PCR-RFLP method for identifying venenum bufonis and application of PCR-RFLP method

Abstract

The invention discloses a method for identifying polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of venenum bufonis, and the method comprises the following steps: (1) DNA (deoxyribonucleic acid) extraction; (2) carrying out PCR amplification; (3) purifying a PCR product; (4) enzyme digestion; and (5) identifying the protogen of the venenum bufonis based on the number and the positions of the enzyme digestion bands in the enzyme digestion map. According to the method, a PCR technology is combined with nucleic acid restriction enzyme digestion, specific cutting is carried out based on single nucleic acid polymorphism (SNP) sites of a sibling substance, protogen identification and adulterants identification of the venenum bufonis can be realized through an enzyme digestion map, and the method has the advantages that high-quality DNA is not needed, sequencing is not needed, the specificity is good, and the result is more visual.

Description

Technical Field

[0001] The invention relates to the technical field of medicine, and in particular to a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for identifying toad venom and an application thereof. Background Art

[0002] Toad venom is derived from the dried secretions of Bufo bufo gargarizans Cantor and Bufo melanostictus Schneider, both of the Toad family. It is pungent, warm, toxic, and enters the heart meridian. It has the effects of detoxification, analgesia, and awakening the mind. It is used for carbuncle, furuncle, sore throat, heatstroke, coma, abdominal pain, vomiting and diarrhea. Modern pharmacological studies have shown that it has pharmacological activities such as anti-tumor, cardiotonic, antibacterial, anti-inflammatory, immunomodulatory, and analgesic.

[0003] However, due to factors such as the shortage of toad resources, modern collection and processing methods, and local medicinal habits, the origin of commercial toad venom in the current market is mixed, and illegal varieties of toad venom are commonly used as medicine. According to current animal taxonomy, Chinese toad Bufo gargarizans Cantor belongs to the class of amphibians, the order of Anura, the order of Procoela, the family of Bufonidae, the genus Bufo, and is divided into three subspecies: the nominate subspecies of Chinese toad B. gargarizansgargarizans Cantor, the Chinese toad B. gargarizans andrewsi Schmidt, and the Chinese toad B. gargarizans minshanicus Stejneger. Comparing the pharmacopoeial source items, the following problems were found: (1) The Chinese giant toad Bufo bufo gargarizans Cantor is inconsistent with animal taxonomy, and it is not clear whether it is used as medicine at the species level or the subspecies level; the "Chinese Medicinal Animals" records that all three subspecies of Chinese toad B. gargarizans can be used as medicine. Some researchers have suggested changing the name of "Chinese giant toad Bufo bufo gargarizans Cantor" under the pharmacopoeia source to "Chinese toad Bufogargarizans Cantor", but the equivalence of the quality of the toad venom of each subspecies needs further study. (2) According to the current classification of the Toad Family, the black-rimmed toad is distantly related to the Chinese toad, and its chemical composition is quite different from that of the Chinese toad venom. According to the toad venom content limit of the 2020 edition of the Chinese Pharmacopoeia, the black-rimmed toad venom is difficult to meet the requirements, so the rationality of its equal use as medicine with the Chinese toad venom is questionable. (3) Recently, some scholars have conducted phylogenetic analysis based on the mitochondrial gene CR-ND2 and the nuclear gene POMC-RAG-1-Rho, and suggested that the evolutionary branch of the Chinese toad distributed in northern East Asia (including Northeast China) be upgraded to the species level and named Northeast toad Bufo sachalinensis. The difference between the toad venom (Northeast Toad Venom) derived from this species of toad and the legal original species Chinese toad venom and black-rimmed toad venom remains to be revealed.

[0004] Based on the above considerations, it is necessary to establish a method for identifying the origin of toad venom, in order to further clarify the quality differences between the legal origin toad venom and closely related toad venom, and between toad venom of each subspecies, and to lay the foundation for expanding the medicinal resources of toad venom. As a typical representative of secretory animal medicine, toad venom lacks the morphological characteristics of its original origin animal after processing, while the chemical composition of closely related species of the same genus is similar and is easily affected by the origin, processing, and preparation process. Therefore, there are certain limitations in clarifying the origin of toad venom by methods such as trait identification and chemical identification. As a carrier of genetic information, DNA has certain intraspecific stability and interspecific diversity, and is not affected by external factors such as growth and development stages, environmental conditions, and human activities. It has obvious advantages in species identification. DNA molecular identification includes identification technology based on molecular hybridization (RFLP and DNA chips), identification technology based on PCR amplification (RAPD, ISSR, SSR, specific PCR), identification technology based on DNA sequence analysis (DNA barcode), etc. Among them, specific PCR and DNA barcode technology are most widely used in the identification of closely related mixed products of Chinese medicinal materials and decoction pieces, multi-gene identification, genetic diversity analysis, germplasm resource research, etc., and has entered the practical stage. For example, since 2010, the "Chinese Pharmacopoeia" has successively included the specific PCR identification method of Acuta acutus and Agama rubrum, the PCR-RFLP identification method of Fritillaria cirrhosa and Dendrobium huoshanense, and the "Guiding Principles of DNA Barcode Molecular Identification Method for Chinese Medicinal Materials".

[0005] Based on the considerations of mixed origin sample detection and improving experimental efficiency, the present invention establishes a PCR-RFLP method for the origin identification of toad venom based on the analysis of COI sequence restriction endonuclease cleavage sites. Summary of the invention

[0006] Based on this, the present invention provides a method for identifying toad venom, the method comprising the following steps:

[0007] (1) DNA extraction: extracting genomic DNA from different species of toad venom samples; (2) PCR amplification: using Chmf4 / Chmr4 as an amplification primer pair and the genomic DNA as a template, PCR amplification is performed to obtain an unpurified PCR product; (3) Purification of PCR product: adding binding buffer to the unpurified PCR product, mixing it well, adding it to a centrifuge column, standing it, centrifuging it, and discarding the effluent; adding washing buffer, centrifuging it, discarding the effluent; centrifuging it to remove the residual washing buffer; placing the centrifuge column in a clean centrifuge tube, adding elution buffer or deionized water, centrifuging it, and obtaining a purified PCR product; (4) Enzyme digestion: using a restriction endonuclease to perform an enzyme digestion reaction on the purified PCR product, after the enzyme digestion reaction is completed, adding a sample loading buffer to mix it well to obtain a mixed solution, taking an appropriate amount of the mixed solution, and performing agarose gel electrophoresis detection to obtain an enzyme digestion map of the purified PCR product; and (5) based on the number and position of the enzyme digestion bands in the enzyme digestion map, the origin of toad venom is identified.

