Primer and probe for identifying semen plantaginis or plantago-based antigen, application of primer and probe and identification method

Abstract

The invention relates to the technical field of traditional Chinese medicine identification, and particularly discloses a primer and a probe for identifying plantain seed or plantain herb-based antigen as well as application and an identification method of the primer and the probe. Wherein the primer and the probe for identifying the plantain seed or the plantain-based antigen comprise a pre-plantain forward primer (SEQ ID NO: 1), a pre-plantain reverse primer (SEQ ID NO: 2), a pre-plantain fluorescent probe (SEQ ID NO: 3), a pre-plantain forward primer (SEQ ID NO: 4), a pre-plantain reverse primer (SEQ ID NO: 5) and a pre-plantain fluorescent probe (SEQ ID NO: 6); the modified fluorophore at the 5'end of the pre-plantar fluorescence probe is FAM, the modified fluorophore at the 3 'end of the pre-plantar fluorescence probe is MGB, the modified fluorophore at the 5' end of the pre-plantar fluorescence probe is VIC, and the modified fluorophore at the 3 'end of the pre-plantar fluorescence probe is MGB. By implementing the method, pre-plantar and pre-plantar can be effectively distinguished.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine identification, and in particular to a primer and a probe for identifying plantago seed or plantago asiatica, and an application and identification method thereof. Background Art

[0002] The 2020 edition of the Chinese Pharmacopoeia stipulates that Plantago is the dried mature seeds of Plantago asiatica L. or Plantago depressa Willd. of the Plantaginaceae family. The fruit ears are harvested when the seeds are mature in summer and autumn, dried in the sun, the seeds are rubbed out, and impurities are removed. Plantain is the dried whole grass of Plantago or Plantago depressa Willd. of the Plantaginaceae family. It is dug in summer, the sand is removed, and it is dried in the sun. Plantago (grass) is sweet and cold in nature. It enters the liver, kidney, lung, and small intestine meridians. It is mainly used to clear heat, promote diuresis, relieve stranguria, infiltrate dampness and stop diarrhea, improve eyesight, and eliminate phlegm. It is used for hot stranguria, edema, diarrhea caused by summer heat, red and swollen eyes, and cough caused by phlegm and heat. Since Plantago and Plantago asiatica have different origins, their chemical composition and content are quite different. The morphology and medicinal characteristics of different origins of Plantago are relatively similar, and the properties of Plantago are more difficult to distinguish. Although traditional identification methods can be used for identification through properties and microscopy, they cannot accurately identify medicinal materials with a certain proportion of Plantago (grass) mixed in. At present, the literature "Application of ITS2 sequence to identify Sichuan Plantago, Plantago asiatica, and Plantago asiatica" and "Comparison of identification of small-seed medicinal materials Plantago based on ITS2 and psbA-trnH sequences" use DNA barcodes to identify Plantago origins and their counterfeits, but they are also unable to detect adulterated medicinal materials. Therefore, in order to ensure the safety and clinical efficacy of traditional Chinese medicine, it is necessary to establish a method to identify the origins of Plantago and Plantago. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a primer and a probe for identifying plantago or plantago asiatica, which have good specificity and can effectively identify plantago and plantain.

[0004] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned primers and probes.

[0005] The technical problem that the present invention needs to solve is to provide a kit for identifying plantago or psyllium root, which can effectively identify plantago and psyllium.

[0006] The technical problem that the present invention needs to solve is to provide a method for identifying plantago or plantago asiatica.

