Gel electrophoresis identification method for leeches, leeches and related adulterants thereof and application of gel electrophoresis identification method

Abstract

The invention discloses a gel electrophoresis identification method for leeches, leeches and related adulterants of leeches and leeches and application of the gel electrophoresis identification method. The method comprises the following steps: (1) pretreating a leech sample to be detected to obtain a test solution of the leech sample to be detected, and pretreating a reference medicinal material to obtain a reference medicinal material solution; wherein the reference medicinal materials are leeches, leeches and / or related adulterants thereof; (2) performing lauryl sodium sulfate polyacrylamide gel electrophoresis and gel imaging analysis on the to-be-detected leech sample test solution and the reference medicinal material solution to obtain a to-be-detected leech sample gel electrophoretogram and a reference medicinal material gel electrophoretogram; and (3) comparing the maps, and determining whether the to-be-detected leeches are leeches, leeches or related adulterants of the leeches or not according to a comparison result, the to-be-detected leeches are selected from one or more of leeches, leeches, poecilobdella manillensis, poecilobdella japonica and poecilobdella corymbosa. The method is intuitive and accurate in detection result and easy to identify.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a gel electrophoresis identification method and application for leeches, blood-sucking leeches and their related adulterants and counterfeits. Background Art

[0002] Traditional Chinese medicine leech is derived from the dried bodies of animals of the family Hirudinidae, namely Whitmania pigra Whitman, Hirudo nipponica Whitman or Whitmania acranulata Whitman, and has the effects of promoting blood circulation to remove blood stasis and dispelling stasis to relieve swelling. Modern pharmacological studies have shown that traditional Chinese medicine leech has various effects such as anticoagulation, antithrombosis, anti-atherosclerosis, anti-platelet aggregation, anti-tumor, anti-inflammatory, improving hemorheology, and protecting against cerebral ischemia-reperfusion injury. There are more than 680 species of leech animals distributed all over the world, among which there are more than 100 species in China. However, there are only three legal origins of traditional Chinese medicine leech, namely leech, Whitmania pigra Whitman and Whitmania acranulata Whitman. Moreover, Whitmania pigra Whitman is the mainstream variety in the market, leech is rare, and Whitmania acranulata Whitman is extremely rare.

[0003] In recent years, with the continuous expansion of the demand for leech medicinal materials, its price has been rising year by year, and the supply falls short of demand. The phenomenon of adulteration and counterfeiting is extremely common. The adulterated leeches are mainly Poecilobdella manillensis Lesson, Poecilobdella javanica Wahlberg and Mimobdella japonica Blanchard. Although the above animals with different origins can be distinguished morphologically, traditional Chinese medicine leech is often used as leech segments or powder for medicine, and it is difficult to distinguish the true from the false through morphological characteristics. Therefore, there is an urgent need to establish a simple and effective identification method to ensure the quality of leech medicinal materials in the circulation link.

[0004] As an animal medicinal material, in addition to small molecule compounds, leech contains a large amount of protein polypeptides and free amino acid components. At present, more than 60 proteins have been isolated and identified from leeches, which have activities such as anticoagulation, fibrinolysis and trypsin inhibition. In addition, leeches are also rich in polypeptides related to anticoagulation, antithrombosis, anti-platelet aggregation and neuroprotection characteristics. At present, there are few studies on using the macromolecular components in leeches to identify the authenticity of medicinal materials. Electrophoresis technology is a commonly used method for analyzing protein components and has been applied in the identification of macromolecular components of various animal medicinal materials such as donkey-hide gelatin, pilose antler, oviductus ranae and lizard. However, in the existing studies, there has been no gel electrophoresis identification method and application for distinguishing (identifying) leeches, blood-sucking leeches and their related adulterants and counterfeits.

[0005] Therefore, the present invention characterizes macromolecular compounds through a gel electrophoresis identification method for identifying leeches, blood-sucking leeches and their related adulterants and counterfeits. Summary of the Invention

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

[0007] (1) Pretreat the test leech sample to obtain a test leech sample test solution, and pretreat the control medicinal material to obtain a control medicinal material solution; wherein, the control medicinal material is leech, Whitmania pigra and / or their closely related adulterants; wherein, the leech and the Whitmania pigra are Hirudo nipponica Whitman of the family Hirudinidae and Whitmania pigra Whitman of the family Hirudinidae, and the closely related adulterants are Poecilobdella manillensis Lesson of the family Hirudinidae, Poecilobdella javanica Wahlberg of the family Hirudinidae and / or Mimobdella japonica Blanchard of the family Glossiphoniidae;

[0008] (2) Perform sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel imaging analysis on the test leech sample test solution and the control medicinal material solution to obtain a gel electrophoresis pattern of the test leech sample and a gel electrophoresis pattern of the control medicinal material; and

[0009] (3) Compare the gel electrophoresis pattern of the test leech sample with the gel electrophoresis pattern of the control medicinal material, and determine whether the test leech is leech, Whitmania pigra or their closely related adulterants according to the comparison result;

[0010] Wherein, the test leech is selected from one or more of the following: leech, Whitmania pigra, Poecilobdella manillensis, Poecilobdella javanica and Mimobdella japonica.

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

[0012] Further, the preparation method of the control medicinal material solution comprises: taking an appropriate amount of the control medicinal material, pulverizing, sieving to obtain the control medicinal material powder, adding a solvent to the control medicinal material powder for extraction, shaking well, centrifuging, taking the supernatant, and diluting the supernatant to obtain the control medicinal material solution.

[0013] Further, the sieve is a No. 3 sieve.

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

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

[0016] Further, the extraction is maceration extraction.

[0017] Further, the time for maceration extraction is 20 to 40 min, such as about 30 min.

[0018] Further, the rotation speed for centrifugation is 10,000 to 15,000 r / min, such as about 12,000 r / min.

[0019] Further, the time for centrifugation is 5 to 20 min, such as about 10 min.

[0020] Further, the dilution multiple is 5 to 120, such as about 10 or about 100.

[0021] Further, the method for preparing the test leech sample solution for test includes: taking an appropriate amount of the test leech sample, pulverizing, sieving to obtain the powder of the test leech sample, adding a solvent for extraction to the powder of the test leech sample, shaking well, centrifuging, taking the supernatant, and diluting the supernatant to obtain the test leech sample solution for test.

[0022] Further, the sieve is a No. 3 sieve.

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

[0024] Further, the concentration of the sodium chloride solution is 0.5% to 2.0%, such as about 0.9%.

[0025] Further, the extraction is maceration extraction.

[0026] Further, the time for maceration extraction is 20 to 40 min, such as about 30 min.

[0027] Further, the rotation speed for centrifugation is 10,000 to 15,000 r / min, such as about 12,000 r / min.

[0028] Further, the time for centrifugation is 5 to 20 min, such as about 10 min.

[0029] Further, the dilution multiple is 5 to 120, such as about 10 or about 100.

[0030] Further, when the test leech is Hirudo nipponica Whitman or Poecilobdella manillensis Lesson, the dilution multiple is about 100 times.

