Gel electrophoresis identification method for leeches and limnoria and their close relatives and application thereof

Abstract

The application discloses a gel electrophoresis identification method for leeches and leech worms and their close relatives and application of the method, and relates to the technical field of traditional Chinese medicine identification. The method comprises the following steps: (1) pre-treating a leech sample to be detected to obtain a sample solution of the leech sample to be detected, and pre-treating a control medicinal material to obtain a solution of the control medicinal material; wherein the control medicinal material is a leech, a leech worm and / or a close relative and fake product thereof; (2) performing sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel imaging analysis on the sample solution of the leech sample to be detected and the solution of the control medicinal material to obtain a gel electrophoresis spectrum of the leech sample to be detected and a gel electrophoresis spectrum of the control medicinal material; and (3) comparing the above spectra, and determining whether the leech sample to be detected is a leech, a leech worm or a close relative and fake product thereof according to a comparison result; and the leech sample to be detected is selected from one or more of the following: a leech, a leech worm, a limnatis filamentaria, a haemopis japonica and a haemopis sanguinea. The method is intuitive, accurate and easy to identify.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a gel electrophoresis method for the identification of leeches and their closely related adulterants, and its application. Background Technology

[0002] Traditional Chinese medicine leeches are derived from the dried bodies of three species of leeches: *Whitmania pigra* Whitman, *Hirudonipponica* Whitman, or *Whitmania acranulata* Whitman. They are believed to have the effects of breaking up blood stasis, promoting menstruation, and eliminating masses. Modern pharmacological studies have shown that leeches possess various effects, including anticoagulation, antithrombosis, anti-atherosclerosis, antiplatelet aggregation, antitumor, anti-inflammatory, improving blood rheology, and protecting against cerebral ischemia-reperfusion injury. While there are over 680 species of leeches worldwide, with over 100 species found in China, only three are legally recognized as medicinal leeches: *Hirudonipponica*, *Hirudonipponica*, and *Whitmania acranulata*. *Hirudonipponica* is the mainstream species in the market, while *Hirudonipponica* is rare, and *Whitmania acranulata* is extremely uncommon.

[0003] In recent years, with the continuous expansion of demand for leeches as medicinal materials, their prices have risen year by year, and supply has fallen short of demand, leading to widespread adulteration and counterfeiting. The main adulterated leeches are *Poecilobdella manillensis* Lesson, *Poecilobdella javanica* Wahlberg, and *Mimobdella japonica* Blanchard. Although these different protozoa are morphologically distinguishable, traditional Chinese medicine leeches are often used in the form of leech segments or powder, making it difficult to identify genuine products based on morphological characteristics. Therefore, it is urgent to establish a simple and effective identification method to ensure the quality of leech medicinal materials in the distribution process.

[0004] As an animal-derived medicinal material, leeches contain a large number of protein polypeptides and free amino acids, in addition to small molecule compounds. Currently, over 60 proteins have been isolated and identified from leeches, exhibiting activities such as anticoagulation, fibrinolysis, and trypsin inhibition. Furthermore, leeches are rich in polypeptides related to anticoagulation, antithrombosis, antiplatelet aggregation, and neuroprotective properties. Currently, there are few studies using the macromolecular components of leeches to identify the authenticity of medicinal materials. Electrophoresis is a commonly used method for analyzing protein components and has been applied in the identification of macromolecular components in various animal-derived medicinal materials such as donkey-hide gelatin, deer antler, toad oil, and lizard. However, existing research has not yet found a gel electrophoresis method or its application for distinguishing (identifying) leeches from bloodworms and their closely related adulterants.

[0005] Therefore, this invention uses gel electrophoresis to characterize macromolecular compounds for the identification of leeches and their closely related adulterants. Summary of the Invention

[0006] Based on this, the present invention provides a gel electrophoresis identification method for leeches and bloodsuckers and their closely related adulterants, the identification method comprising the following steps:

[0007] (1) The leech sample to be tested is pretreated to obtain the leech sample test solution, and the reference medicinal material is pretreated to obtain the reference medicinal material solution; wherein, the reference medicinal material is leech, leech and / or its closely related adulterants; wherein, the leech and the leech are Hirudo nipponica Whitman and Whitmania pigra Whitman of the Hirudinaceae family, and the closely related adulterants are Poecilobdella manillensis Lesson, Poecilobdella javanica Wahlberg and / or Mimobdella japonica Blanchard of the Hirudinaceae family;

[0008] (2) The test solution of the leech sample and the control medicinal material solution were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel imaging analysis to obtain the gel electrophoresis patterns of the leech sample and the control medicinal material; and

[0009] (3) Compare the gel electrophoresis pattern of the leech sample to be tested with the gel electrophoresis pattern of the control medicinal material, and determine whether the leech to be tested is a leech, blood leech or its closely related adulterant based on the comparison results;

[0010] The leech to be tested is selected from one or more of the following: leech, blood leech, horny leech, barn leech, and Japanese leech.

[0011] Furthermore, this comparison is a comparison of the number and position of protein bands.

[0012] Furthermore, the preparation method of the reference medicinal material solution includes: taking an appropriate amount of reference medicinal material, crushing it, sieving it to obtain the reference medicinal material powder, adding a solvent to the reference medicinal material powder for extraction, shaking and mixing, centrifuging, taking the supernatant, and diluting the supernatant to obtain the reference medicinal material solution.

[0013] Furthermore, this sieve is a No. 3 sieve.

[0014] Furthermore, the solvent is a sodium chloride solution.

[0015] Furthermore, the concentration of the sodium chloride solution is 0.5% to 2.0%, for example, about 0.9%.

[0016] Furthermore, the extraction is an maceration extraction.

[0017] Furthermore, the immersion extraction time is 20–40 minutes, for example, about 30 minutes.

[0018] Furthermore, the centrifuge speed is 10,000 to 15,000 r / min, for example, about 12,000 r / min.

[0019] Furthermore, the centrifugation time is 5 to 20 minutes, for example, about 10 minutes.

[0020] Furthermore, the dilution factor is 5 to 120, for example, about 10 or about 100.

[0021] Furthermore, the preparation method of the test solution of the leech sample includes: taking an appropriate amount of the leech sample to be tested, crushing it, sieving it to obtain the leech sample powder to be tested, adding solvent to the leech sample powder to be tested for extraction, shaking and mixing, centrifuging, taking the supernatant, and diluting the supernatant to obtain the test solution of the leech sample to be tested.

[0022] Furthermore, this sieve is a No. 3 sieve.

[0023] Furthermore, the solvent is a sodium chloride solution.

[0024] Furthermore, the concentration of the sodium chloride solution is 0.5% to 2.0%, for example, about 0.9%.

[0025] Furthermore, the extraction is an maceration extraction.

[0026] Furthermore, the immersion extraction time is 20–40 minutes, for example, about 30 minutes.

[0027] Furthermore, the centrifuge speed is 10,000 to 15,000 r / min, for example, about 12,000 r / min.

[0028] Furthermore, the centrifugation time is 5 to 20 minutes, for example, about 10 minutes.

[0029] Furthermore, the dilution factor is 5 to 120, for example, about 10 or about 100.

[0030] Furthermore, when the leech to be tested is a leech (Hirudo nipponica Whitman) or a pheasant leech, the dilution factor is approximately 100 times.

[0031] Furthermore, when the leech to be tested is Hirudo medicinalis or Hirudo nipponia, the dilution factor is approximately 10 times.

[0032] Furthermore, the gel electrophoresis method includes the following steps:

[0033] (a) Add protein loading buffer to the test sample solution, mix well, boil to denature the protein, centrifuge to obtain the test solution;

[0034] (b) Install the glue dispensing device, prepare the separating glue solution and the concentrated glue solution, dispense the glue, and obtain a gel plate;

[0035] (c) Add electrode buffer to the outer tank of the electrophoresis tank, transfer the gel plate to the electrophoresis tank, fill the inner tank of the electrophoresis tank with electrode buffer, remove the electrophoresis comb, and load the electrophoresis marker and the test solution respectively.

[0036] (d) Use the first constant voltage vertical electrophoresis. When the indicator front reaches the separating gel, switch to the second constant voltage vertical electrophoresis. When the indicator front reaches the bottom of the gel plate, stop electrophoresis and remove the gel.

[0037] (e) Add dye to the gel to stain it, obtaining a stained gel; and

[0038] (f) Add destaining solution to the staining gel and destain until the bands are clear to obtain an electrophoresis gel.

[0039] Furthermore, the gel imaging analysis method includes: scanning the electrophoretic gel using a gel imaging analyzer, analyzing the scanning results using Gel-ProAnalyzer software, and obtaining the gel electrophoresis pattern of the leech sample and the leech gel electrophoresis pattern.

[0040] Further, in step (a), the volume ratio of the test sample solution to the protein loading buffer is 3 to 5, for example, about 4.

[0041] Furthermore, in step (a), the boiling temperature is 100°C.

[0042] Furthermore, in step (a), the boiling time is 1 to 10 minutes, for example, about 5 minutes.

[0043] Further, in step (a), the centrifugation speed is 10,000 to 15,000 r / min, for example, about 12,000 r / min.

[0044] Furthermore, in step (a), the centrifugation time is 1 to 10 minutes, for example, about 5 minutes.

