DNA bar code, primer, kit and method for identifying panda

Abstract

The invention belongs to the field of animal molecular biology, and discloses a DNA bar code, a primer, a kit and a method for identifying a bear animal. The DNA bar code is a gene segment of mitochondrial cytochrome c oxidase 2 (COX2) of a bear animal, the length of the DNA bar code is 790bp, and the nucleotide sequence of a primer for amplifying the DNA bar code is as follows: the nucleotide sequence of an upstream primer is as shown in SEQ ID NO: 1, and the nucleotide sequence of a downstream primer is as shown in SEQ ID NO: 2. The method disclosed by the invention can be used for simultaneously detecting and identifying all eight species of the existing bear family, is simple and easy to implement, low in detection and identification cost, high in speed, strong in specificity and high in sensitivity (0.001 ng / mu L), and can be widely applied to detection and identification of bear animals.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a DNA barcode, a primer, a kit and a method for identifying bear animals. Background Art

[0002] Bears are classified into the phylum Chordata, subphylum Vertebrata, class Mammalia, order Carnivora, family Ursidae. There are 5 genera and 8 species in existence, namely, the Asiatic black bear (Ursus thibetanus), the Sun bear (Helarctosmalayanus), the spectacled bear (Tremarctos ornatus), the sloth bear (Melursus ursinus), the American black bear (Ursusamericanus), the brown bear (Ursus arctos), the polar bear (Ursus maritimus) and the giant panda (Ailuropodamelanoleuca), all of which are listed in the Red List of the World Conservation Union (IUCN). Bears are large mammals at the top of the food chain. The stability of their population is crucial to the stability of the ecological balance. Many countries around the world attach great importance to the protection of bears. However, due to the impact of environmental and climate change, the habitats of various populations continue to be lost in large quantities, and their living environment has been seriously threatened for a long time. Data published by the IUCN Red List show that, except for the increase in the number of giant pandas and American black bears and the stability of the number of brown bears, the number of the other five bear species has continued to decrease, indicating that current measures have not been able to effectively alleviate the endangered plight of bears in general. Secondly, bear bile is considered to have special medicinal value in traditional Chinese medicine culture, which can be traced back to Zhen Quan's "Medicine Nature Theory" in the Tang Dynasty. Although relevant documents also analyze the feasibility of using bear bile substitutes through experimental data and clinical observation results, put forward policies and suggestions, and call for the abolition of live bear bile extraction, illegal hunting of black bears and trafficking of bear tissues still occur from time to time. Furthermore, the evolutionary radiation of various bear species is relatively fast and the differentiation interval is relatively short, and the developmental relationship is still controversial.

[0003] Traditional identification methods such as morphological identification, microscopic observation, fluorescence observation, thin-layer chromatography, infrared spectroscopy, high performance and ultra-high performance liquid chromatography, amino acid and trace element determination have great limitations. The determination process is highly dependent on the experience and perception of the identification personnel and is highly subjective. The accuracy of the determination results is greatly affected by the uniformity of the target sample, making it difficult to standardize and impossible to identify the species. Thanks to the rapid development of biological technology, molecular biology has been applied to species identification as early as the 1970s. Today, it has become the core technical means of species identification. Only a small amount of sample is needed to obtain accurate results. It is not affected by the morphological characteristics, developmental conditions and subjective factors of the identification object, and its results are more convincing. With the promotion and popularization of PCR technology, various molecular biological detection and identification methods based on the principle of PCR technology have gradually replaced traditional physical and chemical methods in the past two decades and become the backbone of molecular identification. However, previous studies on molecular biological detection and identification methods for bears were mostly limited to the identification of bear bile powder - using the gene sequence of black bears (established species) as the target to design primers and probes, which cannot meet the identification needs of other bear species at the same time, nor can it clarify the applicability to other types of bear tissue samples. Therefore, it is urgent to establish a method that can quickly and accurately identify all existing bears.

[0004] DNA barcoding technology is a biological identification method that emerged in the early 21st century. This method screens and designs universal or specific primers to amplify the DNA of the target sample, then sequences the PCR amplification products and uses bioinformatics analysis methods to perform base sequence comparison analysis to identify the species of the target sample. Its application range covers the protection of endangered wildlife, identification of invasive species, identification of food and traditional Chinese medicine, etc., but a DNA barcode that can accurately identify all bear species has not yet been found.

