Molecular identification marker and method for sambucus chinensis and sambucus adnata

Abstract

The invention relates to the technical field of molecular biology, and discloses a molecular identification marker and method for sambucus chinensis and sambucus adnata, and the molecular identification marker comprises difference ITS2 sequences of sambucus chinensis and sambucus adnata as shown in SEQ ID NO.1 and SEQ ID NO.2. The difference ITS sequence can accurately distinguish the sambucus chinensis and the sambucus adnata, is not influenced by the form of a medicine, whether the medicine is processed or not and the state, can detect the molecular level to distinguish the sambucus chinensis and the sambucus adnata, makes up the inaccuracy of naked eye distinguishing, and reduces the medication safety risk.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a molecular identification marker and method for *Erigeron breviscapus* and *Hedyotis diffusa*. Background Technology

[0002] The statements in this section provide only background information relevant to the disclosure of this application and may not constitute prior art.

[0003] Bone-setting herb ( Sambucus javanica Lindl. belongs to the genus *Eleutherococcus* of the family Caprifoliaceae. Sambucus This plant, also known as Stinkweed, Eight-angled Hemp, Lu Ying, Seven-leaf Star, etc., has the effects of treating bruises and sprains, dispelling wind and dampness, dispersing blood stasis and reducing swelling, promoting blood circulation and activating collaterals, and detoxifying and reducing inflammation. It is distributed in Guangdong, Guangxi, Sichuan, Guizhou, Yunnan, Tibet and other regions, and is also distributed in Japan. It grows on mountain slopes, under forests, along ditches and in grassy areas at altitudes of 300-2600 meters.

[0004] Blood-filled Grass ( S. adnata Wall. ex DC. is a closely related plant of the same genus as *Hedyotis diffusa*. It is used in folk medicine for treating injuries from falls and blows, and has the effects of promoting blood circulation, removing blood stasis, diuresis, removing dampness, and promoting blood circulation and unblocking collaterals. It is distributed in Ningxia, Qinghai, Sichuan, Guizhou, Yunnan and Tibet. It grows in forests, ditches, thickets, wetlands on mountain slopes and alpine grasslands at altitudes of 1600-3600 meters.

[0005] Elderberry plants have attracted much attention in medicinal research. Because elderberry and bloodwort are extremely similar in morphological characteristics, they are easily confused in folk medicine. In addition, traditional methods of identifying medicinal materials have certain limitations, especially when key characteristics such as the absence of flowers and fruits are not easily distinguished, which can easily lead to misidentification and pose potential risks to the safety and effectiveness of clinical use.

[0006] With the development of DNA molecular identification technology, molecular identification has become an effective means of distinguishing different species. However, the phylogenetic relationship between *Hedyotis diffusa* and *Sambucus chinensis* is extremely close. Therefore, finding stable gene-difference fragments between *Hedyotis diffusa* and *Sambucus chinensis* is the key to distinguishing and identifying them. Summary of the Invention

[0007] The purpose of this invention is to address the problem that the identification of closely related species, such as *Sambucus chinensis* and *Hedyotis diffusa*, is difficult and prone to errors that affect the safety of medication. This invention provides a molecular identification marker and method for *Sambucus chinensis* and *Hedyotis diffusa*, and has screened out stable and differential ITS2 sequences for *Sambucus chinensis* and *Hedyotis diffusa*, which can accurately distinguish between the two and ensure the accuracy and safety of medication.

[0008] The technical solution of the present invention is as follows: The present invention provides a molecular identification marker for *Sambucus chinensis* and *Hedyotis diffusa*, including differential sequences of *Sambucus chinensis* and *Hedyotis diffusa* as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0009] According to a preferred embodiment, the sequences SEQ ID NO.1 and SEQ ID NO.2 differ as follows: The 46th bit of SEQ ID NO.1 is A, the 50th bit is G, the 178th bit is G, and the 209th bit is C; The 46th bit of SEQ ID NO.2 is G, the 50th bit is T, the 178th bit is C, and the 209th bit is T.

[0010] According to a preferred embodiment, SEQ ID NO.1 and SEQ ID NO.2 are the ITS2 sequences of *Hedyotis diffusa* and *Hedyotis diffusa*.

[0011] According to a preferred embodiment, the differentiation is made based on the amplification results by amplifying the ITS2 sequence of *Hedyotis diffusa* as shown in SEQ ID NO.1 or the ITS2 sequence of *Hedyotis diffusa* as shown in SEQ ID NO.2.