[0008] Furthermore, in step (1), the steps of DNA extraction are: (a) taking an appropriate amount of dried toad venom powder, adding buffer GA, inverting and mixing, adding proteinase K, inverting and mixing, reacting and digesting, and inverting and mixing 2 to 3 times every about 1 hour; (b) adding chloroform:isoamyl alcohol (24:1), inverting and mixing, centrifuging, transferring the upper aqueous phase to another centrifuge tube, and repeating this step once; (c) adding buffer GB, inverting and mixing, standing, adding anhydrous ethanol, and shaking and mixing; (d) transferring to an adsorption column, centrifuging, discarding the waste liquid, adding buffer GD, centrifuging, and discarding the waste liquid; (e) adding rinse solution PW, centrifuging, discarding the waste liquid, and repeating this step once; (f) centrifuging, discarding the waste liquid, standing and drying; and (g) placing the adsorption column in a 1.5 mL centrifuge tube, adding eluent PE dropwise to the middle position, standing, centrifuging, collecting the eluate, and obtaining the genomic DNA.

[0009] Further, the volume of the buffer GA in the step (a) is about 400 μL. Further, the volume of the proteinase K in the step (a) is about 40 μL. Further, the temperature of the reaction digestion in the step (a) is about 56°C, and the time of the reaction digestion is about 3h. Further, the volume of the chloroform:isoamyl alcohol (24:1) in the step (b) is about 700 μL. Further, the speed of the centrifugation in the step (b) is about 12000r / min, and the time of the centrifugation is about 5min. Further, the volume of the buffer GB in the step (c) is about 400 μL. Further, the temperature of the standing in the step (c) is about 70°C, and the standing time is about 10min. Further, the volume of the anhydrous ethanol in the step (c) is about 400 μL. Further, the speed of the centrifugation in the step (d) is about 12000r / min, and the time of the centrifugation is about 30s. Further, the volume of the buffer GD in the step (d) is about 500μL. Further, the volume of the rinse liquid PW in the step (e) is about 700μL. Further, the speed of the centrifugation in the step (e) is about 12000r / min, and the time of the centrifugation is about 30s. Further, the speed of the centrifugation in the step (f) is about 12000r / min, and the time of the centrifugation is about 2min. Further, the temperature of the standing and drying in the step (f) is room temperature, and the time of the standing and drying is about 30min. Further, the volume of the eluent PE in the step (g) is about 60μL. Further, the temperature of the standing in the step (g) is room temperature, and the time of the standing is about 2min. Furthermore, the centrifugal speed in step (g) is about 12000 r / min, and the centrifugal time is about 2 min.

[0010] Furthermore, in step (2), the PCR amplification reaction system comprises a template, a PCR premix solution, primers Chmf4 / Chmr4 and ddH 2 O. Further, the volume of the total system is about 20 μL. Further, the volume of the template is about 1 μL. Further, the total DNA mass of the template is >50 ng. Further, the volume of the PCR premix solution is about 10 μL. Further, the concentration of the primers Chmf4 / Chmr4 is about 10 μmol / L. Further, the volume of the primers Chmf4 / Chmr4 is about 1 μL.

[0011] Further, in step (3), 4 to 6 volumes of binding buffer are added to the unpurified PCR product, for example, about 5 volumes. Further, the standing time is 0.5 to 2 min, for example, about 1 min. Further, the centrifugal speed is about 12000 r / min, and the time is 0.5 to 2.5 min, for example, about 1 min or about 2 min. Further, the volume of the washing buffer is about 650 μL. Further, the centrifuge tube is a 1.5 mL centrifuge tube. Further, the temperature of the elution buffer or deionized water is about 65°C, and the volume of the elution buffer or deionized water is about 40 μL. Further, use The concentration of the purified PCR product was determined using an Epoch microplate spectrophotometer.

[0012] Further, in step (4), the conditions of the enzyme digestion reaction are: adding the purified PCR product, rCutSmart buffer, restriction endonuclease and deionized water to the system, and incubating at about 37°C for about 1 hour. Further, the volume of the system is about 25 μL. Further, the mass of the purified PCR product is about 0.5 μg. Further, the volume of the rCutSmart buffer is about 2.5 μL. Further, the volume of the restriction endonuclease is about 1 μL. Further, the loading buffer is 6×DNA loading buffer. Further, the 6×DNA loading buffer is about 4 μL. Further, the volume of the mixed solution is about 10 μL. Further, the concentration of the agarose gel is about 3%. Further, the voltage of the electrophoresis detection is about 140V, and the time of the electrophoresis detection is about 30 min.

[0013] Furthermore, the restriction endonuclease in step (4) is restriction endonuclease HhaI, restriction endonuclease NspI and / or restriction endonuclease EaeI.

[0014] Further, the enzyme cleavage pattern of the Chinese toad venom is that in the enzyme cleavage pattern of the restriction endonuclease HhaI, there are only two bands at about 500bp and about 250bp. Further, the enzyme cleavage pattern of the black-orbital toad venom is that in the enzyme cleavage pattern of the restriction endonuclease NspI, there are only two bands at about 400bp and about 300bp. Further, the enzyme cleavage pattern of the northeastern toad venom is that in the enzyme cleavage pattern of the restriction endonuclease EaeI, there are only two bands between about 300 and 400bp.

[0015] Further, the toad venom sample is selected from one or more of the following: Chinese toad venom, black-spotted toad venom, northeastern toad venom and other toad venom different from Chinese toad venom, black-spotted toad venom and northeastern toad venom.

[0016] Furthermore, the toad venom sample is commercial toad venom.

[0017] According to another aspect of the present invention, a kit for identifying toad venom is provided, comprising the above primer pair and restriction endonuclease, and instructions for use of the above method.

[0018] According to another aspect of the present invention, there is provided a use of the above method or the above kit in identifying or assisting in identifying toad venom.

