SSR molecular markers of medicinal Codonopsis pilosula and its application, identification method of medicinal Codonopsis pilosula

Abstract

The present invention discloses an SSR molecular marker of medicinal Codonopsis pilosula, wherein the SSR molecular marker of the medicinal Codonopsis pilosula is amplified by an upstream primer having a sequence as shown in SEQ ID NO: 1 and a downstream primer having a sequence as shown in SEQ ID NO: 2. The present invention also discloses the application of the SSR molecular marker and a method for identifying medicinal Codonopsis pilosula. The present invention can effectively distinguish medicinal Codonopsis pilosula medicinal materials from Leopardus leucophylla medicinal materials.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine identification, and in particular to an SSR molecular marker of medicinal Codonopsis pilosula and an application thereof, and an identification method of medicinal Codonopsis pilosula. Background Art

[0002] Codonopsis pilosula is the dried root of Codonopsis pilosula (Franch.) Nannf., Codonopsis pilosula Nannf. var. modesta (Nannf.) LTShen or Codonopsis tangshen Oliv. of the Campanulaceae family. It is mainly distributed in Lingchuan, Jincheng, Shanxi, Weiyuan, Dingxi, Gansu, Longnan, Gansu, Gangu, Tianshui, Gansu, Weining, Bijie, Guizhou and other places. The three are closely related in origin and have similar medicinal values. They all have the effects of strengthening the spleen and lungs, nourishing blood and promoting body fluid, and are widely used in clinical practice.

[0003] In recent years, the demand for Codonopsis pilosula has increased dramatically, but the amount of medicinal resources is limited, resulting in the roots of other plants in the same family also entering the market as Codonopsis pilosula in some areas. Codonopsis pilosula is easily confused with Codonopsis pilosula, and its efficacy is very different from that of Codonopsis pilosula, resulting in uneven quality of Codonopsis pilosula on the market, affecting the safety of medication. Codonopsis pilosula is easily confused with the fake varieties of Codonopsis pilosula, which is the dried root of Campanumoea javanica Bl., a plant of the Campanulaceae family. It is commonly known as "Tu Codonopsis pilosula", "Wild Codonopsis pilosula fruit", "Tu Renshen", etc. It is used as "Tu Codonopsis pilosula" in Guizhou and other regions, but the efficacy of the two is quite different. The relationship between Codonopsis pilosula and Panthera genus is relatively close, and the plant morphology of most species, especially the morphology of the roots, is relatively similar, which leads to the confusion of Codonopsis pilosula medicinal materials. In addition, Codonopsis pilosula medicinal materials are often processed into decoction pieces or powdered for use as medicine. Because its properties have changed significantly, it is difficult to accurately identify it through traditional identification methods such as properties or microscopic characteristics.

[0004] At present, molecular identification technologies for Codonopsis pilosula include DNA barcodes, SSR markers, ISSR markers, etc., which are mostly used to study the genetic diversity of species among Codonopsis pilosula species. However, due to the fact that there is a certain degree of mixing and impurity in the cultivation of Codonopsis pilosula, it is difficult to ensure the purity of its varieties by appearance morphology and physical and chemical indicators. At present, there are few literatures related to SSR markers for Codonopsis pilosula. The relevant patents include cpSSR markers for Codonopsis pilosula species. Through 5 pairs of cpSSR markers, Codonopsis pilosula can be distinguished from wild and cultivated Codonopsis pilosula by agarose electrophoresis; in the patents of Codonopsis lanceolata and Adenophora triphylla, SSR markers are mostly used to distinguish Codonopsis lanceolata, Codonopsis pilosula, Adenophora triphylla and Codonopsis ussuriensis, as well as the different years of Codonopsis pilosula.

[0005] However, none of the above public materials involve the identification of different origins of medicinal Codonopsis and other counterfeit medicinal materials, and do not provide SSR molecular marker methods that can be used to identify medicinal Codonopsis and its mixed counterfeit Panthera parvata. Therefore, the present invention can distinguish medicinal Codonopsis medicinal materials through SSR markers, which is of great significance for the identification of medicinal Codonopsis medicinal materials and germplasm purity, and the safety of medicinal Codonopsis medicinal materials. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide an SSR molecular marker of medicinal Codonopsis pilosula, which can effectively identify the authenticity of medicinal Codonopsis pilosula.

