Specific primer pair for identifying turtle blood radix bupleuri and application of specific primer pair

Abstract

The invention discloses a specific primer pair for identifying authenticity of turtle blood radix bupleuri and application of the specific primer pair. The specific primer B-ACT can be amplified in DNA (deoxyribonucleic acid) extracted from the radix bupleuri in the turtle blood to obtain a 167bp target fragment, the primer B-HBA can be amplified in DNA extracted from the radix bupleuri in the turtle blood to obtain a 113bp target fragment, the primer B-COI can be amplified in DNA extracted from the radix bupleuri in the turtle blood to obtain a 142bp target fragment, and the primer B-CYT can be amplified in DNA extracted from the radix bupleuri in the turtle blood to obtain a 147bp target fragment. The PCR method can accurately detect whether the turtle blood radix bupleuri contains turtle blood components or not. The identification method is accurate and rapid, and has higher specificity in the aspect of identifying counterfeit species of turtle blood radix bupleuri and common animal blood processed radix bupleuri.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and mainly relates to a specific primer pair for identifying whether turtle blood components are contained in the Chinese herbal medicine decoction pieces - turtle blood Bupleuri Radix, and its application. Background Art

[0002] The turtle blood Bupleuri Radix has been detailedly recorded in "Ben Cao Hai Li": "If using Bupleuri Radix and adding a few spoons of turtle blood, it will not over-activate the superficies." It is recorded in "Leng Lu Yi Hua" in the Qing Dynasty that "stir-frying Bupleuri Radix with turtle blood" soothes the liver without depleting the liver yin; it is also recorded in "Chang Sha Fang Ge Kuo" in the Qing Dynasty about the processing method of "mixing and steaming with turtle blood"; it has also been detailedly recorded in the Chinese Pharmacopoeia 1963 edition: "For turtle blood Bupleuri Radix, take Bupleuri Radix slices, place them in a large basin, pour in turtle blood diluted with a little warm water, mix well, moisten, stir-fry gently over a slow fire in a pan, take out, and let it cool." In local processing, there are slight differences in the processing method of turtle blood Bupleuri Radix. For example, in Zhejiang and Jiangsu, it is always mixed and steamed with turtle blood. Take Bupleuri Radix turtle blood slices, mix well with turtle blood, yellow rice wine or clear water, slightly moisten, place in a pan, heat over a slow fire, stir-fry until dry, take out and let it cool. For every 100 kg of Bupleuri Radix slices, use 12.5 kg of turtle blood and 12.5 kg of yellow rice wine.

[0003] As a traditional Chinese medicine compound combining animal medicine and plant medicine, turtle blood Bupleuri Radix has significant medicinal value and is widely used in clinical treatment of various diseases. Turtle blood Bupleuri Radix is composed of two medicinal materials, turtle blood and Bupleuri Radix. Turtle blood, taken from the blood of turtles (soft-shelled turtles), is traditionally considered to have the effects of nourishing yin, clearing heat, and cooling blood, and is commonly used to treat symptoms such as hyperactivity of fire due to yin deficiency and hematemesis due to blood heat. Bupleuri Radix is the dried root of the Bupleurum chinense DC. of the Umbelliferae family, and has the effects of soothing the liver and relieving depression, and expelling pathogenic factors and relieving exterior syndrome. It is a commonly used drug for treating stagnation of liver qi and exterior syndrome of fever. Combining Bupleuri Radix with turtle blood not only enhances the effect of Bupleuri Radix in soothing the liver and relieving depression, but also endows it with the effect of nourishing yin and blood, forming a unique combination of medicinal effects.

[0004] In traditional Chinese medicine theory, the liver is in charge of dredging and regulating qi movement. Stagnation of liver qi often leads to symptoms such as distending pain in the chest and hypochondrium and emotional depression. As a representative drug for soothing the liver and relieving depression, Bupleuri Radix is widely used in the treatment of diseases related to stagnation of liver qi. However, simply using Bupleuri Radix may not be sufficient to deal with complex pathological conditions in some cases, especially for patients with symptoms of hyperactivity of fire due to yin deficiency or blood heat. The addition of turtle blood not only makes up for the deficiency of Bupleuri Radix in nourishing yin and blood, but also enhances the efficacy of the overall prescription through its characteristics of clearing heat and cooling blood.