[0019] Beneficial effects of the present invention:

[0020] The method of the present invention combines PCR technology with nucleic acid restriction enzyme digestion, performs specific cutting based on the single nucleotide polymorphism (SNP) site of closely related products, and can realize the identification of the origin of toad venom and the identification of mixed and counterfeit products through the enzyme digestion pattern of endonucleases HhaI, NspI and EaeI. The advantages are that high-quality DNA and sequencing are not required, the specificity is good, the result presentation is more intuitive, and it has the feasibility of popularization and application. Compared with the COI barcode method, the PCR-RFLP method can identify toad venom samples with mixed origins, and the enzyme digestion pattern shows 3 bands, while the toad venom samples with single origin have only 2 bands in the enzyme digestion pattern. The sensitivity test is carried out by making a homemade mixed origin toad venom sample, and the Chinese toad venom, the black orbital toad venom and the northeastern toad venom can be detected when the proportion is 5%, 5% and 40% respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0022] Figure 1 Schematic diagram of the restriction site results of endonucleases HhaI and HinP1I.

[0023] Figure 2 This is a schematic diagram of the results of the restriction site of the endonuclease NspI.

[0024] Figure 3 This is a schematic diagram of the results of the restriction site of the endonuclease NmeAIII.

[0025] Figure 4 It is a schematic diagram of the restriction site results of the endonuclease MmeI.

[0026] Figure 5 This is a schematic diagram of the restriction site results of the endonuclease EaeI.

[0027] Figure 6 This is a schematic diagram of the restriction site results of the endonuclease PaqCI.

[0028] Figure 7 The figure is a 3% agarose gel electrophoresis diagram of the digestion products of toad samples using endonuclease HhaI.

[0029] Among them, 1-26: Chinese toad; 27-37: black-spotted toad; 38-48: Northeast toad.

[0030] Figure 8 The 3% agarose gel electrophoresis diagram of the digestion products of toad samples based on endonuclease NspI. Among them, 1-26: Chinese toad; 27-37: black-spotted toad; 38-48: Northeast toad.

[0031] Fig. 9 The 3% agarose gel electrophoresis diagram of the digestion products of toad samples based on the endonuclease EaeI. Among them, 1-26: Chinese toad; 27-37: black-spotted toad; 38-48: Northeast toad.

[0032] Fig.10 The 3% agarose gel electrophoresis diagram of the digestion product of the toad venom sample based on the endonuclease HhaI, wherein the number is consistent with the sample number in Table 1.

[0033] Fig.11 The 3% agarose gel electrophoresis diagram of the digestion product of the toad venom sample based on the endonuclease NspI, wherein the number is consistent with the sample number in Table 1.

[0034] Fig.12 The 3% agarose gel electrophoresis diagram of the digestion product of the toad venom sample based on the endonuclease EaeI, wherein the number is consistent with the sample number in Table 1.

[0035] Fig.13 The 3% agarose gel electrophoresis diagram of the digestion products of different proportions of Chinese toad venom samples based on the endonuclease HhaI.

[0036] Fig.14 The 3% agarose gel electrophoresis diagram of the digestion products of different proportions of black-orbital toad venom samples based on endonuclease NspI.

[0037] Fig.15 The 3% agarose gel electrophoresis diagram of the digestion products of different proportions of Northeast Toad Venenum samples based on the endonuclease EaeI.

[0038] Fig.16 The 3% agarose gel electrophoresis diagram of the digestion product of commercial toad venom based on endonuclease HhaI. The numbers are consistent with the sample numbers in Table 2.

[0039] Fig.17The 3% agarose gel electrophoresis diagram of the digestion product of commercial toad venom based on endonuclease NspI. The numbers are consistent with the sample numbers in Table 2.

[0040] Fig.18 The 3% agarose gel electrophoresis diagram of the digestion product of commercial toad venom based on the endonuclease EaeI. The numbers are consistent with the sample numbers in Table 2. DETAILED DESCRIPTION

[0041] The technical solutions in the embodiments of the present invention are described clearly and completely below. 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, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0042] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0043] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0044] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0045] As described in the background technology section, as a typical representative of secretory animal drugs, toad venom lacks the morphological characteristics of its original source animal after processing, while the chemical composition of closely related species of the same genus is similar and is easily affected by the origin, processing, and preparation process. Therefore, there are certain limitations in clarifying the origin of toad venom by methods such as property identification and chemical identification. In order to solve the above problems, the present invention provides a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for identifying toad venom, which comprises the following steps: (1) DNA extraction: extracting genomic DNA from toad venom samples of different species; (2) PCR amplification: using Chmf4 / Chmr4 as an amplification primer pair and the genomic DNA as a template, PCR amplification is performed to obtain an unpurified PCR product; (3) purification of the PCR product: adding binding buffer to the unpurified PCR product, mixing it, adding it to a centrifugal column, standing, centrifuging, and discarding the effluent; adding washing buffer, centrifuging, and discarding. The effluent is centrifuged to remove the residual washing buffer; the centrifuge column is placed in a clean centrifuge tube, an elution buffer or deionized water is added, and the elution buffer is centrifuged to obtain a purified PCR product; (4) enzyme digestion: the purified PCR product is subjected to an enzyme digestion reaction using a restriction endonuclease, and after the enzyme digestion reaction is completed, a sample loading buffer is added to mix the mixture to obtain a mixed solution, and an appropriate amount of the mixed solution is taken for agarose gel electrophoresis detection to obtain an enzyme digestion map of the purified PCR product; and (5) based on the number and position of the enzyme digestion bands in the enzyme digestion map, the origin of toad venom is identified.

[0046] In a preferred embodiment, in step (1), the DNA extraction step is as follows: (a) taking an appropriate amount of dried toad venom powder, adding buffer GA, inverting and mixing, adding proteinase K, inverting and mixing, reacting and digesting, and inverting and mixing 2 to 3 times every about 1 hour; (b) adding chloroform:isoamyl alcohol (24:1), inverting and mixing, centrifuging, transferring the upper aqueous phase to another centrifuge tube, and repeating this step once; (c) adding buffer GB, inverting and mixing, standing, adding anhydrous ethanol, and shaking and mixing; (d) transferring to an adsorption column, centrifuging, discarding the waste liquid, adding buffer GD, centrifuging, and discarding the waste liquid; (e) adding rinse solution PW, centrifuging, discarding the waste liquid, and repeating this step once; (f) centrifuging, discarding the waste liquid, standing and drying; and (g) placing the adsorption column in a 1.5 mL centrifuge tube, adding eluent PE dropwise to the middle position, standing, centrifuging, collecting the eluate, and obtaining the genomic DNA.

