Primer, detection method and kit for identifying lysimachia christinae by enzyme digestion and application of primer, detection method and kit

Abstract

The invention belongs to the technical field of plant species identification, and relates to a primer, a detection method and a kit for identifying cymbidium turcicum through enzyme digestion and application of the primer, the detection primer is composed of a nucleotide sequence shown in SEQ ID NO.1 and a nucleotide sequence shown in SEQ ID NO.2, genome DNA of cymbidium turcicum serves as a template, PCR amplification is conducted through the primer, an amplification product is subjected to enzyme digestion through restriction endonuclease EcoRV, and the amplification product is subjected to PCR amplification through enzyme digestion. After the enzyme digestion product is subjected to agarose gel electrophoresis, two bands of 265bp and 216bp are obtained, so that the cymbidium sibiricum can be quickly identified according to the number and the size of the electrophoretic bands. The method disclosed by the invention is high in specificity and visual in identification result, and realizes rapid and accurate detection and identification of the cymbidium turcicum.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant species identification, and particularly relates to a primer for identifying Anthogonium gracile by enzyme digestion, a detection method, a kit and application thereof. BACKGROUND

[0002] Anthogonium gracile belongs to Orchidaceae Anthogonium, and is an endangered protected land orchid variety with ornamental value and ecological function. Anthogonium gracile contains amino acids, sugars, alkaloids, flavones and other substances, and has medicinal research value and market prospect. Due to the continuous exploitation of the economic value of Anthogonium gracile, human interference and destruction, the wild resources of Anthogonium gracile are close to extinction.

[0003] At present, the detection and identification of plant species resources at home and abroad are mainly carried out by observing and analyzing morphological characteristics. It is difficult to make accurate detection only by morphological identification, especially when the plant development is incomplete or only part of the plant material is available, and when the plant is processed. Orchid plants are various in species, and the molecular level difference between species with similar geographical distribution is small. Therefore, it is a technical problem to be solved in the field to design a specific detection method. SUMMARY

[0004] The purpose of the present application is to provide a primer for identifying Anthogonium gracile by enzyme digestion, a detection method, a kit and application thereof. The detection method solves the technical problems of small difference in gene sequences of Anthogonium gracile similar species, difficult positioning of Anthogonium gracile specific fragments and easy false positive amplification, so as to quickly and accurately detect and identify Anthogonium gracile.

[0005] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0006] The present application also provides a detection method for quickly identifying Anthogonium gracile, which comprises: using sample DNA as a template, using a primer composed of a nucleotide sequence shown in SEQ ID NO. 1 and a nucleotide sequence shown in SEQ ID NO. 2 for PCR amplification, using a restriction endonuclease to perform enzyme digestion on the PCR amplification product, performing agarose gel electrophoresis on the enzyme digestion product, and identifying Anthogonium gracile according to the number and size of electrophoresis bands.

[0007] Specifically, the primer comprises an upstream primer SEQ ID NO. 1 and a downstream primer SEQ ID NO. 2, and the sequences are as follows:

[0008] The sequence of the upstream primer SEQ ID NO. 1 is 5'-GAGAACCCGTGAAGCAA-3';

[0009] The sequence of the downstream primer SEQ ID NO. 2 is 5'-CCAACGAGGCGATAGCA-3';

[0010] Specifically, the PCR reaction system, calculated in 25 μL volumes, includes 10× PCR buffer (containing Mg). 2+ 2.5 μL of dNTP mixture (2.5 mmol / L), 0.4 μL of Taq DNA polymerase (5 U / μL), 1 μL of upstream primer SEQ ID NO.1 (10 μmol / L), 1 μL of downstream primer SEQ ID NO.2 (10 μmol / L), 1 μL of DNA template, and 17.1 μL of ddH2O.

[0011] The PCR reaction conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 54℃ annealing for 45 s, 72℃ extension for 1 min, for 30 cycles; and 72℃ extension for 10 min.

