Probe, primer and method for identifying meduralis

Abstract

The invention belongs to the field of plant detection and identification, in particular relates to the technical field of job detection and identification by utilizing a molecular biological technology, and more particularly relates to a probe, a primer and a method for identifying medustar. Aiming at the technical blank in the field of non-morphological identification of astrophe plants in China at present and the identification problem that the morphological identification of the astrophe plants depends on seed morphology and is difficult to accurately distinguish, the invention provides a rapid and accurate method for identifying the astrophe plants, and by virtue of a specific primer and a fluorescent probe disclosed by the invention, the rapid and accurate identification of the astrophe plants can be realized. Therefore, a real-time fluorescent quantitative PCR technology is utilized to quickly and accurately obtain the identification result of the meduscens. The method has the characteristics of high sensitivity, strong specificity, good repeatability, high throughput and the like, so that a new method and thought are provided for identification of endangered species.

Description

Technical Field

[0001] The present invention belongs to the field of plant detection and identification, and in particular to the technical field of plant detection and identification using molecular biology techniques, and more specifically to probes, primers and methods for identifying the planet Medusa. Background Art

[0002] Planet Medusa Astrophytum caput-medusae ) is a plant of the genus Medusa in the Cactaceae family, native to Nuevo León, Mexico. Medusa is listed in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (ITES). my country joined the convention on December 25, 1980 and became one of the contracting parties.

[0003] Because the whole plant of the genus Astragali has white star-shaped dots, its dignified posture, dark green skin inlaid with white dots, and elegant warty hairs on the plant, it has extremely high ornamental value, so it is loved and sought after by domestic enthusiasts. The identification of endangered species at customs ports is an important means of protecting endangered wild animals and plants, and is of great significance to the protection of endangered wild animal and plant resources.

[0004] However, the current identification of Astrophytum plants in China is mainly based on morphology, and morphological identification is highly dependent on professional identification personnel, which results in the inability of customs ports to deploy a large number of morphological identification experts during the identification process. At the same time, the seed morphology of Astrophytum plants is very similar and difficult to distinguish, so it is urgent to find a convenient, fast and accurate identification method to provide technical support for the identification of endangered species at customs ports. Summary of the invention

[0005] The present invention aims at the current domestic technical gap in the field of non-morphological identification of Medusa plants, and the identification problem that the morphological identification of Medusa plants relies on similar seed morphology and is difficult to accurately distinguish, and proposes a fast and accurate Medusa identification method, thereby providing a new method and idea for the identification of endangered species.

[0006] In order to solve the above technical problems and achieve the above invention objectives, the present invention discloses a probe for identifying the Medusa planet, wherein the probe YCF-ACP sequence is shown in SEQ ID NO: 1, the 5' end of the probe YCF-ACP is modified by a fluorescent group, and the 3' end of the probe YCF-ACP is modified by a fluorescence quenching group.

[0007] Preferably, the fluorescent group is FAM, and the fluorescence quenching group is BHQ.

[0008] At the same time, the present invention also discloses primers YCF-ACFP and YCF-ACRP for identifying the planet Medusa, wherein the sequence of the primer YCF-ACFP is shown in SEQ ID NO: 2, and the sequence of the primer YCF-ACRP is shown in SEQ ID NO: 3.

[0009] The present invention also discloses a primer probe set consisting of the above probe and primer.

[0010] At the same time, the present invention further discloses a kit for identifying the Medusa planet, wherein the kit includes a primer probe set consisting of a probe YCF-ACP, a primer YCF-ACFP and a primer YCF-ACRP.

[0011] In addition, the present invention also discloses a reagent for identifying the Medusa planet, wherein the reagent contains a primer pair and a probe YCF-ACP, wherein the primer pair consists of a primer YCF-ACFP and a primer YCF-ACRP.

[0012] Furthermore, the present invention also discloses a method for identifying the Medusa planet, comprising the following steps: (1) Extracting template DNA from the sample to be tested; (2) preparing a PCR reaction system using primers YCF-ACFP, primers YCF-ACRP, and probe YCF-ACP; (3) The extracted template DNA of the sample to be tested is subjected to real-time fluorescence quantitative PCR reaction, and a qualitative test is performed based on the PCR results. The judgment method is that if a logarithmic growth (CT < 40) occurs, it is positive, indicating that the sample to be tested is the planet Medusa; if no logarithmic growth occurs, it is negative, indicating that the sample to be tested is not the planet Medusa.