[0047] In a preferred embodiment, the volume of the buffer GA in the step (a) is about 400 μL. In a preferred embodiment, the volume of the proteinase K in the step (a) is about 40 μL. In a preferred embodiment, the temperature of the reaction digestion in the step (a) is about 56°C, and the time of the reaction digestion is about 3h. In a preferred embodiment, the volume of the chloroform:isoamyl alcohol (24:1) in the step (b) is about 700 μL. In a preferred embodiment, the speed of the centrifugation in the step (b) is about 12000 r / min, and the time of the centrifugation is about 5min. In a preferred embodiment, the volume of the buffer GB in the step (c) is about 400 μL. In a preferred embodiment, the temperature of the standing in the step (c) is about 70°C, and the standing time is about 10min. In a preferred embodiment, the volume of the anhydrous ethanol in the step (c) is about 400 μL. In a preferred embodiment, the centrifugal speed in the step (d) is about 12000r / min, and the centrifugal time is about 30s. In a preferred embodiment, the volume of the buffer GD in the step (d) is about 500μL. In a preferred embodiment, the volume of the rinse solution PW in the step (e) is about 700μL. In a preferred embodiment, the centrifugal speed in the step (e) is about 12000r / min, and the centrifugal time is about 30s. In a preferred embodiment, the centrifugal speed in the step (f) is about 12000r / min, and the centrifugal time is about 2min. In a preferred embodiment, the temperature of the standing and drying in the step (f) is room temperature, and the standing and drying time is about 30min. In a preferred embodiment, the volume of the eluent PE in the step (g) is about 60μL. In a preferred embodiment, the standing temperature in the step (g) is room temperature, and the standing time is about 2min. In a preferred embodiment, the centrifugal rotation speed in the step (g) is about 12000 r / min, and the centrifugal time is about 2 min.

[0048] In a preferred embodiment, in step (2), the PCR amplification reaction system comprises a template, a PCR premix solution, primers Chmf4 / Chmr4 and ddH 2O. In a preferred embodiment, the volume of the total system is about 20 μL. In a preferred embodiment, the volume of the template is about 1 μL. In a preferred embodiment, the total DNA mass of the template is >50 ng. In a preferred embodiment, the volume of the PCR premix solution is about 10 μL. In a preferred embodiment, the concentration of the primers Chmf4 / Chmr4 is about 10 μmol / L. In a preferred embodiment, the volume of the primers Chmf4 / Chmr4 is about 1 μL.

[0049] In a preferred embodiment, in step (3), 4 to 6 volumes of binding buffer, for example, about 5 volumes, are added to the unpurified PCR product. In a preferred embodiment, the standing time is 0.5 to 2 min, for example, about 1 min. In a preferred embodiment, the centrifugal speed is about 12000 r / min, and the time is 0.5 to 2.5 min, for example, about 1 min or about 2 min. In a preferred embodiment, the volume of the wash buffer is about 650 μL. In a preferred embodiment, the centrifuge tube is a 1.5 mL centrifuge tube. In a preferred embodiment, the temperature of the elution buffer or deionized water is about 65°C, and the volume of the elution buffer or deionized water is about 40 μL. In a preferred embodiment, use The concentration of the purified PCR product was determined using an Epoch microplate spectrophotometer.

[0050] In a preferred embodiment, in step (4), the conditions of the enzyme digestion reaction are: adding the purified PCR product, rCutSmart buffer, restriction endonuclease and deionized water to the system, and incubating at about 37°C for about 1 hour. In a preferred embodiment, the volume of the system is about 25 μL. In a preferred embodiment, the mass of the purified PCR product is about 0.5 μg. In a preferred embodiment, the volume of the rCutSmart buffer is about 2.5 μL. In a preferred embodiment, the volume of the restriction endonuclease is about 1 μL. In a preferred embodiment, the loading buffer is 6×DNA loading buffer. In a preferred embodiment, the 6×DNA loading buffer is about 4 μL. In a preferred embodiment, the volume of the mixed solution is about 10 μL. In a preferred embodiment, the concentration of the agarose gel is about 3%. In a preferred embodiment, the voltage of the electrophoresis detection is about 140V, and the time of the electrophoresis detection is about 30min.

[0051] In a preferred embodiment, the restriction endonuclease in step (4) is restriction endonuclease HhaI, restriction endonuclease NspI and / or restriction endonuclease EaeI.

[0052] In a preferred embodiment, the enzyme cleavage pattern of the Chinese toad venom is that in the enzyme cleavage pattern of the restriction endonuclease HhaI, there are only two bands at about 500bp and about 250bp. In a preferred embodiment, the enzyme cleavage pattern of the black-orbital toad venom is that in the enzyme cleavage pattern of the restriction endonuclease NspI, there are only two bands at about 400bp and about 300bp. In a preferred embodiment, the enzyme cleavage pattern of the northeastern toad venom is that in the enzyme cleavage pattern of the restriction endonuclease EaeI, there are only two bands between 300 and 400bp.

[0053] That is, in the enzyme cleavage map of the restriction endonuclease HhaI of the present invention, when there are only two bands at about 500bp and about 250bp, it is identified as Chinese toad venom; in the enzyme cleavage map of the restriction endonuclease NspI, when there are only two bands at about 400bp and about 300bp, it is identified as black-orbital toad venom; in the enzyme cleavage map of the restriction endonuclease EaeI, when there are only two bands between 300 and 400bp, it is identified as Northeast toad venom; when other bands are present in the above enzyme cleavage map, it indicates that other species of toad venom are mixed in the toad venom sample.