[0005] In recent years, there has been a phenomenon in the market of using the blood of other animals (such as pigs, cows, sheep, chickens, ducks, rabbits, donkeys, etc.) to replace turtle blood in the processing of Bupleuri Radix Praeparatus Cum Sanguine Trionycis. The adulteration of Bupleuri Radix Praeparatus Cum Sanguine Trionycis not only reduces the efficacy of the medicinal material, but also may introduce potential safety hazards. There are significant differences in the active ingredients of the blood of different animals compared with turtle blood. After adulteration, the effects of soothing the liver and relieving depression, nourishing yin and blood of Bupleuri Radix Praeparatus Cum Sanguine Trionycis are greatly reduced, affecting the clinical treatment effect. In addition, the blood of other animals may carry different pathogens, which may cause new health risks after adulteration. More seriously, the frequent occurrence of adulteration will damage consumers' trust in the Chinese herbal medicine market, and thus affect the healthy development of the entire industry.

[0006] Polymerase chain reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments, that is, a specific in vitro amplification process of DNA fragments. Its specificity depends on oligonucleotide primers complementary to both ends of the target DNA fragment. The basic principle of PCR is that double-stranded DNA denatures and unwinds into single-stranded DNA at high temperature, and then can renature into double-stranded DNA when the temperature decreases. By controlling the denaturation and renaturation of DNA through temperature changes, adding primers, DNA polymerase, deoxynucleoside triphosphate (dNTP) and the corresponding buffer solution, the in vitro amplification of specific DNA fragments is completed. By designing species-specific primers based on single nucleotide polymorphisms (SNP) sites, PCR reaction is carried out to amplify the target band for species identification, which has the advantages of high specificity, fast analysis speed and accurate results. At present, this method has been included in the Chinese Pharmacopoeia (2020 Edition), and this technology has been used for species identification of animal- and plant-derived Chinese herbal medicines and decoction pieces, raw materials, intermediates, bulk drugs and excipients, etc., and can also be used for the determination of characteristic DNA fragments in the quality control of other drugs.

[0007] Regarding the scientific identification methods for animal blood excipients in Bupleuri Radix Praeparatus Cum Sanguine Trionycis, there are currently various detection techniques and methods, such as DNA barcoding technology, proteomics analysis, spectroscopic techniques (near-infrared spectroscopy and Raman spectroscopy), and chemical fingerprinting (high-performance liquid chromatography and gas chromatography-mass spectrometry). However, the above techniques all have problems such as slow detection speed, high detection cost, complex operation and insufficient standardization. Therefore, it has become an urgent task to establish a molecular biological characteristic identification method for turtle blood products using high technology. In order to solve the problem of identifying whether Bupleuri Radix Praeparatus Cum Sanguine Trionycis is processed with turtle blood, the present invention designs a specific primer pair for turtle blood DNA, and can identify Bupleuri Radix Praeparatus Cum Sanguine Trionycis from Bupleuri Radix Praeparatus Cum Sanguine Sus, Bovis, Ovis, Gallus, Anas, Lepus, and Equus Asinus by PCR amplification. The identification of the present invention has the advantages of low cost, fast speed and high efficiency, and has important application value. Summary of the Invention

[0008] The object of the present invention is to overcome the limitations of morphological feature analysis, physicochemical identification and microscopic identification methods in the prior art, and to propose a method for identifying the DNA of turtle blood based on turtle blood Bupleurum samples. Compared with traditional identification means, the molecular biology technology adopted by the present invention is more efficient, accurate and objective.

[0009] To solve the above technical problems, the present invention adopts the following technical solutions:

[0010] Specific primers for amplifying the nuclear gene β-actin are designed for turtle blood DNA:

[0011] B-ACT forward primer: 5’-CCATCTTGGCGTCTCTCTCT-3’,

[0012] B-ACT reverse primer: 5’-TCCAGTTTTATGAGGCTAGCAT-3’

[0013] Specifically, the amplification product of the above primers is 167bp, and the PCR amplification program is: 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 35 cycles; incubation at 72°C for 5 minutes.

[0014] Specific primers for amplifying the nuclear gene HBA are designed for turtle blood DNA:

[0015] B-HBA forward primer: 5’-CAAGGCAAGAAGGTGCTGAG-3’,

[0016] B-HBA reverse primer: 5’-GGATCCACACGCAGGTTCTT-3’

[0017] Specifically, the amplification product of the above primers is 113bp, and the PCR amplification program is: 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 35 cycles; incubation at 72°C for 5 minutes.