[0012] According to a preferred embodiment, the primer sequences for amplifying SEQ ID NO.1 and SEQ ID NO.2 are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0013] According to a preferred embodiment, DNA is extracted from processed and shredded Chinese medicinal herbs or powder and then amplified.

[0014] Another aspect of the present invention provides a kit for distinguishing between *Sambucus chinensis* and *Hedyotis diffusa*, comprising primers for amplifying SEQ ID NO.1 and SEQ ID NO.2.

[0015] According to a preferred embodiment, the kit is a CAPS / dCAPS kit.

[0016] Preferably, it also includes other auxiliary reagents required for PCR amplification, including ddH2O, Taq DNA polymerase, or restriction endonuclease.

[0017] Another aspect of the present invention provides a molecularly specific identification method for *Sambucus chinensis* and *Hedyotis diffusa*, comprising the following steps: The ITS2 sequence of the plant to be identified is amplified. The numbers and letters of the bases at positions 46, 50, 178 and 209 of the ITS2 sequence of the plant to be identified are combined to generate a molecular identity card for the plant. Then, this molecular identity card is compared with the molecular identity cards of *Eleutherococcus senticosus* and *Hemiberlesia stramonium*. The plant that matches the comparison is the corresponding variety.

[0018] According to a preferred embodiment, the molecular identification number of the herb *Eleutherococcus senticosus* is: JGC46A50G178G209C; and the molecular identification number of the herb *Hedyotis diffusa* is: XMC46G50T178C209T.

[0019] Compared with existing technologies, the advantages of this invention are: 1. A molecular identification marker and method for Sambucus chinensis and Smilax china. This application discovers stable interspecific differential loci between the closely related plants Sambucus chinensis and Smilax china, which can accurately distinguish Sambucus chinensis and Smilax china based on the differences between these multiple loci. This provides strong technical support for the identification of germplasm resources of Sambucus chinensis and Smilax china, the identification of the authenticity of medicinal materials, the quality monitoring of processed medicinal materials, and the rational use of drugs in clinical practice, ensuring the safety and effectiveness of drugs for the public; it can be applied to gene chips, microfluidic chips, detection kits, etc. 2. A molecular identification marker and method for *Clematis armandii* and *Hedyotis diffusa*, which can be directly identified at the molecular genetic level, without being limited by the environment, sample morphology, or processing method (such as powder or extract), and has extremely high accuracy. This method can extract DNA from trace amounts of plant tissue and dried medicinal materials, and detect it by polymerase chain reaction (PCR) combined with high-throughput sequencing technology to obtain identification conclusions. It can effectively identify drugs of various forms and states available on the market. Attached Figure Description

[0020] Figure 1 Morphological identification of *Sambucus chinensis* and *Hemiberlesia javanica*; A-1 and A-2 are front and back views of *Sambucus chinensis* leaves; B-1 and B-2 are front and back views of *Hemiberlesia javanica* leaves; A-3 and A-4 are inflorescence and partial views of *Sambucus chinensis* inflorescence; B-3 and B-4 are inflorescence and partial views of *Hemiberlesia javanica* inflorescence; A-5 and A-6 are immature and mature fruits of *Sambucus chinensis*; B-5 and B-6 are immature and mature fruits of *Hemiberlesia javanica*. Figure 2 Image showing the characteristics of broken stems of *Sambucus chinensis* and *Hemiberlesia lingua*. Figure 3 Legend for electrophoresis diagrams of DNA quality detection using ITS, psbA, and matK primers for samples of *Eleutherococcus senticosus* and *Hemiberlesia lingua*. Figure 4 A comparison diagram of ITS sequences between samples of *Sambucus chinensis* and *Hedyotis diffusa*. Figure 5 The matK sequence alignment diagram for samples of *Eleutherococcus senticosus* and *Hedyotis diffusa*. Figure 6 Legend for DNA quality testing of extracted samples; Figure 7 PCR amplification diagrams for the ITS primers of the screening group (left) and the ITS2 primers of the validation group (right); Figure 8 An example of the phylogenetic tree of the ITS system for the screening group; Figure 9 Legend for the phylogenetic tree of the ITS2 system in the screening group; Figure 10 This is a phylogenetic tree diagram of the ITS2 system for the validation group. Detailed Implementation

[0021] The specific embodiments listed in this invention are merely examples, and the invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of this invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of this invention should be covered within its scope. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments whose manufacturers are not specified are commercially available conventional products. To better illustrate this invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that this invention can be practiced even without certain specific details. In other embodiments, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of this invention.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors unavoidable in industrial production.