[0007] In order to solve the above technical problems, the present invention provides a primer and a probe for identifying plantago seed or plantago asiatica, which include a plantago forward primer, a plantago reverse primer, a plantago fluorescent probe, a plantain forward primer, a plantain reverse primer and a plantain fluorescent probe;

[0008] The nucleotide sequence of the forward primer of Plantago is shown in SEQ ID NO: 1, the nucleotide sequence of the reverse primer of Plantago is shown in SEQ ID NO: 2, the nucleotide sequence of the fluorescent probe of Plantago is shown in SEQ ID NO: 3, the nucleotide sequence of the forward primer of Plantago is shown in SEQ ID NO: 4, the nucleotide sequence of the reverse primer of Plantago is shown in SEQ ID NO: 5, and the nucleotide sequence of the fluorescent probe of Plantago is shown in SEQ ID NO: 6;

[0009] The modified fluorescent group at the 5' end of the Plantago fluorescent probe is FAM, and the modified fluorescent group at the 3' end is MGB;

[0010] The modified fluorescent group at the 5' end of the Plantago asiatica fluorescent probe is VIC, and the modified fluorescent group at the 3' end is MGB.

[0011] Accordingly, the present invention also discloses the use of the above-mentioned primers and probes for identifying Plantago or Plantago asiatica in any one of (1) to (6):

[0012] (1) Identify the origin of Plantago;

[0013] (2) preparing a kit for identifying the origin of Plantago;

[0014] (3) Identify the origin of Plantain;

[0015] (4) preparing a kit for identifying the origin of Plantago;

[0016] (5) Identify whether the sample to be tested contains Plantago asiatica or Plantago asiatica;

[0017] (6) Prepare a kit for identifying whether a sample to be tested contains Plantago asiatica or Plantago asiatica.

[0018] Correspondingly, the present invention also discloses a kit for identifying plantago or plantago rhizome, which comprises the above-mentioned primers and probes for identifying plantago or plantago rhizome.

[0019] As an improvement of the above technical solution, it also includes PCR reaction solution and DNA extraction solution;

[0020] The PCR reaction solution includes sterile water, DNA polymerase, dNTPs, buffer, Mg 2+ of solution and fluorescent dye.

[0021] As an improvement of the above technical solution, the PCR reaction solution includes Probe qPCR Mix, sterile water and 50×ROX.

[0022] As an improvement of the above technical solution, the PCR reaction solution includes:

[0023] Probe qPCR Mix 10.0 μL, 50×ROX 0.4 μL and sterile water 6.4 μL.

[0024] Accordingly, the present invention also discloses a method for identifying plantago or plantago asiatica, which comprises:

[0025] Extracting genomic DNA from samples to be tested;

[0026] Using the genomic DNA as a DNA template, amplification is performed using the primers and probes for identifying plantago or plantago asiatica;

[0027] The fluorescence value of the amplified product is read, and whether the sample to be detected contains Plantago and / or Plantago asiatica is determined according to the fluorescence value.

[0028] As an improvement of the above technical solution, the amplification reaction system includes: Probe qPCR Mix 10.0 μL, 50×ROX 0.4 μL, Plantago forward primer 0.2 μL, Plantago reverse primer 0.2 μL, Plantago fluorescent probe 0.2 μL, Plantago forward primer 0.2 μL, Plantago reverse primer 0.2 μL, Plantago fluorescent probe 0.2 μL, DNA template 2.0 μL and sterile water 6.4 μL.

[0029] As an improvement of the above technical solution, the amplification procedure includes: pre-denaturation at 95°C for 20s; denaturation at 95°C for 1s, annealing at 60°C for 20s, for a total of 35 cycles.

[0030] As an improvement of the above technical solution, the concentration of the DNA template is 50 ng / μL to 70 ng / μL.

[0031] The implementation of the present invention has the following beneficial effects:

[0032] The invention designs primers and probes for Plantago and Plantago asiatica. Based on the primers and probes, a method for identifying the origin of Plantago or Plantago asiatica can be established by a multiple TaqMan-MGB fluorescence quantitative PCR method. The origins of Plantago and Plantago asiatica, Plantago and Plantago asiatica, can be simultaneously identified under the same amplification conditions. The identification efficiency is high and the specificity is good, thus providing a basis for the quality control of Plantago and Plantago asiatica. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1It is a diagram of the fluorescent quantitative PCR identification results of Plantago and Plantago asiatica when different primer and probe combinations are used in Example 1, wherein the primers and probes used in A are CQ1-F, CQ1-R and CQ1-P, the primers and probes used in B are PCQ3-F, PCQ3-R and PCQ3-P, the primers and probes used in C are CQ2-F, CQ2-R, CQ2-P, and the primers and probes used in D are PCQ4-F, PCQ4-R and PCQ4-P.