[0018] Specific primers for amplifying the mitochondrial gene COⅠ are designed for turtle blood DNA:

[0019] B-COⅠ forward primer for amplifying the nuclear gene: 5’-TACTACCTTTTTTGATCCTTCT-3’

[0020] B-COⅠ reverse primer for amplifying the nuclear gene: 5’-ATTAGCATAGTACGTTACTACA-3’

[0021] Specifically, the above primer amplification product is 142 bp, and the PCR amplification procedure is 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 35 cycles; incubation at 72°C for 5 minutes.

[0022] For soft-shelled turtle blood DNA, specific primers for amplifying mitochondrial gene CYT were designed:

[0023] B-CYT forward primer: 5’-TCACTAATTGACCTACCAAGT-3’,

[0024] B-CYT reverse primer: 5’-TGACGAGAATGCTGTTGAGA-3’

[0025] Specifically, the above primer amplification product is 147 bp, and the PCR amplification procedure is as follows: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 54°C for 30 seconds, extension at 72°C for 20 seconds, for 35 cycles; incubation at 72°C for 5 minutes.

[0026] A method for identifying whether soft-shelled turtle blood Bupleurum contains soft-shelled turtle blood components uses the above primers to detect samples, and the specific steps include:

[0027] Extract the genomic DNA of the sample to be tested, and use any pair of the above specific primers to perform PCR amplification on the DNA of the sample to be tested to obtain an amplification product.

[0028] Perform gel electrophoresis detection on the amplification product, and determine whether the sample to be tested contains soft-shelled turtle blood according to the detection results in the following manner:

[0029] Perform PCR amplification on the DNA of the sample to be tested with the specific primer B-ACT. If the amplification product has a single DNA band between 100 - 250 bp (167 bp), then the sample to be tested contains soft-shelled turtle blood components; if the amplification product does not have a single DNA band between 100 - 250 bp (167 bp), then the sample to be tested does not contain soft-shelled turtle blood components.

[0030] Perform PCR amplification on the DNA of the sample to be tested with the specific primer B-HBA. If the amplification product has a single DNA band between 100 - 250 bp (113 bp), then the sample to be tested contains soft-shelled turtle blood components; if the amplification product does not have a single DNA band between 100 - 250 bp (113 bp), then the sample to be tested does not contain soft-shelled turtle blood components.

[0031] Use the specific primer B-COⅠ to perform PCR amplification on the DNA of the sample to be tested. If the amplification product has a single DNA band between 100 and 250 bp (142 bp), then the sample to be tested contains turtle blood components; if the amplification product does not have a single DNA band between 100 and 250 bp (142 bp), then the sample to be tested does not contain turtle blood components.

[0032] Use the specific primer B-CYT to perform PCR amplification on the DNA of the sample to be tested. If the amplification product has a single DNA band between 100 and 250 bp (147 bp), then the sample to be tested contains turtle blood components; if the amplification product does not have a single DNA band between 100 and 250 bp (147 bp), then the sample to be tested does not contain turtle blood components.

[0033] The application of this technology in any of the following:

[0034] (1) Application in the identification or auxiliary identification of turtle blood and common counterfeits of turtle blood, and the common counterfeits of turtle blood are pig blood, sheep blood, cattle blood, chicken blood, duck blood, rabbit blood and donkey blood;

[0035] (2) Application in the products for identifying or auxiliary identifying turtle blood and common counterfeits of turtle blood in the processed Bupleurum with animal blood, and the common counterfeits of turtle blood in Bupleurum are pig blood in Bupleurum, sheep blood in Bupleurum, cattle blood in Bupleurum, chicken blood in Bupleurum, duck blood in Bupleurum, rabbit blood in Bupleurum and donkey blood in Bupleurum.

[0036] (3) Application in the preparation of products for identifying or auxiliary identifying whether the sample to be tested contains turtle blood; Description of the Drawings

[0037] Figure 1 It is the gel electrophoresis diagram of the DNA of various animal blood amplified by the B-ACT primer in Example 1 of the present invention. In the figure, lane M: DL2000 Marker; 1: turtle blood DNA; 2: pig blood DNA; 3: cattle blood DNA; 4: sheep blood DNA; 5: chicken blood DNA; 6: duck blood DNA; 7: rabbit blood DNA; 8: donkey blood DNA.

[0038] Figure 2 It is the gel electrophoresis diagram of the DNA of various animal blood amplified by the B-HBA primer in Example 1 of the present invention. In the figure, lane M: DL2000 Marker; 1: turtle blood DNA; 2: pig blood DNA; 3: cattle blood DNA; 4: sheep blood DNA; 5: chicken blood DNA; 6: duck blood DNA; 7: rabbit blood DNA; 8: donkey blood DNA.