[0023] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0024] Example 1 1. Materials and Methods 1.1 Experimental Materials This study collected 60 samples of two plant species from Yunnan Province (collection information is shown in Table 1), all of which were identified by Professor Pu Chunxia as *Hypericum esculentum*, a plant in the Caprifoliaceae family. Sambucus javanica Blood-filled Grass S. adnata;The samples were identified and confirmed by the Plant Resources Survey, Assessment and Identification Center of the Kunming Institute of Botany, Chinese Academy of Sciences. Fresh, intact, and pest-free leaves were selected for plant sample collection. After being removed with tweezers, the leaves were placed in a resealable bag containing color-changing silica gel for rapid drying and then stored at low temperature as the screening group.

[0025] Table 1. Source of Sample Information

[0026] Subsequently, 33 samples of commercially available, unidentified *Eleutherococcus senticosus* and *Hedyotis diffusa* were purchased as the application validation group (Table 2).

[0027] Table 2 Information on Commercially Available Samples

[0028] 1.2 Total DNA Extraction Total DNA was extracted using a novel rapid plant genomic DNA extraction kit (Bio Teke). Dry leaves were ground into a fine powder using liquid nitrogen, and preheated reagent (65°C) was added. Then, 4 μL of Nase A was added to remove RNA interference, and the mixture was thoroughly mixed. The remaining steps were strictly performed according to the instructions of the plant genomic DNA extraction kit. The extracted total DNA was labeled and stored at -80°C.

[0029] 1.3 ITS trnH-psbA and matK Primer selection The ITS sequence of the *Eleutherococcus* genus exhibits relatively small variation, making sequence alignment relatively easy. The primers used in this experiment for the ITS region were "ITS4" and "ITS5"; additionally, two chloroplast DNA regions were utilized. trn H- psb A and mat The samples were amplified using primers for the K region and primers for the ITS2 region (ITS2-F, ITS2-R) (Table 3).

[0030] Table 3 Primer Information

[0031] 1.4 Sequence amplification and sequencing Using leaf DNA from *Eleutherococcus senticosus* and *Hemiberlesia lataniae* as templates, and employing ITS, trn H -psb A and matPCR amplification was performed using K and ITS2 primers. The PCR amplification system is shown in Table 4. The PCR amplification program for ITS primers was as follows: pre-denaturation at 94℃ for 2 min; then 40 cycles were performed, each cycle consisting of denaturation at 94℃ for 30 s, annealing at 48℃ for 30 s, extension at 72℃ for 90 s; and finally extension at 72℃ for 7 min. trn H -psb A and mat PCR amplification program for primer K: pre-denaturation at 94℃ for 3 min; then 35 cycles, each cycle consisting of denaturation at 94℃ for 30 s, annealing at 55℃ for 60 s, extension at 72℃ for 90 s; and a final extension at 72℃ for 10 min. PCR amplification program for primer ITS2: pre-denaturation at 95℃ for 5 min; then 35 cycles, each cycle consisting of denaturation at 95℃ for 30 s, annealing at 48℃ for 30 s, extension at 72℃ for 30 s; and a final extension at 72℃ for 10 min.

[0032] After analysis by agarose gel electrophoresis, the PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for one-way sequencing.

[0033] Table 4 PCR amplification system

[0034] 1.5 Data Processing The 60 sample sequences that underwent unidirectional sequencing were verified and analyzed using DNAMAN software to ensure the accuracy of the experimental data. ITS sequences of the *Eleutherococcus* genus were downloaded from GenBank and phylogenetic tree (NJ tree) analysis was performed using MEGA11.

[0035] 1.6. Molecular ID Card Construction The differentially stable sites obtained from the ITS sequence alignment results were encoded, and then the molecular identification codes, variety names and other relevant information of *Eleutherococcus senticosus* and *Hedyotis diffusa* were uploaded to an online QR code converter (https: / / cli.im / ) to be converted into molecular data QR codes.