[0045] Further, in step (b), the separating gel solution comprises water, 30% acrylamide-methylenebisacrylamide solution, 1.5M Tris, 10% SDS, 10% APS and TEMED.

[0046] Furthermore, in the separating gel solution, the volume ratio of the water, the 30% acrylamide-methylenebisacrylamide solution, the 1.5M Tris, the 10% SDS, the 10% APS and the TEMED is approximately 800: approximately 1000: approximately 650: approximately 25: approximately 25: approximately 1.

[0047] Further, in step (b), the concentrated gel solution comprises water, 30% acrylamide-methylenebisacrylamide solution, 1.0M Tris, 10% SDS, 10% APS and TEMED.

[0048] Furthermore, in the concentrated gel solution, the volume ratio of the water, the 30% acrylamide-methylenebisacrylamide solution, the 1.0M Tris, the 10% SDS, the 10% APS and the TEMED is approximately 700: approximately 165: approximately 125: approximately 10: approximately 10: approximately 1.

[0049] Further, in step (b), the gel pouring method includes: adding the separating gel solution to one side of the gel pouring device up to 1.5 to 2 cm from the top of the gel pouring device, adding anhydrous ethanol to seal the top, letting it stand at room temperature for about 30 minutes, removing the anhydrous ethanol, continuing to add the concentrated gel solution to one side of the gel pouring device until it is sealed, inserting an electrophoresis comb, letting it stand at room temperature for about 30 minutes, and obtaining the gel plate.

[0050] Further, in step (c), the electrode buffer solution is prepared by weighing appropriate amounts of glycine, Tris, and SDS, adding water to make up the volume, and diluting it by about 10 times to obtain the electrode buffer solution.

[0051] Furthermore, the mass of the glycine is 100–200g, for example, about 144g.

[0052] Furthermore, the mass of the Tris is 20–40g, for example, about 30g.

[0053] Furthermore, the mass of the SDS is 5–15g, for example, about 10g.

[0054] Furthermore, the volume of the final volume is 800–1200 mL, for example, about 1000 mL.

[0055] Further, in step (c), the loading volume of the electrophoresis marker is 1 to 10 μl, for example, about 5 μL.

[0056] Further, in step (c), the sample volume of the test solution is 5 to 15 μL, for example, about 10 μL.

[0057] Furthermore, in step (d), the first constant voltage is 70 to 90V, for example, about 80V.

[0058] Furthermore, in step (d), the second constant voltage is 110 to 130V, for example, about 120V.

[0059] Further, in step (e), the dye solution is Coomassie Brilliant Blue dye solution.

[0060] Furthermore, in step (e), the staining is performed on a shaker.

[0061] Furthermore, in step (e), the staining time is 30 to 60 minutes, for example, about 40 minutes.

[0062] Further, in step (f), the decolorizing solution is a mixed solution composed of methanol, glacial acetic acid and water.

[0063] Furthermore, in this mixed solution, the volume ratio of the methanol, the glacial acetic acid, and the water is (2-4):(0.5-2):(4-8).

[0064] Furthermore, in the mixed solution, the volume ratio of the methanol, the glacial acetic acid, and the water is approximately 3:approximately 1:approximately 6.

[0065] Furthermore, the preparation method of the 30% acrylamide-methylenebisacrylamide solution includes: weighing appropriate amounts of acrylamide and methylenebisacrylamide, diluting with water to about 50 mL, and thus obtaining the solution.

[0066] Furthermore, the acrylamide has a mass of 10–20 g, for example, about 14.55 g.

[0067] Furthermore, the mass of the methylene acrylamide is 0.1 to 1.0 g, for example, about 0.45 g.

[0068] Furthermore, the preparation method of the 1.5M Tris includes: weighing an appropriate amount of Tris, adding water to dissolve it for the first time, adding hydrochloric acid to adjust the pH, and adding water again to make up to about 250 mL.

[0069] Furthermore, the mass of the Tris is 30–60 g, for example, about 45.4 g.

[0070] Furthermore, the volume of the first batch of water is 200–300 mL, for example, about 220 mL.

[0071] Furthermore, the pH is adjusted to approximately 8.8.

[0072] Furthermore, the preparation method of the 1.0M Tris includes: weighing an appropriate amount of Tris, adding water to dissolve it for the first time, adding hydrochloric acid to adjust the pH, and adding water again to make up to about 250 mL.

[0073] Furthermore, the mass of the Tris is 20–40 g, for example, about 30.3 g.

[0074] Furthermore, the volume of the first batch of water is 200–300 ml, for example, about 220 mL.

[0075] Furthermore, the pH is adjusted to approximately 6.8.

[0076] Furthermore, the preparation method of the 10% APS includes: weighing an appropriate amount of APS, adding water to dilute it, and thus obtaining the product.

[0077] Furthermore, the mass of the APS is 0.5 to 2g, for example, about 1g.

[0078] Furthermore, the volume of the water is 5 to 20 mL, for example, about 10 mL.

[0079] Furthermore, the preparation method of the 10% SDS includes: weighing an appropriate amount of SDS, adding water to dilute it, and thus obtaining the product.

[0080] Furthermore, the mass of the SDS is 1 to 10g, for example, about 5g.

[0081] Furthermore, the volume of the water is 40–60 mL, for example, about 50 mL.

[0082] Furthermore, the comparison results include any of the following:

[0083] (1) If the gel electrophoresis pattern of the leech sample to be tested is consistent with or similar to the gel electrophoresis pattern of the leech control material, for example, if the gel electrophoresis pattern of the leech sample to be tested shows three bands at 25-34kDa, 17-25kDa and 11kDa, then the leech to be tested is a leech.

[0084] (2) If the gel electrophoresis pattern of the leech sample to be tested is consistent with or similar to the gel electrophoresis pattern of the leech control material or the gel electrophoresis pattern of the horny leech control material, for example, the gel electrophoresis pattern of the leech sample to be tested shows two bands at 60-75kDa and 25-34kDa, and two bands at 11kDa, then the leech to be tested is a leech or a horny leech;

[0085] (3) If the gel electrophoresis pattern of the leech sample to be tested is consistent with or similar to the gel electrophoresis pattern of the reference herb Hirudo medicinal material, for example, if the gel electrophoresis pattern of the leech sample to be tested shows four bands at 60-75kDa, 25-34kDa, 17-25kDa and 11kDa, then the leech to be tested is Hirudo medicinal material.

[0086] (4) If the gel electrophoresis pattern of the leech sample to be tested is consistent with or similar to the gel electrophoresis pattern of the Japanese leech reference material, for example, if the gel electrophoresis pattern of the leech sample to be tested shows two bands at 25-34kDa and 11kDa, then the leech to be tested is Japanese leech.

[0087] (5) If the gel electrophoresis pattern of the leech sample to be tested is inconsistent or dissimilar to the gel electrophoresis pattern of the above-mentioned control medicinal materials, then the leech to be tested is another adulterant.

[0088] According to another aspect of the invention, the above-described identification method is provided for the use of distinguishing leeches, blood leeches and / or their closely related adulterants, wherein the closely related adulterants are selected from one or more of the following: Hirudo nipponia, Hirudo barbatulata, Hirudo nipponia, and other adulterants.

[0089] The beneficial effects of this invention are:

[0090] This invention allows for the identification of samples by saline extraction and electrophoretic analysis, based on the number and position of protein bands on the gel. The results are intuitive, accurate, and easy to identify. Attached Figure Description

[0091] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0092] Figure 1 This is a schematic diagram showing the results of the separation gel concentration investigation under SDS-PAGE electrophoresis conditions in Example 1 of the present invention.

[0093] Figure 2 This is a schematic diagram showing the results of investigating the dilution factor of the test sample solution under SDS-PAGE electrophoresis conditions in Example 2 of the present invention.

[0094] Figure 3 This is the SDS-PAGE spectrum of Example 3 of the present invention. Detailed Implementation

[0095] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0096] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0097] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0098] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0099] In this invention, the term "comprising" and "including" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0100] As described in the background section, no gel electrophoretic identification method for leeches and blood leeches and their closely related adulterants, as described in this invention, is available in the prior art. To address the above problem, this invention provides a gel electrophoretic identification method for leeches and blood leeches and their closely related adulterants, comprising the following steps:

[0101] (1) The leech sample to be tested is pretreated to obtain the leech sample test solution, and the reference medicinal material is pretreated to obtain the reference medicinal material solution; wherein, the reference medicinal material is leech, leech and / or its closely related adulterants; wherein, the leech and the leech are Hirudo nipponica Whitman and Whitmania pigra Whitman of the Hirudinaceae family, and the closely related adulterants are Poecilobdella manillensis Lesson, Poecilobdella javanica Wahlberg and / or Mimobdella japonica Blanchard of the Hirudinaceae family;

[0102] (2) The test solution of the leech sample and the control medicinal material solution were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel imaging analysis to obtain the gel electrophoresis patterns of the leech sample and the control medicinal material; and

[0103] (3) Compare the gel electrophoresis pattern of the leech sample to be tested with the gel electrophoresis pattern of the control medicinal material, and determine whether the leech to be tested is a leech, blood leech or its closely related adulterant based on the comparison results;

[0104] The leech to be tested is selected from one or more of the following: leech, blood leech, horny leech, barn leech, and Japanese leech.