[0039] Figure 3It is the gel electrophoresis diagram of amplifying the DNA of various animal blood with the B-COⅠ primer in Example 1 of the present invention. In the figure, lane M: DL2000 Marker; 1: soft-shelled turtle blood DNA; 2: pig blood DNA; 3: cattle blood DNA; 4: sheep blood DNA; 5: chicken blood DNA; 6: duck blood DNA; 7: rabbit blood DNA; 8: donkey blood DNA.

[0040] Figure 4 It is the gel electrophoresis diagram of amplifying the DNA of various animal blood with the B-CYT primer in Example 1 of the present invention. In the figure, lane M: DL2000 Marker; 1: soft-shelled turtle blood DNA; 2: pig blood DNA; 3: cattle blood DNA; 4: sheep blood DNA; 5: chicken blood DNA; 6: duck blood DNA; 7: rabbit blood DNA; 8: donkey blood DNA.

[0041] Figure 5 It is the gel electrophoresis diagram of amplifying B-ACT after gradient dilution of soft-shelled turtle blood DNA in Example 2. In the figure, lane M: DL2000 Marker; the added amounts of soft-shelled turtle blood DNA in lanes 1-5 are 1: 50 ng; 2: 25 ng; 3: 10 ng; 4: 5 ng; 5: 2 ng respectively.

[0042] Figure 6 It is the gel electrophoresis diagram of amplifying B-HBA after gradient dilution of soft-shelled turtle blood DNA in Example 2. In the figure, lane M: DL2000 Marker; the added amounts of soft-shelled turtle blood DNA in lanes 1-5 are 1: 50 ng; 2: 25 ng; 3: 10 ng; 4: 5 ng; 5: 2 ng respectively.

[0043] Figure 7 It is the gel electrophoresis diagram of amplifying B-COⅠ after gradient dilution of soft-shelled turtle blood DNA in Example 2. In the figure, lane M: DL2000 Marker; the added amounts of soft-shelled turtle blood DNA in lanes 1-5 are 1: 50 ng; 2: 25 ng; 3: 10 ng; 4: 5 ng; 5: 2 ng respectively.

[0044] Figure 8 It is the gel electrophoresis diagram of amplifying B-CYT after gradient dilution of soft-shelled turtle blood DNA in Example 2. In the figure, lane M: DL2000 Marker; the added amounts of soft-shelled turtle blood DNA in lanes 1-5 are 1: 50 ng; 2: 25 ng; 3: 10 ng; 4: 5 ng; 5: 2 ng respectively.

[0045] Figure 9It is the gel electrophoresis diagram of DNA extracted from Bupleurum processed with various animal blood amplified by B-ACT primer in Example 3 of the present invention. In the figure, lane M: DL2000 Marker; 1: DNA of Bupleurum processed with turtle blood; 2: DNA of Bupleurum processed with pig blood; 3: DNA of Bupleurum processed with cattle blood; 4: DNA of Bupleurum processed with sheep blood; 5: DNA of Bupleurum processed with chicken blood; 6: DNA of Bupleurum processed with duck blood; 7: DNA of Bupleurum processed with rabbit blood; 8: DNA of Bupleurum processed with donkey blood.

[0046] Figure 10 It is the gel electrophoresis diagram of DNA extracted from Bupleurum processed with various animal blood amplified by B-HBA primer in Example 3 of the present invention. In the figure, lane M: DL2000 Marker; 1: DNA of Bupleurum processed with turtle blood; 2: DNA of Bupleurum processed with pig blood; 3: DNA of Bupleurum processed with cattle blood; 4: DNA of Bupleurum processed with sheep blood; 5: DNA of Bupleurum processed with chicken blood; 6: DNA of Bupleurum processed with duck blood; 7: DNA of Bupleurum processed with rabbit blood; 8: DNA of Bupleurum processed with donkey blood.

[0047] Figure 11 It is the gel electrophoresis diagram of DNA extracted from Bupleurum processed with various animal blood amplified by B-COⅠ primer in Example 3 of the present invention. In the figure, lane M: DL2000 Marker; 1: DNA of Bupleurum processed with turtle blood; 2: DNA of Bupleurum processed with pig blood; 3: DNA of Bupleurum processed with cattle blood; 4: DNA of Bupleurum processed with sheep blood; 5: DNA of Bupleurum processed with chicken blood; 6: DNA of Bupleurum processed with duck blood; 7: DNA of Bupleurum processed with rabbit blood; 8: DNA of Bupleurum processed with donkey blood.