[0031] Further, when the test leech is Poecilobdella javanica or Hemiclepsis japonica, the dilution multiple is about 10 times.

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

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

[0034] (b) Install the gel-casting device, prepare the separating gel solution and the stacking gel solution, and pour the gel to 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) Perform the first constant-voltage vertical electrophoresis, change to the second constant-voltage vertical electrophoresis when the indicating front reaches the separating gel, stop the electrophoresis when the indicating front reaches the bottom of the gel plate, and take out the gel;

[0037] (e) Add staining solution to the gel for staining to obtain a stained gel; and

[0038] (f) Add decolorizing solution to the stained gel and decolorize until the bands are clear to obtain an electrophoretic gel.

[0039] Furthermore, the method for gel imaging analysis includes: scanning the electrophoretic gel with a gel imaging analyzer, and analyzing the scanning result with Gel-Pro Analyzer software to obtain the gel electrophoresis map of the test leech sample and the gel electrophoresis map of the leech.

[0040] Furthermore, 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 min, for example, about 5 min.

[0043] Furthermore, in step (a), the centrifugation speed is 10000 to 15000 r / min, for example, about 12000 r / min.

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

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

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

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

[0048] Further, in the stacking gel solution, the volume ratio of the water, the 30% acrylamide - methylene bisacrylamide solution, the 1.0M Tris, the 10% SDS, the 10% APS, and the TEMED is about 700: about 165: about 125: about 10: about 10: about 1.

[0049] Further, in step (b), the method of pouring the gel includes: adding the separating gel solution to one side of the gel - pouring device to a position 1.5 - 2 cm away from the upper end of the gel - pouring device, adding anhydrous ethanol to seal the top, standing at room temperature for about 30 min, removing the anhydrous ethanol, then continuing to add the stacking gel solution to one side of the gel - pouring device until it is full, inserting an electrophoresis comb, and standing at room temperature for about 30 min to obtain the gel plate.

[0050] Further, in step (c), the method for preparing the electrode buffer solution is: weighing an appropriate amount of glycine, Tris, and SDS, adding water to make up the volume, and diluting by about 10 times to obtain the electrode buffer solution.

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

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

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

[0054] Further, the volume for making up the volume is 800 - 1200 mL, for example, about 1000 mL.

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

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

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

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

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

[0060] Further, in step (e), the staining is carried out on a shaker.

[0061] Further, in step (e), the staining time is 30 - 60 min, for example, about 40 min.

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

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

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

[0065] Further, the preparation method of the 30% acrylamide - methylene bisacrylamide solution includes: weighing appropriate amounts of acrylamide and methylene bisacrylamide, diluting with water to about 50 mL, and then obtaining it.

[0066] Further, the mass of acrylamide is 10 - 20 g, for example, about 14.55 g.

[0067] Further, the mass of methylene bisacrylamide is 0.1 - 1.0 g, for example, about 0.45 g.

[0068] Further, the preparation method of the 1.5M Tris includes: weighing appropriate amounts of Tris, dissolving it with the first water, adjusting the pH with hydrochloric acid, and then adding water again to make the volume up to about 250 mL, and then obtaining it.

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

[0070] Further, the volume of the first water is 200 - 300 mL, for example, about 220 mL.

[0071] Further, adjusting the pH means adjusting the pH to about 8.8.

[0072] Further, the preparation method of the 1.0M Tris includes: weighing appropriate amounts of Tris, dissolving it with the first water, adjusting the pH with hydrochloric acid, and then adding water again to make the volume up to about 250 mL, and then obtaining it.

[0073] Further, the mass of the Tris is 20 - 40 g, such as about 30.3 g.

[0074] Further, the volume of the first water is 200 - 300 ml, such as about 220 mL.

[0075] Further, adjusting the pH means adjusting the pH to about 6.8.

[0076] Further, the preparation method of the 10% APS includes: weighing an appropriate amount of APS, adding water for dilution, and then obtaining it.

[0077] Further, the mass of the APS is 0.5 - 2 g, such as about 1 g.

[0078] Further, the volume of the water is 5 - 20 mL, such as about 10 mL.

[0079] Further, the preparation method of the 10% SDS includes: weighing an appropriate amount of SDS, adding water for dilution, and then obtaining it.

[0080] Further, the mass of the SDS is 1 - 10 g, such as about 5 g.

[0081] Further, the volume of the water is 40 - 60 mL, such as about 50 mL.

[0082] Further, the comparison result includes any one of the following:

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

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

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

[0086] (4) If the gel electrophoresis pattern of the leech sample to be tested is identical or similar to that of the reference medicinal material of Japanese leech, for example, if there are two bands at 25 - 34 kDa and 11 kDa in the gel electrophoresis pattern of the leech sample to be tested, then the leech sample to be tested is Japanese leech;

[0087] (5) If the gel electrophoresis pattern of the leech sample to be tested is not identical or not similar to that of the above reference medicinal materials, then the leech sample to be tested is other adulterants.

[0088] According to another aspect of the present invention, there is provided a use of the above identification method in differentiating leech, Whitmania pigra and / or their related adulterants from each other, wherein the related adulterants are selected from one or more of the following: Poecilobdella manillensis, Poecilobdella javanica, Japanese leech and other adulterants.

[0089] Advantages of the present invention:

[0090] By subjecting the sample to saline extraction and electrophoresis analysis, the present invention can identify the sample to be tested according to the number and position of protein bands on the gel, and the test results are intuitive, accurate and easy to identify. Description of the Drawings

[0091] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained according to these drawings without exceeding the scope of the present invention.

[0092] Figure 1 It is a schematic diagram of the results of the investigation on the concentration of the separating gel in the SDS - PAGE electrophoresis conditions of Example 1 of the present invention.

[0093] Figure 2 It is a schematic diagram of the results of the investigation on the dilution factor of the test solution in the SDS - PAGE electrophoresis conditions of Example 2 of the present invention.

[0094] Figure 3 It is the SDS - PAGE pattern of Example 3 of the present invention. Detailed Embodiments

[0095] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

[0096] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains or in the field in which the term is applied. Although any methods, conditions, substances or materials similar or equivalent to those disclosed herein may be used in the practice of the present invention, the preferred methods, conditions, substances or materials are described herein.

[0097] The present invention is expected to cover all alternatives, variations and equivalents, which may be included in the existing field of invention as defined in the claims. Those skilled in the art will recognize many methods and substances similar or equivalent to those described herein, which can be applied to the practice of the present invention. The present invention is not limited to the description of methods and substances.

[0098] 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.

[0099] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "including", "comprising", "having", "containing" or any other variation thereof are intended to cover non-exclusive inclusion. For example, a composition, step, method, article or device containing the recited elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article or device.