[0105] In a preferred embodiment, the comparison is a comparison of the number and position of protein bands.

[0106] In a preferred embodiment, the method for preparing the reference medicinal material solution includes: taking an appropriate amount of reference medicinal material, crushing it, sieving it to obtain the reference medicinal material powder, adding a solvent to the reference medicinal material powder for extraction, shaking and mixing, centrifuging, taking the supernatant, and diluting the supernatant to obtain the reference medicinal material solution.

[0107] In a preferred embodiment, the sieve is a No. 3 sieve.

[0108] In a preferred embodiment, the solvent is a sodium chloride solution.

[0109] In this invention, when concentration, time, rotation speed, multiple, mass, volume, voltage, ratio, molecular weight, or other values ​​or parameters are expressed as ranges, preferred ranges, or a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “0.5% to 2.0%” is disclosed, the described range should be interpreted as including ranges “0.5% to 2.0%”, “0.5% to 1.5%”, “0.5% to 1.0%”, “1.0% to 2.0%”, “1.0% to 1.5%”, “1.5% to 2.0%”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0110] In a preferred embodiment, the concentration of the sodium chloride solution is 0.5% to 2.0%, for example, about 0.9%.

[0111] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.9%" includes 0.9% ± 5%, or from 0.855% to 0.945%.

[0112] In a preferred embodiment, the extraction is an immersion extraction.

[0113] In a preferred embodiment, the immersion extraction time is 20 to 40 minutes, for example, about 30 minutes.

[0114] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes 30 ±5%, or from 28.5 to 31.5.

[0115] In a preferred embodiment, the centrifugation speed is 10,000 to 15,000 r / min, for example, about 12,000 r / min.

[0116] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 12000" includes ±5% of 12000, or from 11400 to 12600.

[0117] In a preferred embodiment, the centrifugation time is 5 to 20 minutes, for example, about 10 minutes.

[0118] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0119] In a preferred embodiment, the dilution factor is 5 to 120, for example about 10 or about 100.

[0120] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5; "about 100" includes ±5% of 100, or from 95 to 105.

[0121] In a preferred embodiment, the preparation method of the test leech sample solution includes: taking an appropriate amount of the test leech sample, crushing it, sieving it to obtain the test leech sample powder, adding solvent to the test leech sample powder for extraction, shaking and mixing, centrifuging, taking the supernatant, and diluting the supernatant to obtain the test leech sample solution.

[0122] In a preferred embodiment, the sieve is a No. 3 sieve.

[0123] In a preferred embodiment, the solvent is a sodium chloride solution.

[0124] In a preferred embodiment, the concentration of the sodium chloride solution is 0.5% to 2.0%, for example, about 0.9%.

[0125] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.9%" includes 0.9% ± 5%, or from 0.855% to 0.945%.

[0126] In a preferred embodiment, the extraction is an immersion extraction.

[0127] In a preferred embodiment, the immersion extraction time is 20 to 40 minutes, for example, about 30 minutes.

[0128] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes 30 ±5%, or from 28.5 to 31.5.

[0129] In a preferred embodiment, the centrifugation speed is 10,000 to 15,000 r / min, for example, about 12,000 r / min.

[0130] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 12000" includes ±5% of 12000, or from 11400 to 12600.

[0131] In a preferred embodiment, the centrifugation time is 5 to 20 minutes, for example, about 10 minutes.

[0132] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0133] In a preferred embodiment, the dilution factor is 5 to 120, for example about 10 or about 100.

[0134] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5; "about 100" includes ±5% of 100, or from 95 to 105.

[0135] In a preferred embodiment, when the leech to be tested is a leech (Hirudo nipponica Whitman) or a pheasant leech, the dilution factor is approximately 100 times.

[0136] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 100" includes ±5% of 100, or from 95 to 105.

[0137] In a preferred embodiment, when the leech to be tested is Hirudo medicinalis or Hirudo nipponia, the dilution factor is approximately 10 times.

[0138] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0139] In a preferred embodiment, the gel electrophoresis method includes the following steps:

[0140] (a) Add protein loading buffer to the test sample solution, mix well, boil to denature the protein, centrifuge to obtain the test solution;

[0141] (b) Install the glue dispensing device, prepare the separating glue solution and the concentrated glue solution, dispense the glue, and obtain a gel plate;

[0142] (c) Add electrode buffer to the outer tank of the electrophoresis tank, transfer the gel plate to the electrophoresis tank, fill the inner tank of the electrophoresis tank with electrode buffer, remove the electrophoresis comb, and load the electrophoresis marker and the test solution respectively.

[0143] (d) Use the first constant voltage vertical electrophoresis. When the indicator front reaches the separating gel, switch to the second constant voltage vertical electrophoresis. When the indicator front reaches the bottom of the gel plate, stop electrophoresis and remove the gel.

[0144] (e) Add dye to the gel to stain it, obtaining a stained gel; and

[0145] (f) Add destaining solution to the staining gel and destain until the bands are clear to obtain an electrophoresis gel.

[0146] In a preferred embodiment, the gel imaging analysis method includes: scanning the electrophoretic gel using a gel imaging analyzer, analyzing the scanning results using Gel-Pro Analyzer software, and obtaining the gel electrophoresis pattern of the leech sample and the leech gel electrophoresis pattern.

[0147] In a preferred embodiment, in step (a), the volume ratio of the test sample solution to the protein loading buffer is 3 to 5, for example, about 4.

[0148] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2.

[0149] In a preferred embodiment, in step (a), the boiling temperature is 100°C.

[0150] In a preferred embodiment, in step (a), the boiling time is 1 to 10 minutes, for example, about 5 minutes.

[0151] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5" includes 5 ± 5%, or from 4.75 to 5.25.

[0152] In a preferred embodiment, in step (a), the centrifugation speed is 10,000 to 15,000 r / min, for example, about 12,000 r / min.

[0153] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 12000" includes ±5% of 12000, or from 11400 to 12600.

[0154] In a preferred embodiment, in step (a), the centrifugation time is 1 to 10 minutes, for example, about 5 minutes.

[0155] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5" includes 5 ± 5%, or from 4.75 to 5.25.

[0156] In a preferred embodiment, in step (b), the separating gel solution comprises water, 30% acrylamide-methylenebisacrylamide solution, 1.5M Tris, 10% SDS, 10% APS and TEMED.

[0157] In a preferred embodiment, the volume ratio of the water, the 30% acrylamide-methylenebisacrylamide solution, the 1.5M Tris, the 10% SDS, the 10% APS and the TEMED in the separating gel solution is about 800: about 1000: about 650: about 25: about 25: about 1.

[0158] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 800" includes ±5% of 800, or from 760 to 840; "about 1000" includes ±5% of 1000, or from 950 to 1050; "about 650" includes ±5% of 650, or from 617.5 to 682.5; "about 25" includes ±5% of 25, or from 23.75 to 26.25; and "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0159] In a preferred embodiment, in step (b), the concentrated gel solution comprises water, 30% acrylamide-methylenebisacrylamide solution, 1.0M Tris, 10% SDS, 10% APS and TEMED.

[0160] In a preferred embodiment, the volume ratio of the water, the 30% acrylamide-methylenebisacrylamide solution, the 1.0M Tris, the 10% SDS, the 10% APS and the TEMED in the concentrated gel solution is about 700: about 165: about 125: about 10: about 10: about 1.

[0161] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 700" includes ±5% of 700, or from 665 to 735; "about 165" includes ±5% of 165, or from 156.75 to 173.25; "about 125" includes ±5% of 125, or from 118.75 to 131.25; "about 10" includes ±5% of 10, or from 9.5 to 10.5; and "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0162] In a preferred embodiment, in step (b), the gel pouring method includes: adding the separating gel solution to one side of the gel pouring device up to 1.5-2 cm from the top of the gel pouring device, adding anhydrous ethanol to seal the top, letting it stand at room temperature for about 30 minutes, removing the anhydrous ethanol, continuing to add the concentrated gel solution to one side of the gel pouring device until it seals the top, inserting an electrophoresis comb, letting it stand at room temperature for about 30 minutes, and obtaining the gel plate.

[0163] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes 30 ±5%, or from 28.5 to 31.5.

[0164] In a preferred embodiment, in step (c), the electrode buffer solution is prepared by weighing appropriate amounts of glycine, Tris, and SDS, adding water to make up the volume, and diluting it by about 10 times to obtain the electrode buffer solution.

[0165] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0166] In a preferred embodiment, the glycine has a mass of 100-200g, for example, about 144g.

[0167] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 144" includes ±5% of 144, or from 136.8 to 151.2.

[0168] In a preferred embodiment, the mass of the Tris is 20 to 40 g, for example, about 30 g.

[0169] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes 30 ±5%, or from 28.5 to 31.5.

[0170] In a preferred embodiment, the mass of the SDS is 5 to 15 g, for example, about 10 g.

[0171] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0172] In a preferred embodiment, the volume of the final volume is 800-1200 ml, for example, about 1000 ml.

[0173] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1000" includes ±5% of 1000, or from 950 to 1050.

[0174] In a preferred embodiment, in step (c), the loading volume of the electrophoresis marker is 1 to 10 μL, for example, about 5 μL.

