Bar code, primer, kit and method for cornu bubali identification

Abstract

The invention relates to a bar code, a primer, a kit and a method for cornu bubali identification. The invention firstly provides a DNA bar code, and the nucleotide sequence of the DNA bar code is as shown in SEQ ID NO. 1. The invention further provides a DNA bar code composition containing the DNA bar code and a detection primer of the DNA bar code composition. The selected small-fragment DNA bar code sequence has high specificity, can be used for effectively distinguishing buffalo horn, yak horn and yellow horn samples, and overcomes the problem that buffalo horn subjected to processing such as high-temperature treatment cannot be effectively identified by a long DNA bar code. The primer provided by the invention has strong specificity, and can reduce non-specific sequencing results caused by exogenous DNA pollution in the process of identifying an ox horn sample.

Description

Technical Field

[0001] The invention belongs to the technical field of Chinese medicinal material species identification, and particularly relates to a barcode, a primer, a kit and a method for identifying buffalo horns. Background Art

[0002] Buffalo horn is the horn of the bovine animal Bubalus bubalis. Its medicinal use was first recorded in the "Famous Doctors' Records" and was listed as a medium-grade product, "which can treat headaches caused by seasonal colds and heat." [1][2] . Modern research shows that buffalo horns mainly contain proteins, peptides, amino acids, sulfur-containing compounds, nucleosides, inorganic elements and other compounds, which have the effects of clearing heat and cooling blood, detoxifying, and calming the nerves. In clinical Chinese medicine, buffalo horns and their prescriptions are often used to treat heat syndromes, with definite efficacy. Buffalo horns are used as substitutes for rhinoceros horns because of their similar and definite effects as rhinoceros horns. With people's recognition of buffalo horns and their Chinese patent medicine formulas, the resource is being used more widely. Buffaloes are raised in most parts of my country, especially in the southern rice-growing areas. [3] . According to the investigation, the commercial specifications of buffalo horns on the market are mainly buffalo horn silk, buffalo horn tips, etc., which lack complete identification characteristics and are difficult to accurately identify the species. Among the buffalo horns on the market, yak horns and yellow cattle horns are sold as buffalo horns, and they account for a large proportion. Therefore, it is urgent to establish a method that can accurately identify the origin of buffalo horns.

[0003] The 2020 edition of the Chinese Pharmacopoeia only has properties and powder microscopic identification items under buffalo horn, and properties, inspections, extracts, and content indicators under buffalo horn concentrated powder. However, most animal medicinal materials have been destroyed in appearance, their microscopic features are not obvious, and their chemical components are not specific. The above methods and indicators cannot fully evaluate the quality of samples such as buffalo horn medicinal materials, standard decoctions, formula granules, and Chinese patent medicines containing buffalo horns, and cannot distinguish between buffalo horns and their closely related species. Therefore, the quality standards for buffalo horns are not perfect, and there is a lack of specific identification methods for buffalo horn medicinal materials, buffalo horn granules, and formula preparations containing buffalo horns.

[0004] At present, there are also public research on the identification of buffalo horns and their similar products. [4] Comparison of thin-layer chromatography images of antelope horns and buffalo horns showed that the spots on antelope horns and buffalo horns were the same in color and position, with no obvious difference. [5] The HPLC method was used to identify buffalo horns and antelope horns. The results showed that the types of amino acids in antelope horns were basically the same as those in buffalo horns, but the amino acid content was quite different. [6]Fourier transform infrared spectroscopy (FTIR) combined with second-order derivative spectrum analysis was used to identify four kinds of horn animal drugs: velvet antler, deer horn, antelope horn, and buffalo horn. The results showed that buffalo horn and antelope horn have great similarities in infrared original spectra and derivative spectra. From the peak intensity, the content of concentrated components in antelope horn is higher than that in buffalo horn. [7] Using CO I sequence as DNA barcode, samples of buffalo horns and their easily confused products were taken to establish a method for identifying buffalo horns and their easily confused counterfeits based on DNA barcode technology. [8] Mathematical geometry and Labelfree quantitative peptidomics analysis and screening were used to identify three characteristic peptides from bovine horns. Characteristic peptides 1 and 3 were detected from buffalo horns, characteristic peptides 1 and 2 were detected from ox horns, and characteristic peptides 2 and 3 were detected from yak horns.