[0007] The technical problem that the present invention needs to solve is to provide the application of the SSR molecular marker of the medicinal Codonopsis pilosula.

[0008] The technical problem that the present invention also aims to solve is to provide a method for identifying medicinal Codonopsis pilosula, which has good accuracy and high specificity.

[0009] In order to solve the above technical problems, the present invention provides an SSR molecular marker of medicinal Codonopsis pilosula, which is amplified by an upstream primer with a sequence as shown in SEQ ID NO: 1 and a downstream primer with a sequence as shown in SEQ ID NO: 2.

[0010] The 5' end of the upstream primer is mounted with a fluorescent marker group, and the fluorescent marker group is selected from one of fluorescein FAM, fluorescein ROX, fluorescein HEX and fluorescein TAMARA.

[0011] Table 1 shows the specific sequences of the upstream primer and the downstream primer.

[0012]

[0013] In order to solve the above technical problems, the present invention provides the use of the SSR molecular marker of the medicinal Codonopsis pilosula in (1) or (2):

[0014] (1) Identify the authenticity of medicinal Codonopsis pilosula;

[0015] (2) Differentiate between medicinal Codonopsis pilosula and medicinal Panthera chinensis.

[0016] In one embodiment, the steps include:

[0017] Extracting genomic DNA from the sample to be tested;

[0018] Using the genomic DNA of the sample to be tested as a template, PCR amplification is performed using the upstream primers and downstream primers of the SSR molecular marker of medicinal Codonopsis pilosula as described in Table 1 to obtain a PCR amplification product;

[0019] Conducting capillary electrophoresis detection on the PCR amplification product;

[0020] In one embodiment, if the characteristic double peaks of 163bp-164bp and 166bp-167bp fragments or the characteristic double peaks of 166bp-167bp and 169bp-170bp fragments are detected, the sample to be tested is a medicinal Codonopsis pilosula;

[0021] If the detection shows a 133bp-134bp fragment peak or a 150bp-151bp fragment peak, the sample to be tested is the golden leopard medicinal material.

[0022] In order to solve the above technical problems, the present invention provides a method for identifying medicinal Codonopsis pilosula, comprising the following steps:

[0023] Extracting genomic DNA from the sample to be tested;

[0024] Using the genomic DNA of the sample to be tested as a template, PCR amplification is performed using the upstream primers and downstream primers of the SSR molecular marker of medicinal Codonopsis pilosula as described in Table 1 to obtain a PCR amplification product;

[0025] The PCR amplification product is subjected to capillary electrophoresis detection, and whether the sample to be tested is a medicinal Codonopsis pilosula is determined according to the detection result.

[0026] In one embodiment, the procedure of PCR amplification is: pre-denaturing the amplification system at a temperature of 94° C.-96° C. for 2 min-4 min;

[0027] Denature at 94°C-96°C for 5s-15s, then anneal at 50°C-60°C for 5s-15s, and then extend at 70°C-75°C for 5s-15s, and repeat the above operation 33-36 times;

[0028] After extension at 70°C-75°C for 4min-6min, storage was completed at 3°C-5°C.

[0029] In one embodiment, the PCR amplification reaction system includes: 2×M5 Supper FastTaq PCR 9μL-11μL, the upstream primer 0.2μL-0.8μL, the downstream primer 0.2μL-0.8μL, the DNA template 0.5μL-1.5μL, and sterile double distilled water to 20μL.

[0030] In one embodiment, the concentration of the DNA template is 25 ng / μl-35 ng / μl;

[0031] The concentration of the upstream primer is 8 μmol / L-12 μmol / L;

[0032] The concentration of the downstream primer is 8 μmol / L-12 μmol / L.

[0033] In one embodiment, the electrophoretic detection comprises:

[0034] The amplified sequences were detected by capillary electrophoresis.

[0035] In one embodiment, if the characteristic double peaks of 163bp-164bp and 166bp-167bp fragments or the characteristic double peaks of 166bp-167bp and 169bp-170bp fragments are detected, the sample to be tested is a medicinal Codonopsis pilosula; otherwise, the sample to be tested is a counterfeit product.

[0036] In one embodiment, if a 133bp-134bp fragment peak or a 150bp-151bp fragment peak is detected, the sample to be tested is Panthera pardus medicinal material.