[0175] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5" includes 5 ± 5%, or from 4.75 to 5.25.

[0176] In a preferred embodiment, in step (c), the sample volume of the test solution is 5 to 15 μL, for example, about 10 μL.

[0177] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0178] In a preferred embodiment, in step (d), the first constant voltage is 70 to 90V, for example, about 80V.

[0179] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 80" includes ±5% of 80, or from 76 to 84.

[0180] In a preferred embodiment, in step (d), the second constant voltage is 110 to 130V, for example, about 120V.

[0181] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 120" includes ±5% of 120, or from 114 to 126.

[0182] In a preferred embodiment, in step (e), the dye solution is Coomassie Brilliant Blue dye solution.

[0183] In a preferred embodiment, in step (e), the staining is performed on a shaker.

[0184] In a preferred embodiment, in step (e), the staining time is 30 to 60 minutes, for example, about 40 minutes.

[0185] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 40" includes ±5% of 40, or from 38 to 42.

[0186] In a preferred embodiment, in step (f), the decolorizing solution is a mixed solution composed of methanol, glacial acetic acid and water.

[0187] In a preferred embodiment, the volume ratio of methanol, glacial acetic acid and water in the mixed solution is (2-4):(0.5-2):(4-8).

[0188] In a preferred embodiment, the volume ratio of methanol, glacial acetic acid, and water in the mixed solution is about 3: about 1: about 6.

[0189] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3" includes 3 ±5%, or from 2.85 to 3.15; "about 1" includes 1 ±5%, or from 0.95 to 1.05; and "about 6" includes 6 ±5%, or from 5.7 to 6.3.

[0190] In a preferred embodiment, the preparation method of the 30% acrylamide-methylenebisacrylamide solution includes: weighing an appropriate amount of acrylamide and methylenebisacrylamide, diluting with water to about 50 ml, and obtaining the solution.

[0191] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 50" includes 50 ± 5%, or from 47.5 to 52.5.

[0192] In a preferred embodiment, the acrylamide has a mass of 10 to 20 g, for example, about 14.55 g.

[0193] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 14.55" includes ±5% of 14.55, or from 13.8225 to 15.2775.

[0194] In a preferred embodiment, the mass of the methylene acrylamide is 0.1 to 1.0 g, for example, about 0.45 g.

[0195] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.45" includes ±5% of 0.45, or from 0.4275 to 0.4725.

[0196] In a preferred embodiment, the preparation method of the 1.5M Tris includes: weighing an appropriate amount of Tris, adding water to dissolve it for the first time, adding hydrochloric acid to adjust the pH, and adding water again to make up to about 250 ml.

[0197] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 250" includes ±5% of 250, or from 237.5 to 262.5.

[0198] In a preferred embodiment, the mass of the Tris is 30 to 60 g, for example, about 45.4 g.

[0199] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 45.4" includes ±5% of 45.4, or from 43.13 to 47.67.

[0200] In a preferred embodiment, the volume of the first water is 200-300 mL, for example, about 220 mL.

[0201] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 220" includes ±5% of 220, or from 209 to 231.

[0202] In a preferred embodiment, the pH adjustment is to adjust the pH to approximately 8.8.

[0203] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 8.8" includes ±5% of 8.8, or from 8.36 to 9.24.

[0204] In a preferred embodiment, the preparation method of the 1.0M Tris includes: weighing an appropriate amount of Tris, adding water to dissolve it for the first time, adding hydrochloric acid to adjust the pH, and adding water again to make up to about 250 ml.

[0205] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 250" includes ±5% of 250, or from 237.5 to 262.5.

[0206] In a preferred embodiment, the mass of the Tris is 20 to 40 g, for example, about 30.3 g.

[0207] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30.3" includes ±5% of 30.3, or from 28.785 to 31.815.

[0208] In a preferred embodiment, the volume of the first water is 200-300 mL, for example, about 220 mL.

[0209] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 220" includes ±5% of 220, or from 209 to 231.

[0210] In a preferred embodiment, the pH adjustment is to adjust the pH to approximately 6.8.

[0211] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 6.8" includes ±5% of 6.8, or from 6.46 to 7.14.

[0212] In a preferred embodiment, the method for preparing the 10% APS includes: weighing an appropriate amount of APS, adding water to dilute it, and thus obtaining the product.

[0213] In a preferred embodiment, the mass of the APS is 0.5 to 2 g, for example, about 1 g.

[0214] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0215] In a preferred embodiment, the volume of the water is 5 to 20 mL, for example, about 10 mL.

[0216] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0217] In a preferred embodiment, the method for preparing the 10% SDS includes: weighing an appropriate amount of SDS, adding water to dilute it, and thus obtaining the desired product.

[0218] In a preferred embodiment, the mass of the SDS is 1 to 10 g, for example, about 5 g.

[0219] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5" includes 5 ± 5%, or from 4.75 to 5.25.

[0220] In a preferred embodiment, the volume of the water is 40 to 60 mL, for example, about 50 mL.

[0221] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 50" includes 50 ± 5%, or from 47.5 to 52.5.

[0222] In a preferred embodiment, the leech medicinal material gel electrophoresis pattern includes three protein bands located at 25–34 kDa, 17–25 kDa, and 11 kDa, respectively.

[0223] In a preferred embodiment, the comparison result includes any of the following:

[0224] (1) If the gel electrophoresis pattern of the leech sample to be tested is consistent with or similar to the gel electrophoresis pattern of the leech control material, for example, if the gel electrophoresis pattern of the leech sample to be tested shows three bands at 25-34kDa, 17-25kDa and 11kDa, then the leech to be tested is a leech.

[0225] (2) If the gel electrophoresis pattern of the leech sample to be tested is consistent with or similar to the gel electrophoresis pattern of the leech control material or the gel electrophoresis pattern of the horny leech control material, for example, the gel electrophoresis pattern of the leech sample to be tested shows two bands at 60-75kDa and 25-34kDa, and two bands at 11kDa, then the leech to be tested is a leech or a horny leech;

[0226] (3) If the gel electrophoresis pattern of the leech sample to be tested is consistent with or similar to the gel electrophoresis pattern of the reference herb Hirudo medicinal material, for example, if the gel electrophoresis pattern of the leech sample to be tested shows four bands at 60-75kDa, 25-34kDa, 17-25kDa and 11kDa, then the leech to be tested is Hirudo medicinal material.

[0227] (4) If the gel electrophoresis pattern of the leech sample to be tested is consistent with or similar to the gel electrophoresis pattern of the Japanese leech reference material, for example, if the gel electrophoresis pattern of the leech sample to be tested shows two bands at 25-34kDa and 11kDa, then the leech to be tested is Japanese leech.

[0228] (5) If the gel electrophoresis pattern of the leech sample to be tested is inconsistent or dissimilar to the gel electrophoresis pattern of the above-mentioned control medicinal materials, then the leech to be tested is another adulterant.

[0229] According to another aspect of the present invention, a gel electrophoresis method is provided for distinguishing leeches, blood leeches, and / or their closely related adulterants, characterized in that it comprises:

[0230] Step 1: Take the reference medicinal material sample and the sample to be tested, crush them, and sieve them to obtain the reference medicinal material sample powder and the sample to be tested powder. Weigh equal amounts of the reference medicinal material sample powder and the sample to be tested powder respectively.

[0231] Step 2: Extract the above powders separately by soaking in saline solution, shaking frequently, centrifuging, and taking the supernatant to obtain the reference medicinal material sample solution and the test sample solution;

[0232] Step 3: The above test solutions (leech and pheasant test solutions diluted 100 times, and leech, bar-shaped leech, Japanese leech, and unknown leech test solutions diluted 10 times) are analyzed by gel electrophoresis to obtain gels. The gels are then scanned using a gel imaging analyzer and analyzed using Gel-Pro Analyzer software. The samples to be tested are identified based on the number and position of protein bands on the gel.

[0233] In a preferred embodiment, the brine solution is a sodium chloride solution with a concentration of 0.9% (M / V).

[0234] In a preferred embodiment, the immersion extraction time is 30 min; the centrifugation conditions are 12000 r / min for 10 min.

[0235] In a preferred embodiment, the gel electrophoresis is sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); the SDS-PAGE consists of a 12% separating gel and a 5% stacking gel; 3 μL of marker sample and 10 μL of test sample are loaded. Electrode buffer is added to the inner and outer electrophoresis tanks, and electrophoresis is started at a constant voltage of 80V. Once the indicator front reaches the separating gel, the voltage is increased to 120V, and electrophoresis is stopped when the indicator front reaches the bottom of the glass plate. The gel is stained with Coomassie brilliant blue staining solution, and repeatedly rinsed with destaining solution until the bands are clearly visible. The gel is scanned using a gel imaging analyzer and analyzed using Gel-ProAnalyzer software.

[0236] In a preferred embodiment, if the gel electrophoresis pattern of the sample to be tested shows a single band at 25-34 kDa, 17-25 kDa, and 11 kDa, then the sample to be tested is determined to be a leech.

[0237] According to another aspect of the invention, the above-described identification method is provided for the use of distinguishing leeches, blood leeches and / or their closely related adulterants, wherein the closely related adulterants are selected from one or more of the following: Hirudo nipponia, Hirudo barbatulata, Hirudo nipponia, and other adulterants.