[0013] In a specific technical solution, the PCR reaction system is: a total volume of 20 μL, including 10 μL of fluorescent PCR reaction mixture, 0.8 μL of upstream and downstream primers with a concentration of 10 mol / L, 0.4 μL of probe with a concentration of 10 mol / L, 2.0 μL of sample template DNA to be detected, 0.4 μL of ROX reagent, and the balance is water.

[0014] In a specific technical solution, the reaction parameters of real-time fluorescence quantitative PCR are: 94° C. for 15 s, 60° C. for 1 min, and 40 cycles.

[0015] The inventors of the present invention sequenced and analyzed the chloroplast genome of the genus Astrophytum and found that the genus Astrophytum exists ycf1(hypothetical chloroplast open reading frame 1) functional gene sequence (SEQID NO: 4), and compared with other genus plants, there are 6 mutation sites in the functional gene sequence of Medusa, such as Figure 1 As shown, the six base mutation sites are marked with red boxes.

[0016] against Figure 1 We designed and obtained specific primers and fluorescent probes for the gene sequence shown in the figure, so that we can quickly and accurately obtain the identification results of the Medusa Planet using real-time fluorescence quantitative PCR technology. This method has the characteristics of high sensitivity, strong specificity, good repeatability and high throughput. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 For the planet Medusa ycf1 Schematic diagram of base mutation sites.

[0018] Figure 2 This is a schematic diagram of the real-time fluorescence PCR amplification results of the endogenous genes of the sample to be tested.

[0019] Figure 3 This is a schematic diagram of the real-time fluorescence quantitative PCR specific amplification results of the Medusa planet.

[0020] Figure 4 Schematic diagram of the real-time fluorescence PCR sensitivity results of the sensitivity experiment on the Medusa planet sample As13.

[0021] Figure 5 This is the standard curve of real-time fluorescence PCR amplification of the sensitivity experiment of Medusa planet sample As13. DETAILED DESCRIPTION

[0022] In order to better understand the present invention, the present invention is further described below in conjunction with specific examples. The experimental methods used in the following examples are all conventional experimental operation methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0023] TaKaRa Premix Ex TapTM (Probe qPCR) was purchased from Nanjing E-Tep Instrument Co., Ltd., and the plant genomic DNA extraction kit was purchased from Beijing Tiangen Biochemical Technology Co., Ltd. The real-time fluorescence PCR instrument was ABI 7500 FAST fluorescence quantitative PCR instrument.

[0024] A total of 21 test materials were collected, including 17 samples of 6 species of Echinocactus and 4 samples of 4 species of Echinocactus plants. The test materials were intercepted from Nanjing, Jiangsu, Zhangzhou, Fujian, and ports. They were all reviewed and identified by experts from Nanjing Zhongshan Botanical Garden, and fresh plant tissues were collected for testing. Detailed information on the samples is shown in Table 1: Table 1 Plant materials and sources Example 1

[0025] The samples in Table 1 were surface disinfected respectively, the plant tissues were ground into powder using a mortar, and the genomic DNA of the experimental samples was extracted according to the steps and methods in the instructions of the plant genomic DNA extraction kit. The extracted genomic DNA was stored at 4°C for later use.

[0026] According to the conserved gene sequence unique to Medusa, the specific primers and probes for Medusa were designed using Primer Express 3.0 software, and their sequences are shown in Table 2. The 5′ end of the probe contains a FAM fluorescent reporter group, and the 3′ end contains a non-fluorescent quencher group BHQ1.

[0027] Table 2 Primers and probes for real-time fluorescence RT-PCR detection of Medusa

[0028] The real-time fluorescence PCR test was performed on the extracted DNA template using the universal plant endogenous gene 18SrRNA. The upstream and downstream primers and probes of the endogenous gene were designed and synthesized with reference to the 18SrRNA sequence in SN / T 1204-2016. The sample to be tested should have a typical amplification curve. If no real-time fluorescence PCR amplification curve appears, it means that there is a problem with the quality of DNA extraction, or there are factors that inhibit the PCR reaction in the DNA extract. The DNA should be re-extracted until the PCR product is amplified. The amplification conditions are shown in Table 3; Table 3 Real-time fluorescence PCR reaction system

[0029] The reaction parameters of real-time fluorescence quantitative PCR were: 94°C for 15s, 60°C for 1min, and 40 cycles.

[0030] It should be noted that different instruments can adjust the reaction parameters appropriately according to the requirements of the instrument and the reaction reagents.