[0005] However, the methods for identifying genuine and counterfeit buffalo horns in the above literature have the following main defects: (1) Thin layer chromatography, infrared method, high performance liquid chromatography, etc. have been applied to the identification of buffalo horns and distinguishing other horns, but due to weak specificity or low detection sensitivity, they cannot be effectively used for the identification of buffalo horns. (2) The CO I barcode identification amplification fragment is relatively long, and the high temperature extraction process of traditional Chinese medicine will affect the integrity of DNA, thereby affecting the accuracy of identification. The above method cannot meet the requirements of the identification of processed buffalo horns, such as formula granules. (3) The CO I barcode universal primers used above have poor specificity and are easily contaminated by exogenous DNA. Animal medicinal materials contain a large amount of nutrients, which are prone to breed bacteria, fungi, parasites and storage pests during processing, storage and transportation, leading to DNA contamination. Summary of the invention

[0006] Problem that the invention aims to solve

[0007] In view of the problems existing in the prior art, such as the high similarity of DNA sequences of buffalo and its closely related species (including yaks and cattle), the low specificity of universal DNA barcodes and universal primers, and the easy contamination by exogenous DNA, the present invention provides a small-fragment DNA barcode and primers thereof, as well as a method for identifying buffalo horns using the DNA barcode and primers, which can effectively distinguish buffalo horns, yak horns and cattle horns, and overcome the problems of long amplified fragments and high-temperature extraction damaging DNA, and reduce the influence of exogenous DNA contamination on the identification results. It provides a reference for the quality control of buffalo horns and their preparations, and provides a basis for the improvement of buffalo horn quality standards.

[0008] Solutions for solving problems

[0009] The first aspect of the present invention provides a DNA barcode, wherein the nucleotide sequence of the DNA barcode is shown as SEQ ID NO.1.

[0010] The second aspect of the present invention provides a DNA barcode composition, wherein the DNA barcode composition comprises:

[0011] A first DNA barcode, wherein the nucleotide sequence of the first DNA barcode is shown as SEQ ID NO.1;

[0012] a second DNA barcode, the nucleotide sequence of which is shown in SEQ ID NO.2; and

[0013] A third DNA barcode, the nucleotide sequence of the third DNA barcode is shown as SEQ ID NO.3.

[0014] The third aspect of the present invention provides a primer composition, wherein the primer composition comprises detection primers designed based on the DNA barcode according to the first aspect of the present invention and / or based on the DNA barcode composition according to the second aspect of the present invention.

[0015] In some embodiments, the primer composition comprises a detection primer as described below:

[0016] NJ161-F, the nucleotide sequence of which is shown in SEQ ID NO.35;

[0017] NJ161-R, the nucleotide sequence of which is shown in SEQ ID NO.36.

[0018] The fourth aspect of the present invention provides use of the DNA barcode according to the first aspect of the present invention, the DNA barcode composition according to the second aspect of the present invention, or the primer composition according to the third aspect of the present invention in identifying buffalo horns and / or constructing a phylogenetic tree of buffalo horns.

[0019] A fifth aspect of the present invention provides a method for identifying buffalo horns, wherein the method comprises the following steps (i) to (iii):

[0020] (i) extracting genomic DNA from the bovine horn sample to be tested;

[0021] (ii) using the genomic DNA of the horn sample to be tested extracted in step (i) as a template, and performing PCR amplification using the primer combination described in the third aspect of the present invention;

[0022] (iii) sequencing the amplified product obtained in step (ii), comparing the sequencing result with the DNA barcode described in the first aspect of the present invention and / or the DNA barcode composition described in the second aspect of the present invention, and / or applying the sequencing result to construct a phylogenetic tree of bovine horns.