[0100] As described in the background art section, there is no method for gel electrophoresis identification of a leech and its near-related adulterants in the prior art. To solve the above problems, the present invention provides a method for gel electrophoresis identification of a leech and its near-related adulterants, and the identification method comprises the following steps:

[0101] (1) Pretreat the test leech sample to obtain a test leech sample test solution, and pretreat the control medicinal material to obtain a control medicinal material solution; wherein, the control medicinal material is a leech, a horse leech and / or its near-related adulterants; wherein, the leech and the horse leech are Hirudo nipponica Whitman of the family Hirudinidae and WhitmaniapigraWhitman of the family Hirudinidae, and the near-related adulterants are Poecilobdella manillensis Lesson of the family Hirudinidae, Poecilobdellajavanica Wahlberg of the family Hirudinidae and / or Mimobdellajaponica Blanchard of the family Glossiphoniidae;

[0102] (2) Perform sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel imaging analysis on the test leech sample test solution and the control medicinal material solution to obtain the gel electrophoresis pattern of the test leech sample and the gel electrophoresis pattern of the control medicinal material; and

[0103] (3) Compare the gel electrophoresis pattern of the test leech sample with the gel electrophoresis pattern of the control medicinal material, and determine whether the test leech is Hirudo, Whitmania, or its closely related adulterants according to the comparison result;

[0104] Among them, the test leech is selected from one or more of the following: Hirudo, Whitmania, Poecilobdella manillensis, Poecilobdella javanica, and Haemadipsa japonica.

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

[0106] In a preferred embodiment, the preparation method of the control medicinal material solution includes: taking an appropriate amount of the control medicinal material, pulverizing, sieving to obtain the powder of the control medicinal material, adding a solvent to the powder of the control medicinal material for extraction, shaking well, centrifuging, taking the supernatant, and diluting the supernatant to obtain the control 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 the present invention, when a concentration, time, rotation speed, multiple, mass, volume, voltage, ratio, molecular weight, or other value or parameter is expressed in a range, a preferred range, or a range defined by a series of upper limit preferred values and lower limit preferred values, it should be understood that all ranges formed by any pairing of any range upper limit or preferred value and any range lower limit or preferred value are specifically disclosed, regardless of whether the range is separately disclosed. For example, when the range "0.5% to 2.0%" is disclosed, the described range should be interpreted as including the 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 a numerical range is described herein, unless otherwise specified, the range is intended to include its end values and all integers and fractions within the 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 the present invention, "about" means a value within a range of ±5% of a specific value. For example, "about 0.9%" includes ±5% of 0.9%, or from 0.855% to 0.945%.

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

[0113] In a preferred embodiment, the time for maceration extraction is 20 to 40 min, such as about 30 min.

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

[0115] In a preferred embodiment, the rotational speed for centrifugation is 10000 to 15000 r / min, such as about 12000 r / min.

[0116] In the present invention, "about" refers to a value within the 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 time for centrifugation is 5 to 20 min, such as about 10 min.

[0118] In the present invention, "about" refers to a value within the 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 multiple is 5 to 120, such as about 10 or about 100.

[0120] In the present invention, "about" refers to a value within the 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 method for preparing the test leech sample test solution includes: taking an appropriate amount of the test leech sample, pulverizing it, sieving it, obtaining the powder of the test leech sample, adding a solvent for extraction to the powder of the test leech sample, shaking it well, centrifuging it, taking the supernatant, and diluting the supernatant to obtain the test leech sample test 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%, such as about 0.9%.

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

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

[0127] In a preferred embodiment, the time for maceration extraction is 20 - 40 min, such as about 30 min.

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

[0129] In a preferred embodiment, the rotational speed of the centrifugation is 10000 - 15000 r / min, such as about 12000 r / min.

[0130] In the present invention, "about" refers to a value within the 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 time for centrifugation is 5 - 20 min, such as about 10 min.

[0132] In the present invention, "about" refers to a value within the 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 multiple is 5 - 120, such as about 10 or about 100.

[0134] In the present invention, "about" refers to a value within the 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 Hirudo nipponica Whitman or Poecilobdella manillensis Lesson, the dilution multiple is about 100 times.

[0136] In the present invention, "about" refers to a value within the 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 Haemadipsa picta or Haemadipsa japonica, the dilution multiple is about 10 times.

[0138] In the present invention, "about" refers to a value within the 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 method of gel electrophoresis comprises the following steps:

[0140] (a) Adding protein loading buffer to the test sample solution, mixing well, boiling to denature the protein, and centrifuging to obtain a test solution;

[0141] (b) Installing a gel-casting device, preparing a separating gel solution and a stacking gel solution, and casting the gel to obtain a gel plate;

[0142] (c) Adding electrode buffer to the outer tank of the electrophoresis tank, transferring the gel plate to the electrophoresis tank, filling the inner tank of the electrophoresis tank with electrode buffer, removing the electrophoresis comb, and loading the electrophoresis marker and the test solution respectively;

[0143] (d) Performing the first constant voltage vertical electrophoresis, changing to the second constant voltage vertical electrophoresis when the indicating front reaches the separating gel, stopping the electrophoresis when the indicating front reaches the bottom of the gel plate, and taking out the gel;

[0144] (e) Adding a staining solution to the gel for staining to obtain a stained gel; and

[0145] (f) Adding a decolorizing solution to the stained gel, decolorizing until the bands are clear to obtain an electrophoretic gel.

[0146] In a preferred embodiment, the method of gel imaging analysis comprises: scanning the electrophoretic gel using a gel imaging analyzer, and analyzing the scanning result using Gel-Pro Analyzer software to obtain the gel electrophoresis map of the test leech sample and the gel electrophoresis map of the leech.

[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 the present invention, "about" refers to a value within the 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 min, for example about 5 min.

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

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

[0153] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 12,000" includes ±5% of 12,000, or from 11,400 to 12,600.

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

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

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

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

[0158] In the present invention, "about" refers to a value within the 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; "about 1" includes ±5% of 1, or from 0.95 to 1.05.

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

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

[0161] In the present invention, "about" refers to a value within the 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; "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0162] In a preferred embodiment, in step (b), the method of pouring the gel includes: adding the separating gel solution to one side of the gel - pouring device to a position 1.5 - 2 cm from the upper end of the gel - pouring device, adding anhydrous ethanol to cap it, standing at room temperature for about 30 min, removing the anhydrous ethanol, then continuing to add the concentrated gel solution to one side of the gel - pouring device until it is capped, inserting an electrophoresis comb, and standing at room temperature for about 30 min to obtain the gel plate.

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

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

[0165] In the present invention, "about" refers to a value within the 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 mass of the glycine is 100 - 200 g, for example, about 144 g.

[0167] In the present invention, "about" refers to a value within the 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 - 40 g, for example, about 30 g.

[0169] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 30" includes ±5% of 30, 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 the present invention, "about" refers to a value within the 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 constant volume is 800 to 1200 ml, for example about 1000 ml.

[0173] In the present invention, "about" refers to a value within the 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 the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 5" includes ±5% of 5, or from 4.75 to 5.25.

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

[0177] In the present invention, "about" refers to a value within the 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 90 V, for example about 80 V.

[0179] In the present invention, "about" refers to a value within the 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 130 V, for example about 120 V.

[0181] In the present invention, "about" refers to a value within the 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 staining solution is Coomassie Brilliant Blue staining solution.

