bp. The gel electrophoresis pattern of the PCR product is shown in Figure 4 。

[0078] PCR was performed using the pig blood DNA obtained in Example 2. The PCR program was: pre-denaturation at 94 °C for 3 minutes; denaturation at 94 °C for 30 seconds, annealing at 56 °C for 30 seconds, extension at 72 °C for 20 seconds, for 40 cycles. To amplify the pig nuclear gene fragment, the primers were ZH-F: 5’-CAAGAGCATCGACAATGTGTC-3’; ZH-R: 5’-GTGGGCTTCGGCCGTTAG-3’, and the product size was 155 bp. The gel electrophoresis pattern of the PCR product is shown in Figure 5 。

[0079] PCR was performed using the bovine blood DNA obtained in Example 2. The PCR program was: pre-denaturation at 94 °C for 3 minutes; denaturation at 94 °C for 30 seconds, annealing at 51 °C for 30 seconds, extension at 72 °C for 20 seconds, for 40 cycles. To amplify the bovine mitochondrial gene fragment, the primers were NX-F: 5’-GAGTAATCCTTCTGCTCACA-3’; NX-R: 5’-GATTCATTCGACTAAATTTGTG-3’, and the product size was 170 bp. The gel electrophoresis pattern of the PCR product is shown in Figure 6 。

[0080] PCR was performed on the sheep blood DNA obtained in Example 2. The PCR program was as follows: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 51°C for 30 seconds, extension at 72°C for 20 seconds, for 40 cycles. The mitochondrial gene fragment of sheep was amplified. The primers were YX-F: 5’-TTTGACTAAGCCATGTTGAC-3’; YX-R: 5’-TTAACTCTATTTGATGTAGTGC-3’. The product size was 124 bp. The gel electrophoresis pattern of the PCR product is shown in Figure 7 .

[0081] PCR was performed on the chicken blood DNA obtained in Example 2. The PCR program was as follows: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 57°C for 30 seconds, extension at 72°C for 20 seconds, for 40 cycles. The nuclear gene fragment of chicken was amplified. The primers were JH-F: 5’-ACAGATAAATGGCTTCCACACT-3’; JH-R: 5’-ATTTATTGTGGTCTGCAGGTGG-3’. The product size was 132 bp. The gel electrophoresis pattern of the PCR product is shown in Figure 8 .

[0082] PCR was performed on the duck blood DNA obtained in Example 2. The PCR program was as follows: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 59°C for 30 seconds, extension at 72°C for 20 seconds, for 40 cycles. The nuclear gene fragment of duck was amplified. The primers were YaH-F: 5’-AGATAAACGGCCTCCACAGA-3’; YaH-R: 5’-ATTTATTGTGGTCTGCAGGTGG-3’. The product size was 138 bp. The gel electrophoresis pattern of the PCR product is shown in Figure 9 .

[0083] PCR was performed on the donkey blood DNA obtained in Example 2. The PCR program was as follows: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 20 seconds, for 40 cycles. The mitochondrial gene fragment of donkey was amplified. The primers were LvX-F: 5’-AGCGTTCAAGCTCAACGTC-3’; LvX-R: 5’-TGAGTATTCGTTCTGATATAGGC-3’. The product size was 119 bp. The gel electrophoresis pattern of the PCR product is shown in Figure 10 ;

[0084] The method flow of extracting the turtle blood DNA in the turtle blood Bupleuri Radix tablet of the present invention is as shown in Figure 11 shown.

[0085] Although embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for rapidly extracting turtle blood DNA from turtle blood and bupleurum slices, characterized in that: The following steps are involved: The turtle blood bupleurum sample is soaked in the soaking liquid to wash the turtle blood from the turtle blood bupleurum bupleurum, and the phenolic and polysaccharide plant components in the sample are removed to obtain the turtle blood bupleurum soaking liquid; The obtained turtle blood and bupleurum soaking liquid is subjected to low-speed centrifugation to remove granular impurities in the soaking liquid, and then subjected to high-speed centrifugation to obtain a precipitate containing turtle blood components; The DNA in the precipitate of the turtle blood component is extracted by using an improved blood DNA extracting solution to obtain turtle blood DNA.

2. A method for rapidly extracting turtle blood DNA from turtle blood and bupleurum slices according to claim 1, characterized in that: The method for soaking the turtle blood bupleurum sample is: take 1g of turtle blood bupleurum and soak it in 3mL of soaking solution, and manually shake and mix for 20 minutes to dissolve the turtle blood in the turtle blood bupleurum into the soaking solution.

3. A method for rapidly extracting turtle blood DNA from turtle blood and bupleurum slices according to claim 1, characterized in that: The turtle blood and bupleurum immersion solution was first centrifuged at 1000 rpm for 1 min to obtain a supernatant and a precipitate layer; The supernatant was transferred to a new centrifuge tube and then centrifuged at 12000 rpm for 5 min to obtain a precipitate containing turtle blood components.

4. A method for rapidly extracting turtle blood DNA from turtle blood and bupleurum slices according to claim 3, characterized in that: Add 200 μL of the preheated improved blood DNA extraction solution to the precipitate containing turtle blood components, and place it in a 56° C. water bath for 30 minutes, shaking it every 10 minutes to fully release the DNA in the precipitate, thereby obtaining the turtle blood DNA.

5. A method for rapidly extracting turtle blood DNA from turtle blood and bupleurum slices according to claim 1, characterized in that: The turtle blood bupleurum immersion solution contains ascorbic acid, polyvinyl pyrrolidone and Tris-HCl.

6. A method for rapidly extracting turtle blood DNA from turtle blood and bupleurum slices according to claim 1, characterized in that: The DNA extraction solution contains Chelex100, polyvinyl pyrrolidone and proteinase K.

7. Application of the method for rapidly extracting turtle blood DNA from turtle blood and bupleurum slices according to any one of claims 1 to 6 in extracting blood DNA from animal blood processed bupleurum, comprising: Isolation and identification of pig blood DNA from processed pig blood Bupleurum chinense; Isolation and identification of bovine blood DNA from processed bovine blood Bupleurum chinense; Separation and identification of sheep blood DNA from processed sheep blood Bupleurum chinense; Isolation and identification of chicken blood DNA from processed chicken blood Bupleurum chinense; Isolation and identification of duck blood DNA from processed duck blood Bupleurum chinense; Isolation and identification of donkey blood DNA from processed donkey blood Bupleurum chinense.

8. Use of turtle blood DNA prepared by the method for rapidly extracting turtle blood DNA from turtle blood and bupleurum slices according to any one of claims 1 to 6 in mitochondrial gene amplification, characterized in that: The extracted DNA was used as a template, and the forward and reverse primers were selected to configure the PCR system. The conditions of 94°C pre-denaturation for 3 min, 94°C denaturation for 30 s, 53.5°C annealing for 30 s, 72°C extension for 20 s, and 40 amplification cycles were set to amplify the mitochondrial gene BX.

9. The use according to claim 8, characterized in that: The PCR system is as follows: 20 μL of total reaction system, 2 μL of genomic DNA with a concentration of 3-7 ng / μL, 0.5 μL of upstream primer and downstream primer respectively, 10 μL of 2×Es Taq Master Mix, and 7 μL of ultrapure water.

10. The use according to claim 9, The invention is characterized in that: The upstream primer is BX-F: 5'-TACTACCTTTTTTGATCCTTCT-3', The downstream primer is BX-R: 5'-ATTAGCATAGTACGTTACTACA-3'; The characteristic band obtained by primer amplification of Turtle Blood Bupleurum was 147bp.

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