[0005] Based on the above difficulties, the present invention establishes a non-established species identification method for identifying bears, accurately identifying the species origin of bears and their products, so as to further combat the illegal and criminal activities of hunting and trafficking in wild protected animals, fulfill the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), and help maintain ecological security. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a DNA barcode for identifying bears, which can simultaneously identify 8 existing bear species, has the characteristics of low cost, high speed and high sensitivity, the maximum intraspecific genetic distance calculated is 0.014%, the minimum interspecific genetic distance is 0.024%, the minimum interspecific genetic distance is greater than the maximum intraspecific genetic distance, and there is a significant barcode interval, which can identify bears to species, fills the current gap and has wide application value.

[0007] The present invention also provides a primer for amplifying the DNA barcode of the bear family animal.

[0008] The invention also provides a kit containing the primers.

[0009] The present invention also provides the application of the DNA barcode, primer and kit.

[0010] The invention also provides a detection method for identifying bear species.

[0011] In a first aspect of the present invention, the present invention proposes a DNA barcode for identifying bear species, wherein the DNA barcode is a gene fragment of mitochondrial cytochrome c oxidase 2 (COX2) of bear animals, and the length is 790 bp.

[0012] In some embodiments of the present invention, the bear family includes all eight existing species of the bear family: Asiatic black bear, Malayan bear, spectacled bear, sloth bear, American black bear, brown bear, polar bear and / or giant panda.

[0013] In the second aspect of the present invention, the present invention proposes a primer for identifying bear animals, which can amplify the DNA barcode, and the primer includes an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in SEQ ID NO:1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:2.

[0014] In some embodiments of the present invention, the Tm value of the primer is 58°C.

[0015] In some embodiments of the present invention, the target fragment amplified using the primers is 790 bp in length.

[0016] The primers designed by the scheme of the present invention are obtained by using DNAMAN software to perform multiple comparisons on the mitochondrial gene sequences of existing bear species obtained from the National Center for Biotechnology Information (NCBI) of the United States. After long-term screening and optimization, a pair of universal PCR primers with high sensitivity and good specificity are designed on the COX2 gene using Oligo software.

[0017] In the third aspect of the present invention, the present invention provides a kit for identifying bear animals, wherein the kit comprises the above-mentioned primers.

[0018] In some embodiments of the present invention, the kit further comprises Taq PCR Master mix.

[0019] In some embodiments of the present invention, the Taq PCR Master mix comprises Taq buffer, MgCl2 , dNTP mix and Taq DNA polymerase.

[0020] In the fourth aspect of the present invention, the present invention proposes the use of the above-mentioned DNA barcode, primer and kit in any of the following:

[0021] 1) Detection of bears;

[0022] 2) Identify the species origin of bears;

[0023] 3) Identify the authenticity of products containing bear ingredients.

[0024] In a fifth aspect of the present invention, the present invention provides a method for identifying bears, comprising the following steps:

[0025] (1) Extracting DNA from the sample to be tested;

[0026] (2) using the DNA extracted in step (1) as a template, and using the above-mentioned primers or kit to perform PCR amplification on the template to obtain an amplified product;

[0027] (3) sequencing the amplified product;

[0028] (4) performing sequence consistency comparison analysis on the sequence information of the amplified product obtained after sequencing in step (3) and a gene database to identify the species to which the animal sample to be tested belongs.

[0029] In some embodiments of the present invention, the step of removing foreign matter from the sample to be tested is also included before extracting the DNA from the sample to be tested.

[0030] In some embodiments of the present invention, the foreign body removal treatment includes the following steps: wiping the sample surface with anhydrous ethanol. The successful extraction of DNA is the basis of molecular identification. Since the types of wild protected animal samples are relatively complex, the present invention can remove foreign matter attached to the animal samples to be tested that may affect the test results by performing foreign body removal treatment, thereby improving the accuracy of the test results.

[0031] In some embodiments of the present invention, the animal sample in step (1) includes animal fur, muscle, internal organs or claws. In the present invention, all available animal samples can be used as detection and identification materials, so the scheme of the present invention has good applicability.

[0032] In some embodiments of the present invention, when the animal sample is a sample that is difficult to dissolve, the animal sample is subjected to the foreign matter removal treatment and then pulverized to obtain animal sample powder.

[0033] In some embodiments of the present invention, the difficult-to-dissolve samples include animal claws, teeth, and horns. Since claws and other samples are hard and easily contaminated, conventional methods are difficult to effectively extract mitochondrial DNA from the above types of animal samples. Therefore, they can be refined to facilitate hydrolysis and digestion, which is helpful for subsequent DNA extraction operations.