[0012] Specifically, the restriction endonuclease mentioned is EcoRV.

[0013] This invention provides a rapid identification method for *Cymbidium goeringii*. DNA is extracted from the plant sample, and *Cymbidium goeringii* is identified using a combination of specific sequences, primers, and restriction enzymes. This invention rapidly identifies *Cymbidium goeringii* based on the number and size of electrophoretic bands. PCR amplification is performed using specific primers SEQ ID NO.1 and SEQ ID NO.2. *Cymbidium goeringii* amplifies a single band of 481 bp, while other similar species do not produce amplification products. Furthermore, because the PCR product of *Cymbidium goeringii* contains the EcoRV restriction site, digestion and electrophoresis result in two bands of 265 bp and 216 bp in size, respectively.

[0014] The present invention also provides a rapid identification kit for Cymbidium goeringii, the kit comprising: upstream primer SEQ ID NO.1 and downstream primer SEQ ID NO.2.

[0015] Specifically, the upstream primer SEQ ID NO.1 sequence is: 5'-GAGAACCCGT GAAGCAA-3';

[0016] The downstream primer SEQ ID NO.2 sequence is: 5'-CCAACGAGGCGATAGCA-3';

[0017] Specifically, the restriction endonuclease mentioned is EcoRV.

[0018] Compared with the prior art, the technical solution provided by this invention has the following beneficial effects or advantages:

[0019] (1) By determining the target gene sequence of orchid plants, sequence alignment analysis of the difference sites of Anthogonium gracile and its approximate species, and according to the design of specific primers and restriction endonucleases of the target gene sequence of Anthogonium gracile, the technical problems of small difference in gene sequence of Anthogonium gracile approximate species and difficult positioning of Anthogonium gracile specific fragments are solved, and Anthogonium gracile and its approximate species can be effectively distinguished.

[0020] (2) According to the difference of the target gene sequence of Anthogonium gracile, the specific region is selected for specific PCR amplification, and then the specific recognition enzyme cutting of GATATC by restriction endonuclease EcoRV is used, and the number and size of bands produced by agarose gel electrophoresis are used to identify Anthogonium gracile, which has high accuracy and is used to identify Anthogonium gracile at different development stages. Especially when the plant development is not complete or only part of the plant material, and after processing, accurate detection and identification can be made.

[0021] (3) The PCR amplification and enzyme cutting technology used in the present application are traditional molecular biology technologies, which are simple and fast by skillfully using the combination of specific sequences + primers + endonucleases. Compared with the gene sequencing method, it saves time and can get the detection result within one day. It can realize rapid and accurate identification and tracing of Anthogonium gracile, has high amplification efficiency, and solves the technical problem of false positive amplification. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is a schematic diagram of the position of specific primers SEQ ID NO. 1, SEQ ID NO. 2 and EcoRV enzyme cutting site on the target gene sequence of Anthogonium gracile.

[0023] Figure 2 It is the amplification result of the PCR product of Anthogonium gracile. Among them, A is Anthogonium gracile, B is Thunia alba, C is Phalaenopsis equestris, D is Acanthephippium sylhetense, E is Nervilia fordii, F is Spathoglottis pubescens, G is Bletilla striata, and H is ddH2O.

[0024] Figure 3 It is the enzyme cutting identification result of the PCR product of Anthogonium gracile. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope of the present application.

[0026] Embodiment 1

[0027] In this embodiment, a combination site of a specific sequence, a primer and an endonuclease used in the detection method of the Cymbidium lancifolium is designed and synthesized.

[0028] By comparing and analyzing the gene sequences of the orchid plants, a combination site of a specific sequence, a primer and an endonuclease is designed to achieve the purpose of specifically identifying the Cymbidium lancifolium. The combination site needs to meet the specificity requirement, and the site in the target sequence needs to be accurately appropriate, so as to cut the target sequence into a band size meeting the identification requirement, thereby rapidly identifying the Cymbidium lancifolium according to the number and size of the electrophoresis bands.