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

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

[0185] In the present invention, "about" means a value within the 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, in the mixed solution, the volume ratio of methanol, glacial acetic acid and water is (2 - 4):(0.5 - 2):(4 - 8).

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

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

[0190] In a preferred embodiment, the method for preparing the 30% acrylamide - methylene bisacrylamide solution includes: weighing an appropriate amount of acrylamide and methylene bisacrylamide, and diluting with water to about 50 ml to obtain it.

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

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

[0193] In the present invention, "about" means a value within the 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 bisacrylamide is 0.1 - 1.0 g, for example about 0.45 g.

[0195] In the present invention, "about" refers to a value within the 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 method for preparing 1.5M Tris includes: weighing an appropriate amount of Tris, dissolving it in water for the first time, adjusting the pH with hydrochloric acid, and then adding water to make the volume up to about 250 ml.

[0197] In the present invention, "about" refers to a value within the 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 - 60 g, such as about 45.4 g.

[0199] In the present invention, "about" refers to a value within the 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 water for the first time is 200 - 300 mL, such as about 220 mL.

[0201] In the present invention, "about" refers to a value within the 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, adjusting the pH means adjusting the pH to about 8.8.

[0203] In the present invention, "about" refers to a value within the 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 method for preparing 1.0M Tris includes: weighing an appropriate amount of Tris, dissolving it in water for the first time, adjusting the pH with hydrochloric acid, and then adding water to make the volume up to about 250 ml.

[0205] In the present invention, "about" refers to a value within the 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 - 40 g, such as about 30.3 g.

[0207] In the present invention, "about" refers to a value within the 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 the present invention, "about" refers to a value within the 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 about 6.8.

[0211] In the present invention, "about" refers to a value within the 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 preparation method of the 10% APS includes: weighing an appropriate amount of APS, adding water for dilution, and then obtaining it.

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

[0214] In the present invention, "about" refers to a value within the 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 - 20 mL, for example, about 10 mL.

[0216] In the present invention, "about" refers to a value within the 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 preparation method of the 10% SDS includes: weighing an appropriate amount of SDS, adding water for dilution, and then obtaining it.

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

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

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

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

[0222] In a preferred embodiment, the gel electrophoresis pattern of the medicinal leech material includes 3 protein bands, located at 25 - 34 kDa, 17 - 25 kDa, and 11 kDa respectively.

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

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

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

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

[0227] (4) If the gel electrophoresis pattern of the leech sample to be tested is consistent or similar to the gel electrophoresis pattern of the control medicinal Hemiclepsis japonica material, for example, if the gel electrophoresis pattern of the leech sample to be tested shows two bands at 25 - 34 kDa and 11 kDa, then the leech sample to be tested is a Hemiclepsis japonica;

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

[0229] According to another aspect of the present invention, there is provided a gel electrophoresis identification method for differentiating leeches, medicinal leeches, and / or their related adulterated or fake products from each other, characterized by including:

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

[0231] Step 2: Immerse and extract the above-mentioned powders with saline solution respectively, shake constantly, centrifuge, and take the supernatant to obtain the reference medicinal material sample solution and the test sample solution for the sample to be detected.

[0232] Step 3: Analyze the above-mentioned test sample solutions (the test sample solutions of Hirudo nipponia, Poecilobdella manillensis are diluted 100 times, and the test sample solutions of Whitmania pigra, Poecilobdella javanica, Whitmania nipponia, and unknown leeches are diluted 10 times) by gel electrophoresis to obtain a gel, scan the gel with a gel imaging analyzer, and analyze it with Gel-Pro Analyzer software. Identify the sample to be detected according to the number and position of protein bands on the gel.

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

[0234] In a preferred embodiment, the extraction time for the immersion extraction is 30 min; the centrifugation conditions are centrifugation at 12,000 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 is loaded, and 10 μL of the test sample is loaded. When performing electrophoresis, add electrode buffer to the inner and outer electrophoresis tanks, start electrophoresis at a constant voltage of 80 V, increase the voltage to 120 V after the leading edge reaches the separating gel, and stop electrophoresis when the leading edge reaches the bottom of the glass plate. Stain the gel with Coomassie Brilliant Blue staining solution, and repeatedly rinse the gel with decolorizing solution until the bands are clearly visible. Scan the gel with a gel imaging analyzer and analyze it with Gel-Pro Analyzer software.

[0236] In a preferred embodiment, if there are single bands at 25 - 34 kDa, 17 - 25 kDa, and 11 kDa in the gel electrophoresis pattern of the sample to be detected, it is determined that the sample to be detected is Whitmania pigra.

[0237] According to another aspect of the present invention, there is provided a use of the above-mentioned identification method in differentiating Hirudo nipponia, Whitmania pigra and / or their closely related adulterants, wherein the closely related adulterants are selected from one or more of the following: Poecilobdella manillensis, Poecilobdella javanica, Whitmania nipponia, and other adulterants.

[0238] The following further elaborates the present invention in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or the conditions recommended by the manufacturer.

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

[0240] The above-mentioned features mentioned in the present invention, or the features mentioned in the embodiments, can be combined arbitrarily. All the features disclosed in this patent specification can be used in combination with any composition 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 only general examples of equivalent or similar features.

[0241] Examples

[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 tabletop high-speed refrigerated centrifuge (Huxi Instrument Equipment Co., Ltd., Hunan), S20 SevenEasy TM pH meter (Mettler-Toledo International Co., Ltd.), BSA-124S-CW ten-thousandth electronic balance (Beijing Sartorius Scientific Instruments Co., Ltd.).

[0244] Rainbow 180 broad-spectrum protein Marker (11-180KD), tris (hydroxymethyl) aminomethane (Tris) (Solarbio Science & Technology Co., Ltd.), SDS-PAGE protein loading buffer (5X), Coomassie brilliant blue staining solution (product number: P0017B) (Beyotime Biotechnology Co., Ltd.), sodium dodecyl sulfate (SDS), acrylamide, methylene bisacrylamide, ammonium persulfate (APS), N,N,N',N'-tetramethylethylenediamine (TEMED), glycine (Sigma, USA), physiological sodium chloride solution (Shijiazhuang No. 4 Pharmaceutical Co., Ltd.), ethanol (Sinopharm Chemical Reagent Co., Ltd.).