[0054] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05; "about 400" includes ±5% of 400, or from 380 to 420; "about 40" includes ±5% of 40, or from 38 to 42; "about 56" includes ±5% of 56, or from 53.2 to 58.8; "about 3" includes ±5% of 3, or from 2.85 to 3.15; "about 700" includes ±5% of 700. 5%, or from 665 to 735; "about 12000" includes ±5% of 12000, or from 11400 to 12600; "about 5" includes ±5% of 5, or from 4.75 to 5.25; "about 70" includes ±5% of 70, or from 66.5 to 73.5; "about 10" includes ±5% of 10, or from 9.5 to 10.5; "about 30" includes ±5% of 30, or from 28.5 to 31.5; "about 500" includes ±5% of 500, or from 475 to 525; "about 2" includes ±5% of 2, or from 1.9 to 2.1; "about 60" includes ±5% of 60, or from 57 to 63; "about 20" includes ±5% of 20, or from 19 to 21; "about 650" includes ±5% of 650, or from 617.5 to 682.5; "about 65" includes ±5% of 650, or from 617.5 to 682.5; "about 65" includes ±5% of 650, or from 617.5 to 682.5. “About 37” includes ±5% of 37, or from 35.15 to 38.85; “about 25” includes ±5% of 25, or from 23.75 to 26.25; “about 2.5” includes ±5% of 2.5, or from 2.375 to 2.625; “about 0.5” includes ±5% of 0.5, or from 0.475 to 0.525.

[0055] In a preferred embodiment, the toad venom sample is selected from one or more of the following: Chinese toad venom, black-spotted toad venom, northeastern toad venom and other toad venom different from Chinese toad venom, black-spotted toad venom and northeastern toad venom.

[0056] In a preferred embodiment, the toad venom sample is commercial toad venom.

[0057] According to another aspect of the present invention, a kit for identifying toad venom is provided, comprising the above primer pair and restriction endonuclease, and instructions for use of the above method.

[0058] According to another aspect of the present invention, there is provided a use of the above method or the above kit in identifying or assisting in identifying toad venom.

[0059] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0060] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0061] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0062] Example

[0063] 1. Experimental instruments and reagents

[0064] JXFSTPRP-II-01 cryo-grinder (Shanghai Jingxin Industrial Development Co., Ltd.), SCILOGEX MS-X vortex mixer, SCILOGEX MS-RD-Pro CNC disc rotating mixer, DLAB D1008E centrifuge, DLAB D1008 centrifuge, DLAB HB120-S metal bath, LongGene T series Multi-Block Thermal Cycler (Hangzhou Longji Scientific Instrument Co., Ltd.), DYY-6C electrophoresis apparatus (Beijing Liuyi Instrument Factory), BIO-RAD Gel Doc EZ gel imager, Epoch microplate spectrophotometer (Berteng Instrument Co., Ltd., USA); TIANamp GenomeDNA kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.), PCR Master Mix (With Dye) (Yishen Biotechnology (Shanghai) Co., Ltd.), agarose (Qingke Biotechnology Co., Ltd.), D2000 DNA Ladder, green fluorescent nucleic acid dye (Solabao Biotechnology Co., Ltd.), primers were synthesized by Beijing Liuhe BGI Gene Technology Co., Ltd.; 100 bp DNA Ladder (Solabao Biotechnology Co., Ltd.), EasyPure PCR Purification Kit (Beijing Quanshijin Biotechnology Co., Ltd.), HhaI, NspI, EaeI (New England Biolabs) were purchased from Beijing Bailingke Biotechnology Co., Ltd.

[0065] 2. Experimental samples

[0066] From 2015 to 2022, the research team collected a total of 53 batches of toad venom samples of different origins, including the nominate subspecies of Chinese toad venom, the West Asian species of Chinese toad venom, the Minshan subspecies of Chinese toad venom, the black-rimmed toad venom and the northeastern toad venom. Among them, 24 batches collected corresponding toad animals as voucher specimens, which were identified by Gao Huimin, a researcher at the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, as the nominate subspecies of Chinese toad B. gargarizans gargarizans Cantor, the West Asian species of Chinese toad B. gargarizansandrewsi Schmidt, the Minshan subspecies of Chinese toad B. gargarizans minshanicus Stejneger, the black-rimmed toad B. melanostictus Schneider and the northeastern toad B. sachalinensis Nikolskii. The collection information of the above 53 batches of toad venom samples is shown in Table 1. In addition, the research team also collected 17 batches of commercial toad venom, and the sample information is shown in Table 2. All the above samples are stored in the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences.

[0067] Table 1 Collection information of toad venom samples

[0068]

[0069]

[0070] Note: "√" indicates that there are voucher animals for this batch, and "-" indicates that voucher animals for this batch have not been collected. "ZH, HX, MS, HK, DB" in the number refers to the origin, and the subsequent letters refer to the place of origin, such as "ZH-JLLY" means the nominate subspecies of Chinese toad, which comes from Liaoyuan, Jilin.

[0071] Table 2 Information on commercial toad venom samples

[0072]

[0073]

[0074] 3. Experimental methods

[0075] 3.1. Total DNA extraction from toad and toad venom samples

[0076] Take about 30 mg of dried leg muscle tissue or toad venom powder from toads and place it in a 2 mL centrifuge tube. Use a high-throughput tissue grinder to grind and pulverize (60 Hz, 3 min). Use the TIANamp Genome DNA kit to extract total DNA according to the instructions and the standard operating procedures for DNA barcode molecular identification of animal medicinal materials in the "Standard Sequences of DNA Barcodes for Chinese Pharmacopoeia of Traditional Chinese Medicines". The specific operation is as follows: (1) Add 400 μL of buffer GA to each sample, invert and mix, add 40 μL of proteinase K, invert and mix, and digest at 56°C for 3 h, invert and mix 2 to 3 times every 1 h; (2) Add 700 μL of chloroform:isoamyl alcohol (24:1), invert and mix, and spin at 12000 r·min -1 Centrifuge for 5 min, transfer the upper aqueous phase to a new centrifuge tube, and repeat this step once; (3) add 400 μL of buffer GB, invert and mix, place at 70°C for 10 min, add 400 μL of anhydrous ethanol, and shake to mix; (4) transfer to the adsorption column and spin at 12,000 r / min -1 Centrifuge for 30 seconds, discard the waste liquid, add 500 μL of buffer GD, and spin at 12000 r·min -1 Centrifuge for 30 seconds and discard the waste liquid; (5) Add 700 μL of rinsing solution PW and centrifuge at 12000 r·min -1 Centrifuge for 30 seconds, discard the waste liquid, and repeat this step once; (6) 12000 r·min -1 Centrifuge for 2 min, discard the waste liquid, and place at room temperature for 30 min to dry the rinse liquid; (7) Place the adsorption column in a 1.5 mL centrifuge tube, add 60 μL of eluent PE to the middle position, place at room temperature for 2 min, and rotate at 12000 r / min. -1 Centrifuge for 2 minutes and collect the eluate. The total DNA obtained was detected by 1.0% agarose gel electrophoresis (140V, 30min) and used The concentration and purity were determined by Epoch microplate spectrophotometer and stored at -20°C.

