Vanda albicans detection and identification primer, detection method, kit and application of Vanda albicans detection and identification primer

Abstract

The invention belongs to the technical field of plant species identification, and relates to Vanda albicans detection and identification primers, a detection method, a kit and application of the Vanda albicans detection and identification primers, the detection method and the kit, the detection primers are composed of a nucleotide sequence shown in SEQ ID NO.1 and a nucleotide sequence shown in SEQ ID NO.2, genome DNA of Vanda albicans is used as a template, the primers are used for PCR amplification, an amplification product is subjected to enzyme digestion with restriction enzyme AclI, and the amplification product is subjected to PCR amplification. After the enzyme digestion product is subjected to agarose gel electrophoresis, two bands of 300bp and 110bp are obtained, so that the vanda albicans can be quickly identified according to the number and the size of the electrophoretic bands. The method is high in specificity and visual in identification result, and rapid and accurate identification of vanda albicans is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant species identification, and particularly relates to primers for detecting and identifying Vanda brunnea, a detection method, a kit and their applications. Background Art

[0002] Vanda brunnea belongs to the genus Vanda of the Orchidaceae family. It is produced in Yunnan, and is also distributed in Myanmar and Thailand. It is a rare and endangered orchid plant species. Vanda is a monopodial epiphytic orchid with narrow ribbon-shaped leaves having beautiful lines and well-developed fleshy aerial roots; its flower posture is graceful and unrestrained, and its flower color is bright and rich, with excellent ornamental value. It can be cultivated and applied as cut flowers, potted flowers or landscape plants, and is a typical tropical epiphytic orchid.

[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 often difficult to make accurate detections only relying on morphological identification, especially when the plants are not fully developed or there is only part of the plant material. There are many species of Orchidaceae plants, and the molecular level differences of species with relatively close geographical distributions are small. Designing a specific detection method is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0004] The purpose of the present invention is to provide primers for detecting and identifying Vanda brunnea, a detection method, a kit and their applications. This detection method solves the technical problems that the gene sequence differences of similar species of Vanda brunnea are small, it is difficult to locate the specific fragments of Vanda brunnea, and false positive amplification is likely to occur, so as to quickly and accurately detect and identify Vanda brunnea.

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

[0006] The present invention provides a pair of PCR primers for identifying Vanda brunnea, including: the nucleotide sequence shown in SEQ ID NO.1 and the nucleotide sequence shown in SEQ ID NO.2, and their sequences are:

[0007] The sequence of the upstream primer SEQ ID NO.1 is: 5'-CGAAATGCGATACGTGGTGC-3';

[0008] The sequence of the downstream primer SEQ ID NO.2 is: 5'-CTTAAACTCGGCGGGTGGTC-3'.

[0009] The present invention also provides a detection method for quickly identifying Vanda brunnea, including: using the sample DNA as a template, performing PCR amplification with the above primers, digesting the PCR amplification product with a restriction endonuclease, subjecting the digested product to agarose gel electrophoresis, and identifying Vanda brunnea according to the number and size of the electrophoresis bands.

[0010] Specifically, the primers include the upstream primer SEQ ID NO.1 and the downstream primer SEQ ID NO.2, and their sequences are as follows:

[0011] The sequence of the upstream primer SEQ ID NO.1 is: 5'-CGAAATGCGATACGTGGTGC-3';

[0012] The sequence of the downstream primer SEQ ID NO.2 is: 5'-CTTAAACTCGGCGGGTGGTC-3';

[0013] Specifically, taking 25 μL as the calculation basis, the PCR reaction system includes 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, ddH 2 O 17.1 μL.

[0014] The PCR reaction conditions are as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 59°C for 45 s, extension at 72°C for 1 min, for 30 cycles; extension at 72°C for 10 min.

[0015] Specifically, the restriction endonuclease is AclI.

[0016] A detection method for rapid identification of Vanda brunnea provided by the present invention extracts the DNA of the plant sample to be detected, and uses the combination site of specific sequence + primer + endonuclease to detect and identify Vanda brunnea. The present invention rapidly identifies Vanda brunnea according to the number and size of electrophoresis bands. PCR amplification is carried out using the specific primers SEQ ID NO.1 and SEQ ID NO.2. Since the PCR product of Vanda brunnea has an AclI restriction site, two bands appear after its enzymatic digestion and electrophoresis, with sizes of 300 bp and 110 bp respectively; there is no AclI restriction site in the PCR products of other similar species, and only one band appears after enzymatic digestion and electrophoresis, with a size of 410 bp.

[0017] The present invention also provides a detection kit for rapid identification of Vanda brunnea, and the detection kit includes: upstream primer SEQ ID NO.1, downstream primer SEQ ID NO.2.

[0018] Specifically, the sequence of the upstream primer SEQ ID NO.1 is: 5'-CGAAATGCGATACGTGGTGC-3';

[0019] The sequence of the downstream primer SEQ ID NO.2 is: 5'-CTTAAACTCGGCGGGTGG TC-3';

[0020] Specifically, the restriction endonuclease is AclI.

[0021] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects or advantages:

[0022] (1) By measuring and comparing the gene sequences of Orchidaceae plants, a combination site conforming to the specific sequence + primer + restriction endonuclease is designed. Specific primers and restriction endonucleases are designed according to the specific target sequence, solving the technical problems of small differences in the gene sequences of closely related species of Vanda brunnea and difficulty in locating the specific fragments of Vanda brunnea, and can effectively distinguish Vanda brunnea and its closely related species.