[0048] Figure 12 It is the gel electrophoresis diagram of DNA extracted from Bupleurum processed with various animal blood amplified by B-CYT primer in Example 3 of the present invention. In the figure, lane M: DL2000 Marker; 1: DNA of Bupleurum processed with donkey blood; 2: DNA of Bupleurum processed with rabbit blood; 3: DNA of Bupleurum processed with duck blood; 4: DNA of Bupleurum processed with chicken blood; 5: DNA of Bupleurum processed with sheep blood; 6: DNA of Bupleurum processed with cattle blood; 7: DNA of Bupleurum processed with pig blood; 8: DNA of Bupleurum processed with turtle blood. Detailed implementation methods

[0049] Combined with the accompanying drawings in the embodiments of the present invention, the technical solutions of the present invention will be further described in detail below. The following described embodiments are only partial embodiments of the present invention, rather than all embodiments. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0050] The experimental methods in the following implementation cases are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0051] Example 1 Design and verification of DNA identification primers for turtle blood

[0052] I. Design of Specific Primers for Nuclear Genes of Soft-Shelled Turtle Blood

[0053] First, download the β-actin and HBA gene sequences of several animals including soft-shelled turtle, pig, cow, sheep, chicken, duck, rabbit, and donkey from NCBI, and use MEGA11 software for Clustal W multiple sequence alignment to analyze the differential SNP (Single Nucleotide Polymorphisms) sites. SNP refers to the DNA sequence diversity caused by the variation of a single nucleotide at the genomic level. By designing species-specific primers based on SNP sites and performing PCR reactions to amplify the target bands for species identification, it has the advantages of high specificity, fast analysis speed, and accurate results.

[0054] Taking the nucleic acid sequence of soft-shelled turtle β-actin in Genbank (accession number: XM_006134860.1) as a reference, through alignment and analysis with the corresponding genes of other animals, it is found that there are 5 specific SNP sites between nucleotides 1047 and 1066, which can be selected as the upstream primer; there are 6 specific SNP sites between nucleotides 1193 and 1214, which can be selected as the downstream primer.

[0055] Taking the nucleic acid sequence of soft-shelled turtle HBA in Genbank (accession number: NW_005855496.1) as a reference, through alignment and analysis with the corresponding genes of other animals, it is found that there are 5 specific SNP sites between nucleotides 408 and 427, which are selected as the upstream primer; there are 3 specific SNP sites between nucleotides 501 and 520, which are selected as the downstream primer.

[0056] Align the mRNA sequences of β-actin and HBA genes of soft-shelled turtle with the corresponding genomic β-actin and HBA DNA sequences, and design specific primers in combination with SNP differential sites so that the amplified fragments of the upstream and downstream primers are in one exon.

[0057] Design the upstream and downstream primers for soft-shelled turtle β-actin according to the above conditions as follows: B-ACT-F: 5’-CCATCTTGGCGTCTCTCTCT-3’, B-ACT-R: 5’-TCCAGTTTTATGAGGCTAGCAT-3’. The amplified product is 167bp. Design the upstream and downstream primers for soft-shelled turtle HBA as follows: B-HBA-F: 5’-CAAGGCAAGAAGGTGCTGAG-3’, B-HBA-R: 5'-GGATCCACACGCAGGTTCTT-3', and the amplification product is 113 bp.

[0058] II. Design of Specific Primers for Soft-Shelled Turtle Blood Mitochondrial Genes

[0059] Download the mitochondrial genome DNA sequences of several animals including soft-shelled turtle, pig, cow, sheep, chicken, duck, rabbit, and donkey from NCBI, and use MEGA11 for Clustal W multiple sequence alignment to analyze the differential SNP sites.

[0060] Taking the soft-shelled turtle mitochondrial DNA sequence (accession number: AY687385.1) in Genbank as a reference, it was found through analysis that there are 3 specific SNP sites between nucleotides 6060 and 6081 within the mitochondrial genes COⅠ and CYT gene regions, which can be selected as the upstream primer for COⅠ; there are also 4 specific SNP sites between nucleotides 6180 and 6201, which can be selected as the downstream primer for COⅠ. There are 4 specific SNP sites between nucleotides 14284 and 14304, which are selected as the upstream primer for CYT; there are also 4 specific SNP sites between nucleotides 14411 and 14430, which are selected as the downstream primer for CYT.