[0077] 3.2 PCR amplification of toad and toad venom genomic DNA

[0078] The amphibian COⅠ barcoded universal primers Chmf4 / Chmr4 were used to amplify the genomic DNA of toads and toad venom, respectively. The PCR reaction was carried out in a 20 μL system, including 1 μL template (total DNA mass > 50 ng), 10 μL PCR premix solution, forward and reverse primers (10 μmol·L -1 ) 1 μL each, add ddHO 2 The PCR primer sequences and reaction procedures are shown in Table 3.

[0079] Table 3 PCR amplification primers and amplification program

[0080]

[0081] Among them, for the 24 batches of toad animals in Table 1, 3 animals were selected from each batch, and the PCR amplification products of a total of 72 toad animal samples were subjected to Sanger sequencing (Beijing Nosai Genome Research Center Co., Ltd.), and the sequencing primers were COI-CO1 / COI-CO3 (F: TYTCWACWAAY CAYAAAGAYATTGG, R: ACYTCYGGRTGACCAAARAAYCA), and the COⅠ sequences of different species of toads were obtained for the screening of restriction endonucleases.

[0082] 3.3 Purification of PCR products from toad and toad venom samples

[0083] The PCR products of toad and toad venom genomic DNA were purified using the TRAN EasyPure PCR Purification Kit according to the instructions. The specific steps were as follows: (1) PCR products were added to 5 volumes of binding buffer, mixed and added to the centrifuge column, allowed to stand for 1 min, and centrifuged at 12000 r / min. -1 Centrifuge for 1 min and discard the effluent; (2) Add 650 μL of washing buffer and centrifuge at 12000 r / min -1 Centrifuge for 1 min and discard the effluent; (3) 12000 r / min -1 Centrifuge for 2 min to remove the residual washing buffer; (4) Place the centrifuge column in a clean 1.5 mL centrifuge tube, add 40 μL of 65°C preheated deionized water to the center of the column, and centrifuge at 12000 r·min -1 Centrifuge for 2 minutes to obtain the purified PCR product. The concentration was measured using an Epoch microplate spectrophotometer and stored at -20°C.

[0084] 3.4. Screening of restriction endonucleases based on the COⅠ sequence of toad

[0085] The COⅠ sequences of different species of toads were imported into the NEBcutter V2.0 website (http: / / nc2.neb.com / NEBcutter2 / ) for restriction endonuclease cleavage site analysis. All sequences of the same species of toads were compared to find the stable restriction endonuclease sites within the species, and then species-specific restriction endonucleases were selected based on the SNP sites.

[0086] 3.5 Enzyme Digestion

[0087] The enzyme digestion reaction conditions are as follows: 25 μL of total system, add about 0.5 μg of purified PCR product, 2.5 μL of rCutSmart buffer, 1 μL of restriction endonuclease, make up the volume with deionized water, and incubate at 37°C for 1 hour. After the reaction is completed, add 4 μL of 6× DNA loading buffer and mix quickly, and take 10 μL for detection by 3% agarose gel electrophoresis (140V, 30min).

[0088] 4. Experimental results

[0089] 4.1. Restriction endonuclease selection

[0090] According to the methods in 3.1 and 3.2, DNA extraction, PCR amplification and sequencing of amplified products were performed on toad samples. Restriction endonuclease screening was performed according to the method in 3.4. It was found that the sequence of about 430bp of the COⅠ full sequence of Chinese toad (no SNP site was found between the subspecies of Chinese toad) was GCGC ( Figure 1 ), which is the restriction endonuclease cleavage site (GCGC) of the restriction endonucleases HhaI and HinP1I. The sequence of the black-orbiting toad at around 400 bp is ACATGC ( Figure 2 ), which is the restriction endonuclease NspⅠ cleavage site (RCATGY); the sequence around 100bp is CTCGGC ( Figure 3 ), which is the restriction endonuclease NmeAIII cleavage site (GCCGAG (21 / 19)); the sequence around 350 bp is GTTGGA ( Figure 4 ), which is the restriction endonuclease MmeI cleavage site (TCCRAC (20 / 18)), all three are specific endonucleases of Toad nigropunctata. The Northeast Toad sequence at around 370bp is TGGCCG ( Figure 5 ), which is the restriction endonuclease EaeI cleavage site (YGGCCR); the sequence around 430bp is GCAGGTG ( Figure 6 ), which is the restriction endonuclease PaqCI cleavage site (CACCTGC (4 / 8)). Considering the detectability and cost of the digestion products, the endonucleases HhaI, NspI, and EaeI were selected as the specific endonucleases of Bufo gargarizans (Bufo gargarizans), Bufo nigropunctatus (Bufo nigropunctatus), and Bufo gargarizans (Bufo gargarizans), respectively. Toad samples were selected based on haplotype and origin for specificity verification.

[0091] 4.2. Verification of restriction endonuclease specificity

[0092] Of the 24 batches of toads in Table 1, 3 animals were selected from each batch, totaling 72 toad samples for Sanger sequencing. On this basis, 48 ​​toad samples were selected based on haplotype and origin (different haplotypes, same haplotype but different origins) for specificity verification of the three endonucleases HhaI, NspI and EaeI.

[0093] According to the methods in 3.1, 3.2 and 3.3, DNA was extracted from toad samples, PCR amplified and PCR products were purified. According to the conditions in 3.5, restriction enzymes HhaI, NspI and EaeI were used for restriction enzyme digestion. The results of restriction enzyme digestion are shown in the following table. Figure 7 , Figure 8 and Fig. 9 shown.

[0094] The Chinese toad was specifically cut by the endonuclease HhaI, and there were only two bands at about 500bp and 250bp; the black-spotted toad was specifically cut by the endonuclease NspI, and there were only two bands at about 400bp and 300bp; the northeastern toad was specifically cut by the endonuclease EaeI, and there were only two bands between about 300 and 400bp. At the same time, the uncut ones produced a single band at about 700bp. The above results show that the endonucleases HhaI, NspI, and EaeI have good specificity and can specifically cut the COI sequences of the Chinese toad, black-spotted toad, and northeastern toad, respectively. The enzyme cutting patterns show two bands of significantly different sizes.