[0036] 2. Results and Analysis 2.1. Morphological Analysis Morphological observation and analysis were conducted on the collected plants of Sambucus javanica and S. adnata, with a focus on comparing the morphological characteristics of their leaves, flowers, and mature fruits. Figure 1 In the flora, the roots and rhizomes of *Hedyotis diffusa* are found to exude red sap when broken. However, during sample collection, it was observed that the stems of *Sambucus chinensis* exude red sap when broken, while the red sap characteristic of *Hedyotis diffusa* stems is not obvious. Figure 2Therefore, the red sap cannot be used as a basis for identification. *Sambucus chinensis* has yellow, cup-shaped sterile flowers, and its mature fruit is black with small wart-like protrusions; while *Hedyotis diffusa* does not have cup-shaped sterile flowers, and its leaflets' stipules are reduced to bottle-shaped glandular protrusions, and its mature fruit is orange-red. Figure 1 Therefore, flowers and mature fruits are key morphological features for distinguishing between two species.

[0037] In traditional medicine, the whole plant or rhizome of *Sambucus chinensis* and *Hedyotis diffusa* are commonly used. However, *Sambucus chinensis* and *Hedyotis diffusa* are morphologically very similar, and their geographical areas overlap in some regions, making them easy to confuse in traditional medicine. Therefore, molecular identification methods are needed to distinguish between the two.

[0038] Based on the professional morphological identification by botanists, this application accurately divided 60 samples from 6 production areas into 30 samples of *Hedyotis diffusa* and 30 samples of *Hedyotis diffusa* for subsequent screening of molecular identification markers.

[0039] 2.2. ITS sequence identification of *Sambucus chinensis* and *Hedyotis diffusa* In preliminary experiments, five individuals were selected from samples from each origin for molecular identification pre-tests. ITS was used... trn H -psb A and mat K primers were used to amplify and sequence the DNA of these 5 individuals using PCR. Figure 3 Comparison revealed that the ITS sequences of *Sambucus chinensis* and *Hedyotis diffusa* exhibited differentially stable fragments between the species. Figure 4 ); psbA During the sequencing process, most samples exhibited poly structures. matK Small sequence differences (see) Figure 5 ).

[0040] Preliminary experimental results showed that the sequence amplified by the ITS primers was of discriminative significance; therefore, in subsequent experiments, we will detect ITS in 60 individual samples. Total DNA was extracted from 60 individuals, and the quality of the extracted DNA was determined by agarose gel electrophoresis. Figure 6 Using qualified DNA as a template, PCR amplification was performed using ITS primers. The quality of the amplified products was then assessed by agarose gel electrophoresis. Figure 7 (Left). The sample with clear and bright bands was subjected to unidirectional sequencing.

[0041] DNAMAN alignment analysis of the 60 samples that were sequenced showed that the ITS sequence length of *Eleutherococcus senticosus* was 604 bp and that of *Hedyotis diffusa* was 603 bp; there were a total of 18 variant sites (Table 5).

[0042] As shown in Table 5, gene coding sorting based on the full length of the ITS sequence revealed 18 variation sites. Eleven of these sites were interspecific (9, 73, 172, 204, 218, 420, 423, 426, 430, 558, 589), while seven were intraspecific. Specifically, the ITS sequence of *Sambucus chinensis* contained two intraspecific variation sites (63, 79), and the ITS sequence of *Hedyotis diffusa* contained five intraspecific variation sites (76, 169, 506, 518, 590). The intraspecific individual differences between *Sambucus chinensis* and *Hedyotis diffusa* are small and cannot be used for origin identification.

[0043] This experiment used MEGA11 to perform interspecific analysis of the ITS sequences of 30 *Sambucus chinensis* and 30 *Hedyotis diffusa* samples, identifying interspecific ITS sequence variation sites. Nine variation sites were found in the ITS1 region and nine in the ITS2 region. The stable interspecific variation sites were 9, 73, 172, 204, 218, 420, 423, 426, 430, 558, and 589. These stable interspecific variation sites can be used as molecular codes for constructing molecular identity cards.

[0044] Table 5. Variant Site Information

[0045] The experiment combined 10 ITS sequences from different species of the genus *Sambucus* obtained from GenBank (GenBank numbers are shown in Table 6) and performed phylogenetic tree (NJ tree) analysis using MEGA11 (see [link to analysis]). Figure 8 The results showed that the identification results precisely corresponded to the morphological identification results. The 30 *Sambucus chinensis* samples and 30 *Sambucus terrestris* samples each clustered into a single monophyletic clade, demonstrating good monophyletic characteristics. Furthermore, comparisons with downloaded sequences from databases showed that *Sambucus chinensis* clustered with *Sambucus chinensis* (GenBank U88201.1) in a single clade with *Sambucus terrestris* (GenBank HQ7144441.1) in a single clade with *Sambucus terrestris*. For other *Sambucus* species, the *Sambucus chinensis* and *Sambucus terrestris* clades could be clearly distinguished. This indicates that the interspecific stable variation sites proposed in this application can accurately distinguish *Sambucus chinensis* and *Sambucus terrestris* from other *Sambucus* species, and can also accurately distinguish *Sambucus chinensis* and *Sambucus terrestris*. These interspecific stable variation sites can be used as molecular codes for constructing molecular identity cards.