[0037] The implementation of the present invention has the following beneficial effects:

[0038] The SSR molecular marker of the medicinal Codonopsis pilosula provided by the present invention can distinguish the medicinal Codonopsis pilosula medicinal materials and the Golden Leopard medicinal materials from Codonopsis pilosula, Codonopsis pilosula, Codonopsis pilosula and Golden Leopard medicinal materials.

[0039] The identification method of medicinal Codonopsis pilosula provided by the present invention can be used to distinguish medicinal Codonopsis pilosula. It includes extracting genomic DNA, using the above genomic DNA as a template, using designed primers to perform PCR amplification, and detecting the amplified sequence by capillary electrophoresis. The allele size is determined by referring to the GS-LIZ500 molecular weight internal standard and using GeneMarker. If the characteristic double peaks of 163bp-164bp and 166bp-167bp fragments or the characteristic double peaks of 166bp-167bp and 169bp-170bp fragments are detected, the sample to be tested is a medicinal Codonopsis pilosula medicinal material; if the 133bp-134bp fragment peak or the 150bp-151bp fragment peak is detected, the sample to be tested is a golden leopard medicinal material. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The primer screening results in Example 1 of the present invention are shown in FIG. 1 ; wherein M: 20 DNA Ladder; 1-4: Codonopsis pilosula; 5-8: Codonopsis pilosula; 9-12: Codonopsis pilosula;

[0041] Figure 2 This is the sample detection result in Example 1 of the present invention. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is described in further detail below.

[0043] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0044] In the present invention, “preferred” is only used to describe an implementation method or example with better effects, and it should be understood that it does not constitute a limitation on the protection scope of the present invention.

[0045] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0046] In the present invention, reference to a numerical range includes both endpoints of the numerical range unless otherwise specified.

[0047] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0048] In order to solve the above technical problems, the present invention provides an SSR molecular marker of medicinal Codonopsis pilosula, which is amplified by an upstream primer with a sequence as shown in SEQ ID NO: 1 and a downstream primer with a sequence as shown in SEQ ID NO: 2.

[0049] The 5' end of the upstream primer is mounted with a fluorescent marker group, and the fluorescent marker group is selected from one of fluorescein FAM, fluorescein ROX, fluorescein HEX and fluorescein TAMARA.

[0050] Table 1 shows the specific sequences of the upstream primer and the downstream primer.

[0051]

[0052] Accordingly, the present invention provides the use of the SSR molecular marker of the medicinal Codonopsis pilosula in (1) or (2):

[0053] (1) Identify the authenticity of medicinal Codonopsis pilosula;

[0054] (2) Differentiate between medicinal Codonopsis pilosula and medicinal Panthera chinensis.

[0055] In one embodiment, the application of the SSR molecular marker of medicinal Codonopsis pilosula comprises the following steps:

[0056] Extracting genomic DNA from the sample to be tested;

[0057] Using the genomic DNA of the sample to be tested as a template, PCR amplification is performed using the upstream primers and downstream primers of the SSR molecular marker of medicinal Codonopsis pilosula as described in Table 1 to obtain a PCR amplification product;

[0058] Conducting capillary electrophoresis detection on the PCR amplification product;

[0059] If the characteristic double peaks of 163bp-164bp and 166bp-167bp fragments or the characteristic double peaks of 166bp-167bp and 169bp-170bp fragments appear in the test, the sample to be tested is a medicinal Codonopsis pilosula;

[0060] If the detection shows a 133bp-134bp fragment peak or a 150bp-151bp fragment peak, the sample to be tested is the golden leopard medicinal material.

[0061] Accordingly, the present invention provides a method for identifying medicinal Codonopsis pilosula, comprising the following steps:

[0062] S1, extracting genomic DNA of the sample to be tested;

[0063] In one embodiment, genomic DNA was extracted using Wizard Genomic DNA Purification System Extraction Kit (A2361) (Promega, USA), DNA concentration was measured using Drop-nano micro-volume UV spectrophotometer, A260 / A280 and A260 / A230 were recorded simultaneously, and the concentration was adjusted to 30 ng / μl. The adjusted samples were stored in a 4°C refrigerator.