[0095] 4.3 Identification of Toad Venom

[0096] 4.3.1. Identification of different origin toad venom samples

[0097] According to the methods in 3.1, 3.2 and 3.3, DNA was extracted from the toad venom sample, PCR amplified and PCR product purified. According to the conditions in 3.5, restriction enzymes HhaI, NspI and EaeI were used for restriction digestion reaction. The results are as follows: Fig.10 , Fig.11 and Fig.12 shown.

[0098] The Chinese toad venom samples (2-5, 7, 9-14, 17-23, 25-34) were specifically cut by the endonuclease HhaI, and there were only two bands at about 500bp and about 250bp, indicating that they were a single origin without any other origin of toad venom mixed in; the black-spotted toad venom samples (35, 37, 39-40, 43-44) were specifically cut by the endonuclease NspI, and there were only two bands at about 400bp and about 300bp, indicating that they were a single origin without any other origin of toad venom mixed in; the northeastern toad venom samples (45-53) were specifically cut by the endonuclease EaeI, and there were only two bands between 300-400bp, indicating that they were a single origin without any other origin of toad venom mixed in.

[0099] In addition, the enzyme digestion patterns of some toad venom samples showed three bands: samples 1, 6, 8, 15, 16, 24, and 38 had three bands at about 700bp, 500bp, and 250bp after digestion with endonuclease HhaI; samples 36, 38, and 42 had three bands at about 700bp, 400bp, and 300bp after digestion with endonuclease NspI; samples 1, 6, 15, and 16 had three bands at about 700bp and between about 300 and 400bp after digestion with endonuclease EaeI. This indicates that the above toad venom samples are not single-origin, and other origins are mixed in. Among them, sample 38 can be cut by both endonucleases HhaI and NspI, and is a mixture of Chinese toad venom and black-circle toad venom; samples 1, 6, 15, and 16 can be cut by both endonucleases HhaI and EaeI, and are a mixture of Chinese toad venom and northeastern toad venom; while samples 8 and 24 are only cut by endonuclease HhaI, and samples 36 and 42 are only cut by NspI, and all have three bands, indicating that other original toad venoms except Chinese toad venom, black-circle toad venom and northeastern toad venom are mixed in.

[0100] The above results show that the PCR-RFLP method can be used to identify the origin of toad venom and distinguish between mixed and counterfeit products with high specificity.

[0101] 4.3.2 Identification of homemade mixed-origin toad venom samples

[0102] In order to investigate the specificity and sensitivity of the PCR-RFLP method for detecting toad venom of different origins, the same batch of toad venom of Chinese toad venom, toad venom of black eye and toad venom of northeast China with clear origins were mixed in different proportions to obtain a total of 13 toad venom samples (Table 4).

[0103] Table 4 Mixing ratio of homemade mixed original toad venom samples

[0104]

[0105] According to the methods in 3.1, 3.2 and 3.3, DNA extraction, PCR amplification and PCR product purification were performed on the homemade mixed toad venom sample. According to the conditions in 3.5, restriction enzyme digestion was performed using endonucleases HhaI, NspI and EaeI, respectively. The results are as follows: Fig.13 , Fig.14 and Fig.15 shown.

[0106] When the proportion of Chinese toad venom in the mixed sample is 5%, it can be detected, and fuzzy bands can be seen at about 500bp and 250bp. As the proportion increases, the bands become more obvious; when the proportion of black-spotted toad venom is 5%, it can be detected, and two fuzzy bands can be seen at about 300bp and 400bp; when the proportion of northeastern toad venom is 40%, it can be detected, and two fuzzy bands can be seen between about 300 and 400bp. It can be seen that the detection sensitivity of northeastern toad venom is low. The total DNA concentration extracted from the above 13 samples is about 200ng / μL, and the concentration of purified DNA is about 100ng / μL. The concentration and purity are good. It can be seen that the detection sensitivity is not related to the total DNA concentration, which may be related to the amount of DNA contained in the selected sample, the DNA extraction rate or the PCR amplification efficiency.

[0107] The above experimental results further verified the specificity of the three restriction endonucleases HhaI, NspI, and EaeI, indicating that the PCR-RFLP method can be used to identify the origin of toad venom and distinguish between mixed and counterfeit products, but the detection sensitivity for different origins is different.

[0108] 4.3.3 Identification of commercial toad venom

[0109] According to the methods in 3.1, 3.2 and 3.3, DNA extraction, PCR amplification and PCR product purification were performed on 17 commercial toad venom samples. According to the conditions in 3.5, restriction enzyme digestion was performed using endonucleases HhaI, NspI and EaeI, respectively. The results are as follows: Fig.16 , Fig.17 and Fig.18 shown.

[0110] All 17 commercial toad venom samples were specifically cut by the endonuclease HhaI, and there were only two bands at about 500bp and about 250bp, indicating that the commercial toad venom samples were all single-origin, that is, Chinese toad venom, without any other origin toad venom mixed in. The above results further show that the PCR-RFLP method established in this experiment can be used to identify the origin of commercially available toad venom and identify mixed and counterfeit products.

[0111] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A polymerase chain reaction-restriction fragment length polymorphism method for identifying toad venom, characterized in that: The method comprises the following steps: (1) DNA extraction: Extract genomic DNA from different species of toad venom samples; (2) PCR amplification: using Chmf4 / Chmr4 as an amplification primer pair and the genomic DNA as a template, PCR amplification is performed to obtain an unpurified PCR product; (3) Purification of PCR products: adding binding buffer to the unpurified PCR product, mixing well and adding to a centrifuge column, standing, centrifuging, and discarding the effluent; adding washing buffer, centrifuging, and discarding the effluent; centrifuging to remove residual washing buffer; placing the centrifuge column in a clean centrifuge tube, adding elution buffer or deionized water, and centrifuging to obtain a purified PCR product; (4) Enzyme digestion: using restriction endonucleases to digest the purified PCR product, adding a sample loading buffer to mix the mixture after the digestion reaction is completed to obtain a mixed solution, taking an appropriate amount of the mixed solution and performing agarose gel electrophoresis to obtain a digestion map of the purified PCR product; as well as (5) Based on the number and position of the enzyme cleavage bands in the enzyme cleavage pattern, the origin of toad venom is identified.