[0034] Figure 2 This is a graph showing the sensitivity test results of Plantago and Plantago asiatica by fluorescence quantitative PCR in Example 1, wherein the primers and probes used in A are CQ1-F, CQ1-R and CQ1-P, and the primers and probes used in B are PCQ3-F, PCQ3-R and PCQ3-P.

[0035] Figure 3 This is a diagram of the suitability test results of fluorescent quantitative PCR of Plantago and Plantago asiatica in Example 1, wherein the primers and probes used in A are CQ1-F, CQ1-R and CQ1-P, and the primers and probes used in B are PCQ3-F, PCQ3-R and PCQ3-P.

[0036] Figure 4 This is a fluorescence quantitative PCR test result of Plantago mixed with Plantago asiatica in Example 1. DETAILED DESCRIPTION

[0037] For ease of understanding of the present invention, the present invention will be described in more detail below. However, it should be understood that the present invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. On the contrary, the purpose of providing these embodiments or examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific implementation or embodiment, and are not intended to limit the present invention. The optional scope of the term "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of related listed items, and the combinations include any two related listed items, any more related listed items, or all related listed items.

[0039] The following examples are given for the purpose of illustrating various embodiments of the present invention and are not intended to limit the present invention in any way.

[0040] The present invention. Those skilled in the art will understand that the changes and other uses thereof are within the spirit of the present invention as defined by the scope of the claims. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources. The promoter and terminator sequences mentioned in the examples can also be downloaded from NCBI, and the specific sequence start positions can be obtained based on the primers in the primer table. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0041] In the present invention, "first aspect", "second aspect" and the like are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features.

[0042] 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.

[0043] The percentage contents involved in the present invention, unless otherwise specified, refer to mass percentage for solid-liquid mixing and solid-solid mixing, and refer to volume percentage for liquid-liquid mixing.

[0044] The percentage concentrations involved in the present invention, unless otherwise specified, refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of the component.

[0045] The temperature parameters in the present invention, if not specifically limited, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0046] The invention provides a primer and a probe for identifying plantago seed or plantago herbaceous origin, which specifically comprise a plantago forward primer, a plantago reverse primer, a plantago fluorescent probe, a plantago forward primer, a plantago reverse primer and a plantago fluorescent probe; the nucleotide sequence of the plantago forward primer is shown in SEQ ID NO: 1, the nucleotide sequence of the plantago reverse primer is shown in SEQ ID NO: 2, the nucleotide sequence of the plantago fluorescent probe is shown in SEQ ID NO: 3, the nucleotide sequence of the plantago forward primer is shown in SEQ ID NO: 4, the nucleotide sequence of the plantago reverse primer is shown in SEQ ID NO: 5, and the nucleotide sequence of the plantago fluorescent probe is shown in SEQ ID NO: 6; the modified fluorescent group at the 5' end of the plantago fluorescent probe is FAM, and the modified fluorescent group at the 3' end is MGB; the modified fluorescent group at the 5' end of the plantago fluorescent probe is VIC, and the modified fluorescent group at the 3' end is MGB.

[0047] Based on the primers and probes, a method for identifying the origin of Plantago or Plantago can be established through the multiplex TaqMan-MGB fluorescence quantitative PCR method. The origins of Plantago and Plantago, Plantago asiatica and Plantago asiatica, can be simultaneously identified under the same amplification conditions, with high identification efficiency and good specificity, providing a good basis for the identification of Plantago and Plantago.

