Specific primers for identifying Herba Chrysanthemi Indici by molecular biology method and identification method of Herba Chrysanthemi Indici

Abstract

The present invention discloses a specific primer for identifying Herba Chrysanthemi Morifolii from Bozhou by a molecular biology method and a method for identifying Herba Chrysanthemi Morifolii from Bozhou, belonging to the technical field of molecular identification. By widely investigating the information of the main cultivation areas of Herba Chrysanthemi Morifolii from Bozhou, the co-distributed species in the wild and the related medicinal species, and then sampling as many varieties as possible, and then through comparison, the specific information sites of the ITS sequence unique to Herba Chrysanthemi Morifolii from Bozhou are found, and further specific primers are designed: the specific primers are SEQ ID NO.1 and SEQ ID NO.2, and the use of the specific primers will greatly facilitate the rapid identification of the Chinese medicinal material Herba Chrysanthemi Morifolii from Bozhou.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology. By means of site-specific polymerase chain reaction, variable sites with stable differences are found in the ITS sequence of Chrysanthemum morifolium Ramat. cv. 'Boju', and specific primers for identifying Chrysanthemum morifolium Ramat. cv. 'Boju' are designed. Background Art

[0002] Chrysanthemum is a perennial herbaceous plant of the genus Chrysanthemum L. in the family Asteraceae. Its dried capitulum is often used as traditional Chinese medicine, and its main efficacy and function are to disperse wind-heat, soothe the liver and improve eyesight, clear heat and detoxify. It is used for symptoms such as cold headache, hyperactivity of liver-fire, high blood pressure, furuncle and carbuncle, etc. It is an important ingredient in many prescriptions and a raw material for the production of Chinese patent medicines. Among them, Chrysanthemum morifolium Ramat. cv. 'Boju' native to Bozhou City, Anhui Province, is one of the four famous chrysanthemums in China. In recent years, the market demand for the Chinese medicinal material chrysanthemum has increased sharply. However, the planting, harvesting, production and processing processes of the Chinese medicinal material chrysanthemum lack management. Due to historical and regional medication habits and other reasons, closely related species of the same genus are often mixed with the original species of Chinese medicinal materials either because their medicinal properties and functions are similar or because their appearances are similar. There are many substitutes and adulterants, resulting in particularly difficult identification work.

[0003] At present, molecular identification technology has been widely used in the identification of Chinese medicinal material species. Molecular identification technology uses standard, highly variable, easily amplified and relatively short DNA fragments to quickly and accurately identify species automatically. This technology is not affected by the characteristics of the medicinal materials themselves and external factors, and has the advantages of simplicity, high efficiency, good repeatability, the ability to identify unknown species, and accurate identification results. Therefore, in this study, the nuclear gene ITS fragment was designed, and the ITS fragments of Chrysanthemum morifolium Ramat. cv. 'Boju' and its easily confused varieties were amplified, sequenced and analyzed using DNA barcoding technology to provide a scientific basis for the accurate identification of Chrysanthemum morifolium Ramat. cv. 'Boju' and its closely related species of the same genus.

[0004] In eukaryotes, ribosomal DNA is composed of ribosomal genes and adjacent spacer regions, and its genomic sequence includes 18S rDNA gene, ITS1, 5.8S gene, ITS2, and 26S / 28S gene. The genomic sequences of 18S, 5.8S, and 28S in ribosomal DNA tend to be conserved in most organisms, with small changes among species. As non-coding regions, ITS1 and ITS2 are under less selective pressure, with relatively larger changes, and can provide heritable traits required for detailed systematic analysis. ITS has been used as a universal barcode to distinguish more than 21,722 plant species and is recommended for verifying the authenticity of Chinese medicinal materials. This gene is widely distributed and exists in almost all plants. However, so far, there have been no reports on the molecular identification of Chrysanthemum morifolium Ramat. var. boju Makino. Therefore, it is of great significance to select Chrysanthemum morifolium Ramat. var. boju Makino and its several related species for molecular identification research and establish a rapid and effective molecular identification method for the identification of Chrysanthemum morifolium in Chinese medicinal materials. Summary of the Invention

[0005] The purpose of the present invention is precisely to solve the above problems. First, the variable sites in the ITS sequence of Chrysanthemum morifolium Ramat. var. boju Makino are discovered, and then specific primers for identifying Chrysanthemum morifolium Ramat. var. boju Makino are designed based on these variable sites, as well as a method for identifying Chrysanthemum morifolium Ramat. var. boju Makino using these specific primers, so as to achieve rapid identification and improve the reliability and accuracy of the identification results.