[0023] In some embodiments, in step (i), the buffalo horn sample to be tested includes any one or more of buffalo horn medicinal materials, buffalo horn standard decoctions, buffalo horn formula granules, and Chinese patent medicines containing buffalo horns.

[0024] In some embodiments, in step (ii), the reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 61°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles; extension at 72°C for 5 min.

[0025] The sixth aspect of the present invention provides a kit for identifying buffalo horns, wherein the kit comprises the primer composition described in the third aspect of the present invention.

[0026] In some embodiments, the kit further comprises PCR reaction reagents.

[0027] Effects of the Invention

[0028] Through the implementation of the above technical solution, the present invention achieves the following technical effects:

[0029] Through a large number of analyses, the present invention first obtains a gene fragment that can be used to distinguish buffalo horns, yak horns and yellow cattle horns. In this gene fragment, there are many different sites among buffalo horns, yak horns and yellow cattle horns. Further, the present invention provides corresponding small-fragment DNA barcodes and DNA barcode compositions. The small-fragment DNA barcode sequence selected by the present invention has high specificity and can be used to effectively distinguish buffalo horn, yak horn and yellow cattle horn samples, and overcomes the problem that longer DNA barcodes cannot effectively identify buffalo horns that have been processed, such as high-temperature treatment.

[0030] Secondly, through extensive research, the present invention has screened out universal primers for amplifying the DNA barcodes of the above-mentioned buffalo horns, yak horns, and yellow cattle horns. The primers provided by the present invention have strong specificity and can reduce non-specific sequencing results caused by exogenous DNA contamination during the identification of cattle horn samples.

[0031] Furthermore, the present invention provides an identification method and a kit for buffalo horn, which provides an effective reference for the quality control of buffalo horn and its preparations, and provides a basis for the improvement of the quality standard of buffalo horn. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the phylogenetic tree diagram of buffalo horns, yak horns, and ox horns.

[0033] Figure 2 This is a peak overlap result diagram obtained when the sample MN01 in the embodiment is sequenced using universal primers LCO1490 and HCO2198.

[0034] Figure 3 For the general Figure 2 The result of blast comparison of the peak sequencing results.

[0035] Figure 4 This is a single peak result diagram obtained when the sample MN01 in the embodiment is sequenced using the specific primers NJ161-F and NJ161-R provided by the present invention.

[0036] Figure 5 This is a comparison chart of the barcode sequences of buffalo horn, yak horn and cattle horn.

[0037] Figure 6 This is a systematic evolutionary tree diagram of Chinese herbal formula granules and Chinese patent medicines. DETAILED DESCRIPTION

[0038] The following describes the embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various structures described below, and various changes can be made within the scope of the invention claim, and the embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention.

[0039] In the present invention, the term "a" or "an" or "the" may mean "one", and may also mean "one or more", "at least one" and "one or more than one".

[0040] In the present invention, the term "about" may mean that a value includes the standard deviation of the error of the device or method used to determine the value. The numerical ranges and parameters used to define the present invention are all approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value essentially inevitably contains standard deviations caused by the aforementioned testing devices or methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in the present invention are modified by "about". Here, "about" generally means that the actual value is within ±2%, ±1% or ±0.5% of a particular value or range.

[0041] In the present invention, the terms "comprising", "having", "including" or "containing" may mean inclusive or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising", "having", "including" or "containing" may also mean closed, excluding additional, uncited elements or method steps.

[0042] In the present invention, "water buffalo" is Bubalus bubalis, "yak" is Bos grunniens, and "cattle" is Bostaurus.

[0043] In the present invention, "drug material" or "raw drug" refers to raw materials of traditional Chinese medicine that have not been processed or made into finished products.

[0044] In the present invention, "standard decoction" refers to a single-flavor Chinese medicine decoction prepared by standardized process under the guidance of traditional Chinese medicine theory, based on clinical application, and with reference to modern extraction methods.

[0045] In the present invention, "formulated granules" are granules prepared from single-flavor Chinese herbal medicine pieces according to traditional standards, extracted and concentrated, and used for clinical prescriptions of traditional Chinese medicine.