[0245] Experimental materials

[0246] Collect 28 batches of samples from different regions of China, including 11 batches of Whitmania pigra, 12 batches of Hirudo nipponia, 1 batch of Poecilobdella javanica, 1 batch of Poecilobdella manillensis, 2 batches of Haemadipsa japonica, and 1 batch of unknown leech. The specific information is as follows:

[0247] Sample 1: Whitmania pigra, produced in Shandong (purchased from Anguo Chinese herbal medicine market);

[0248] Sample 2: Whitmania pigra, produced in Jining County, Jining City, Shandong;

[0249] Sample 3: Whitmania pigra, produced in Suyu District, Suqian City, Jiangsu Province;

[0250] Sample 4 leeches are from Bamen Town, Baodi District, Tianjin City;

[0251] Sample 5 leeches are from Xihua County, Zhoukou City, Henan Province;

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

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

[0254] Sample 8 leeches are from De'an County, Jiujiang City, Jiangxi Province;

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

[0256] Sample 10 leeches are from Gong'an County, Jingzhou City, Hubei Province;

[0257] Sample 11 leeches are from Gong'an County, Jingzhou City, Hubei Province;

[0258] Sample 12 leeches, from Jining County, Jining City, Shandong Province;

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

[0260] Sample 14 leeches, from Bamen Town, Baodi District, Tianjin City;

[0261] Sample 15 leeches are from Xihua County, Zhoukou City, Henan Province;

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

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

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

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

[0266] Sample 20 leeches are from Jining County, Jining City, Shandong Province;

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

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

[0269] Sample 23 leeches are from Bamen Town, Baodi District, Tianjin City;

[0270] Sample 24 Haemadipsa papillosa are from Qinzhou South District, Qinzhou City, Guangxi Zhuang Autonomous Region;

[0271] Sample 25 Hirudinaria manillensis are from Zhongshan City, Guangdong Province;

[0272] Sample 26: Japanese leech-like organism, origin: Xihua County, Zhoukou City, Henan Province;

[0273] Sample 27: Japanese leech-like organism, origin: Xinhui District, Jiangmen City, Guangdong Province.

[0274] Sample 28: Unknown leech, origin: De'an County, Jiujiang City, Jiangxi Province.

[0275] Example 1

[0276] Gel electrophoresis identification method for leeches, horse leeches and their closely related adulterants, investigation of separating gel concentration, including:

[0277] Reagent and gel preparation

[0278] 30% acrylamide - N,N'-methylenebisacrylamide solution (29:1): Weigh 14.55 g of acrylamide and 0.45 g of N,N'-methylenebisacrylamide, dilute with water to 50 mL, and store in the dark at 4°C.

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

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

[0281] 10% APS: Weigh 1.00 g of APS, add 10 mL of water, dispense, and store at -20°C.

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

[0283] Separating gels with different concentrations are shown in Table 1.

[0284] Table 1 Separating gels with different concentrations

[0285]

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

[0287] Electrode buffer (10×): Weigh 144 g of glycine, 30 g of Tris, and 10 g of SDS, make up the volume to 1 L with water, and dilute 10 times before use to obtain electrode buffer (1×).

[0288] Electrophoresis staining solution: Coomassie Brilliant Blue staining solution is purchased from Beyotime Biotechnology Co., Ltd.

[0289] Electrophoretic decolorizing solution: A mixed solution of methanol - glacial acetic acid - water (3:1:6).

[0290] Preparation of test solution

[0291] Take leeches (sample 11), Whitmania pigra (sample 20), Poecilobdella javanica (sample 24), Hirudinaria manillensis (sample 25), Haementeria japonica (sample 26), unknown leech (sample 28)), crush them, pass through a No. 3 sieve, and obtain powder samples of Whitmania pigra, leeches or their related adulterants respectively. Weigh equal amounts of the powder samples of Whitmania pigra, leeches or their related adulterants respectively.

[0292] Immerse and extract the above powders with 5 mL of physiological saline solution for 30 minutes, shaking constantly, centrifuge at 12000 r / min for 10 minutes, and take the supernatant. Dilute the supernatant 10 times to obtain the test solution.

[0293] Protein loading pretreatment: Mix according to the volume of the test solution: protein loading buffer (5×) = 4:1, boil at 100 °C for 5 minutes, and centrifuge at 12000 rpm for 5 minutes.

[0294] SDS - PAGE procedure

[0295] (1) Install the gel - casting device.

[0296] (2) Use a pipette to add the prepared separating gel solution to the gel - casting device from one side, add it to a position 1.5 - 2 cm from the upper end of the glass plate, add absolute ethanol to the upper end of the glass plate, let it stand at room temperature for 30 minutes, tilt the gel - casting device to pour out the absolute ethanol, and blot the residual absolute ethanol with filter paper.

[0297] (3) Use a pipette to continue adding the prepared stacking gel solution to the gel - casting device from one side until it reaches the upper end 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 clamped 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, start electrophoresis at a constant voltage of 80 V, and increase the voltage to 120 V after the bromophenol blue front reaches the separating gel.

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

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

[0302] (8) Decolorization: Place the gel in a decolorization box, add an appropriate amount of decolorizing solution, and replace the fresh decolorizing solution midway until the bands are clear after decolorization on a shaker.

[0303] (9) Scanning: Scan the gel using a gel imaging analyzer, save the scanning results, and analyze them with Gel-Pro Analyzer software. The results are as Figure 1 shown. Figure 1 In it, M is the protein molecular weight standard (Marker), 20 is Hirudo nipponica (HN), 11 is Whitmania pigra, 24 is Poecilobdella javanica, 25 is Pontobdella muricata, 26 is Metabdella nipponica, and 28 is an unknown leech.

[0304] The electrophoresis diagrams of separating gels with different concentrations are as Figure 1 shown. In the electrophoresis of 8% separating gel, the separation effect of Marker protein bands is poor, not all unfolded, only 8 bands appear, and the main protein bands of the sample are not all unfolded (there are protein bands at 11 kDa), as Figure 1 shown in A. In the electrophoresis of 10% separating gel, the separation effect of Marker protein bands is poor, not all unfolded, only 9 bands appear, and the main protein bands of the sample are not all unfolded (there are protein bands at 11 kDa), as Figure 1 shown in B. In the electrophoresis of 15% separating gel, the separation effect of Marker protein bands is poor, not all unfolded, only 9 bands appear, and the protein bands of the sample are distributed in the upper part of the gel electrophoresis, and there may be overlap, as Figure 1 shown in D. The separation effect of 12% separating gel is the best, all 10 protein bands of Marker are unfolded, and the main protein bands of the sample are clear, as Figure 1 shown in C. Therefore, 12% separating gel is selected.

[0305] Example 2

[0306] A method for identifying Hirudo nipponica, Whitmania pigra and their closely related adulterants by gel electrophoresis, investigation on the dilution multiple of test samples, including:

[0307] Reagent and gel preparation

[0308] 30% acrylamide - methylene bisacrylamide solution (29:1): Weigh 14.55 g of acrylamide and 0.45 g of methylene bisacrylamide, dilute with water to 50 mL, and store in the dark at 4°C.

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

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

[0311] 10% APS: Weigh 1.00 g of APS, add 10 mL of water, dispense it, and store it at -20 °C.

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

[0313] 12% polyacrylamide gel electrophoresis separating gel: 1.6 mL of water, 2.0 mL of 30% acrylamide - methylene bisacrylamide solution, 1.3 mL of 1.5M Tris (pH 8.8), 50 μL of 10% SDS, 50 μL of 10% APS, 2 μL of TEMED.

[0314] 5% polyacrylamide gel stacking gel: 1.4 mL of water, 0.33 mL of 30% acrylamide - methylene bisacrylamide solution, 0.25 mL of 1.0M Tris (pH 6.8), 20 μL of 10% SDS, 20 μL of 10% APS, 2 μL of TEMED.