[0061] Design the soft-shelled turtle COⅠ primers according to the above conditions as follows: B-COⅠ-F: 5'-TACTACCTTTTTTGATCCTTCT-3', B-COⅠ-R: 5'-ATTAGCATAGTACGTTACTACA-3', and the amplification product is 142 bp. Design the soft-shelled turtle CYT primers as follows: B-CYT-F: 5'-TCACTAATTGACCTACCAAGT-3', B-CYT-R: 5'-TGACGAGAATGCTGTTGAGA-3', and the amplification product is 147 bp.

[0062] III. Primer Specificity Detection

[0063] Extract the DNA of soft-shelled turtle blood, pig blood, cow blood, sheep blood, chicken blood, duck blood, rabbit blood, and donkey blood using a blood extraction kit.

[0064] Perform PCR amplification on the blood DNA of 8 animals using the four pairs of designed primers.

[0065] The total volume of the PCR reaction system is 20 μl, including 10 μl of 2×PCRMix, 0.5 μl each of the upstream and downstream primers (10 μmol / L), 1 μl of DNA template, and made up to 20 μl with sterile double-distilled water.

[0066] Primers B-ACT and B-HBA, and the PCR amplification procedure 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 35 cycles; incubation at 72°C for 5 minutes.

[0067] Primer B-COⅠ, and the PCR amplification procedure 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 35 cycles; incubation at 72°C for 5 minutes.

[0068] Primer B-CYT, and the PCR amplification procedure was as follows: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 54°C for 30 seconds, extension at 72°C for 20 seconds, for 35 cycles; incubation at 72°C for 5 minutes.

[0069] After the amplification results were subjected to gel electrophoresis as Figures 1-4 The results showed that when the four primers were used for amplification in various blood DNAs, a single bright target band was amplified only in soft-shelled turtle blood DNA, and no amplification occurred in other animal blood DNAs.

[0070] When the amplification conditions of the four primers were increased to 40 cycles, non-specific bands could be amplified in other animal bloods by the two primers B-HBA and B-CYT.

[0071] Example 2 Sensitivity Detection of Specific Primers B-HBA, B-ACT, B-CYT and B-COⅠ

[0072] The initial concentration of soft-shelled turtle blood DNA was adjusted to 10 ng / μL.

[0073] The amount of template DNA in the PCR reaction system was adjusted. In a 20-μL PCR reaction system, the DNA addition amounts were set to 50 ng, 25 ng, 10 ng, 5 ng, and 2 ng respectively.

[0074] The DNA amounts in a 20-μL PCR reaction system were 5.0, 2.5, 1.0, 0.5, and 0.2 μL respectively.

[0075] Four pairs of specific primers B-HBA, B-ACT, B-CYT and B-COⅠ were used to amplify the gradient-diluted soft-shelled turtle blood DNA. The amplification system and PCR procedure were the same as in Example 1.

[0076] The amplification products were subjected to agarose gel electrophoresis, and the results showed that:

[0077] B-HBA successfully amplified a target gene fragment with a size of 113 bp in soft-shelled turtle blood DNA at different concentrations. As the concentration decreased, the brightness of the amplified target band changed little, indicating that primer B-HBA had high sensitivity.

[0078] The target gene fragment with a size of 167 bp was successfully amplified from turtle blood DNA at different concentrations. As the concentration decreased, the amplified target band became lighter. When the content of turtle blood DNA in the PCR system was less than 5 ng, the deficiency in content was identified by this primer.

[0079] B-CYT could successfully amplify a target fragment with a size of 147 bp from turtle blood DNA at different concentrations, and the brightness of the band did not change with the change of DNA concentration, indicating that the primer B-CYT had high sensitivity.

[0080] B-COⅠ could successfully amplify a target fragment with a size of 142 bp from turtle blood DNA at different concentrations, and the brightness of the band did not change with the change of DNA concentration, indicating that the primer B-COⅠ had high sensitivity.

[0081] Example 3 Design the application of turtle blood primers in identifying turtle blood Bupleuri Radix.

[0082] I. DNA Extraction Reagents and Materials

[0083] Soaked solution of turtle blood Bupleuri Radix: Ascorbic acid 0.09 g / L, Tris-HCl 0.1 mol / L, Polyvinylpyrrolidone 0.1 g / L.