[0034] In some embodiments of the present invention, the pulverizing process includes adding liquid nitrogen for grinding.

[0035] In some embodiments of the present invention, the A260 / A280 of the DNA extracted in step (1) is 1.7-1.9. When the A260 / A280 value of the DNA is 1.7-1.9, it indicates that the extracted DNA contains fewer impurities and has a higher purity; if it exceeds this range, it indicates that the DNA sample is contaminated by carbohydrates, salts, proteins or organic solvents, which will have an adverse effect on the subsequent PCR amplification. Therefore, when the DNA sample does not fall within the above range, the DNA extraction operation needs to be repeated.

[0036] In some embodiments of the present invention, the system for PCR amplification in step (2) is:

[0037]

[0038]

[0039] Make up to 25 μL with water.

[0040] In some embodiments of the present invention, the system for PCR amplification in step (2) is:

[0041]

[0042] Make up to 25 μL with water.

[0043] In some embodiments of the present invention, the reaction procedure of the PCR amplification in step (2) is: pre-denaturation at 92-96°C for 3-5 min; then entering the cycling stage: denaturation at 92-96°C for 25-35 s, annealing at 50-60°C for 25-35 s, extension at 70-75°C for 50-70 s, 30-40 cycles; extension at 70-75°C for 8-12 min.

[0044] In some embodiments of the present invention, the reaction procedure of the PCR amplification in step (2) is: pre-denaturation at 94°C for 4 min; then entering the cycling stage: denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 60 s, 35 cycles; extension at 72°C for 10 min.

[0045] In some embodiments of the present invention, before sequencing the amplified product, the step of performing electrophoresis analysis using agarose gel and selecting a bright amplified product with an electrophoresis band fragment size of 790 bp for sequencing.

[0046] In some embodiments of the present invention, the gene database includes the National Center for Biotechnology Information (NCBI) and the Barcode of Life Database (BOLD).

[0047] In some embodiments of the present invention, the method further comprises the step of analyzing and aligning the obtained sequences using MEGA7.0 software, and calculating the intra-species and inter-species genetic distances of each species based on Kimura-2-parameter (Kimura two-parameter model).

[0048] In some embodiments of the present invention, the identification method further comprises the step of performing cluster analysis on the sample sequences.

[0049] In some embodiments of the present invention, the cluster analysis is to construct a phylogenetic tree.

[0050] The present invention also proposes the application of the above method in identifying bears. The identification method established by the present invention is applicable to the identification of all existing bear species and has broad application prospects.

[0051] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0052] (1) The primers designed by the present invention have been verified to be applicable to the identification of animal samples of different tissues and different states, including air-dried tissues and shed hair, and have high reliability in detection and identification and a wide range of applications;

[0053] (2) The method adopted by the present invention has been verified to be applicable to the identification of all existing bear species, with the maximum intraspecific genetic distance being 0.014% and the minimum interspecific genetic distance being 0.024%. The minimum interspecific genetic distance is greater than the maximum intraspecific genetic distance, and there is a significant barcode interval. Bear animals can be identified to species, and have the advantages of high accuracy, fast speed and low cost, filling the current gap and providing a fast and accurate species identification method for law enforcement departments such as customs, forest police, maritime police, and market supervision and management, which is of great significance for protecting endangered animals and combating illegal activities. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0055] Figure 1The electrophoresis results of the DNA amplification products of 9 bear tissue samples in the embodiment of the present invention, wherein M is DL2000 DNA Marker, 1-9 are samples with sample numbers 1-9 in Table 1, N is a negative control; B is a blank control;

[0056] Figure 2 The electrophoresis results of 4 synthetic plasmid amplification products of bears in the examples of the present invention are shown in FIG. 1 , wherein M is DL2000 DNA Marker, 1-4 are plasmid numbers 1-4 in Table 3, N is a negative control, and B is a blank control;

[0057] Figure 3 The electrophoresis results of the amplified DNA products of 4 bear tissue samples and 16 terrestrial and aquatic animal tissue samples in the embodiment of the present invention, wherein M is DL2000 DNA Marker, 1-4 are the sample numbers 1, 2, 4, 3 in Table 1, 5-20 are the serial numbers 1-16 in Table 2, N is the negative control, and B is the blank control;