[0046] In the present invention, "Chinese patent medicine" refers to Chinese medicinal materials as raw materials, which are processed into Chinese medicinal products of certain dosage forms according to prescribed prescriptions and preparation processes under the guidance of traditional Chinese medicine theory for the purpose of preventing and treating diseases.

[0047] Unless otherwise defined, other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0048] The present invention is mainly realized based on the following research:

[0049] Buffalo horns, fake yellow cattle horns and yak horns currently on the market were collected and DNA was extracted. The COI barcodes of the collected samples were amplified using universal primers in the prior art and sequenced. The mitochondrial sequences of buffalo horns, yellow cattle horns and yak horns were downloaded from the NCBI gene library. The COI sequences and mitochondrial DNA sequences amplified by the collected samples were imported into the BioEdit software and compared respectively. In the mitochondrial genome and COI sequences, the difference regions with more difference sites among buffalo horns, yak horns and yellow cattle horns were searched, and in this sequence, the stable conserved regions of the three types of horns were simultaneously searched for the design of universal primers for the three types of horns. The collected samples were amplified and sequenced using the designed primers. The sequencing results of all samples were blasted in GENBANK to verify the accuracy of the method. All sample sequences were imported into mega7.0 to construct a phylogenetic tree, and the accuracy of the method was verified twice.

[0050] DNA barcode and DNA barcode composition

[0051] The present invention provides a DNA barcode through a large number of analyses and comparisons, and the nucleotide sequence of the DNA barcode is shown in SEQ ID NO.1.

[0052] Meanwhile, the present invention provides a barcode composition comprising the above-mentioned DNA barcode. In some embodiments, the DNA barcode composition comprises: a first DNA barcode, the nucleotide sequence of the first DNA barcode is shown as SEQ ID NO.1; a second DNA barcode, the nucleotide sequence of the second DNA barcode is shown as SEQ ID NO.2; and a third DNA barcode, the nucleotide sequence of the third DNA barcode is shown as SEQ ID NO.3.

[0053] The DNA barcode and composition provided by the present invention have good versatility when applied to species identification systems because they include conservative region sequences; on the other hand, they have high accuracy when used for species determination because they have multiple nucleotide difference sites. Therefore, the present invention provides an effective molecular marker for the authenticity identification of buffalo horns.

[0054] Primer composition

[0055] In order to identify buffalo horns by means of PCR, the present invention provides detection primers designed based on the above-mentioned DNA barcode and / or DNA barcode composition.

[0056] In some embodiments, the primer composition provided by the present invention comprises the following detection primers: NJ161-F, whose nucleotide sequence is shown in SEQ ID NO.35; NJ161-R, whose nucleotide sequence is shown in SEQ ID NO.36.

[0057] The primer combination provided by the present invention has strong specificity and can reduce non-specific sequencing results caused by exogenous DNA contamination during the identification of horn samples, thereby improving the accuracy of the identification results.

[0058] Identification method of buffalo horn

[0059] The present invention provides a method for identifying buffalo horns, which comprises the following steps (i) to (iii):

[0060] (i) extracting genomic DNA from the bovine horn sample to be tested;

[0061] (ii) using the genomic DNA of the horn sample to be tested extracted in step (i) as a template, and performing PCR amplification using the above primer combination;

[0062] (iii) sequencing the amplified product obtained in step (ii), comparing the sequencing result with the above-mentioned DNA barcode and / or DNA barcode combination, and / or applying the sequencing result to construct a phylogenetic tree of bovine horns.

[0063] Since the DNA barcode sequence provided by the present invention is relatively short, when used for identification of horn samples, the problem that the long amplified fragment in the prior art makes it impossible to accurately identify processed horn samples, such as those treated at high temperatures, can be overcome, so that the method provided by the present invention can identify a variety of horn samples. In some embodiments, in step (i), the horn sample to be tested includes any one or more of buffalo horn medicinal materials, buffalo horn standard decoctions, buffalo horn formula granules, and Chinese patent medicines containing buffalo horns.