[0315] Electrode buffer (10×): Weigh 144 g of glycine, 30 g of Tris, and 10 g of SDS, make up the volume to 1 L with water, and dilute it 10 - fold for use as electrode buffer (1×).

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

[0317] Electrophoresis decolorizing solution: A mixed solution of methanol - glacial acetic acid - water (3:1:6).

[0318] Preparation of test solution

[0319] Take leeches (sample 11), Hirudo nipponia (sample 20), Poecilobdella javanica (sample 24), Hirudinaria manillensis (sample 25), Whitmania japonica (sample 26), unknown leeches (sample 28)), crush them, pass through a No. 3 sieve to obtain the control medicinal material powders of leeches, Hirudo nipponia or their related adulterants, and weigh equal amounts of the control medicinal material powders of leeches, Hirudo nipponia or their related adulterants respectively.

[0320] Immerse and extract the above - mentioned powders with 5 mL of normal saline solution for 30 min, shake constantly, centrifuge at 12000 r / min for 10 min, and take the supernatant. Dilute the supernatant 10 - fold and 100 - fold to obtain the test solution.

[0321] Protein loading pretreatment: Mix according to the volume of the test solution: protein loading buffer (5×) = 4:1, boil at 100 °C for 5 min, and centrifuge at 12000 rpm for 5 min.

[0322] SDS-PAGE procedure

[0323] (1) Install the gel-casting device.

[0324] (2) Use a pipette to add the prepared separating gel solution to the gel-casting device from one side, add it to a position 1.5 - 2 cm from the upper end of the glass plate, add absolute ethanol to the upper end of the glass plate, let it stand at room temperature for 30 min, tilt the gel-casting device to pour out the absolute ethanol, and use filter paper to absorb the remaining absolute ethanol.

[0325] (3) Use a pipette to continue adding the prepared stacking gel solution to the gel-casting device from one side until it reaches the upper end of the glass plate. Quickly insert the sample comb and let it stand at room temperature for 30 min.

[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, start electrophoresis at a constant voltage of 80 V, and increase the voltage to 120 V after the bromophenol blue front reaches the separating gel.

[0328] (6) Stop electrophoresis when the bromophenol blue front reaches the bottom of the glass plate, and use a gel opener to take out the gel.

[0329] (7) Staining: Put the gel into a staining box, add an appropriate amount of staining solution, and stain it on a shaker for 40 min.

[0330] (8) Decolorization: Put the gel into a decolorization box, add an appropriate amount of decolorizing solution, change the fresh decolorizing solution halfway until it is decolorized to clear bands on the shaker.

[0331] (9) Scanning: Scan the gel through a gel imaging analyzer, save the scanning results, and analyze them with Gel-Pro Analyzer software. The results are as Figure 2 shown. Figure 2 In it, M is the protein molecular weight standard (Marker), 20 is Hirudo nipponia (HN), 11 is Whitmania pigra, 24 is Poecilobdella javanica, 25 is Pontobdella muricata, 26 is Haemadipsa japonica, 28 is an unknown leech.

[0332] Investigation of different dilution multiples of sample supernatant: As shown in the gel electrophoresis pattern, when the supernatant of Hirudo nipponia and Poecilobdella manillensis was diluted 10 times, the two protein bands at 11 kDa were difficult to observe, and the background of the gel electrophoresis pattern was dark. When the supernatant was diluted 100 times, the two protein bands at 11 kDa were easy to observe, and the background of the gel electrophoresis pattern was clear. When the supernatant of Whitmania pigra, Whitmania acranulata, Haemadipsa japonica, and unknown leeches was diluted 10 times, the protein bands were easy to observe. When diluted 100 times, the protein bands became blurred. Therefore, the supernatant of Hirudo nipponia and Poecilobdella manillensis was diluted 100 times, and the supernatant of Whitmania pigra, Whitmania acranulata, Haemadipsa japonica, and unknown leeches was diluted 10 times.

[0333] Example 3

[0334] A method for identifying Hirudo nipponia, Whitmania acranulata and their closely related adulterants by gel electrophoresis, including:

[0335] Reagent and gel preparation

[0336] 30% acrylamide - methylene bisacrylamide solution (29:1): Weigh 14.55 g of acrylamide and 0.45 g of methylene bisacrylamide, dilute with water to 50 mL, and store in the dark at 4°C.

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

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

[0339] 10% APS: Weigh 1.00 g of APS, add 10 mL of water, dispense, and store at -20°C.

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

[0341] 12% polyacrylamide gel electrophoresis separating gel: 1.6 mL of water, 2.0 mL of 30% acrylamide - methylene bisacrylamide solution, 1.3 mL of 1.5 M Tris (pH 8.8), 50 μL of 10% SDS, 50 μL of 10% APS, 2 μL of TEMED.

[0342] 5% polyacrylamide gel stacking gel: 1.4 mL of water, 0.33 mL of 30% acrylamide - methylene bisacrylamide solution, 0.25 mL of 1.0 M Tris (pH 6.8), 20 μL of 10% SDS, 20 μL of 10% APS, 2 μL of TEMED.

[0343] Electrode buffer (10×): Weigh 144 g of glycine, 30 g of Tris, and 10 g of SDS, add water to make up to 1 L. Dilute 10-fold before use to obtain electrode buffer (1×).

[0344] Electrophoresis staining solution: Coomassie Brilliant Blue staining solution was purchased from Beyotime Biotechnology Co., Ltd.

[0345] Electrophoresis decolorizing solution: A mixed solution of methanol - glacial acetic acid - water (3:1:6).

[0346] Preparation of test sample solution

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

[0348] Immerse the above powders in 5 mL of normal saline solution for extraction for 30 min, shake constantly, centrifuge at 12,000 r / min for 10 min, and take the supernatant. Dilute the supernatant 10-fold and conduct comparative analysis of the electrophoresis patterns to identify whether the sample is leech, horse leech, or their adulterated products; if the electrophoresis pattern has no clear bands due to too high a concentration of the test sample solution, it means that the concentration of the test leech sample solution is too high, then dilute the test leech sample solution to 100-fold and then conduct detection and identification.

[0349] Protein loading pretreatment: Mix according to the volume of the test sample solution: protein loading buffer (5×) = 4:1, boil at 100 °C for 5 min, and centrifuge at 12,000 rpm for 5 min.

[0350] SDS-PAGE procedure

[0351] (1) Install the gel-casting device.

[0352] (2) Use a pipette to add the prepared separating gel solution to the gel-casting device from one side, add it to a position 1.5 - 2 cm from the upper end of the glass plate, add anhydrous ethanol to the upper end of the glass plate, let it stand at room temperature for 30 min, tilt the gel-casting device to pour out the anhydrous ethanol, and blot the residual anhydrous ethanol with filter paper.

[0353] (3) Use a pipette to continue adding the prepared stacking gel solution to the gel-casting device from one side until it reaches the upper end of the glass plate. Quickly insert the sample comb and let it stand at room temperature for 30 min.