[0048] Based on the above characteristics, the primers in the present invention can be used in the following applications:

[0049] (1) Identify the origin of Plantago;

[0050] (2) preparing a kit for identifying the origin of Plantago;

[0051] (3) Identify the origin of Plantain;

[0052] (4) preparing a kit for identifying the origin of Plantago;

[0053] (5) Identify whether the sample to be tested contains Plantago asiatica or Plantago asiatica;

[0054] (6) Prepare a kit for identifying whether a sample to be tested contains Plantago asiatica or Plantago asiatica.

[0055] It should be noted that the Plantago seeds and Plantago in the present invention can be raw medicinal materials, or processed medicinal pieces, or standard decoctions, Chinese medicine formula granules, etc. prepared through other processing, but are not limited thereto.

[0056] Accordingly, the present invention also discloses a kit for identifying the origin of Plantago or Herba Plantaginis, which comprises the above-mentioned primers and probes for identifying the origin of Plantago or Herba Plantaginis. Through the above-mentioned primers and probes, the origin of Plantago / Herba Plantaginis, Plantago and Plantago asiatica, can be effectively identified.

[0057] Preferably, in some embodiments, the kit further comprises a PCR reaction solution and a DNA extracting solution, wherein the DNA extracting solution is used to extract the DNA of the sample to be tested, and the DNA extracting solution may include a CTAB extracting solution, an SDS extracting solution, a phenol-chloroform-isopropanol extracting solution or an extracting solution in other commercial DNA kits commonly used in the art, but is not limited thereto. The PCR reaction solution comprises sterile water, DNA polymerase, dNTPs, a buffer solution, and a Mg-containing 2+ Preferably, in some embodiments, the kit further includes a positive control, a negative control, etc., but is not limited thereto.

[0058] More preferably, in some embodiments, the PCR reaction solution includes Probe qPCR Mix, sterile water and 50×ROX. More specifically, the composition of the PCR reaction solution is as follows: Probe qPCR Mix 10.0 μL, 50×ROX 0.4 μL and sterile water 6.4 μL.

[0059] Accordingly, the present invention also discloses a method for identifying plantago or plantago asiatica, which comprises:

[0060] (1) extracting genomic DNA from the sample to be tested;

[0061] Specifically, the extraction can be performed using a DNA extracting solution, which may be a CTAB extracting solution, an SDS extracting solution, a phenol-chloroform-isopropanol extracting solution, or an extracting solution in other commercial DNA kits commonly used in the art, but is not limited thereto.

[0062] (2) forming a PCR amplification system and performing amplification to obtain an amplification product;

[0063] Specifically, the DNA template, the Plantago forward primer, the Plantago reverse primer, the Plantago fluorescent probe, the Plantago asiatica forward primer, the Plantago asiatica reverse primer, the Plantago asiatica fluorescent probe, the PCR reaction solution and sterile water are mixed evenly to obtain a PCR amplification system.

[0064] Preferably, in one embodiment, the total volume of the PCR amplification system is 20 μL, specifically including: Probe qPCRMix 10.0 μL, 50×ROX 0.4 μL, Plantago forward primer 0.2 μL, Plantago reverse primer 0.2 μL, Plantago fluorescent probe 0.2 μL, Plantago forward primer 0.2 μL, Plantago reverse primer 0.2 μL, Plantago fluorescent probe 0.2 μL, DNA template 2.0 μL and sterile water 6.4 μL.

[0065] Specifically, in one embodiment, the amplification program includes 95° C. pre-denaturation for 20 s; 95° C. denaturation for 1 s, and 60° C. annealing for 20 s, for a total of 35 cycles.

[0066] (3) analyzing the amplified products;

[0067] Specifically, the fluorescence value of the amplified product is read, and whether the sample to be tested contains Plantago and Plantago asiatica is determined according to the fluorescence value. Specifically, when the fluorescence value (Ct value) of the Plantago probe is ≤35, the sample to be tested contains Plantago; when the fluorescence value (Ct value) of the Plantago asiatica probe is ≤35, the sample to be tested contains Plantago.