[0064] In order to improve the accuracy of the detection results, in some embodiments, in step (ii), the reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 61°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles; extension at 72°C for 5 min.

[0065] Kit for identification of buffalo horns

[0066] In order to improve the convenience of identifying buffalo horns, the present invention provides a kit for identifying buffalo horns, and the kit includes the above-mentioned primer combination.

[0067] By using the kit provided by the present invention, the authenticity of buffalo horns can be effectively identified by the above-mentioned identification method of buffalo horns. In some embodiments, the kit further comprises a PCR reaction reagent.

[0068] In some specific embodiments, the PCR reaction reagents include buffer, dNTP, DNA polymerase and sterile double distilled water.

[0069] Example

[0070] The embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out under normal conditions or conditions recommended by the manufacturer. Unless otherwise specified, the materials or instruments used are all commercially available conventional products.

[0071] Embodiment 1

[0072] 1 Instruments and samples

[0073] 1.1 Instruments

[0074] Electric constant temperature water bath (Shanghai Boxun Medical Biological Instrument Co., Ltd.), centrifuge (Thermo Fisher Scientific Inc.), high-throughput tissue grinder (Suizhen Biotechnology Co., Ltd.), sterilizer (Shanghai Shen'an Medical Equipment Factory), PCR instrument (Bio-Rad Biotechnology Co., Ltd., USA), WIX electrophoresis system (Wex Technology Co., Ltd.), gel imaging system (Azure Biotechnology Co., Ltd., USA).

[0075] 1.2 Reagents

[0076] Blood / cell / tissue genomic DNA extraction kit (DP304) and GenRed nucleic acid dye (RT211) were purchased from Tiangen Biochemical Technology Co., Ltd., DTT (Solabo Biotechnology Co., Ltd.), rTaqDNA polymerase (Baoriyi Biotechnology Co., Ltd., batch number R001A), and 2000bp DNA marker (Adamas Biotechnology Co., Ltd.).

[0077] 1.3 Samples

[0078] The samples used in this study were purchased from larger farms or medicinal material markets across the country. The buffalo horns all met the requirements of the 2020 edition of the Chinese Pharmacopoeia. The medicinal material information is shown in Table 1.

[0079] Table 1 Sample information table

[0080] Serial number batch number Material Name Origin Serial number batch number Material Name Origin 1 SN01 buffalo City A, Jiangxi Province 15 HN06 cattle City A, Anhui Province 2 SN02 buffalo City A, Jiangxi Province 16 HN07 cattle Anhui Province B City 3 SN03 buffalo City A, Jiangxi Province 17 HN08 cattle Anhui Province B City 4 SN04 buffalo City A, Guangxi Zhuang Autonomous Region 18 HN09 cattle City A, Anhui Province 5 SN05 buffalo City A, Guangxi Zhuang Autonomous Region 19 MN01 Yak City A, Hunan Province 6 SN06 buffalo City A, Guangxi Zhuang Autonomous Region 20 MN02 Yak City A, Hunan Province 7 SN07 buffalo City A, Guangdong Province 21 MN03 Yak City B, Guangdong Province 8 SN08 buffalo City A, Guangdong Province 22 MN04 Yak City A, Hainan Province 9 SN09 buffalo City B, Jiangxi Province 23 MN05 Yak City B, Jiangxi Province 10 HN01 cattle City B, Guangxi Zhuang Autonomous Region 24 MN06 Yak City B, Jiangxi Province 11 HN02 cattle City B, Guangxi Zhuang Autonomous Region 25 MN07 Yak City B, Jiangxi Province 12 HN03 cattle City B, Jiangxi Province 26 MN08 Yak City C, Jiangxi Province 13 HN04 cattle City B, Guangxi Zhuang Autonomous Region 27 MN09 Yak City C, Jiangxi Province 14 HN05 cattle City A, Anhui Province

[0081] 2 Experimental methods

[0082] 2.1 Primer design

[0083] The mitochondrial sequences and barcode CO I sequences of buffalo horns, yellow cattle horns, and yak horns were compared to find the variable regions with more interspecific variable sites in the three horns, and the conserved regions outside the variable regions were used to design universal primers for the three horns. The universal primers for the three horns were designed using premier Primer 5.0 software, with forward primer NJ161-F: 5'-AACGTAGTTGTAACCGCACACGCAT-3' (SEQ ID NO.35) and reverse primer NJ161-R: 5'-GAGGGAGGGAGGAGTCAGAAGCTTAT-3' (SEQ ID NO.36).