[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, start electrophoresis at a constant voltage of 80 V, and increase the voltage to 120 V after the bromophenol blue front reaches the separating gel.

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

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

[0358] (8) Decolorization: Place the gel in a decolorization box, add an appropriate amount of decolorizing solution, and replace the fresh decolorizing solution midway until the bands are clear after decolorization on a shaker.

[0359] (9) Scanning: Scan the gel with a gel imaging analyzer, save the scanning results, and analyze with Gel-Pro Analyzer software. The results are as Figure 3 shown. Figure 3 In it, M is the protein molecular weight standard (Marker), WP is the control medicinal material of Hirudo, 1 - 11 are Hirudo, 12 - 23 are Whitmania pigra (HN), 24 is Poecilobdella javanica, 25 is Whitmania acranulata, 26 - 27 are Japanese leeches, 28 is an unknown leech, and PM is the control medicinal material of Whitmania acranulata.

[0360] In the gel electrophoresis pattern, check the number and position of the protein bands of the test leech sample in the electrophoresis pattern. If the test leech sample is similar to the gel electrophoresis pattern of the control medicinal material of Hirudo, especially showing three bands at 25 - 34 kDa, 17 - 25 kDa, and 11 kDa, then the test leech is Hirudo. Therefore, 1 - 11 are Hirudo, as Figure 3 shown in Figure A; if the test leech sample is similar to the gel electrophoresis pattern of Whitmania pigra or Whitmania acranulata, especially showing two bands at 60 - 75 kDa and 25 - 34 kDa respectively, and two bands at 11 kDa, then the test leech is Whitmania pigra or Whitmania acranulata. Therefore, 12 - 23 and 25 are Whitmania pigra or Whitmania acranulata, as Figure 3 shown in Figures B and C; if the test leech sample is similar to the gel electrophoresis pattern of the control medicinal material of Poecilobdella javanica, especially showing four bands at 60 - 75 kDa, 25 - 34 kDa, 17 - 25 kDa, and 11 kDa, then the test leech is Poecilobdella javanica. Therefore, 24 is Poecilobdella javanica, as Figure 3 shown in Figure C; if the test leech sample is similar to the gel electrophoresis pattern of the control medicinal material of Japanese leeches, especially showing two bands at 25 - 34 kDa and 11 kDa, the test leech is a Japanese leech. Therefore, 26 and 27 are Japanese leeches, as Figure 3 shown in Figure C; if the gel electrophoresis pattern of the test leech sample is not similar to the gel electrophoresis patterns of the above control medicinal materials, it is other adulterated products.

[0361] The above has introduced the embodiments of the present invention in detail. Specific examples are used in this text to elaborate on the principle and implementation manner 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, those skilled in the art, based on the idea of the present invention, the changes or deformations made in the specific implementation manner and application scope of the present invention all belong to the protection scope of the present invention. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. A gel electrophoresis identification method for leeches and their closely related counterfeits, characterized in that: The identification method comprises the following steps: (1) Pre-treating a leech sample to be tested to obtain a leech sample test solution, and pre-treating a control medicinal material to obtain a control medicinal material solution; wherein the control medicinal material is a leech, a leech, and / or a closely related mixed product thereof; wherein the leech and the leech are a leech (Hirudo nipponica Whitman) of the Hirudinidae family and a leech (Whitmania pigra Whitman) of the Hirudinidae family, and the closely related mixed product is a leech (Poecilobdella manillensis Lesson), a leech (Poecilobdella javanica Wahlberg) of the Hirudinidae family, and / or a leech (Mimobdella japonica Blanchard) of the Hirudinidae family; (2) subjecting the leech sample solution to be tested and the control medicinal material solution to sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel imaging analysis to obtain a gel electrophoresis map of the leech sample to be tested and a gel electrophoresis map of the control medicinal material; and (3) comparing the gel electrophoresis pattern of the leech sample to be tested with the gel electrophoresis pattern of the reference medicinal material, and determining whether the leech to be tested is a leech, a leech, or a closely related mixed product thereof according to the comparison result; Wherein, the leeches to be tested are selected from one or more of the following: leeches, leeches, Philippine leeches, bar-grazing leeches and Japanese leeches.

2. The identification method according to claim 1, characterized in that: The comparison is the comparison of the number and position of protein bands; Preferably, the preparation method of the control medicinal material solution comprises: taking an appropriate amount of control medicinal material, crushing, sieving, obtaining the control medicinal material powder, adding a solvent to the control medicinal material powder for extraction, shaking, centrifuging, taking a supernatant, and diluting the supernatant to obtain the control medicinal material solution; Preferably, the sieve is a No. 3 sieve; Preferably, the solvent is sodium chloride solution; More preferably, the concentration of the sodium chloride solution is 0.5% to 2.0%, for example about 0.9%; More preferably, the extraction is maceration extraction; Preferably, the immersion extraction time is 20 to 40 minutes, for example, about 30 minutes; Preferably, the centrifugal speed is 10000-15000 r / min, for example, about 12000 r / min; Preferably, the centrifugation time is 5 to 20 min, for example, about 10 min; More preferably, the dilution factor is 5-120, such as about 10 or about 100.

3. The identification method according to claim 1, characterized in that: The preparation method of the leech sample test solution comprises: taking an appropriate amount of the leech sample to be tested, crushing, sieving, obtaining the leech sample powder to be tested, adding a solvent to the leech sample powder to extract, shaking and evenly mixing, centrifuging, taking a supernatant, and diluting the supernatant to obtain the leech sample test solution to be tested; Preferably, the sieve is a No. 3 sieve; Preferably, the solvent is sodium chloride solution; More preferably, the concentration of the sodium chloride solution is 0.5% to 2.0%, for example about 0.9%; More preferably, the extraction is maceration extraction; Preferably, the immersion extraction time is 20 to 40 minutes, for example, about 30 minutes; Preferably, the centrifugal speed is 10000-15000 r / min, for example, about 12000 r / min; Preferably, the centrifugation time is 5 to 20 min, for example, about 10 min; More preferably, the dilution factor is 5 to 120, such as about 10 or about 100; Particularly more preferably, when the leech to be tested is Hirudo nipponica Whitman or Hirudo nipponica Whitman, the dilution multiple is about 100 times; Particularly more preferably, when the leech to be tested is Hirudo clavata or Hirudo japonica, the dilution multiple is about 10 times.