[0023] (2) According to the differences in the target gene sequences of Vanda brunnea, specific regions are selected for specific PCR amplification, and then the restriction endonuclease AclI is used to specifically recognize and digest AACGTT. Vanda brunnea is identified by the number and size of the bands generated by agarose gel electrophoresis, and the identification accuracy is high, which can be used to identify Vanda brunnea at different developmental stages. Especially when the plant development is incomplete or only partial plant materials are available, accurate detection and identification can also be made.

[0024] (3) The PCR amplification and digestion techniques used in the present invention are both traditional molecular biology techniques. By ingeniously adopting the combination site of specific sequence + primer + restriction endonuclease, the operation is simple and fast, saving more time compared with the traditional gene sequencing method, and the detection results can be obtained within one day. It can realize rapid and accurate identification and traceability of Vanda brunnea, and solve the technical problem of easy false positive amplification. Description of the Drawings

[0025] Figure 1 It is a schematic diagram of the positions of the specific primers SEQ ID NO.1, SEQ ID NO.2 and the AclI digestion site on the target gene sequence of Vanda brunnea in the present invention.

[0026] Figure 2 It is the amplification result of the PCR product of Vanda brunnea.

[0027] Figure 3 It is the digestion identification result of the PCR product of Vanda brunnea.

[0028] Description of the attached drawing reference signs: A is Vanda brunnea, B is Vanda pumila, C is Vanda cristata, D is Vanda alpina, E is Vanda subconcolor, F is Vanda coerulea, G is Vanda concolor, H is ddH 2 O. Detailed implementation manners

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0030] Embodiment 1

[0031] In this embodiment, a combination site of a specific sequence + primer + restriction enzyme used for the detection method of the Vanda brunnea was designed and synthesized.

[0032] By measuring and comparing the gene sequences of Orchidaceae plants, the differential sites of Vanda brunnea and its similar species were determined, and a combination site conforming to the specific sequence + primer + restriction enzyme was designed to achieve the purpose of specifically identifying Vanda brunnea. This combination should meet the specificity requirements, and its site in the target sequence should be accurate and appropriate, and the target sequence can be enzymatically cleaved into band sizes that meet the identification requirements, so as to quickly identify Vanda brunnea according to the number and size of the electrophoresis bands.

[0033] After screening and comparison, a group of specific primers and restriction enzymes for identifying Vanda brunnea were obtained, and their positions on the gene sequence of Vanda brunnea are as Figure 1 shown, and the specific sequences are as follows:

[0034] The sequence of the upstream primer SEQ ID NO.1 is: 5'-CGAAATGCGATACGTGGT GC-3';

[0035] The sequence of the downstream primer SEQ ID NO.2 is: 5'-CTTAAACTCGGCGGGTGG TC-3';

[0036] The specific restriction enzyme is: AclI.

[0037] Embodiment 2

[0038] In this embodiment, a detection method for identifying Vanda brunnea was established. Calculated based on a 25 μL PCR reaction system, it includes 2.5 μL of 10×PCR buffer (containing Mg 2+ 2+), 2 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 ddH 2 2O.

[0039] The PCR reaction conditions are as follows: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 59 °C for 45 s, extension at 72 °C for 1 min, for 30 cycles; extension at 72 °C for 10 min.

[0040] The present invention also provides a detection kit for rapidly identifying Vanda brunnea. This detection kit includes: upstream primer SEQ ID NO.1 and downstream primer SEQ ID NO.2.

[0041] Using the primer combination of SEQ ID NO.1 and SEQ ID NO.2 obtained in Example 1 for PCR amplification, the amplification result of the PCR product is as Figure 2 shown. For Vanda brunnea, Vanda pumila, Vanda cristata, Vanda alpina, Vanda subconcolor, Vanda coerulea, and Vanda erythraeae, a band with a size of 410 bp was amplified.

[0042] Using a restriction endonuclease to digest the PCR amplification product, and subjecting the digested product to agarose gel electrophoresis, the digestion identification result of the PCR product is as Figure 3 shown. Since the PCR product of Vanda brunnea has an AclI digestion site, after digestion and electrophoresis, two bands appear, with sizes of 300 bp and 110 bp respectively. Vanda brunnea is identified based on the number and size of the electrophoresis bands.

[0043] The above details the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In summary, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made using conventional techniques known in the art. Some basic features can be applied according to the scope of the following appended claims.

Claims

1. A PCR primer for identifying Vanda leucopsis, characterized in that: include: The nucleotide sequence shown in SEQ ID NO.1 and the nucleotide sequence shown in SEQ ID NO.2 are as follows: The sequence of the upstream primer SEQ ID NO.1 is: 5'-CGAAATGCGATACGTGGT GC-3'; The sequence of the downstream primer SEQ ID NO.2 is: 5'-CTTAAACTCGGCGGGTGG TC-3'.

2. A method for rapid identification of Vanda leucopsis, characterized in that: include: The sample DNA is used as a template, PCR amplification is performed using the primers described in claim 1, the PCR amplification product is digested with a restriction endonuclease, the digestion product is subjected to agarose gel electrophoresis, and the white column Vanda orchid is identified according to the number and size of the electrophoresis bands.

3. The detection method according to claim 2, characterized in that: The restriction endonuclease is AcII.

4. The detection method according to claim 2, characterized in that: The number and size of electrophoretic bands were used to identify Vanda alba, and the positive result was determined based on two specific bands of 300 bp and 110 bp.

5. Use of the detection method according to any one of claims 2 to 4 in detecting and identifying Vanda alba.

6. A detection kit for rapid identification of Vanda leucopsis, characterized in that: include: The primer according to claim 1.

7. The rapid identification kit for Vanda leucopsis according to claim 6, characterized in that: Also included is a restriction endonuclease, wherein the restriction endonuclease is AcII.

8. Use of the kit according to any one of claims 6 to 7 in detecting and identifying Vanda alba.

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