[0064] S2, using the genomic DNA of the sample to be tested as a template, performing PCR amplification using the upstream primer and downstream primer of the SSR molecular marker of medicinal Codonopsis pilosula as described in Table 1 to obtain a PCR amplification product;

[0065] In one embodiment, the procedure of the PCR amplification is:

[0066] A. Denature the amplification system at 94°C-96°C for 2-4 minutes;

[0067] B. Denature at 94°C-96°C for 5s-15s, then anneal at 50°C-60°C for 5s-15s, and then extend at 70°C-75°C for 5s-15s, and repeat the above operation 33-36 times;

[0068] C. After extension at 70℃-75℃ for 4min-6min, store at 3℃-5℃.

[0069] In one embodiment, the PCR amplification reaction system includes: 2×M5 Supper FastTaq PCR 9μL-11μL, the upstream primer 0.2μL-0.8μL, the downstream primer 0.2μL-0.8μL, the DNA template 0.5μL-1.5μL, and sterile double distilled water to 20μL.

[0070] Preferably, the procedure of the PCR amplification is:

[0071] A. Denature the amplification system at 95°C for 3 min.

[0072] B. Denature at 95°C for 10 seconds, then anneal at 55°C for 10 seconds, and then extend at 72°C for 10 seconds, and repeat the above steps 35 times;

[0073] C. After extension at 72°C for 5 min, the cells were stored at 4°C.

[0074] The PCR amplification reaction system includes: 2×M5 Supper FastTaq PCR 10 μL, the upstream primer 0.5 μL, the downstream primer 0.5 μL, the DNA template 1 μL, and sterilized double distilled water to 20 μL.

[0075] S3, performing capillary electrophoresis detection on the PCR amplification product, and judging whether the sample to be tested is a medicinal Codonopsis pilosula according to the detection result.

[0076] In one embodiment, the electrophoretic detection comprises:

[0077] The amplified sequences were detected by capillary electrophoresis.

[0078] If the characteristic double peaks of 163bp-164bp and 166bp-167bp fragments or the characteristic double peaks of 166bp-167bp and 169bp-170bp fragments appear in the test, the sample to be tested is a medicinal Codonopsis pilosula;

[0079] If the detection shows a 133bp-134bp fragment peak or a 150bp-151bp fragment peak, the sample to be tested is the golden leopard medicinal material.

[0080] The present invention is described below with reference to specific embodiments.

[0081] Example 1

[0082] The identification method of medicinal Codonopsis pilosula specifically comprises the following steps:

[0083] S1, extracting genomic DNA of the sample to be tested;

[0084] Prepare samples according to Table 2, and extract genomic DNA from samples according to the following method.

[0085] Table 2 is a list of samples

[0086]

[0087]

[0088] Then, the genomic DNA of the above samples was extracted using Wizard Genomic DNA Purification System Extraction Kit (A2361) (Promega, USA), and the DNA concentration was determined using Drop-nano micro-volume UV spectrophotometer, while A260 / A280 and A260 / A230 were recorded, and the concentration was adjusted to 30 ng / μl. The adjusted samples were stored in a 4°C refrigerator.

[0089] S2, using the genomic DNA of the sample to be tested as a template, performing PCR amplification using the upstream primer and downstream primer of the SSR molecular marker of medicinal Codonopsis pilosula as described in Table 1 to obtain a PCR amplification product;

[0090] 2.1 Primer design

[0091] SSR loci were mined and primers were designed using Primer 5.0. The obtained sequences are shown in Table 3. The synthesis of primers was completed by Shanghai Sangon Biotechnology Co., Ltd.

[0092] Table 3 is the primer information table

[0093]

[0094] 2.2 PCR amplification

[0095] The PCR reaction system was as follows: 2×M5 Supper FastTaq PCR 10 μl, upstream and downstream primers (10 μM) 0.5 μl each, template DNA (30 ng / μl) 1 μl, and sterile water was added to 20 μl system.

[0096] PCR reaction program: pre-denaturation at 95°C for 3 min; 35 cycles: denaturation at 95°C for 10 s, annealing at 55°C for 10 s, extension at 72°C for 10 s; then extension at 72°C for 5 min, and storage at 4°C.

[0097] S3. Detect differential bands of the PCR amplification products by polyacrylamide gel electrophoresis, label the target sequence with a detectable fluorescent substance, detect the amplified sequence by capillary electrophoresis, and determine whether the sample to be tested is a medicinal Codonopsis pilosula according to the detection result.