[0084] DNA extraction buffer: CTAB 0.1 g / L, Tris-HCl 0.1 mol / L, Polyvinylpyrrolidone 0.1 g / L. Sodium chloride 0.41 g / L, EDTA 2.86 g / L, Polyvinylpyrrolidone 0.01 g / L.

[0085] Chloroform:isoamyl alcohol mixed solution: Prepared according to the volume ratio of 24:1.

[0086] 75% (v / v) ethanol solution: Mix absolute ethanol and double-distilled water according to the volume ratio of 3:1

[0087] Proteinase K solution: The storage solution contains proteinase K at a concentration of 20 mg / ml, and the working concentration of proteinase K is 400 μg / mL

[0088] Eight kinds of animal blood processed Bupleuri Radix, such as turtle blood Bupleuri Radix, pig blood Bupleuri Radix, cattle blood Bupleuri Radix, sheep blood Bupleuri Radix, chicken blood Bupleuri Radix, goat blood Bupleuri Radix, rabbit blood Bupleuri Radix and donkey blood Bupleuri Radix, were provided by Jiangsu Chengkai Traditional Chinese Medicine Co., Ltd.

[0089] II. Sampling and Improved CTAB Method to Extract DNA of Various Animal Blood in Bupleuri Radix

[0090] S1 Take 1 g of animal blood processed Bupleuri Radix in a 5 mL centrifuge tube, add 3 mL of soaked buffer solution of turtle blood Bupleuri Radix, shake for 20 minutes, and wash out the turtle blood components in the turtle blood Bupleuri Radix;

[0091] S2 Transfer 1.5 mL of the soaking solution into a centrifuge tube, centrifuge at 12,000 rpm for 10 minutes, discard the supernatant, and retain the precipitate.

[0092] S3 Add CTAB buffer and proteinase K to the precipitate to extract DNA, and incubate in a water bath at 56 °C for 30 minutes.

[0093] S4 After cooling, add an equal volume of a mixture of chloroform and isoamyl alcohol (24:1), shake well, and centrifuge at 12,000 rpm for 10 minutes.

[0094] S5 Pipette the upper aqueous phase, transfer it to a new centrifuge tube, add an equal volume of isopropanol, mix well by shaking, place it in a refrigerator at 4 °C for 10 - 20 minutes to precipitate nucleic acids, then centrifuge at 12,000 rpm for 10 minutes, and discard the supernatant.

[0095] S6 Add 1 mL of 75% ethanol to the precipitate, gently pipette to wash the precipitate, centrifuge at 12,000 rpm for 5 minutes, discard the supernatant, and air-dry at room temperature.

[0096] S7 Add 30 μL of ultrapure water to the obtained precipitate to dissolve the DNA, and thus obtain the turtle blood DNA solution in turtle blood-treated Bupleurum chinense DC., and this DNA can be used for subsequent gene amplification.

[0097] III. Verification of specific primers in Bupleurum chinense DC. processed with different animal blood

[0098] Perform PCR amplification on the DNA of Bupleurum chinense DC. processed with 8 kinds of animal blood using four pairs of primers. The PCR amplification system and the PCR program for each primer are the same as in Example 1.

[0099] After the amplification results are subjected to gel electrophoresis as Figures 9-12 shown, B-ACT, B-HBA, B-CYT, and B-COⅠ can only amplify a single target band in the DNA of turtle blood-treated Bupleurum chinense DC., and there are no amplification bands in Bupleurum chinense DC. processed with other animal blood. After multiple experimental verifications, it is found that in the DNA of turtle blood-treated Bupleurum chinense DC. extracted in different batches, the two pairs of nuclear gene discrimination primers B-ACT and B-HBA cannot amplify the target band in some cases. Relatively speaking, the amplification effect of the two pairs of mitochondrial gene discrimination primers B-CYT and B-COⅠ is more stable. This may be due to the fact that the copy number of the mitochondrial genome is much more than that of the nuclear genome.

[0100] In the previous research work, multiple pairs of primers were designed for the SNP sites of four genes, namely B-HBA, B-ACT, B-CYT, and B-COⅠ in soft-shelled turtle. The results showed that it was difficult to obtain the target bands by amplification in the DNA of Bupleuri Radix Praeparata Cum Sanguine Soft-Shelled Turtle when the amplification products exceeded 180 bp. It was speculated that during the processing of Bupleuri Radix Praeparata Cum Sanguine Soft-Shelled Turtle, there was a step of stir-frying at high temperature, which caused the DNA to break, and there were few long DNA fragments larger than 200 bp in the DNA of Bupleuri Radix Praeparata Cum Sanguine Soft-Shelled Turtle. Therefore, the amplification products of the four pairs of primers designed in this invention are all between 110 - 170 bp.