[0068] The present invention is described below with reference to specific embodiments.

[0069] Example 1

[0070] 1. Sample List

[0071] Table 1 Sample list

[0072]

[0073] 2. Primer Design

[0074] The ITS2 sequences of Plantago and Plantago asiatica were downloaded from the NCBI (http: / / www.ncbi.nlm.nih.gov) database, and the SNP sites of Plantago and Plantago asiatica were obtained by comparison using BioEdit software. TaqMan-MGB probes and primers of Plantago and Plantago asiatica were designed using Primer Express 3.0.1, and probes and primers with strong specificity were screened. The sequences of primers and probes are shown in Table 2. The primers and probes were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0075] Table 2 Primer and probe sequence list

[0076]

[0077] 3. Extraction of genomic DNA

[0078] Plantago was extracted using a high-efficiency plant genomic DNA extraction kit (DP350-03, Tiangen Biochemical Technology (Beijing) Co., Ltd.), and Plantago was extracted using a plant / seed DNA extraction kit (TD619-200, Jianshi Biotechnology (Beijing) Co., Ltd.).

[0079] 4. DNA Concentration Determination

[0080] The above DNA samples were taken, and the DNA concentration was determined using a BioSpec-nano micro-volume UV spectrophotometer, while OD260 / OD230 and OD260 / OD280 were recorded, and the concentration was adjusted to 50 ng / μL to 70 ng / μL using TB buffer.

[0081] 5. Screening of Taqman-MGB probes and primers

[0082] According to the characteristics of the enzyme and the T m Value, set the qPCR reaction system: in a 100μL centrifuge tube, the total reaction volume is 20μL, the reaction system includes Probe qPCR Mix 10.0μL, 50×ROX 0.4μL, Plantago forward and reverse primers, probe 0.2μL each, Plantago forward and reverse primers, probe 0.2μL each, DNA template 2.0μL, sterile ultrapure water to 20μL. Place the centrifuge tube in the qPCR instrument, set the qPCR reaction parameters: 95℃ pre-denaturation for 20s; 95℃ denaturation for 1s, 60℃ annealing for 20s, a total of 35 cycles, and collect the fluorescence signal. According to the above amplification system and conditions, qPCR amplification of Plantago and Plantago samples was performed with the probes and primers shown in Table 1.

[0083] The results show that Figure 1 ), within 35 cycles, the plantago fluorescent probe CQ1 had a specific amplification curve for the plantago DNA sample, with a Ct mean of 14.15, while the plantain samples had no amplification curve; the plantain fluorescent probe PCQ3 had a specific amplification curve for the plantain DNA sample, with a Ct mean of 14.01, while the plantago samples had no amplification curve; while the plantago fluorescent probe CQ2 and the plantain fluorescent probe PCQ4 both amplified the plantago DNA samples, with poor specificity, indicating that the probes CQ1 and PCQ3 have strong specificity and are suitable for the multiplex qPCR detection of the plantago and plantain genes.

[0084] VI. Evaluation of Taqman-MGB Fluorescence Quantitative PCR Detection Method

[0085] (1) Sensitivity investigation

[0086] To test the sensitivity of primers and probes for Plantago and Psyllium, DNA samples of Plantago (73.87 ng / μL) and Psyllium (60.17 ng / μL) were diluted with TB buffer in a gradient of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7The concentration gradient was repeated 3 times, and the amplification was performed according to the TaqMan-MGB fluorescence quantitative PCR identification system. The data were analyzed by StepOne Software v2.3 and the Ct value was recorded. The results show ( Figure 2 , Table 3), when the DNA concentrations of Plantago and Plantago asiatica were 73.87-0.0074 ng / μL and 60.17-0.0060 ng / μL, respectively, there were positive amplification curves, while no amplification occurred when the DNA concentrations were 0.00074 ng / μL and 0.00060 ng / μL and below, respectively, indicating that the detection limits of the established system for the DNA of Plantago and Plantago asiatica were 0.0074 ng / μL and 0.0060 ng / μL, respectively.