[0029] After screening and comparison, a specific primer and a restriction endonuclease for identifying the Cymbidium lancifolium are obtained, and the position of the specific primer and the restriction endonuclease on the target gene sequence of the Cymbidium lancifolium is as shown in Table 1. Figure 1 The specific sequences are as follows:

[0030] The sequence of the upstream primer SEQ ID NO. 1 is 5'-GAGAACCCGTGAAGCAA-3';

[0031] The sequence of the downstream primer SEQ ID NO. 2 is 5'-CCAACGAGGCGATAGCA-3';

[0032] The specific restriction endonuclease is EcoRV.

[0033] Embodiment 2

[0034] In this embodiment, a detection method for identifying the Cymbidium lancifolium is established. The PCR reaction system is calculated as 25 μL, including 10×PCR buffer (containing Mg 2+ ) 2.5 μL, dNTP mixture (2.5 mmol / L) 2 μL, Taq DNA polymerase (5 U / μL) 0.4 μL, upstream primer SEQ ID NO. 1 (10 μmol / L) 1 μL, downstream primer SEQ ID NO. 2 (10 μmol / L) 1 μL, DNA template 1 μL and ddH2O 17.1 μL.

[0035] The PCR reaction conditions are as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 54℃ annealing for 45 s, 72℃ extension for 1 min, 30 cycles; and 72℃ extension for 10 min.

[0036] The application also provides a detection kit for rapidly identifying Cymbidium lancifolium, which comprises an upstream primer SEQ ID NO. 1 and a downstream primer SEQ ID NO. 2.

[0037] The primer SEQ ID NO. 1 and the primer SEQ ID NO. 2 obtained in Example 1 are combined to perform PCR amplification, and the PCR product amplification result is shown in Figure 2 Cymbidium lancifolium amplifies a band with a size of 481 bp, and other similar species have no amplification product.

[0038] The PCR amplification product is subjected to enzyme cutting by using a restriction enzyme, and the enzyme cutting product is subjected to agarose gel electrophoresis, and the enzyme cutting identification result of the PCR product is shown in Figure 3 As the PCR product of Cymbidium lancifolium has an EcoRV enzyme cutting site, two bands with sizes of 265 bp and 216 bp appear after enzyme cutting and electrophoresis, and Cymbidium lancifolium is identified according to the number and size of the electrophoresis bands.

[0039] The above describes the application in detail. For those skilled in the art, the application can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In general, according to the principle of the application, the application intends to include any change, use or improvement of the application, including changes made by using conventional techniques known in the art, which deviates from the range disclosed in the application. Some basic features can be applied according to the scope of the following attached claims.

Claims

1. A detection method for rapid identification of Acampe, characterized in that, Comprising: Using sample DNA as a template, performing PCR amplification using primers consisting of the nucleotide sequence shown in SEQ ID NO. 1 and the nucleotide sequence shown in SEQ ID NO. 2, performing enzyme cutting on the PCR amplification product by a restriction endonuclease, performing agarose gel electrophoresis on the enzyme cutting product, and identifying the Cypripedium according to the number and size of the electrophoresis bands.

2. The detection method according to claim 1, characterized in that, The restriction endonuclease is EcoRV.

3. The method of claim 1, wherein, Identifying the Cypripedium according to the number and size of the electrophoresis bands, and determining a positive result according to two specific bands of 265 bp and 216 bp.

4. Use of the detection method according to any one of claims 1-3 in identifying Cypripedium.

5. A test kit for rapid identification of Oncidium, characterized by, Comprising: The primers according to claim 1.

6. The test kit for rapid identification of Oncidium according to claim 5, characterized by, The restriction endonuclease is EcoRV.

7. Use of the kit according to any one of claims 5-6 in identifying Cypripedium.

Images