Claims

1. A method for identifying whether there is turtle blood component in Bupleuri Radix Praeparata Cum Sanguine Testudinis, characterized in that, Include two pairs of specific primers for amplifying nuclear genes, namely B-HBAD and B-ACT, and two pairs of specific primers for amplifying mitochondria, namely B-16S and B-CYT.

2. The primer B-ACT according to claim 1, characterized in that: The forward primer is B-ACT-F: 5’-CCATCTTGGCGTCTCTCTCT-3’, The reverse primer is B-ACT-R: 5’-TCCAGTTTTATGAGGCTAGCAT-3’; The characteristic band obtained by amplifying soft-shelled turtle blood DNA with the primer is 167bp.

3. The primer B-HBA according to claim 1, characterized in that: The forward primer is B-HBA-F: 5’-GTTCATGGAAGCTGTCTGCAA-3’, The reverse primer is B-HBA-R: 5’-GTTGGCATCTTTATTGGGTCT-3’; The characteristic band obtained by amplifying soft-shelled turtle blood DNA with the primer is 113bp.

4. The primer B-COⅠ according to claim 1, characterized in that: The forward primer is B-COⅠ-F: 5’-TGAAACATACCCACCAAACCA-3’, The reverse primer is B-COⅠ-R: 5’-CGCCCGTACTAGGATTAATGAG-3’; The characteristic band obtained by amplifying soft-shelled turtle blood DNA with the primer is 142bp.

5. The primer B-CYT according to claim 1, characterized in that: The forward primer is B-CYT-F: 5’-TACTACCTTTTTTGATCCTTCT-3’, The reverse primer is B-CYT-R: 5’-TACTACCTTTTTTGATCCTTCT-3’; The characteristic band obtained by amplifying soft-shelled turtle blood DNA with the primer is 147bp.

6. The method according to claim 1, wherein Include the following steps: (1) Extract DNA of the sample to be tested (2) Use the primer pair described in claim 1 to perform PCR amplification on the extracted DNA, perform agarose gel electrophoresis on the PCR product, and observe, photograph and save it in a gel imager; (3) Analyze according to the situation of the gel electrophoresis amplification product obtained in step (2). If the amplification product is a single target band, it can be judged that there is soft-shelled turtle blood component in the sample to be tested; when there is no target band in the amplification product, it can be judged that there is no soft-shelled turtle blood component in the tested sample.

7. For the primer pairs described in claim 2 and claim 3, the PCR amplification program is: pre-denaturation at 94℃ for 3 minutes; denaturation at 94℃ for 30 seconds, annealing at 55℃ for 30 seconds, extension at 72℃ for 20 seconds, 35 cycles; incubation at 72℃ for 5 minutes.

8. For the primer pair described in claim 4, the PCR amplification program is: pre-denaturation at 94℃ for 3 minutes; denaturation at 94℃ for 30 seconds, annealing at 57℃ for 30 seconds, extension at 72℃ for 20 seconds, 35 cycles; incubation at 72℃ for 5 minutes.

9. For the primer pair described in claim 5, the PCR amplification program is: pre-denaturation at 94℃ for 3 minutes; denaturation at 94℃ for 30 seconds, annealing at 54℃ for 30 seconds, extension at 72℃ for 20 seconds, 35 cycles; incubation at 72℃ for 5 minutes.

10. Use of the specific primer pair for soft-shelled turtle blood according to claims 1-5 and the steps according to claim 6 in any of the following: (1) Use in identifying or assisting in the identification of soft-shelled turtle blood and common adulterated products of soft-shelled turtle blood, wherein the common adulterated products of soft-shelled turtle blood are pig blood, sheep blood, cattle blood, chicken blood, duck blood, rabbit blood and donkey blood; (2) Use in products for identifying or assisting in the identification of soft-shelled turtle blood and common adulterated products of soft-shelled turtle blood by processing Bupleurum with animal blood, wherein the common adulterated products of Bupleurum processed with soft-shelled turtle blood are Bupleurum processed with pig blood, Bupleurum processed with sheep blood, Bupleurum processed with cattle blood, Bupleurum processed with chicken blood, Bupleurum processed with duck blood, Bupleurum processed with rabbit blood and Bupleurum processed with donkey blood. (3) Use in preparing products for identifying or assisting in the identification of whether a test sample contains soft-shelled turtle blood.

Images