[0087] Table 3 Plantago asiatica and Plantago asiatica DNA concentrations and their corresponding Ct values

[0088]

[0089] (2) Repeatability and precision study

[0090] The same sample of Plantago and Plantago asiatica was measured three times, and the Ct values ​​were recorded. The relative standard deviation (RSD) of the Ct values ​​within the group was analyzed to determine the repeatability of the detection method. The results showed (Table 4) that the mean Ct values ​​of Plantago and Plantago asiatica were 11.08 and 13.44, respectively, and the RSDs were 0.63% and 0.31%, respectively, both less than 3%, proving that the method had good repeatability.

[0091] At the same time, 5 samples of the same batch of Plantago and Plantago asiatica were measured in parallel, and the Ct values ​​were recorded. The relative standard deviation (RSD) of the Ct values ​​within the group was analyzed to investigate the precision of the method. The results showed (Table 5) that the mean Ct values ​​of the 5 samples of Plantago and Plantago asiatica in the same batch were 14.30 and 14.07, respectively, and the RSDs were 2.19% and 1.18%, respectively, both less than 3%, proving that the method has good precision.

[0092] Table 4 Repeatability test results of Plantago and Plantago asiatica by fluorescence quantitative PCR

[0093]

[0094] Table 5 Precision test results of Plantago and Plantago asiatica by fluorescence quantitative PCR

[0095]

[0096] VII. Applicability Examination

[0097] The multiplex Taqman-MGB fluorescence quantitative PCR method established above was used to test 11 batches of Plantago (Plantago), 17 batches of Plantago (Plantago) and 10 batches of Plantago (Plantago) from different origins and sources to verify the applicability of the reaction system. The results showed that ( Figure 3 , Table 6 and Table 7), in the multiplex qPCR system, the plant samples were all positively amplified under the plant fluorescent probe, but not amplified under the plantain fluorescent probe, among which, the Ct value of Plantago (Plantago) was 13.30-19.90, the average was 15.00, and the RSD average was 0.28%; the Ct value of Plantago was 11.95-13.99, the average was 13.07, and the RSD average was 0.50%. On the contrary, the plantain samples were all positively amplified under the plantain fluorescent probe, and not amplified under the plantain fluorescent probe, and their Ct values ​​were 10.19-12.45, the average was 10.96, and the RSD average was 0.32%, indicating that the multiplex fluorescence quantitative PCR method has good applicability.

[0098] Table 6 Plantain sample test results

[0099]

[0100] Table 7 Plantago samples test results

[0101]

[0102] 8. Testing of mixing of plankton and flat plankton

[0103] The same batch of Plantago was mixed with 0%, 1%, 2%, 3%, 5%, 10%, 50%, 80%, and 100% of Plantago asiatica respectively, and the established multiple Taqman-MGB fluorescence quantitative PCR method was used for amplification reaction, and the Ct value was recorded to detect the detection limit of Plantago asiatica mixed with Plantago asiatica. It can be seen from the table that when the mixing ratio reaches more than 3%, Plantago asiatica can be detected.

[0104] Table 8 Mixing test results of samples mixed in different proportions

[0105]

[0106] In summary, based on the primers and probes of the present invention, a method for identifying the origin of Plantago or Plantago can be established by a multiplex TaqMan-MGB fluorescent quantitative PCR method. The origins of Plantago and Plantago, Plantago asiatica and Plantago asiatica, can be simultaneously identified under the same amplification conditions, with high identification efficiency and good specificity, which provides a basis for the quality control of Plantago and Plantago.

[0107] The above-mentioned embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical solution of the present invention in detail, but it cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the scope of protection of the present invention.