[0058] Figure 4 NJ evolutionary tree of 25 sequences in the embodiment of the present invention;

[0059] Figure 5 The electrophoresis results of the detection sensitivity of the bear animal DNA barcode identification method established in the embodiment of the present invention; wherein, M is DL2000 DNA Marker, N is negative control, B is blank control; 1-8 are giant panda liver DNA samples with concentrations of 100 ng / μL, 10 ng / μL, 1 ng / μL, 0.1 ng / μL, 0.01 ng / μL, 0.001 ng / μL, 0.0001 ng / μL and 0.00001 ng / μL, respectively. DETAILED DESCRIPTION

[0060] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0061] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained through conventional commercial channels or can be obtained through existing known methods.

[0062] Experimental reagents: EZNATM Tissue DNA Kit (OMEGA, USA) DNA extraction kit, Taq PCR Master mix (TIANGEN, Tiangen Biochemical Technology (Beijing) Co., Ltd.) PCR reaction system, DNA Marker DL 2000 (TaKaRa, Takara Biotechnology (Dalian) Co., Ltd.).

[0063] Experimental equipment: Veriti TM 96-Well Thermal Cycler (Applied Biosystems, USA) PCR instrument; Alpha Imager HP (Alpha Innotech, USA) gel imaging system; Sigma 3-18K (Sartorius, Germany) high-speed centrifuge; NanoDrop-1000 Spectrophotometer (NanoDrop Technologies, USA) spectrophotometer; Dry Block Heater 4 (IKA, USA) dry bath; Sanger Sequencing 3500 Series Genetic Analyzers (Applied Biosystems, USA) gene sequencer.

[0064] Example 1

[0065] This embodiment provides a DNA barcode for identifying Ursidae and primers for amplifying the DNA barcode. This embodiment uses the biological software DNAMAN to perform multiple sequence alignment based on the Ursidae mitochondrial gene sequence published in GenBank of the National Center for Biotechnology Information (NCBI) of the United States, and uses Oligo7.0 to design PCR primers that can identify target species and exclude non-target species in the highly conserved region of the COX2 gene. After continuous screening and optimization of the designed multiple pairs of primers, a pair of primers with excellent performance that can be used to amplify the DNA barcode is finally confirmed and obtained (synthesized by Shanghai Pfizer Biotechnology Co., Ltd.), and the nucleotide sequence of the primer is:

[0066] Upstream primer: 5′-GGTGAAAATCCTTTATCTTTATGGC-3′ (SEQ ID NO: 1);

[0067] Downstream primer 5'-AGTTGTGGCATCTTCATTAAGGAG-3' (SEQ ID NO: 2).

[0068] The Tm value of this primer is 58°C.

[0069] Example 2

[0070] This example provides a kit for identifying bears, comprising the primers (SEQ ID NO: 1-2) prepared in Example 1 and 2×Taq PCR Master mix (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). The primers in Example 1 are prepared in the form of a kit, which is convenient for storage, carrying and use, and can provide convenience for actual detection.

[0071] Example 3

[0072] This embodiment provides a method for identifying bears, which specifically includes the following steps:

[0073] 1. Remove foreign matter

[0074] When the sample is tissue (including bear bile, bear claws, bear meat, bear hair, and bear liver tissue), in order to ensure the quality of the extracted DNA, use anhydrous ethanol to evenly wipe the surface of the above sample several times and then air-dry it at room temperature to remove foreign matter attached to the surface of the sample that may affect the test results, thereby improving the accuracy of the test and identification results.

[0075] 2. Preparation of Animal Sample Powder

[0076] When the animal sample is a hard (difficult to dissolve) sample, such as claws, teeth, and horns, it is difficult to effectively extract mitochondrial DNA from such animal samples using conventional methods. After removing foreign matter, sampling is performed using a suitable sterile sampling device, and the sample is ground into powder in a grinding mortar with the aid of liquid nitrogen. About 50 mg is weighed and placed in a centrifuge tube for inspection.

[0077] 3. DNA Extraction

[0078] The samples treated as above are subjected to conventional DNA extraction methods. If the kit is used for extraction, please refer to the kit instructions. If manual extraction is used, please refer to the following steps:

[0079] (1) Take the treated sample and add 700 μL of lysis buffer, place it in a 65°C water bath for 2-3 hours (if the sample is difficult to lyse, the lysis time can be appropriately extended or overnight), shaking from time to time.