[0006] The technical solution of the present invention is as follows:

[0007] Specific primers for identifying Chrysanthemum morifolium Ramat. var. boju Makino based on molecular biology methods, characterized in that the upstream primer is: 5’-AAACTCAAGAAGGCTCGTTTCA-3’, and the downstream primer is: 5’-TATCCGCCCCCAAAACACAC-3’.

[0008] Use of the above primers in rapidly identifying Chrysanthemum morifolium Ramat. var. boju Makino.

[0009] The present invention also provides a method for rapidly identifying Chrysanthemum morifolium Ramat. var. boju Makino using the above primers, including the following steps:

[0010] (1) Extract DNA from the sample to be tested;

[0011] (2) Using the genomic DNA extracted in step (1) as a template, perform polymerase chain reaction (PCR) amplification using the above upstream primer and downstream primer;

[0012] (3) Perform agarose gel electrophoresis detection and gel imaging on the reaction product obtained in step (2) to achieve specific identification of Chrysanthemum morifolium Ramat. var. boju Makino.

[0013] According to the above scheme, in step (3), specific identification of Flos Chrysanthemi Indici is carried out according to the results of gel imaging. On the premise of effectively extracting the DNA template of the sample to be tested, when a single bright electrophoresis band is observed, the sample to be tested is identified as Flos Chrysanthemi Indici; when a single bright electrophoresis band is not observed, the sample to be tested is identified as not Flos Chrysanthemi Indici.

[0014] Preferably, the amplification system of the PCR is 25 μL, and the component ratio is as follows: 1 μL of each of the forward and reverse primers at 2.5 μmol / L, 12.5 μL of 2×Taq PCR MasterMix, 2.0 μL of template DNA at 50 - 100 ng / L, and 8.5 μL of ddH₂O.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] At present, there are few reports on the molecular identification research of Flos Chrysanthemi Indici. The present invention first widely samples multiple varieties of Flos Chrysanthemi Indici and its related medicinal species of the genus Chrysanthemum in the same place, and then discovers the specific information sites of the ITS sequence of Flos Chrysanthemi Indici through comparison, and further obtains its specific specific primers.

[0017] The present invention provides a pair of specific primers for Flos Chrysanthemi Indici, and achieves the purpose of distinguishing Flos Chrysanthemi Indici from other varieties through specific amplification. The identification result can be more intuitively reflected by observing whether a band is produced in the gel electrophoresis result, and at the same time, the time for sequencing and comparison and analysis of the sequencing results is reduced. The identification of Flos Chrysanthemi Indici by the specific primers of the present invention reduces the operation time of drug inspection personnel, compresses the inspection time, improves the inspection efficiency, and at the same time reduces the professional restrictions on inspection personnel in theory. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a detection result diagram of specific amplification electrophoresis. Among them, M: Maker (DL2000); K: negative control (H₂O). 1 - 10: samples numbered G - 1 to G - 10.

[0019] Figure 2 It is a comparison sequence of samples used for medicinal plants of the genus Chrysanthemum. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] In order to better understand the present invention, the content of the present invention will be further clarified below in conjunction with embodiments, but the present invention is not limited only to the following embodiments.

[0021] Unless otherwise specified, the methods used in the following embodiments are all conventional methods. The primers used are all synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., the sequencing is carried out by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., the plant genomic DNA extraction kit and the DNA gel recovery kit are all purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., and the methods are all carried out according to the instructions.

[0022] In the following embodiments, the fresh samples of Herba Chrysanthemi Indici were all collected from Bozhou City, Anhui Province, and the sample information is shown in Table 1.