[0108] It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the protection scope of the attached claims of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A primer and a probe for identifying plantago or plantago asiatica, characterized in that: Including Plantago forward primer, Plantago reverse primer, Plantago fluorescent probe, Plantago asiatica forward primer, Plantago asiatica reverse primer and Plantago asiatica fluorescent probe; The nucleotide sequence of the forward primer of Plantago is shown in SEQ ID NO: 1, the nucleotide sequence of the reverse primer of Plantago is shown in SEQ ID NO: 2, the nucleotide sequence of the fluorescent probe of Plantago is shown in SEQ ID NO: 3, the nucleotide sequence of the forward primer of Plantago is shown in SEQ ID NO: 4, the nucleotide sequence of the reverse primer of Plantago is shown in SEQ ID NO: 5, and the nucleotide sequence of the fluorescent probe of Plantago is shown in SEQ ID NO: 6; The modified fluorescent group at the 5' end of the Plantago fluorescent probe is FAM, and the modified fluorescent group at the 3' end is MGB; The modified fluorescent group at the 5' end of the Plantago asiatica fluorescent probe is VIC, and the modified fluorescent group at the 3' end is MGB.

2. Use of the primers and probes for identifying Plantago or Plantago asiatica as claimed in claim 1 in any one of (1) to (6): (1) Identify the origin of Plantago; (2) preparing a kit for identifying the origin of Plantago; (3) Identify the origin of Plantain; (4) preparing a kit for identifying the origin of Plantago; (5) Identify whether the sample to be tested contains Plantago asiatica or Plantago asiatica; (6) Prepare a kit for identifying whether a sample to be tested contains Plantago asiatica or Plantago asiatica.

3. A kit for identifying plantago or plantago asiatica, characterized in that: The method comprises the primers and probes for identifying plantago or plantago asiatica as claimed in claim 1.

4. The kit for identifying Plantago or Plantago asiatica as claimed in claim 3, characterized in that: It also includes PCR reaction solution and DNA extraction solution; The PCR reaction solution includes sterile water, DNA polymerase, dNTPs, buffer, Mg 2+ of solution and fluorescent dye.

5. The kit for identifying Plantago or Plantago asiatica as claimed in claim 4, characterized in that: The PCR reaction solution includes Probe qPCR Mix, sterile water and 50×ROX.

6. The kit for identifying Plantago or Plantago asiatica as claimed in claim 4, characterized in that: The PCR reaction solution comprises: Probe qPCR Mix 10.0 μL, 50×ROX 0.4 μL and sterile water 6.4 μL.

7. A method for identifying plantago or plantago asiatica, characterized in that: include: Extracting genomic DNA from samples to be tested; Using the genomic DNA as a DNA template, amplification is performed using the primers and probes for identifying Plantago or Plantago asiatica as claimed in claim 1; The fluorescence value of the amplified product is read, and whether the sample to be detected contains Plantago and / or Plantago asiatica is determined according to the fluorescence value.

8. The method for identifying the plantago seed or plantago asiatica base according to claim 7, characterized in that: The amplification reaction system included: Probe qPCR Mix 10.0 μL, 50×ROX 0.4 μL, Plantago forward primer 0.2 μL, Plantago reverse primer 0.2 μL, Plantago fluorescent probe 0.2 μL, Plantago forward primer 0.2 μL, Plantago reverse primer 0.2 μL, Plantago fluorescent probe 0.2 μL, DNA template 2.0 μL and sterile water 6.4 μL.

9. The method for identifying the plantago seed or plantago asiatica base according to claim 7, characterized in that: The amplification procedure included: pre-denaturation at 95°C for 20 s; denaturation at 95°C for 1 s, and annealing at 60°C for 20 s, for a total of 35 cycles.

10. The method for identifying the plantago seed or plantago asiatica base according to claim 7, characterized in that: The concentration of the DNA template is 50 ng / μL to 70 ng / μL.

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