[0080] (2) After centrifugation at 4°C and 12,000 g for 5 min, remove the supernatant and transfer it to a new 1.5 mL centrifuge tube. Add an equal volume of phenol, mix thoroughly, and centrifuge again at 4°C and 12,000 g for 5 min.

[0081] (3) Take the supernatant, add an equal volume of a mixed solution of chloroform and isoamyl alcohol, mix thoroughly, and centrifuge at 4°C and 12,000 g for 5 min.

[0082] (4) Take the supernatant, add 1 / 10 volume of sodium acetate solution, mix well, and then add 2 volumes of -20°C precooled anhydrous ethanol. Let stand at -20°C for 1 hour. Centrifuge at 4°C and 12,000 g for 5 minutes, and discard the supernatant.

[0083] (5) Wash once with 70% ethanol, dry in a clean bench at room temperature, add 50-100 μL TE to dissolve the precipitate to prepare a DNA solution.

[0084] The A260 / A280 of the extracted DNA should be 1.7-1.9, otherwise it needs to be re-extracted. The extracted DNA can be directly used for PCR amplification or stored at -20℃ for future use.

[0085] 4. PCR Amplification

[0086] The extracted DNA was used as a template and PCR amplification was performed using the upstream and downstream primers shown in SEQ ID NO: 1-2 in Example 1 to obtain an amplified product.

[0087] The PCR amplification system is:

[0088] 2×Taq PCR Master mix 12.5μL;

[0089] 10 μmol / L upstream primer shown in SEQ ID NO: 1 1 μL;

[0090] 10 μmol / L downstream primer shown in SEQ ID NO: 2 1 μL;

[0091] DNA template 2 μL;

[0092] Make up to 25 μL with water.

[0093] Taq PCR Master mix contains Taq buffer, MgCl 2 , dNTP mix and TaqDNA polymerase.

[0094] The reaction procedure for PCR amplification is:

[0095] Pre-denaturation at 94°C for 4 min;

[0096] Denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 60 s were performed for 35 cycles;

[0097] Extension at 72°C for 7 min.

[0098] 5. Electrophoresis and sequencing of amplified products

[0099] Take 5 μL of amplified product and add it to 1.5% agarose gel, perform electrophoresis at 100V for 35 min, observe and analyze it with the gel imaging analysis system, cut and purify the bands that match the size of the target fragment, and sequence the target fragment using the Sanger sequencing method.

[0100] 6. Sequence Analysis

[0101] The sequences obtained by sequencing were compared with the Basic Local Alignment Search Tool (BLAST) of the GenBank database retrieval tool of the National Center for Biotechnology Information (NCBI) of the United States to obtain the species information with the highest consistency with the sequence measured by sequencing and identify the species source. The COX2 gene sequences with the same consistency were downloaded from the GenBank database, and the sequences were aligned using the MEGA software. Based on the Kimura-2-Parameter (K2P) model, the Transition substitution (Ts) substitution type was selected to calculate the genetic distances within and between species.

[0102] Example 4

[0103] In this example, 9 tissue samples of bears, 16 tissue samples of terrestrial and aquatic non-bears, and 4 dilutions of mitochondrial full-gene DNA plasmids of bears were used as research objects, and the method used to identify bears in Example 3 was used for detection and identification. The detailed information of the samples and plasmids used are shown in Tables 1, 2, and 3. All tissue samples were retained by the Animal Quarantine Laboratory of Gongbei Customs Technology Center, and the terrestrial and aquatic non-bear tissue samples were confirmed as listed species using molecular biological identification methods designed by this laboratory and certified by CMA and CNAS; all DNA plasmids were commissioned to be synthesized by Beijing Liuhe BGI Gene Technology Co., Ltd.

[0104] The identification result sequences of 9 bear tissue samples were compared with 16 mitochondrial DNA sequences of 8 selected bear species, a total of 25 sequences, and the genetic distance of each species was calculated using the Kimura-2-parameter (K2P) model of MEGA. The Neighbor-Joining Method (NJ) evolutionary tree was constructed based on the maximum composite likelihood estimation, base conversion, 1000 resampling and complete deletion of empty chains. The detailed information of the selected bear mitochondrial DNA sequences is shown in Table 4.