[0098] Under the conditions of voltage 130V and current 20mA, 10% polyacrylamide gel electrophoresis was performed in 1×TBE buffer, with 20DNAladder and DL1000 Marker (Beijing Bio-Tech Co., Ltd., TAKARA) as references. The PCR products were detected by silver staining, and the target sequence was labeled with a detectable fluorescent substance, and the amplified sequence was detected by capillary electrophoresis. The results are shown in Figure 1 and Figure 2As shown in Table 4, from the above results, it can be seen that the primer combination, amplification system and detection method of the SSR molecular marker provided by the present invention can effectively distinguish medicinal Codonopsis pilosula and Leopardus leopardus medicinal materials from Codonopsis pilosula, Codonopsis pilosula, Codonopsis pilosula and Leopardus leopardus medicinal materials. If the characteristic double peaks of 163bp-164bp and 166bp-167bp fragments or the characteristic double peaks of 166bp-167bp and 169bp-170bp fragments appear in the detection, the sample to be tested is medicinal Codonopsis pilosula medicinal material; if the 133bp-134bp fragment peak or the 150bp-151bp fragment peak appears in the detection, the sample to be tested is Leopardus leopardus medicinal material.

[0099] Table 4 shows the capillary electrophoresis results of different genes

[0100]

[0101]

[0102] Finally, the fluorescent PCR system was established and tested. According to the primer screening results, the primers in Table 3 were synthesized with 5'-end FAM fluorescent label. The primers were synthesized by Shanghai Shenggong Biotechnology Co., Ltd. and purified by HPLC.

[0103] Table 5 is the fluorescent primer information table

[0104]

[0105] The PCR products were sent to Ruibo Biotech Co., Ltd. for testing, and the allele sizes were determined by GeneMarker with reference to the GS-LIZ500 molecular weight internal standard.

[0106] In summary, the identification method of medicinal Codonopsis pilosula provided by the present invention can be used to distinguish medicinal Codonopsis pilosula. It includes extracting genomic DNA, using the above genomic DNA as a template, using the designed primers for PCR amplification, detecting differential bands by polypropylene gel electrophoresis, and the amplified target sequence is labeled with a detectable fluorescent substance, and the amplified sequence is detected by capillary electrophoresis. If the characteristic double peaks of 163bp-164bp and 166bp-167bp fragments or the characteristic double peaks of 166bp-167bp and 169bp-170bp fragments are detected, the sample to be tested is a medicinal Codonopsis pilosula medicinal material; wherein the medicinal Codonopsis pilosula medicinal material includes Codonopsis pilosula (Franch.) Nannf., Codonopsis pilosula Nannf.var.modesta (Nannf.) LTShen and Codonopsis tangshen Oliv. of Campanulaceae. If the detection shows a 133bp-134bp fragment peak or a 150bp-151bp fragment peak, the sample to be tested is the golden leopard medicinal material.

[0107] The above is a preferred embodiment of the invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the invention. These improvements and modifications are also considered to be within the scope of protection of the invention.

Claims

1. A primer set, characterized in that: The primer set includes an upstream primer with a sequence as shown in SEQ ID NO: 1 and a downstream primer with a sequence as shown in SEQ ID NO: 2, wherein the 5' end of the upstream primer is mounted with a fluorescent marker group, and the fluorescent marker group is selected from one of fluorescein FAM, fluorescein ROX, fluorescein HEX and fluorescein TAMARA.

2. Use of the primer set according to claim 1 in (1) or (2): (1) Identification of Codonopsis pilosula Codonopsis pilosula (Franch.) Nannf.) Medicinal materials, Codonopsis pilosula ( Codonopsis pilosula Nannf. var. modesta (Nannf.) LT Shen) medicinal materials and Sichuan Codonopsis pilosula ( Codonopsis tangshen Oliv.) Authenticity of medicinal materials; (2) Differentiate Codonopsis pilosula ( Codonopsis pilosula (Franch.) Nannf.) Medicinal materials, Codonopsis pilosula ( Codonopsis pilosula Nannf. var. modesta (Nannf.) LT Shen) medicinal materials and Sichuan Codonopsis pilosula ( Codonopsis tangshen Oliv.) medicinal materials and leopard medicinal materials.