4. The identification method according to claim 1, characterized in that: The gel electrophoresis method comprises the following steps: (a) adding protein loading buffer to the test solution, mixing, boiling to denature the protein, and centrifuging to obtain a test solution; (b) installing a gel pouring device, preparing a separation gel solution and a concentration gel solution, pouring the gel, and obtaining a gel plate; (c) adding electrode buffer to the outer tank of the electrophoresis tank, transferring the gel plate to the electrophoresis tank, filling the inner tank of the electrophoresis tank with electrode buffer, unplugging the electrophoresis comb, and loading the electrophoresis marker and the test solution respectively; (d) using a first constant voltage vertical electrophoresis, and when the indicator front reaches the separation gel, switching to a second constant voltage vertical electrophoresis, and when the indicator front reaches the bottom of the gel plate, stopping the electrophoresis and taking out the gel; (e) adding a dye solution to the gel for dyeing to obtain a dyed gel; as well as (f) adding a destaining solution to the stained gel to destain until the bands are clear, thereby obtaining an electrophoresis gel; Preferably, the gel imaging analysis method comprises: scanning the electrophoresis gel using a gel imaging analyzer, analyzing the scanning result using Gel-ProAnalyzer software, and obtaining the gel electrophoresis map of the leech sample to be tested and the gel electrophoresis map of the leech.

5. The identification method according to claim 4, characterized in that: In step (a), the volume ratio of the test solution to the protein loading buffer is 3 to 5, for example, about 4; Preferably, in step (a), the boiling temperature is 100°C; Preferably, in step (a), the boiling time is 1 to 10 minutes, for example, about 5 minutes; Preferably, in step (a), the centrifugal speed is 10000-15000 r / min, for example, about 12000 r / min; Preferably, in step (a), the centrifugation time is 1 to 10 min, for example, about 5 min; Preferably, in step (b), the separation gel solution comprises water, 30% acrylamide-methylenebisacrylamide solution, 1.5M Tris, 10% SDS, 10% APS and TEMED; More preferably, in the separation 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 about 800: about 1000: about 650: about 25: about 25: about 1; Also preferably, in step (b), the stacking gel solution comprises water, 30% acrylamide-methylenebisacrylamide solution, 1.0 M Tris, 10% SDS, 10% APS and TEMED; More preferably, in the stacking 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 about 700: about 165: about 125: about 10: about 10: about 1; Particularly preferably, in step (b), the gel casting method comprises: adding the separation gel solution to one side of the gel casting device to a distance of 1.5 to 2 cm from the upper end of the gel casting device, adding anhydrous ethanol to cap the top, leaving it at room temperature for about 30 minutes, removing the anhydrous ethanol, continuing to add the concentrated gel solution to one side of the gel casting device to cap the top, inserting an electrophoresis comb, leaving it at room temperature for about 30 minutes, and obtaining the gel plate; Particularly preferably, in step (c), the electrode buffer is prepared by weighing appropriate amounts of glycine, Tris, and SDS, adding water to a fixed volume, and diluting the solution by about 10 times to obtain the electrode buffer; Most preferably, the mass of glycine is 100-200 g, for example about 144 g; Most preferably, the mass of Tris is 20-40 g, for example about 30 g; Most preferably, the mass of the SDS is 5 to 15 g, for example about 10 g; Most preferably, the volume of the fixed volume is 800-1200 mL, for example about 1000 mL; Most preferably, in step (c), the loading volume of the electrophoresis Marker is 1 to 10 μL, for example, about 5 μL; Most preferably, in step (c), the sample volume of the test solution is 5 to 15 μL, such as about 10 μL.

6. The identification method according to claim 4, characterized in that: In step (d), the first constant voltage is 70 to 90 V, for example, about 80 V; Preferably, in step (d), the second constant voltage is 110-130V, for example, about 120V; More preferably, in step (e), the dye solution is Coomassie Brilliant Blue dye solution; More preferably, in step (e), the dyeing is performed on a shaker; More preferably, in step (e), the dyeing time is 30 to 60 minutes, such as about 40 minutes.

7. The identification method according to claim 4, characterized in that: In step (f), the decolorizing solution is a mixed solution consisting of methanol, glacial acetic acid and water; More preferably, in the mixed solution, the volume ratio of the methanol, the glacial acetic acid and the water is (2-4): (0.5-2): (4-8); Particularly preferably, in the mixed solution, the volume ratio of the methanol, the glacial acetic acid and the water is about 3: about 1: about 6.

8. The identification method according to claim 5, characterized in that: The preparation method of the 30% acrylamide-methylenebisacrylamide solution comprises: weighing appropriate amounts of acrylamide and methylene acrylamide, and diluting with water to about 50 mL to obtain; Preferably, the mass of the acrylamide is 10 to 20 g, for example, about 14.55 g; Preferably, the mass of the methylene acrylamide is 0.1 to 1.0 g, for example, about 0.45 g; More preferably, the preparation method of 1.5M Tris comprises: 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 the volume to about 250 mL, to obtain; Preferably, the mass of Tris is 30-60 g, for example, about 45.4 g; Preferably, the volume of the first water is 200-300 mL, for example, about 220 mL; Preferably, the pH is adjusted to about 8.8; Preferably, the preparation method of 1.0M Tris comprises: 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 the volume to about 250 mL, to obtain; More preferably, the mass of Tris is 20-40 g, for example, about 30.3 g; More preferably, the volume of the first water is 200-300 ml, for example, about 220 ml; More preferably, the pH is adjusted to about 6.8; More preferably, the preparation method of the 10% APS comprises: weighing an appropriate amount of APS, adding water to dilute, and obtaining; Particularly preferably, the mass of the APS is 0.5 to 2 g, for example, about 1 g; Particularly preferably, the volume of the water is 5 to 20 mL, for example about 10 mL; Particularly preferably, the preparation method of the 10% SDS comprises: weighing an appropriate amount of SDS, adding water to dilute, and obtaining; Most preferably, the mass of the SDS is 1 to 10 g, for example about 5 g; Most preferably, the volume of water is 40-60 mL, such as about 50 mL.

9. The identification method according to claim 1, characterized in that: 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 medicinal material, for example, the gel electrophoresis pattern of the leech sample to be tested presents three bands at 25-34 kDa, 17-25 kDa and 11 kDa, 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 medicinal material or the gel electrophoresis pattern of the Philippine Hirudo control medicinal material, for example, the gel electrophoresis pattern of the leech sample to be tested presents two bands at 60-75 kDa and 25-34 kDa, and two bands at 11 kDa, then the leech to be tested is a leech or a Philippine Hirudo; (3) If the gel electrophoresis spectrum of the leech sample to be tested is consistent with or similar to the gel electrophoresis spectrum of the control medicinal material Hirudo clavipes, for example, the gel electrophoresis spectrum of the leech sample to be tested presents four bands at 60-75 kDa, 25-34 kDa, 17-25 kDa and 11 kDa, then the leech to be tested is Hirudo clavipes; (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 medicinal material, for example, the gel electrophoresis pattern of the leech sample to be tested shows two bands at 25-34 kDa and 11 kDa, then the leech to be tested is a Japanese leech; (5) If the gel electrophoresis spectrum of the leech sample to be tested is inconsistent with or dissimilar to the gel electrophoresis spectrum of the reference medicinal material, the leech to be tested is a counterfeit product.

10. Use of the identification method according to any one of claims 1 to 9 in distinguishing leeches, leeches and / or their closely related counterfeits from each other, wherein the closely related counterfeits are selected from one or more of the following: Philippine leech, bar-patterned leech, Japanese leech and other counterfeits.

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