[0238] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0239] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0240] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0241] Example

[0242] Experimental instruments and reagents

[0243] DYY-6D electrophoresis apparatus, WD-9413B gel imaging analyzer, WD-9405 decolorizing shaker, DLAB HB120-S metal bath (Beijing Liuyi Biotechnology Co., Ltd.), TGL20MW benchtop high-speed refrigerated centrifuge (Hunan Hexi Instrument Equipment Co., Ltd.), S20 SevenEasy TM pH meter (Mettler-Toledo International Ltd.), BSA-124S-CW electronic balance (Beijing Sartorius Scientific Instruments Co., Ltd.).

[0244] Rainbow 180 broad-spectrum protein marker (11-180KD), Tris(hydroxymethylaminomethane) (Solepro Science & Technology Co., Ltd.), SDS-PAGE protein loading buffer (5X), Coomassie brilliant blue staining solution (No.: P0017B) (Beyotime Biotechnology Co., Ltd.), sodium dodecyl sulfate (SDS), acrylamide, methylenebisacrylamide, ammonium persulfate (APS), tetramethylethylenediamine (TEMED), glycine (Sigma-Aldrich, USA), physiological sodium chloride solution (Shijiazhuang No. 4 Pharmaceutical Co., Ltd.), ethanol (Sinopharm Chemical Reagent Co., Ltd.).

[0245] Experimental materials

[0246] Twenty-eight batches of samples were collected from different regions of China, including 11 batches of leeches, 12 batches of leeches, 1 batch of bar-shaped leeches, 1 batch of leeches with bark, 2 batches of Japanese leeches, and 1 batch of an unknown leech. Specific information is as follows:

[0247] Sample 1: Leeches originating from Shandong (purchased from Anguo Medicinal Materials Market);

[0248] Sample 2: Leeches originated from Jining County, Jining City, Shandong Province;

[0249] Sample 3, leeches, originated from Sucheng District, Suqian City, Jiangsu Province;

[0250] Sample 4: Leeches originated from Bamen Town, Baodi District, Tianjin.

[0251] Sample 5 was found in Xihua County, Zhoukou City, Henan Province.

[0252] Sample 6, leeches, originated from Jining County, Jining City, Shandong Province;

[0253] Sample 7, leeches, originated from Sucheng District, Suqian City, Jiangsu Province;

[0254] Sample 8: Leeches originated from De'an County, Jiujiang City, Jiangxi Province;

[0255] Sample 9, leeches, originated from Yuanjiang City, Yiyang City, Hunan Province;

[0256] Sample 10: Leeches, originating from Gong'an County, Jingzhou City, Hubei Province;

[0257] Sample 11: Leeches, originating from Gong'an County, Jingzhou City, Hubei Province;

[0258] Sample 12: Leeches, originating from Jining County, Jining City, Shandong Province;

[0259] Sample 13, leeches, originated from Sucheng District, Suqian City, Jiangsu Province;

[0260] Sample 14, leeches, originated from Bamen Town, Baodi District, Tianjin.

[0261] Sample 15: Leeches, originating from Xihua County, Zhoukou City, Henan Province;

[0262] Sample 16, leeches, originated from Jining County, Jining City, Shandong Province;

[0263] Sample 17, leeches, originated from Sucheng District, Suqian City, Jiangsu Province;

[0264] Sample 18, leeches, originated from De'an County, Jiujiang City, Jiangxi Province;

[0265] Sample 19, leeches, originated from Yuanjiang City, Yiyang City, Hunan Province;

[0266] Sample 20: Leeches originating from Jining County, Jining City, Shandong Province;

[0267] Sample 21, leeches, originated from Gong'an County, Jingzhou City, Hubei Province;

[0268] Sample 22, leeches, originated from Gong'an County, Jingzhou City, Hubei Province;

[0269] Sample 23, leeches, originated from Bamen Town, Baodi District, Tianjin.

[0270] Sample 24, a barbed leech, originated from Qinnan District, Qinzhou City, Guangxi Zhuang Autonomous Region;

[0271] Sample 25, *Hirudo medicinalis*, originated in Zhongshan City, Guangdong Province;

[0272] Sample 26, Japanese leech, originated from Xihua County, Zhoukou City, Henan Province;

[0273] Sample 27 is a Japanese leech from Xinhui District, Jiangmen City, Guangdong Province.

[0274] Sample 28, origin of unknown leeches, was from De'an County, Jiujiang City, Jiangxi Province.

[0275] Example 1

[0276] Gel electrophoretic identification method for leeches and bloodsuckers and their closely related adulterants, including determination of separating gel concentration, including:

[0277] Reagent and gel preparation

[0278] 30% Acrylamide-N-methylbisacrylamide solution (29:1): Weigh 14.55g acrylamide and 0.45g methylenebisacrylamide, dilute with water to 50mL, and store at 4℃ protected from light.

[0279] 1.5M Tris (pH 8.8): Weigh 45.4g Tris, dissolve in 220mL of water, adjust the pH to 8.8 with hydrochloric acid, and add water to bring the volume to 250mL.

[0280] 1.0M Tris (pH 6.8): Weigh 30.3g Tris, dissolve in 220mL of water, adjust the pH to 6.8 with hydrochloric acid, and add water to a final volume of 250mL.

[0281] 10% APS: Weigh 1.00g of APS, add 10mL of water, dispense into smaller portions, and store at -20℃.

[0282] 10% SDS: Weigh 5.00g SDS and add 50mL of water.

[0283] The separating gels of different concentrations are shown in Table 1.

[0284] Table 1 Separating gels of different concentrations

[0285]

[0286] 5% polyacrylamide gel stack: 1.4 mL water, 0.33 mL 30% acrylamide-methylenebisacrylamide solution, 0.25 mL 1.0 M Tris (pH 6.8), 20 μ L 10% SDS, 20 μ L 10% APS, 2 μ L TEMED.

[0287] Electrode buffer (10×): Weigh 144g glycine, 30g Tris, and 10g SDS, add water to a final volume of 1L, and dilute 10 times before use to prepare electrode buffer (1×).

[0288] Electrophoretic staining solution: Coomassie brilliant blue staining solution was purchased from Beyotime Biotechnology Co., Ltd.

[0289] Electrophoretic decolorization solution: a mixed solution of methanol, glacial acetic acid and water (3:1:6).

[0290] Preparation of test solution

[0291] Leeches (sample 11), leeches (sample 20), bar-striped leeches (sample 24), leeches (sample 25), Japanese leeches (sample 26), and unknown leeches (sample 28) were crushed and passed through a No. 3 sieve to obtain leech, leech or closely related adulterant sample powders. Equal amounts of leech, leech or closely related adulterant sample powders were weighed.

[0292] The above powders were extracted by soaking in 5 mL of physiological saline solution for 30 min, with occasional shaking. The mixture was then centrifuged at 12000 r / min for 10 min, and the supernatant was collected. The supernatant was diluted 10 times to obtain the test solution.

[0293] Protein loading pretreatment: Mix the sample solution volume with protein loading buffer (5×) at a ratio of 4:1, boil at 100℃ for 5 min, and centrifuge at 12000 rpm for 5 min.

[0294] SDS-PAGE steps

[0295] (1) Install the glue dispensing device.

[0296] (2) Add the prepared separating gel solution to the dispensing device from one side using a pipette, up to 1.5-2 cm from the top of the glass plate. Add anhydrous ethanol to the top of the glass plate and let it stand at room temperature for 30 minutes. Tilt the dispensing device to pour out the anhydrous ethanol and blot the remaining anhydrous ethanol with filter paper.

[0297] (3) Continue to add the prepared concentrated gel solution from one side to the dispensing device using a pipette until it reaches the top of the glass plate. Quickly insert the sample comb and let it stand at room temperature for 30 minutes.

[0298] (4) Add an appropriate volume of electrode buffer (1×) to the outer tank of the electrophoresis tank, transfer the glass plate with the gel to the electrophoresis tank, fill the inner tank with electrode buffer (1×), remove the electrophoresis comb, and load the sample.

[0299] (5) Connect the power supply and start electrophoresis at a constant voltage of 80V. After the bromophenol blue front reaches the separating gel, increase the voltage to 120V.

[0300] (6) Stop electrophoresis when the bromophenol blue front reaches the bottom of the glass plate and remove the gel with a gel opener.

[0301] (7) Staining: Place the gel in the staining box, add an appropriate amount of staining solution, and stain on a shaker for 40 minutes.

[0302] (8) Decolorization: Place the gel in the decolorization box, add an appropriate amount of decolorization solution, and replace the decolorization solution halfway through until the stripes are clear on the shaker.

[0303] (9) Scanning: The gel was scanned using a gel imaging analyzer, the scan results were saved, and analyzed using Gel-ProAnalyzer software. The results are as follows: Figure 1 As shown. Figure 1 In the table, M represents the protein molecular weight standard (Marker), 20 represents leech (HN), 11 represents blood leech, 24 represents barn leech, 25 represents barn leech, 26 represents Japanese leech, and 28 represents an unknown leech.