[0084] 2.2 Genomic DNA extraction

[0085] ① Wipe the sample surface with 75% ethanol and irradiate it under UV light for 30 minutes. Then, use the instructions of the blood / cell / tissue DNA extraction kit and add 40 μL DTT (1 mol / L) to extract the bovine horn DNA. The steps are as follows:

[0086] ②Take an appropriate amount of this product, grind it into fine powder, take about 30 mg, put it in a 2.0 mL centrifuge tube, add 200 μL GA buffer, 20 μL proteinase K, 40 μL DTT (1 mol / L), vortex to mix, heat in a 56°C water bath for 2 to 3 hours, and invert the mixed sample 2-3 times every hour.

[0087] ③Add 200 μL of GB buffer, vortex to mix, and heat in a 70°C water bath for 10 minutes.

[0088] ④ Add 200 μL of anhydrous ethanol and shake thoroughly for 15 seconds.

[0089] ⑤ Add the mixed solution to an adsorption column CB3 (the adsorption column is placed in a collection tube), centrifuge at 12000rpm for 30 seconds, pour out the waste liquid, and put the adsorption column CB3 back into the collection tube.

[0090] ⑥ Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 12000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in the collection tube.

[0091] ⑦ Add 600 μL of rinse solution PW to the adsorption column CB3, centrifuge at 12000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in the collection tube. Repeat once.

[0092] ⑧Put the adsorption column CB3 back into the collection tube, centrifuge at 12000rpm for 2 minutes, and discard the waste liquid. Leave the adsorption column CB3 at room temperature for 5 minutes to dry the rinse liquid.

[0093] ⑨ Place the adsorption column CB3 in a clean centrifuge tube, add 180 μL of elution buffer TE to the center of the adsorption membrane, leave it at room temperature for 2 to 5 minutes, centrifuge it at 12000 rpm for 2 minutes, and collect the solution in the centrifuge tube.

[0094] 2.3 PCR reaction system and amplification conditions

[0095] PCR reaction system: 2.5 μL of 10× buffer, 1.5 μL of dNTP, 0.25 μL of each primer, 0.2 μL of rTaq enzyme, 1 μL of DNA template, add sterile double distilled water to 25 μL. The reaction procedure is 95°C pre-denaturation for 3 min, (95°C denaturation for 30 s, 61°C annealing for 30 s, 72°C extension for 30 s) for a total of 35 cycles, and 72°C final extension for 5 min. After the PCR reaction is completed, take 2 μL of the PCR product, add 1 μL of 6× Loading buffer, mix well, and detect by 1.5% agarose gel electrophoresis, and observe and image on the gel imaging system.

[0096] 2.4 PCR product analysis

[0097] If the product of step 2.3 has a single bright band at 161 bp, perform bidirectional sequencing on the remaining PCR products and compare the differences between the forward and reverse sequences to obtain a sequence of 161 bp in size.

[0098] 3 Results and analysis

[0099] Perform gel electrophoresis on the PCR products separately, and sequence the samples with bright single bands. The sequencing primers are consistent with the PCR primers. To ensure the accuracy of sequencing, forward and reverse sequencing is required, and the bidirectional sequencing results are imported into the BioEdit software. Look at the peak shape on the peak graph interface. If it is not good, re-sequencing is required. If it is good, continue splicing.