2. The method according to claim 1, characterized in that In step (1), the steps of DNA extraction are: (a) Take an appropriate amount of dried toad venom powder, add buffer GA, mix by inversion, add proteinase K, mix by inversion, and digest by reaction, inverting and mixing 2 to 3 times every 1 hour; (b) Add chloroform:isoamyl alcohol (24:1), mix thoroughly by inversion, centrifuge, transfer the upper aqueous phase to another centrifuge tube, and repeat this step once; (c) Add buffer GB, invert to mix, let stand, add anhydrous ethanol, and shake to mix; (d) transferring to an adsorption column, centrifuging, discarding the waste liquid, adding buffer GD, centrifuging, and discarding the waste liquid; (e) adding rinsing solution PW, centrifuging, discarding the waste solution, and repeating this step once; (f) centrifuging, discarding the waste liquid, and standing to dry; and (g) placing the adsorption column in a 1.5 mL centrifuge tube, adding eluent PE dropwise to the middle position, allowing the column to stand, centrifuging, and collecting the eluent to obtain the genomic DNA.

3. The method according to claim 2, characterized in that The volume of the buffer GA in the step (a) is about 400 μL; Preferably, the volume of the proteinase K in step (a) is about 40 μL; Preferably, the reaction digestion temperature in step (a) is about 56° C., and the reaction digestion time is about 3 h; More preferably, the volume of the chloroform:isoamyl alcohol (24:1) in step (b) is about 700 μL; More preferably, the centrifugal speed in step (b) is about 12000 r / min, and the centrifugal time is about 5 min; Also preferably, the volume of the buffer GB in step (c) is about 400 μL; Preferably, the standing temperature in step (c) is about 70° C., and the standing time is about 10 min. Preferably, the volume of the anhydrous ethanol in step (c) is about 400 μL; Particularly preferably, the centrifugal speed in the step (d) is about 12000 r / min, and the centrifugal time is about 30 s; Particularly preferably, the volume of the buffer GD in the step (d) is about 500 μL; Particularly preferably, the volume of the rinse solution PW in step (e) is about 700 μL; Particularly preferably, the centrifugal speed in the step (e) is about 12000 r / min, and the centrifugal time is about 30 s; Particularly preferably, the centrifugal speed in step (f) is about 12000 r / min, and the centrifugal time is about 2 min; Particularly preferably, the temperature of the standing and drying in step (f) is room temperature, and the standing and drying time is about 30 minutes; Particularly preferably, the volume of the eluent PE in step (g) is about 60 μL; Most preferably, the standing temperature in step (g) is room temperature, and the standing time is about 2 minutes; Most preferably, the centrifugal speed in step (g) is about 12000 r / min, and the centrifugal time is about 2 min.

4. The method according to claim 1, characterized in that: In step (2), the PCR amplification reaction system comprises a template, a PCR premix solution, primers Chmf4 / Chmr4 and ddH2O; Preferably, the volume of the total system is about 20 μL; More preferably, the volume of the template is about 1 μL; More preferably, the total DNA mass of the template is >50 ng; Also preferably, the volume of the PCR premix solution is about 10 μL; Particularly preferably, the concentration of the primers Chmf4 / Chmr4 is about 10 μmol / L each; Particularly preferably, the volume of the primers Chmf4 / Chmr4 is about 1 μL each.

5. The method according to claim 1, characterized in that In step (3), 4 to 6 volumes of binding buffer, for example, about 5 volumes, are added to the unpurified PCR product; Preferably, the standing time is 0.5 to 2 minutes, for example, about 1 minute; Preferably, the centrifugal speed is about 12000 r / min, and the time is 0.5 to 2.5 min, such as about 1 min or about 2 min; More preferably, the volume of the wash buffer is about 650 μL; More preferably, the centrifuge tube is a 1.5 mL centrifuge tube; More preferably, the temperature of the elution buffer or deionized water is about 65°C, and the volume of the elution buffer or deionized water is about 40 μL; More preferably, using The concentration of the purified PCR product was measured using an Epoch microplate spectrophotometer.

6. The method according to claim 1, characterized in that In step (4), the conditions of the enzyme digestion reaction are: adding the purified PCR product, rCutSmart buffer, restriction endonuclease and deionized water to the system, and incubating at about 37° C. for about 1 hour; Preferably, the volume of the system is about 25 μL; More preferably, the mass of the purified PCR product is about 0.5 μg; Also preferably, the volume of the rCutSmart buffer is about 2.5 μL; Particularly preferably, the volume of the restriction endonuclease is about 1 μL; Particularly preferably, the loading buffer is 6× DNA loading buffer; Particularly preferably, the 6× DNA loading buffer is about 4 μL; Particularly preferably, the volume of the mixed solution is about 10 μL; Particularly preferably, the concentration of the agarose gel is about 3%; Most preferably, the voltage of the electrophoresis detection is about 140 V, and the time of the electrophoresis detection is about 30 min.

7. The PCR-RFLP method for identifying venom of toad according to claim 1, characterized in that: The restriction endonuclease in step (4) is restriction endonuclease HhaI, restriction endonuclease NspI and / or restriction endonuclease EaeI; Preferably, the restriction enzyme pattern of Toad Venenum chinensis is that in the restriction enzyme pattern of the restriction endonuclease HhaI, there are only two bands at about 500 bp and about 250 bp; Preferably, the enzyme digestion pattern of the venom of Toad venom is that in the enzyme digestion pattern of the restriction endonuclease NspI, there are only two bands at about 400 bp and about 300 bp; Preferably, the enzyme cleavage pattern of the Northeast Bufon venom is that in the enzyme cleavage pattern of the restriction endonuclease EaeI, there are only these two bands between 300 and 400 bp.

8. The PCR-RFLP method for identifying venom of toad according to claim 1, characterized in that: The toad venom sample is selected from one or more of the following: Chinese toad venom, black-spotted toad venom, northeastern toad venom, and other toad venom different from Chinese toad venom, black-spotted toad venom, and northeastern toad venom; Preferably, the toad venom sample is commercial toad venom.

9. A kit for identifying toad venom, comprising the primer pair and restriction endonuclease according to any one of claims 1 to 8, and instructions for use of the method according to any one of claims 1 to 8.

10. Use of the PCR-RFLP method for identifying toad venom according to any one of claims 1 to 8 or the kit according to claim 9 in identifying or assisting in identifying toad venom.

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