[0304] Electrophoresis diagrams of separating gels at different concentrations are shown below. Figure 1 As shown, the marker protein bands were poorly separated in 8% separating gel electrophoresis, with only 8 bands appearing, and the main protein bands of the sample were not fully developed (there was a protein band at 11kDa). Figure 1 As shown in Figure A, the marker protein bands showed poor separation in 10% separating gel electrophoresis, with only 9 bands appearing, and the main protein bands of the sample were not fully developed (a protein band was present at 11 kDa). Figure 1 As shown in B, the marker protein bands in the 15% separating gel electrophoresis were poorly separated, not fully developed, with only 9 bands appearing. Furthermore, the protein bands of the sample were distributed in the upper part of the gel electrophoresis, possibly with overlap. Figure 1 As shown in D, a 12% separating gel provides the best separation effect; all 10 protein bands on the marker expand, and the main protein bands of the sample are clear, as shown in Figure D. Figure 1 As shown in C. Therefore, 12% separating gel is selected.

[0305] Example 2

[0306] A gel electrophoretic identification method for leeches and bloodsuckers and their closely related adulterants, including investigation of sample dilution factors, including:

[0307] Reagent and gel preparation

[0308] 30% Acrylamide-N-methylbisacrylamide solution (29:1): Weigh 14.55g acrylamide and 0.45g methylenebisacrylamide, dilute with water to 50mL, and store at 4℃ protected from light.

[0309] 1.5M Tris (pH 8.8): Weigh 45.4g Tris, dissolve in 220mL of water, adjust the pH to 8.8 with hydrochloric acid, and add water to bring the volume to 250mL.

[0310] 1.0M Tris (pH 6.8): Weigh 30.3g Tris, dissolve in 220mL of water, adjust the pH to 6.8 with hydrochloric acid, and add water to a final volume of 250mL.

[0311] 10% APS: Weigh 1.00g of APS, add 10mL of water, dispense into smaller portions, and store at -20℃.

[0312] 10% SDS: Weigh 5.00g SDS and add 50mL of water.

[0313] 12% polyacrylamide gel electrophoresis separation gel: 1.6 mL water, 2.0 mL 30% acrylamide-methylenebisacrylamide solution, 1.3 mL 1.5 M Tris (pH 8.8), 50 μ L 10% SDS, 50 μ L 10% APS, 2 μ L TEMED.

[0314] 5% polyacrylamide gel stack: 1.4 mL water, 0.33 mL 30% acrylamide-methylenebisacrylamide solution, 0.25 mL 1.0 M Tris (pH 6.8), 20 μ L 10% SDS, 20 μ L 10% APS, 2 μ L TEMED.

[0315] Electrode buffer (10×): Weigh 144g glycine, 30g Tris, and 10g SDS, add water to a final volume of 1L, and dilute 10 times before use to prepare electrode buffer (1×).

[0316] Electrophoretic staining solution: Coomassie brilliant blue staining solution was purchased from Beyotime Biotechnology Co., Ltd.

[0317] Electrophoretic decolorization solution: a mixed solution of methanol, glacial acetic acid and water (3:1:6).

[0318] Preparation of test solution

[0319] Leeches (sample 11), leeches (sample 20), bark leeches (sample 24), leeches (sample 25), Japanese leeches (sample 26), and unknown leeches (sample 28) were crushed and passed through a No. 3 sieve to obtain leech, leech or their closely related adulterant reference medicinal material powders. Equal amounts of leech, leech or their closely related adulterant reference medicinal material powders were weighed out.

[0320] The above powders were extracted by soaking in 5 mL of physiological saline solution for 30 min, with occasional shaking, and centrifuged at 12000 r / min for 10 min. The supernatant was collected. The supernatant was diluted 10 and 100 times to obtain the test solution.

[0321] Protein loading pretreatment: Mix the sample solution volume with protein loading buffer (5×) at a ratio of 4:1, boil at 100℃ for 5 min, and centrifuge at 12000 rpm for 5 min.

[0322] SDS-PAGE steps

[0323] (1) Install the glue dispensing device.

[0324] (2) Add the prepared separating gel solution to the dispensing device from one side using a pipette, up to 1.5-2 cm from the top of the glass plate. Add anhydrous ethanol to the top of the glass plate and let it stand at room temperature for 30 minutes. Tilt the dispensing device to pour out the anhydrous ethanol and blot the remaining anhydrous ethanol with filter paper.

[0325] (3) Continue to add the prepared concentrated gel solution from one side to the dispensing device using a pipette until it reaches the top of the glass plate. Quickly insert the sample comb and let it stand at room temperature for 30 minutes.

[0326] (4) Add an appropriate volume of electrode buffer (1×) to the outer tank of the electrophoresis tank, transfer the glass plate with the gel to the electrophoresis tank, fill the inner tank with electrode buffer (1×), remove the electrophoresis comb, and load the sample.

[0327] (5) Connect the power supply and start electrophoresis at a constant voltage of 80V. After the bromophenol blue front reaches the separating gel, increase the voltage to 120V.

[0328] (6) Stop electrophoresis when the bromophenol blue front reaches the bottom of the glass plate and remove the gel with a gel opener.

[0329] (7) Staining: Place the gel in the staining box, add an appropriate amount of staining solution, and stain on a shaker for 40 minutes.

[0330] (8) Decolorization: Place the gel in the decolorization box, add an appropriate amount of decolorization solution, and replace the decolorization solution halfway through until the stripes are clear on the shaker.

[0331] (9) Scanning: The gel was scanned using a gel imaging analyzer, the scan results were saved, and analyzed using Gel-ProAnalyzer software. The results are as follows: Figure 2 As shown. Figure 2 In the table, M represents the protein molecular weight standard (Marker), 20 represents leech (HN), 11 represents blood leech, 24 represents barn leech, 25 represents barn leech, 26 represents Japanese leech, and 28 represents an unknown leech.

[0332] Investigation of different dilution factors of sample supernatants: Gel electrophoresis patterns showed that when the supernatant of leeches and *Hirudo nipponia* was diluted 10-fold, the two protein bands at 11 kDa were difficult to observe, and the background of the gel electrophoresis image was dark. When the supernatant was diluted 100-fold, the two protein bands at 11 kDa were easily observed, and the background of the gel electrophoresis image was clear. When the supernatant of *Hirudo scabra*, *Hirudo leech*, *Hirudo nipponia*, and *Unknown leech* was diluted 10-fold, the protein bands were easily observed. When diluted 100-fold, the protein bands became blurred. Therefore, the supernatant of leeches and *Hirudo nipponia* should be diluted 100-fold, while the supernatant of *Hirudo scabra*, *Hirudo nipponia*, *Hirudo nipponia*, and *Unknown leech* should be diluted 10-fold.

[0333] Example 3

[0334] Gel electrophoretic identification methods for leeches and bloodsuckers and their closely related adulterants include:

[0335] Reagent and gel preparation

[0336] 30% Acrylamide-N-methylbisacrylamide solution (29:1): Weigh 14.55g acrylamide and 0.45g methylenebisacrylamide, dilute with water to 50mL, and store at 4℃ protected from light.

[0337] 1.5M Tris (pH 8.8): Weigh 45.4g Tris, dissolve in 220mL of water, adjust the pH to 8.8 with hydrochloric acid, and add water to bring the volume to 250mL.

[0338] 1.0M Tris (pH 6.8): Weigh 30.3g Tris, dissolve in 220mL of water, adjust the pH to 6.8 with hydrochloric acid, and add water to a final volume of 250mL.

[0339] 10% APS: Weigh 1.00g of APS, add 10mL of water, dispense into smaller portions, and store at -20℃.

[0340] 10% SDS: Weigh 5.00g SDS and add 50mL of water.

[0341] 12% polyacrylamide gel electrophoresis separation gel: 1.6 mL water, 2.0 mL 30% acrylamide-methylenebisacrylamide solution, 1.3 mL 1.5 M Tris (pH 8.8), 50 μ L 10% SDS, 50 μ L 10% APS, 2 μ L TEMED.

[0342] 5% polyacrylamide gel stack: 1.4 mL water, 0.33 mL 30% acrylamide-methylenebisacrylamide solution, 0.25 mL 1.0 M Tris (pH 6.8), 20 μ L 10% SDS, 20 μ L 10% APS, 2 μ L TEMED.

[0343] Electrode buffer (10×): Weigh 144g glycine, 30g Tris, and 10g SDS, add water to a final volume of 1L, and dilute 10 times before use to prepare electrode buffer (1×).

[0344] Electrophoretic staining solution: Coomassie brilliant blue staining solution was purchased from Beyotime Biotechnology Co., Ltd.

[0345] Electrophoretic decolorization solution: a mixed solution of methanol, glacial acetic acid and water (3:1:6).

[0346] Preparation of test solution

[0347] Take leeches and the sample to be tested, crush them, and pass them through a No. 3 sieve to obtain leech sample powder and sample to be tested powder. Weigh out equal amounts of leech sample powder and sample to be tested powder respectively.

[0348] The above powders were extracted by soaking in 5 mL of physiological saline solution for 30 min, with occasional shaking. The mixture was then centrifuged at 12000 rpm for 10 min, and the supernatant was collected. The supernatant was diluted 10-fold, and electrophoretic patterns were compared to identify the sample as leeches, bloodsuckers, or their adulterants. If the electrophoretic pattern showed no clear bands due to excessively high concentration of the test solution, it indicated that the concentration of the leech sample solution was too high. In this case, the leech sample solution was diluted 100-fold before further testing and identification.