[0031] The results showed that the real-time fluorescence PCR test of the genomic DNA of 21 samples was performed using the universal plant endogenous gene 18SrRNA, and all the samples had typical amplification curves, such as Figure 2As shown in the figure, the Ct value is between 10 and 18, indicating that the DNA extraction quality of all samples is good and subsequent experiments can be carried out.

[0032] Using the specific probe YCF-ACP, primers YCF-ACFP and YCF-ACRP designed by the present invention, real-time fluorescence quantitative PCR was performed on 21 sample DNAs according to the real-time fluorescence PCR reaction system in Table 3 and the aforementioned reaction parameters (94°C 15s, 60°C 1min, 40 cycles). The results are as follows: Figure 3 As shown, it can be seen that only three Medusa samples (As13, As16 and As17) show typical amplification curves, indicating that the primers and fluorescent probes disclosed in the present invention have good specificity for Medusa. Example 2

[0033] The genomic DNA of the Medusa planet sample As13 was extracted and the nucleic acid concentration was determined to be 4.57 ng / μL. The sample DNA was diluted 10 times to obtain 10 -1 ~10 -7 Dilution sample.

[0034] The seven concentration dilution samples were subjected to real-time fluorescence quantitative PCR detection according to the PCR amplification method disclosed in Example 1 to detect the sensitivity of the real-time fluorescence quantitative PCR method disclosed in the present invention.

[0035] The amplification results are as follows Figure 4 As shown, it can be seen that the nucleic acid stock solution and 10 -1 ~10 -5 Typical amplification curves were obtained for samples with 10-fold dilution, and their Ct values ​​were 19.003, 22.651, 25.989, 29.446, 33.760, and 37.167, respectively. -6 and 10 -7 The DNA dilution samples did not produce a typical amplification curve.

[0036] Furthermore, we prepared a standard curve diagram for the sensitivity test based on the amplification results ( Figure 5 ), it can be seen that the standard curve correlation coefficient of the sensitivity test is 0.997. Therefore, the detection limit of the present invention is 4.6×10 -5 ng / μL.

[0037] The above is a specific implementation of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications are also considered to be within the protection scope of the present invention.

Claims

1. Identify the probe of the planet Medusa, its characteristics are, The sequence of the probe YCF-ACP is shown in SEQ ID NO:

1. The 5' end of the probe YCF-ACP is modified with a fluorescent group, and the 3' end of the probe YCF-ACP is modified with a fluorescent quenching group.

2. The probe for identifying the planet Medusa according to claim 1, characterized in that: The fluorescent group is FAM, and the fluorescent quenching group is BHQ.

3. The primer pair for identifying the planet Medusa is characterized by: The primer comprises a YCF-ACFP primer and a YCF-ACRP primer. The YCF-ACFP primer sequence is shown in SEQ ID NO: 2, and the YCF-ACRP primer sequence is shown in SEQ ID NO:

3.

4. A primer-probe set consisting of the probe according to claim 1 and the primer according to claim 3.

5. A kit for identifying the planet Medusa, characterized in that: The kit comprises a primer probe set consisting of the probe YCF-ACP described in claim 1 and the primers YCF-ACFP and YCF-ACRP described in claim 3.

6. The reagent for identifying the planet Medusa is characterized by: The reagent contains the primer pair according to claim 3 and the probe YCF-ACP according to claim 1.

7. The identification method of Medusa Planet is characterized by: The following steps are involved: (1) Extracting template DNA from the sample to be tested; (2) preparing a PCR reaction system using primers YCF-ACFP, primers YCF-ACRP, and probe YCF-ACP; (3) The extracted template DNA of the sample to be tested is subjected to real-time fluorescence quantitative PCR reaction, and a qualitative test is performed based on the PCR results. The judgment method is that if a logarithmic growth (CT < 40) occurs, it is positive, indicating that the sample to be tested is the planet Medusa; if no logarithmic growth occurs, it is negative, indicating that the sample to be tested is not the planet Medusa.

8. The method for identifying the Medusa planet according to claim 7, characterized in that: The PCR reaction system is as follows: a total volume of 20 μL, including 10 μL of fluorescent PCR reaction mixture, 0.8 μL of upstream and downstream primers with a concentration of 10 mol / L, 0.4 μL of probe with a concentration of 10 mol / L, 2.0 μL of sample template DNA to be detected, 0.4 μL of ROX reagent, and the balance is water.

9. The method for identifying the Medusa planet according to claim 7 or 8, characterized in that: In a specific technical solution, the reaction parameters of real-time fluorescence quantitative PCR are: 94° C. 15 s, 60° C. 1 min, and 40 cycles.

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