[0100] The buffalo horn barcode standard detection sequence SEQ ID NO.1 Bubalus bubalis, the yellow cattle horn barcode standard detection sequence SEQ ID NO.2 Bos taurus, the yak horn barcode standard detection sequence SEQ ID NO.3 Bos grunniens (the standard barcode sequences are shown in Table 3) and the sequences of the tested samples (see Table 3) were imported into MEGA software, and the phylogenetic tree was constructed using the NJ method. The support rate of each branch was tested by Bootstap (1000 times), and only the branches with bootstrap support rate ≥50% were displayed. Figure 1 . The 9 buffalo horn samples and the standard barcode of buffalo horn clustered into a large branch, which was clearly distinguished from yak horn and yellow cattle horn, and the node support rate was 100%; the yak horn and yellow cattle horn samples clustered into another large branch, the 9 yak horn samples and the standard barcode of yak horn clustered into one branch, with a node support rate of 98%, and the 9 yellow cattle horn samples and the standard barcode of yellow cattle horn clustered into another branch, with a node support rate of 92%, indicating that the barcodes of various breeds of cattle in this research method have good accuracy and high credibility for molecular identification of buffalo horns. The barcode sequences measured by this method can also be Blast compared in GenBank, and the results are consistent with the universal primer barcode COI sequence comparison.

[0101] Embodiment 2

[0102] Processed beef of unknown origin was purchased from the market, and throat pain granules, compound ling detoxification tablets and concentrated buffalo horn tablets produced by a certain company were purchased from the pharmacy. The relevant experiments were carried out according to the method of Example 1, in which the sugar-coated tablets were scraped off for DNA extraction. The experimental results are shown in Figure 6 The results showed that one batch of processed beef was identified as yellow beef, the throat pain granules were identified as containing yak horn ingredients, and the compound Xiling Jiedu tablets and concentrated buffalo horn tablets were identified as containing authentic buffalo horn ingredients. This shows that the barcodes and primers provided by the present invention can be used to identify samples that have been processed (such as heat treated) and contain yellow cattle, yak or buffalo ingredients.

[0103] At present, there are few literatures related to the barcode research of buffalo horns. The existing technology mainly uses universal primers LCO1490 (5'-GGTCAACAAATCATAAAGATATTGG-3', SEQ ID NO.37) and HCO2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3', SEQ ID NO.38) for amplification and identification. Using this pair of universal primers, exogenous DNA can also be amplified. For example, when Liu Xuchao and others identified 62 batches of buffalo horns, they found that 5 batches of samples could not identify the original species, which had been contaminated by exogenous substances such as oomycetes, ascomycetes, beetles, and moths, accounting for 8.1%. When this study used universal primers LCO1490 and HCO2198 for amplification, it was also found that some samples were contaminated by exogenous gene storage beetles. For example, the sequencing result of sample MN01 was a peak ( Figure 2 ), and when the sequencing results of the peaks were compared, it was found that it was an exogenous animal gene ( Figure 3 ), while after changing the designed specific cow horn primers (NJ161-F and NJ161-R), a single peak shape without nested peaks was obtained ( Figure 4 ).

[0104] Comparison of barcode standard detection sequences of buffalo horn, yellow cattle horn, and yak horn and sequencing results of the tested samples can be found in Figure 5 In this barcode segment, there are no intraspecific variation sites in buffalo horns and yak horns, and only two intraspecific difference sites exist in cattle horns, indicating that the intraspecific sequence is stable in this barcode segment. The statistics of the site differences among buffalo horns, cattle horns, and yak horns are shown in Table 2 below.

[0105] Table 2 Statistics of differences in sequence sites of barcode standard detection of buffalo horn, yellow cattle horn and yak horn

[0106] Location 36 51 57 66 72 75 87 90 102 111 114 120 129 132 135 Buffalo Horn T G C G T C C T C C A C G C T Yellow ox horn C A C A C T T C T T A T A T T Yak horn C G A A C T T T C T G C A C C

[0107] Table 3 Standard barcodes and sample sequencing results of buffalo horns, cattle horns, and yak horns

[0108]

[0109]

[0110]

[0111]

[0112] References:

[0113] [1] Tao Hongjing. Ming Yi Bie Lu (Collected and Edited Edition). Shang Zhijun, Edited and Edited. Beijing: China Traditional Chinese Medicine Press, 2013: 142-143.