[0349] Protein loading pretreatment: Mix the sample solution volume with protein loading buffer (5×) at a ratio of 4:1, boil at 100℃ for 5 min, and centrifuge at 12000 rpm for 5 min.

[0350] SDS-PAGE steps

[0351] (1) Install the glue dispensing device.

[0352] (2) Add the prepared separating gel solution to the dispensing device from one side using a pipette, up to 1.5-2 cm from the top of the glass plate. Add anhydrous ethanol to the top of the glass plate and let it stand at room temperature for 30 minutes. Tilt the dispensing device to pour out the anhydrous ethanol and blot the remaining anhydrous ethanol with filter paper.

[0353] (3) Continue to add the prepared concentrated gel solution from one side to the dispensing device using a pipette until it reaches the top of the glass plate. Quickly insert the sample comb and let it stand at room temperature for 30 minutes.

[0354] (4) Add an appropriate volume of electrode buffer (1×) to the outer tank of the electrophoresis tank, transfer the glass plate with the gel to the electrophoresis tank, fill the inner tank with electrode buffer (1×), remove the electrophoresis comb, and load the sample.

[0355] (5) Connect the power supply and start electrophoresis at a constant voltage of 80V. After the bromophenol blue front reaches the separating gel, increase the voltage to 120V.

[0356] (6) Stop electrophoresis when the bromophenol blue front reaches the bottom of the glass plate and remove the gel with a gel opener.

[0357] (7) Staining: Place the gel in the staining box, add an appropriate amount of staining solution, and stain on a shaker for 40 minutes.

[0358] (8) Decolorization: Place the gel in the decolorization box, add an appropriate amount of decolorization solution, and replace the decolorization solution halfway through until the stripes are clear on the shaker.

[0359] (9) Scanning: The gel was scanned using a gel imaging analyzer, the scan results were saved, and analyzed using Gel-ProAnalyzer software. The results are as follows: Figure 3 As shown. Figure 3 In the table, M represents the protein molecular weight standard (Marker), WP represents the leech reference material, 1-11 represent leeches, 12-23 represent leeches (HN), 24 represents the bar-striped leech, 25 represents the pheasant leech, 26-27 represent the Japanese leech, 28 represents an unknown leech, and PM represents the pheasant leech reference material.

[0360] The gel electrophoresis pattern indicates the presence of protein bands in the leech sample. If the sample's pattern is similar to that of the leech control sample, particularly showing three bands at 25–34 kDa, 17–25 kDa, and 11 kDa, then the leech is a leech. Therefore, bands 1-11 are leeches. Figure 3 As shown in A; if the gel electrophoresis pattern of the leech sample to be tested is similar to that of Hirudo medicinalis or Hirudo phalloides, especially showing two bands at 60–75 kDa and 25–34 kDa respectively, and two bands at 11 kDa, then the leech to be tested is Hirudo medicinalis or Hirudo phalloides. Therefore, bands 12-23 and 25 are Hirudo medicinalis or Hirudo phalloides. Figure 3 As shown in B and 3C; if the gel electrophoresis pattern of the leech sample to be tested is similar to that of the *Hirudo medicinalis* control material, especially showing four bands at 60–75 kDa, 25–34 kDa, 17–25 kDa, and 11 kDa, then the leech to be tested is *Hirudo medicinalis*, therefore 24 is *Hirudo medicinalis*. Figure 3 As shown in C; the gel electrophoresis pattern of the leech sample to be tested is similar to that of the Japanese leech reference material, especially showing two bands at 25-34 kDa and 11 kDa. The leech to be tested is a Japanese leech, therefore 26 and 27 are Japanese leeches, as shown in Figure C. Figure 3 As shown in C; if the gel electrophoresis pattern of the leech sample to be tested is not similar to the gel electrophoresis pattern of the control medicinal material, then it is another adulterant.

[0361] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A gel electrophoretic identification method for leeches and bloodsuckers and their closely related adulterants, characterized in that, The identification method includes the following steps: (1) The leech sample to be tested is pretreated to obtain the leech sample test solution, and the reference medicinal material is pretreated to obtain the reference medicinal material solution; wherein, the reference medicinal material is leech, leech and its closely related adulterants; wherein, the leech and the leech are Hirudo nipponica Whitman and Whitmaniapigra Whitman of the Hirudinaceae family, and the closely related adulterants are Poecilobdella manillensis Lesson, Poecilobdella javanica Wahlberg and Mimobdella japonica Blanchard of the Hirudinaceae family; (2) The test leech sample solution and the control medicinal material solution were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel imaging analysis to obtain the gel electrophoresis patterns of the test leech sample and the control medicinal material; and (3) Compare the gel electrophoresis pattern of the leech sample to be tested with the gel electrophoresis pattern of the control medicinal material, and determine whether the leech to be tested is a leech, blood leech or its closely related adulterant based on the comparison results; The leeches to be tested are selected from one or more of the following: leeches, blood leeches, barn leeches, barn leeches and Japanese leeches; The preparation method of the reference medicinal material solution includes: taking an appropriate amount of reference medicinal material, crushing it, sieving it to obtain reference medicinal material powder, adding a solvent to the reference medicinal material powder for extraction, shaking and mixing, centrifuging, taking the supernatant, and diluting the supernatant to obtain the reference medicinal material solution, wherein the solvent is a sodium chloride solution with a concentration of about 0.9%; The method for preparing the test solution of the leech sample includes: taking an appropriate amount of the leech sample to be tested, crushing it, sieving it to obtain the leech sample powder to be tested, adding a solvent to the leech sample powder to be tested for extraction, shaking and mixing it, centrifuging it, taking the supernatant, and diluting the supernatant to obtain the test solution of the leech sample to be tested, wherein the solvent is a sodium chloride solution with a concentration of about 0.9%; In the preparation methods of the reference medicinal material solution and the test sample solution of the leech to be tested, the supernatant of leech and horny leech is diluted 100 times, and the supernatant of horny leech, leech and Japanese leech is diluted 10 times. The comparison results include any of the following: (1) If the gel electrophoresis pattern of the leech sample to be tested is consistent with or similar to the gel electrophoresis pattern of the leech control material, that is, the gel electrophoresis pattern of the leech sample to be tested shows three bands at 25-34kDa, 17-25kDa and 11kDa, then the leech to be tested is a leech. (2) If the gel electrophoresis pattern of the leech sample to be tested is consistent with or similar to the gel electrophoresis pattern of the leech control material or the gel electrophoresis pattern of the horny leech control material, that is, the gel electrophoresis pattern of the leech sample to be tested shows two bands at 60-75kDa and 25-34kDa, and two bands at 11kDa, then the leech to be tested is a leech or a horny leech; (3) If the gel electrophoresis pattern of the leech sample to be tested is consistent with or similar to the gel electrophoresis pattern of the reference herb Hirudo medicinal material, that is, the gel electrophoresis pattern of the leech sample to be tested shows four bands at 60-75kDa, 25-34kDa, 17-25kDa and 11kDa, then the leech to be tested is Hirudo medicinal material. (4) If the gel electrophoresis pattern of the leech sample to be tested is consistent with or similar to the gel electrophoresis pattern of the Japanese leech reference material, that is, the gel electrophoresis pattern of the leech sample to be tested shows two bands at 25-34kDa and 11kDa, then the leech to be tested is Japanese leech. (5) If the gel electrophoresis pattern of the leech sample to be tested is inconsistent or dissimilar to the gel electrophoresis pattern of the control medicinal material, then the leech to be tested is another adulterant.

2. The identification method according to claim 1, characterized in that, The gel electrophoresis method includes the following steps: (a) Add protein loading buffer to the test solution, mix well, boil to denature the protein, centrifuge to obtain the test solution; (b) Install the glue dispensing device, prepare the separating glue solution and the concentrated glue solution, dispense the glue, and obtain a gel plate; (c) Add electrode buffer to the outer tank of the electrophoresis tank, transfer the gel plate to the electrophoresis tank, fill the inner tank of the electrophoresis tank with electrode buffer, remove the electrophoresis comb, and load the electrophoresis marker and the test solution respectively. (d) Use the first constant voltage vertical electrophoresis. When the indicator front reaches the separating gel, switch to the second constant voltage vertical electrophoresis. Stop electrophoresis when the indicator front reaches the bottom of the gel plate and remove the gel. (e) Add dye solution to the gel to stain it, and obtain a stained gel; as well as (f) Add destaining solution to the staining gel and destain until the bands are clear to obtain an electrophoresis gel.

3. The identification method according to claim 2, characterized in that, In step (b), the separating gel solution comprises water, 30% acrylamide-methylenebisacrylamide solution, 1.5M Tris, 10% SDS, 10% APS, and TEMED; in the separating gel solution, the volume ratio of the water, the 30% acrylamide-methylenebisacrylamide solution, the 1.5M Tris, the 10% SDS, the 10% APS, and the TEMED is (760–840): (950–1050): (617.5–682.5): (23.75–26.25): (23.75–26.25): (0.95–1.05); the separating gel is a 12% concentration separating gel.

4. The identification method according to claim 1, characterized in that, The comparison refers to the comparison of the number and position of protein bands.