[0105] Table 1 Information on tissue samples of bears

[0106]

[0107]

[0108] Table 2 Information on terrestrial and aquatic animal tissue samples

[0109] Serial number organize Species scientific name 1 Skin Reticulated Python Malayopython reticulatus 2 Skin red fox Vulpes vulpes 3 Skin European mink Mustela lutreola 4 hair Asiatic Lion Panthera leo persica 5 hair Siberian Tiger Panthera tigris altaica 6 horn Sabi Saiga tatarica 7 Meat House cat Felis catus Linnaeus 8 Meat House mouse Mus musculus 9 Meat Domestic Pig Sus scrofa domestica 10 Meat Yak Bos mutus 11 Meat buffalo Bubalus bubalis 12 Meat goat Capra hircus 13 Meat sheep Ovis aries 14 Meat Junglefowl Gallus gallus domesticus 15 Meat Muscovy Duck Cairina moschata 16 Meat Grass carp Ctenopharyngodon idella

[0110] Table 3 DNA plasmid information of bear animals

[0111]

[0112]

[0113] Table 4 Mitochondrial DNA sequence information of bears

[0114]

[0115] The electrophoresis results of the sample DNA amplification products are shown in the figure below. Figure 1-3 As shown in the figure, it can be seen that the DNA of bear tissue samples numbered 1 to 9 and the bear DNA plasmid numbered 1-4 all showed significant amplification bands at 790bp, and no amplification bands were found in the negative control and the blank control; 4 bear tissue sample DNA specific amplification bands were significant, and 16 terrestrial and aquatic animal tissue sample DNAs had no amplification bands.

[0116] Further sequence comparison results showed that sample numbers 1-5 were Asian black bears, sample numbers 6-7 were brown bears, sample number 8 was polar bears, and sample number 9 was giant pandas. The consistency of all sequence comparison results was ≥98%, as shown in Table 5. Plasmid numbers 1-4 were Malayan bears, sloth bears, spectacled bears, and American black bears, respectively. The consistency of all sequence comparison results was ≥96%, as shown in Table 6.

[0117] Table 5 BLAST results of sample numbers 1-9

[0118] Sample No. organize scientific name consistency 1 Bear Bile Ursus thibetanus 98.07％～99.11％ 2 Bear Bile Ursus thibetanus 99.41％～99.56％ 3 Bear Claw Ursus thibetanus 98.04％～98.95％ 4 Bear Bile Ursus thibetanus 98.00％～99.50％ 5 Bear meat Ursus thibetanus 99.00％～99.86％ 6 Bear Claw Ursus arctos 99.59％～99.73％ 7 Bear Hair Ursus arctos 99.57％～99.86％ 8 Bear Hair Ursus maritimus 99.42％～99.42％ 9 Bear Liver Ailuropoda melanoleuca 99.69％～100.0％

[0119] Table 6 BLAST results of plasmid sequence numbers 1-4

[0120] Plasmid number scientific name consistency 1 Helarctos malayanus 98.17％～99.72％ 2 Melursus ursinus 98.37％～99.86％ 3 Tremarctos ornatus 99.31％～99.86％ 4 Ursus americanus 96.96％～100.0％

[0121] In terms of genetic distance, the pairwise comparison results of the 25 sequences showed that the maximum intraspecific genetic distance between sequences was 0.014, which appeared in two Asian black bear tissue sample DNA sequences; the minimum interspecific genetic distance was 0.024, which appeared in a polar bear tissue sample DNA sequence and a gene bank brown bear mitochondrial DNA sequence; the genetic distance between the polar bear tissue sample DNA sequence and the brown bear tissue sample DNA sequence was 0.024. The above results suggest that the minimum interspecific genetic distance is greater than the maximum intraspecific genetic distance, indicating that there is a significant gap between the DNA barcode sequences of different bear species, as shown in Table 7, where: 1-9 are sample numbers 1-9 in Table 1, 10 is the American black bear (OQ318937), 11 is the American black bear (OQ318942), 12 is the Sun bear (OQ564480), 13 is the Sun bear (FM177765), 14 is the Asiatic black bear (NC_011118), 15 is the Asiatic black bear (PQ1 82686), 16 is brown bear (OK512947), 17 is brown bear (OK512925), 18 is polar bear (JX196383), 19 is polar bear (OK001275), 20 is giant panda (PP871704), 21 is giant panda (EF196663), 22 is sloth bear (MH931229), 23 is sloth bear (MH910095), 24 is spectacled bear (OR999077), and 25 is spectacled bear (NC_009969).