[0114] [2] Chinese Pharmacopoeia Committee. Pharmacopoeia of the People's Republic of China[M]. China Medical Science and Technology Press, 2020.

[0115] [3] Editorial Committee of Chinese Materia Medica by State Administration of Traditional Chinese Medicine. Chinese Materia Medica-9[M]. Shanghai: Shanghai Science and Technology Press, 1999: 808.

[0116] [4] Hua Hongshun. Comparison of thin layer chromatography of antelope horn and buffalo horn [J]. Journal of Jiangxi University of Traditional Chinese Medicine, 1998, 10(2): 84.

[0117] [5] Li Xiaomeng, He Xinrong. Identification of buffalo horns and antelope horns by HPLC[J]. Journal of Guangdong Pharmaceutical College, 2003, 19(2): 97-98.

[0118] [6] Tang Huiying, Yan Dan, Wu Yanwen, et al. Identification of horn animal medicines based on FTIR technology [J]. Chinese Medicinal Materials, 2009, 32(11): 1658-1660.

[0119] [7] Liu Xuchao. Study on DNA barcoding identification of buffalo horn and earthworm medicinal materials and optimization of DNA extraction method of animal medicinal materials[D]. Jinan: Shandong University of Traditional Chinese Medicine, 2017.

[0120] [8] Liu Rui, Duan Jinli, Qian Dawei, et al. A characteristic peptide fragment of cattle horn and its detection method: CN114395014 A[P]. 2022-06-26.

Claims

1. A DNA barcode, characterized in that: The nucleotide sequence of the DNA barcode is shown as SEQ ID NO.

1.

2. A DNA barcode composition, characterized in that: The DNA barcode composition comprises: A first DNA barcode, wherein the nucleotide sequence of the first DNA barcode is shown as SEQ ID NO.1; a second DNA barcode, the nucleotide sequence of which is shown in SEQ ID NO.2; and A third DNA barcode, the nucleotide sequence of the third DNA barcode is shown as SEQ ID NO.

3.

3. A primer composition, characterized in that The primer composition comprises detection primers designed based on the DNA barcode according to claim 1 and / or based on the DNA barcode composition according to claim 2.

4. The primer composition according to claim 3, characterized in that The primer composition comprises the following detection primers: NJ161-F, the nucleotide sequence of which is shown in SEQ ID NO.35; NJ161-R, the nucleotide sequence of which is shown in SEQ ID NO.

36.

5. Use of the DNA barcode according to claim 1, the DNA barcode composition according to claim 2, or the primer composition according to claim 3 or 4 in identifying buffalo horns and / or constructing a phylogenetic tree of bovine horns.

6. A method for identifying buffalo horns, characterized in that: The method comprises the following steps (i) to (iii): (i) extracting genomic DNA from the bovine horn sample to be tested; (ii) using the genomic DNA of the horn sample to be tested extracted in step (i) as a template, and performing PCR amplification using the primer combination according to claim 3 or 4; (iii) sequencing the amplified product obtained in step (ii), comparing the sequencing result with the DNA barcode according to claim 1 and / or the DNA barcode composition according to claim 2, and / or applying the sequencing result to construct a phylogenetic tree of bovine horns.

7. The identification method according to claim 6, characterized in that: In step (i), the buffalo horn sample to be tested includes any one or more of buffalo horn medicinal materials, buffalo horn standard decoctions, buffalo horn formula granules and Chinese patent medicines containing buffalo horns.

8. The identification method according to claim 6 or 7, characterized in that: In step (ii), the reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 61°C for 30 s, extension at 72°C for 30 s, for a total of 35 cycles; extension at 72°C for 5 min.

9. A kit for identifying buffalo horns, characterized in that: The kit comprises the primer combination according to claim 4 or 5.

10. The kit according to claim 8, characterized in that The kit also includes PCR reaction reagents.

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