3. The use of the primer set according to claim 2, characterized in that: The following steps are involved: Extracting genomic DNA from the sample to be tested; Using the genomic DNA of the sample to be tested as a template, PCR amplification is performed using an upstream primer as shown in SEQ ID NO: 1 and a downstream primer as shown in SEQ ID NO: 2 to obtain a PCR amplification product; Conducting capillary electrophoresis detection on the PCR amplification product; If the characteristic double peaks of 163bp-164bp and 166bp-167bp fragments or the characteristic double peaks of 166bp-167bp and 169bp-170bp fragments appear in the test, the sample to be tested is Codonopsis pilosula ( Codonopsis pilosula (Franch.) Nannf.) Medicinal materials, Codonopsis pilosula ( Codonopsis pilosula Nannf. var. modesta (Nannf.) LT Shen) medicinal materials and Sichuan Codonopsis pilosula ( Codonopsis tangshen Oliv.) One of the medicinal herbs; If the detection shows a 133bp-134bp fragment peak or a 150bp-151bp fragment peak, the sample to be tested is the golden leopard medicinal material.

4. A method for identifying Codonopsis pilosula, Codonopsis pilosula and Codonopsis pilosula, characterized in that: The following steps are involved: Extracting genomic DNA from the sample to be tested; Using the genomic DNA of the sample to be tested as a template, PCR amplification is performed using an upstream primer as shown in SEQ ID NO: 1 and a downstream primer as shown in SEQ ID NO: 2 to obtain a PCR amplification product; The PCR amplification product is subjected to capillary electrophoresis detection, and whether the sample to be tested is Codonopsis pilosula ( Codonopsis pilosula (Franch.) Nannf.) Medicinal materials, Codonopsis pilosula ( Codonopsis pilosula Nannf. var. modesta (Nannf.) LT Shen) medicinal materials and Sichuan Codonopsis pilosula ( Codonopsis tangshen Oliv.) medicinal materials.

5. The method for identifying the medicinal material of Codonopsis pilosula, Codonopsis pilosula and Codonopsis pilosula as claimed in claim 4, characterized in that: The procedure of the PCR amplification is: Pre-denature the amplification system at 94°C-96°C for 2min-4min; Denaturing at 94°C-96°C for 5s-15s, then annealing at 50°C-60°C for 5s-15s, and then extending at 70°C-75°C for 5s-15s, repeating the denaturing, annealing and extending treatments 33-36 times each; After extension at 70°C-75°C for 4min-6min, storage was completed at 3°C-5°C.

6. The method for identifying the medicinal material of Codonopsis pilosula, Codonopsis pilosula and Codonopsis pilosula as claimed in claim 4, characterized in that: The PCR amplification reaction system includes: 2×M5 Supper FastTaq PCR 9μL-11μL, the upstream primer 0.2μL-0.8μL, the downstream primer 0.2μL-0.8μL, the DNA template 0.5μL-1.5μL, and sterile double distilled water to 20μL.

7. The method for identifying the medicinal material of Codonopsis pilosula, Codonopsis pilosula and Codonopsis pilosula as claimed in claim 6, characterized in that: The concentration of the DNA template is 25ng / μl-35ng / μl; The concentration of the upstream primer is 8 μmol / L-12 μmol / L; The concentration of the downstream primer is 8 μmol / L-12 μmol / L.

8. The method for identifying the medicinal material of Codonopsis pilosula, Codonopsis pilosula and Codonopsis pilosula as claimed in claim 4, characterized in that: The electrophoresis detection includes: detecting the amplified sequence by capillary electrophoresis.

9. The method for identifying the medicinal material of Codonopsis pilosula, Codonopsis pilosula and Codonopsis pilosula as claimed in claim 4, characterized in that: If the characteristic double peaks of 163bp-164bp and 166bp-167bp fragments or the characteristic double peaks of 166bp-167bp and 169bp-170bp fragments appear in the test, the sample to be tested is Codonopsis pilosula ( Codonopsis pilosula (Franch.) Nannf.) Medicinal materials, Codonopsis pilosula ( Codonopsis pilosula Nannf. var. modesta (Nannf.) LT Shen) medicinal materials and Sichuan Codonopsis pilosula ( Codonopsis tangshen Oliv.) Medicinal herbs; Otherwise, the sample to be tested is a counterfeit product.

10. The method for identifying the medicinal material of Codonopsis pilosula, Codonopsis pilosula and Codonopsis pilosula as claimed in claim 4, characterized in that: If the detection shows a 133bp-134bp fragment peak or a 150bp-151bp fragment peak, the sample to be tested is the golden leopard medicinal material.

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