[0046] Table 6 GenBank Information

[0047] 3. Verification of commercially available samples 3.1. ITS2 primer amplification To verify whether the ITS sequence differences discovered in this application could be used to identify commercially available *Clematis chinensis* and *Hedyotis diffusa* products, ITS amplification was performed on 33 commercially available samples. The results showed that most of the 33 samples were processed, chopped, or powdered traditional Chinese medicine slices, and the DNA was largely degraded. After DNA extraction, the concentration of the collected DNA was detected using a multi-functional microplate reader, revealing low concentration and poor DNA quality, making it impossible to amplify a complete ITS sequence. Therefore, ITS2 primers were subsequently selected for amplification. The PCR products were analyzed by agarose gel electrophoresis (see...). Figure 7 (Right), the sample with clear and bright bands was sent to Sangon Biotech (Shanghai) Co., Ltd. for one-way sequencing.

[0048] 3.2. ITS2 sequence verification of commercially available samples The ITS sequences of *Elsholtzia ciliata* and *Heliotropium indicum* obtained in previous experiments were annotated using HMMer, removing the 5.8S and 28S regions at both ends, to obtain the ITS2 sequences. Phylogenetic tree (NJ tree) analysis was performed on 60 samples from the screening group using MEGA11 (see [link to MEGA11]). Figure 9 The results showed that the identification results precisely corresponded to the morphological identification results. The 30 *Sambucus chinensis* samples and 30 *Hemiberlesia javanica* samples each clustered into a monophyletic clade, exhibiting good monophyletic characteristics. Although the clade support rate decreased due to reduced information, the interspecific stable variation sites could still accurately distinguish *Sambucus chinensis* and *Hemiberlesia javanica* from other species of the *Sambucus* genus, and could also accurately distinguish *Sambucus chinensis* and *Hemiberlesia javanica*. These interspecific stable variation sites can be used as molecular codes for constructing molecular identity cards. Based on the above results, standard sequences for subsequent identification were determined. *Sambucus chinensis* ITS2 was denoted as JGC-ITS2 (Table 7), and *Hemiberlesia javanica* ITS2 was denoted as XMC-ITS2 (Table 7). The site codes were re-encoded according to the length of the ITS2 sequence, and the interspecific ITS2 sequence variation information of the standard sequences is shown in Table 8. ITS2 amplification was performed on 33 unknown samples, and the successfully sequenced sequences were annotated using HMMer, removing the 5.8S and 28S regions at both ends to obtain the ITS2 sequences.

[0049] Table 7 Standard ITS2 Sequence

[0050] Table 8 Standard ITS2 variant sites

[0051] The ITS2 sequences of commercially available samples were combined with the standard sequences for identification (JGC-ITS2 and XMC-ITS2), and seven ITS sequences of closely related plants of elderberry were downloaded from GenBank. Phylogenetic tree (NJ tree) analysis was performed using MEGA11 (see [link to analysis]). Figure 10 The results showed that AH2, BS, CD, CG2, CQ, DL2, GD, HG, HH, KM, KY, LJ, LQ, LY, MY, NJ, QJ, SM, XY, AH1, CG1, CX1, DL1, GX, JZ, HN, NC, YX, and ZT1 clustered with *Hemiberlesia javanica* ITS2 into a single monophyletic clade, with a branch support rate of 85%; CX2, LWS, ML, and ZT2 clustered with *Hemiberlesia javanica* ITS2 into another monophyletic clade, with a branch support rate of 86%. It is evident that the NJ tree constructed using the ITS2 sequence can accurately distinguish the origins of all 33 commercially available samples and accurately classify them into two monophyletic clades, indicating that the sequences in this application have sufficient identification accuracy. Furthermore, there is no issue of confusion between the two origins in the commercially available samples, which is consistent with the fact that the distribution areas of the two species are largely non-overlapping. NJ tree also shows that the ITS2 sequence of this application can be used to distinguish *Salvia splendens* and *Sambucus chinensis* from closely related plants of the *Sambucus* genus with sufficient accuracy.