[0122] Table 7 Genetic distance of gene sequences based on Kimura two-parameter model

[0123]

[0124]

[0125] In terms of evolutionary relationships, the NJ evolutionary tree results of the 25 sequences are shown in the figure below: Figure 4 As shown in the figure, it can be seen that the 25 sequences led to 7 different evolutionary branches in the NJ evolutionary tree, which were grouped into 8 bear species. Among them, the spectacled bear and giant panda have a relatively distant evolutionary relationship with the other 6 species, while the brown bear and polar bear have a relatively close evolutionary relationship. The 9 samples of 4 bear tissues and the reference sequences of the corresponding species were also grouped into one branch.

[0126] Example 5

[0127] This example is a sensitivity verification test of the bear detection and identification method established in Example 3. Sample No. 9 in Table 1, namely giant panda liver, is used as a representative sample to test the sensitivity of the method of the present invention. After DNA is extracted using the method in Example 3, the DNA concentration of the Asian black bear bile is determined to be 325 ng / μL (A260 / A280=1.85) using a NanoDrop-1000 analyzer. It is diluted to 100 ng / μL and then diluted 10 times in a gradient dilution to prepare 8 different concentrations, namely, a stock solution of 100 ng / μL and dilutions of 10 ng / μL, 1 ng / μL, 0.1 ng / μL, 0.01 ng / μL, 0.001 ng / μL, 0.0001 ng / μL and 0.00001 ng / μL, respectively. They are used as DNA templates, and PCR amplification and electrophoresis detection are performed using the method in Example 3.

[0128] The results are as follows Figure 5 As shown in the figure, it can be seen that the brightness of the electrophoresis band of the amplified product is positively correlated with the concentration of the DNA template. The DNA template band with a concentration of 0.001ng / μL is clearly visible, the DNA template band with a concentration of 0.0001ng / μL is vaguely visible, and the DNA template band with a concentration of 0.00001ng / μL is invisible. This shows that the method proposed in the present invention has a high sensitivity, at least reaching 0.001ng / μL, and has outstanding characteristics and advantages.

[0129] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A DNA barcode for identifying bears, characterized in that: The DNA barcode is a gene fragment of cytochrome c oxidase 2 of mitochondria of bear family, and the length is 790 bp.

2. A primer for amplifying the DNA barcode according to claim 1, characterized in that: The primers include an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in SEQ ID NO:1; the nucleotide sequence of the downstream primer is shown in SEQ ID NO:

2.

3. A kit for identifying bears, characterized in that: The kit comprises the primers according to claim 2; Preferably, the kit further comprises Taq PCR Master mix; Preferably, the Taq PCR Master mix comprises Taq buffer, MgCl2, dNTP mix and Taq DNA polymerase.

4. Use of the DNA barcode according to claim 1, the primer according to claim 2 or the kit according to claim 3 in any of the following: 1) Detection of bears; 2) Identify the species origin of bears; 3) Identify the authenticity of products containing bear ingredients.

5. A method for identifying bears, characterized in that: The identification method comprises the following steps: (1) Extracting DNA from the sample to be tested; (2) using the DNA extracted in step (1) as a template, and using the above-mentioned primers or kit to perform PCR amplification on the template to obtain an amplified product; (3) sequencing the amplified product; (4) performing sequence consistency comparison analysis on the sequence information of the amplified product obtained after sequencing in step (3) and a gene database to identify the species to which the animal sample to be tested belongs.

6. The method according to claim 5, characterized in that The sample to be tested in step (1) includes animal fur, muscle, internal organs or claws.

7. The method according to claim 5, characterized in that The step of removing foreign matter from the sample to be tested is also included before extracting the DNA of the sample to be tested; Preferably, the foreign matter removal process comprises the following steps: wiping the surface of the sample with anhydrous ethanol.

8. The method according to claim 5, characterized in that The system for PCR amplification in step (2) is: And / or, the reaction procedure of the PCR amplification in step (2) is: pre-denaturation at 92-96°C for 3-5 min; then entering the cycling stage: denaturation at 92-96°C for 25-35 s, annealing at 50-60°C for 25-35 s, extension at 70-75°C for 50-70 s, 30-40 cycles; extension at 70-75°C for 8-12 min.

9. The method according to claim 5, characterized in that The gene databases include the National Center for Biotechnology Information (NCBI) and the Barcode of Life Database (BOLD).

10. Use of the method according to any one of claims 5 to 9 in identifying bears.

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