5. The identification method according to claim 1, characterized in that, The sieve in question is a No. 3 sieve.

6. The identification method according to claim 1, characterized in that, The extraction method is maceration extraction.

7. The identification method according to claim 6, characterized in that, The immersion extraction time is 20–40 min.

8. The identification method according to claim 7, characterized in that, The immersion extraction time is approximately 30 minutes.

9. The identification method according to claim 1, characterized in that, The centrifuge speed is 10000-15000 r / min.

10. The identification method according to claim 9, characterized in that, The centrifuge speed is approximately 12,000 r / min.

11. The identification method according to claim 1, characterized in that, The centrifugation time is 5 to 20 minutes.

12. The identification method according to claim 11, characterized in that, The centrifugation time is approximately 10 minutes.

13. The identification method according to claim 1, characterized in that, The gel imaging analysis method includes: scanning the gel electrophoresis with a gel imaging analyzer, analyzing the scanning results with Gel-Pro Analyzer software, and obtaining the gel electrophoresis pattern of the leech sample to be tested and the gel electrophoresis pattern of the control medicinal material.

14. The identification method according to claim 2, characterized in that, In step (a), the volume ratio of the test solution to the protein loading buffer is 3 to 5.

15. The identification method according to claim 14, characterized in that, The volume ratio of the test solution to the protein loading buffer is approximately 4.

16. The identification method according to claim 2, characterized in that, In step (a), the boiling temperature is 100°C.

17. The identification method according to claim 2, characterized in that, In step (a), the boiling time is 1 to 10 minutes.

18. The identification method according to claim 17, characterized in that, The boiling time is approximately 5 minutes.

19. The identification method according to claim 2, characterized in that, In step (a), the centrifugation speed is 10000 to 15000 r / min.

20. The identification method according to claim 19, characterized in that, The centrifuge speed is approximately 12,000 r / min.

21. The identification method according to claim 2, characterized in that, In step (a), the centrifugation time is 1 to 10 minutes.

22. The identification method according to claim 21, characterized in that, The centrifugation time is approximately 5 minutes.

23. The identification method according to claim 2, characterized in that, In step (b), the concentrated gel solution comprises water, 30% acrylamide-methylenebisacrylamide solution, 1.0 M Tris, 10% SDS, 10% APS and TEMED.

24. The identification method according to claim 23, characterized in that, In the concentrated gel solution, the volume ratio of the water, the 30% acrylamide-methylenebisacrylamide solution, the 1.0M Tris, the 10% SDS, the 10% APS and the TEMED is (665-735):(156.75-173.25):(118.75-131.25):(9.5-10.5):(9.5-10.5):(0.95-1.05).

25. The identification method according to claim 2, characterized in that, In step (b), the gel pouring method includes: adding the separating gel solution to one side of the gel pouring device up to 1.5-2 cm from the top of the gel pouring device, adding anhydrous ethanol to seal the top, letting it stand at room temperature for about 30 minutes, removing the anhydrous ethanol, continuing to add the concentrated gel solution to one side of the gel pouring device until it seals the top, inserting an electrophoresis comb, letting it stand at room temperature for about 30 minutes, and obtaining the gel plate.

26. The identification method according to claim 2, characterized in that, In step (c), the electrode buffer solution is prepared by weighing appropriate amounts of glycine, Tris, and SDS, adding water to make up the volume, and diluting it by about 10 times to obtain the electrode buffer solution.

27. The identification method according to claim 26, characterized in that, The mass of the glycine is 100-200g.

28. The identification method according to claim 27, characterized in that, The mass of the glycine is approximately 144g.

29. The identification method according to claim 26, characterized in that, The mass of the Tris is 20–40 g.

30. The identification method according to claim 29, characterized in that, The mass of the Tris is approximately 30g.

31. The identification method according to claim 26, characterized in that, The mass of the SDS is 5-15g.

32. The identification method according to claim 31, characterized in that, The mass of the SDS is approximately 10g.

33. The identification method according to claim 26, characterized in that, The mass of the SDS is 800-1200 mL for the volume of the final volume.

34. The identification method according to claim 33, characterized in that, The mass of the SDS is approximately 1000 mL when the volume is adjusted.

35. The identification method according to claim 2, characterized in that, In step (c), the loading volume of the electrophoresis marker is 1 to 10 μL.

36. The identification method according to claim 35, characterized in that, The loading volume of the electrophoresis marker is approximately 5 μL.

37. The identification method according to claim 2, characterized in that, In step (c), the sample volume of the test solution is 5 to 15 μL.

38. The identification method according to claim 37, characterized in that, The sample volume of the test solution is approximately 10 μL.

39. The identification method according to claim 2, characterized in that, In step (d), the first constant voltage is 70-90V.

40. The identification method according to claim 39, characterized in that, The first constant voltage is approximately 80V.

41. The identification method according to claim 2, characterized in that, In step (d), the second constant voltage is 110 to 130 V.

42. The identification method according to claim 41, characterized in that, The second constant voltage is approximately 120V.

43. The identification method according to claim 2, characterized in that, In step (e), the dye solution is Coomassie Brilliant Blue dye solution.

44. The identification method according to claim 2, characterized in that, In step (e), the staining is performed on a shaker.

45. The identification method according to claim 2, characterized in that, In step (e), the staining time is 30 to 60 minutes.

46. ​​The identification method according to claim 45, characterized in that, The staining time is approximately 40 minutes.

47. The identification method according to claim 2, characterized in that, In step (f), the decolorizing solution is a mixed solution composed of methanol, glacial acetic acid and water.

48. The identification method according to claim 47, characterized in that, In the mixed solution, the volume ratio of methanol, glacial acetic acid and water is (2-4):(0.5-2):(4-8).

49. The identification method according to claim 48, characterized in that, In the mixed solution, the volume ratio of methanol, glacial acetic acid and water is (2.85–3.15):(0.95–1.05):(5.7–6.3).

50. The identification method according to claim 3, characterized in that, The preparation method of the 30% acrylamide-methylenebisacrylamide solution includes: weighing appropriate amounts of acrylamide and methylenebisacrylamide, diluting with water to about 50 mL, and then obtaining the solution.

51. The identification method according to claim 50, characterized in that, The mass of the acrylamide is 10-20g.

52. The identification method according to claim 51, characterized in that, The acrylamide weighs approximately 14.55g.

53. The identification method according to claim 50, characterized in that, The mass of the methylene acrylamide is 0.1 to 1.0 g.

54. The identification method according to claim 53, characterized in that, The mass of the methylene acrylamide is approximately 0.45 g.

55. The identification method according to claim 3, characterized in that, The preparation method of the 1.5M Tris includes: weighing an appropriate amount of Tris, adding water to dissolve it for the first time, adding hydrochloric acid to adjust the pH, and adding water again to make up to about 250 mL.

56. The identification method according to claim 55, characterized in that, The mass of the Tris is 30–60 g.

57. The identification method according to claim 56, characterized in that, The mass of the Tris is approximately 45.4 g.

58. The identification method according to claim 55, characterized in that, The volume of the first water is 200-300 mL.

59. The identification method according to claim 58, characterized in that, The volume of the first batch of water was approximately 220 mL.

60. The identification method according to claim 55, characterized in that, The pH adjustment is to adjust the pH to approximately 8.

8.

61. The identification method according to claim 23, characterized in that, The preparation method of the 1.0M Tris includes: weighing an appropriate amount of Tris, adding water to dissolve it for the first time, adding hydrochloric acid to adjust the pH, and adding water again to make up to about 250mL.

62. The identification method according to claim 61, characterized in that, The mass of the Tris is 20–40 g.

63. The identification method according to claim 62, characterized in that, The mass of the Tris is approximately 30.3g.

64. The identification method according to claim 61, characterized in that, The volume of the first water is 200-300 ml.

65. The identification method according to claim 64, characterized in that, The volume of the first batch of water was approximately 220 mL.

66. The identification method according to claim 61, characterized in that, The pH adjustment is to adjust the pH to approximately 6.

8.

67. The identification method according to claim 3, characterized in that, The preparation method of the 10% APS includes: weighing an appropriate amount of APS, adding water to dilute it, and then obtaining the product.

68. The identification method according to claim 67, characterized in that, The mass of the APS is 0.5–2 g.

69. The identification method according to claim 68, characterized in that, The mass of the APS is approximately 1g.

70. The identification method according to claim 67, characterized in that, The volume of the water is 5 to 20 mL.

71. The identification method according to claim 70, characterized in that, The volume of the water is approximately 10 mL.

72. The identification method according to claim 3, characterized in that, The method for preparing the 10% SDS includes: weighing an appropriate amount of SDS, adding water to dilute it, and then obtaining the desired result.

73. The identification method according to claim 72, characterized in that, The mass of the SDS is 1 to 10 g.

74. The identification method according to claim 73, characterized in that, The mass of the SDS is approximately 5g.

75. The identification method according to claim 72, characterized in that, The volume of the water is 40-60 mL.

76. The identification method according to claim 75, characterized in that, The volume of the water is approximately 50 mL.

77. The use of the identification method according to any one of claims 1 to 76 in distinguishing between leeches, blood leeches and / or their closely related adulterants, wherein the closely related adulterants are selected from one or more of the following: Hirudo nipponia, Hirudo barbatulata, Hirudo nipponia, and other adulterants.

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