[0052] Interspecific analysis of the ITS2 sequences of 33 unknown samples and the standard ITS2 sequence was performed using DNAMAN9. The comparison revealed that the following sites in the 33 samples showed stable differences among species (Table 9), and the aforementioned key variant sites were retained: 46, 50, 178, and 209. These stable interspecific variant sites can be used as molecular codes for constructing molecular identity cards.

[0053] Table 9. Information on ITS2 sequence variation sites among 33 samples.

[0054] 3.3. Construction of Molecular Identity Cards Based on the ITS2 sequence alignment results, four sites exhibited differential stability. These four differentially stable sites were used as distinguishing features for *Sambucus chinensis* and *Hedyotis diffusa*, constructing molecular identification codes. Encoding was performed based on the location of the differentially stable sites, the base letters of those sites, and the first letter of the sample, resulting in two molecular identification codes combining numbers and letters: JGC46A50G178G209C and XMC46G50T178C209T. The basic information of *Sambucus chinensis* and *Hedyotis diffusa*, along with the molecular identification codes, were then entered into a QR code generator (https: / / cli.im / ) to generate scannable QR codes for *Sambucus chinensis* and *Hedyotis diffusa* DNA molecular identification codes.

[0055] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. A molecular identification marker for *Sambucus chinensis* and *Hedyotis diffusa*, characterized in that, This includes the *Hemiberlesia lataniae* sequence shown in SEQ ID NO.1 and the *Hedyotis diffusa* sequence shown in SEQ ID NO.

2.

2. The molecular identification marker for *Sambucus chinensis* and *Hedyotis diffusa* according to claim 1, characterized in that, The sequences SEQ ID NO.1 and SEQ ID NO.2 differ as follows: The 46th bit of SEQ ID NO.1 is A, the 50th bit is G, the 178th bit is G, and the 209th bit is C; The 46th bit of SEQ ID NO.2 is G, the 50th bit is T, the 178th bit is C, and the 209th bit is T.

3. The molecular identification marker for *Sambucus chinensis* and *Hedyotis diffusa* according to claim 2, characterized in that, The SEQ ID NO.1 and SEQ ID NO.2 are the ITS2 sequences of *Hedyotis diffusa* and *Hedyotis diffusa*.

4. A molecular identification method for *Sambucus chinensis* and *Hedyotis diffusa*, characterized in that, The differentiation is made based on the amplification results of the ITS2 sequence of *Hedyotis diffusa* as shown in SEQ ID NO.1 or the ITS2 sequence of *Hedyotis diffusa* as shown in SEQ ID NO.

2.

5. The molecular identification method for *Sambucus chinensis* and *Hedyotis diffusa* according to claim 4, characterized in that, The primer sequences for amplifying SEQ ID NO.1 and SEQ ID NO.2 are shown in SEQ ID NO.3 and SEQ ID NO.

4.

6. The molecular identification method for *Sambucus chinensis* and *Hedyotis diffusa* according to claim 4, characterized in that, DNA was extracted from processed and shredded Chinese medicinal herbs or powder and then amplified.

7. A reagent kit for distinguishing between *Sambucus chinensis* and *Hedyotis diffusa*, characterized in that, Includes primers used to amplify SEQ ID NO.1 and SEQ ID NO.

2.

8. A reagent kit for distinguishing between *Sambucus chinensis* and *Hedyotis diffusa* according to claim 7, characterized in that, The kit is a CAPS / dCAPS kit.

9. A molecularly specific identification method for *Sambucus chinensis* and *Hedyotis diffusa*, characterized in that, Includes the following steps: The ITS2 sequence of the plant to be identified is amplified. The numbers and letters of the bases at positions 46, 50, 178 and 209 of the ITS2 sequence of the plant to be identified are combined to generate a molecular identity card for the plant. Then, this molecular identity card is compared with the molecular identity cards of *Eleutherococcus senticosus* and *Hemiberlesia stramonium*. The plant that matches the comparison is the corresponding variety.

10. The molecularly specific identification method for *Sambucus chinensis* and *Hedyotis diffusa* according to claim 9, characterized in that, The molecular identification number of the herb *Eleutherococcus senticosus* is: JGC46A50G178G209C; the molecular identification number of the herb *Hedyotis diffusa* is: XMC46G50T178C209T.

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