[0023] Table 1 Information Table of Herba Chrysanthemi Indici Samples

[0024]

[0025] Example 1

[0026] A pair of specific primers for identifying Herba Chrysanthemi Indici based on molecular biology specific amplification, the upstream primer is: 5’-AAACTCAAGAAGGCTCGTTTCA-3’, and the downstream primer is: 5’-TATCCGCCCCCAAAACACAC-3’.

[0027] The method for quickly identifying Herba Chrysanthemi Indici using the above primers. The test samples were respectively selected from the chrysanthemum samples numbered G-1 to G-10 in Table 1 for parallel experiments to identify whether the chrysanthemum samples numbered G-1 to G-10 are Herba Chrysanthemi Indici; the specific steps are as follows:

[0028] (1) Extraction of total DNA: Take about 100 mg of fresh plant tissue or about 30 mg of dry weight tissue, and add liquid nitrogen to grind thoroughly. The specific steps are as follows: Use the plant genomic DNA extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract DNA;

[0029] (2) The present invention preferably uses a 25 μL PCR amplification system, and the component ratio is: 1 μL of each of the forward and reverse primers at 2.5 μmol / L, 12.5 μL of 2×TaqPCR MasterMix, 2.0 μL of template DNA at 50 - 100 ng / L, and 8.5 μL of ddH2O. The present invention has no special limitation on the source of reagents in the amplification system, and the reagents used in the amplification system can all be commercial reagents well-known to those skilled in the art.

[0030] The amplification conditions of PCR are: pre-denaturation at 94 °C for 4 min, 1 cycle; denaturation at 94 °C for 15 s, annealing at 63.5 °C for 15 sec, extension at 72 °C for 15 sec, 33 cycles; extension at 72 °C for 5 min. Under the above amplification system and amplification conditions, all the sample DNA sequences to be screened can be successfully amplified. Those skilled in the art can make appropriate and reasonable adjustments to the amplification system and amplification conditions on the basis of the above technical solutions, such as changing the volume of the amplification system, the concentration of components, adjusting the amplification temperature and time, etc., which all belong to the protection scope of the present invention.

[0031] (3) Perform agarose gel electrophoresis detection and gel imaging on the reaction product obtained in step (2). The concentration of the agarose gel is 1% (mass / volume ratio). Prepare 40 mL of agarose gel, add 3 μL of DNA dye GelRed and 3 μL of the reaction product obtained in step (2). The voltage for electrophoresis detection is 120 V, and the electrophoresis time is 30 min. The results of the gel electrophoresis detection are all as Figure 2 shown. Among them, a single bright band was observed for the sample numbered G1, which was identified as Flos Chrysanthemi Indici; no single bright band was observed for the other samples to be tested, which were identified as non-Flos Chrysanthemi Indici.

[0032] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several improvements and modifications can be made, and these all belong to the protection scope of the present invention.

Claims

1. Specific primers for identifying Herba Chrysanthemi Indici by molecular biology methods, characterized in that, The upstream primer is: 5’-AAACTCAAGAAGGCTCGTTTCA-3’, and the downstream primer is: 5’-TATCCGCCCCCAAAACACAC-3’.

2. Use of the specific primer according to claim 1 in the preparation of a reagent for identifying Flos Chrysanthemi Indici.

3. A method for rapidly identifying Flos Chrysanthemi Indici based on specific amplification of molecular biology, characterized in that, The main steps are as follows: (1) Effectively extract the total DNA of the sample to be tested to obtain the extracted genomic DNA; (2) Using the genomic DNA extracted in step (1) as a template, perform polymerase chain reaction amplification with the upstream primer and downstream primer according to claim 1; (3) Perform agarose gel electrophoresis detection and gel imaging on the reaction product obtained in step (2) to achieve specific identification of Flos Chrysanthemi Indici.

4. The method for rapidly identifying Flos Chrysanthemi Indici based on specific amplification of molecular biology according to claim 3, characterized in that, In step (3), specifically identify Flos Chrysanthemi Indici according to the result of gel imaging. When a single bright band is observed, the sample to be tested is identified as Flos Chrysanthemi Indici; when a single bright band is not observed, the sample to be tested is identified as not Flos Chrysanthemi Indici.

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