PCR enzyme typing detection and identification method, kit and application of Rhododendron variegated

Abstract

The present invention belongs to the technical field of plant species identification, and specifically relates to a PCR enzyme cutting typing detection and identification method, a kit and an application of variegated rhododendron orchid. The detection method for identifying variegated rhododendron orchid comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2. The genomic DNA of variegated rhododendron orchid is used as a template, and the primer pair is used for PCR amplification, and the restriction endonuclease is used to Okay I enzyme digestion amplification product, after agarose gel electrophoresis, can obtain two bands of 467bp and 101bp, which are used for rapid identification of variegated rhododendron orchid. The method of the invention has strong specificity, intuitive identification results, and can rapidly and accurately detect and identify variegated rhododendron orchid.

Description

Technical Field

[0001] The invention belongs to the technical field of plant species identification, and specifically relates to a PCR enzyme cutting typing detection and identification method for variegated rhododendron orchid, a kit and an application thereof. Background Art

[0002] Variegated rhododendron orchid Cremastra unguiculata ) is a perennial herbaceous plant belonging to the genus Rhododendron of the Orchidaceae family. Its rhizomes and pseudobulbs have high medicinal value and have the effects of clearing away heat and detoxifying, resolving phlegm and dispersing knots. At the same time, the scape of Rhododendron variegatedum is upright and the flowers are brightly colored, which has a high ornamental value. Due to long-term large-scale excavation, destruction of the growth environment, and difficulty in natural pollination, it has become difficult to breed, and its wild resources have been sharply reduced and are on the verge of extinction. It has been listed in the "National Key Protected Wild Plant List". With the development and utilization of the medicinal effects of Rhododendron variegatedum and its artificial cultivation, it is easy to be confused with varieties and adulterated after processing.

[0003] At present, the detection and identification of variegated azalea orchid and similar species at home and abroad are mainly carried out through observation and analysis of morphological characteristics, such as the morphology of flowers or leaves. It is often difficult to make an accurate identification based solely on morphological identification, especially when the plant is not fully developed or only part of the plant material is available. After processing, the original characteristics of variegated azalea orchid have disappeared, and it will be impossible to effectively identify it through trait identification methods.

[0004] There are many species of orchids, and species with close geographical distribution have small differences at the molecular level. Designing a specific identification method is a technical problem that needs to be solved in this field. At present, there is a lack of technical reports on the effective identification of variegated rhododendron orchids at the molecular level. After inquiry, there are no reports on the rapid identification method of variegated rhododendron orchids, especially the molecular biological identification method, at home and abroad. Summary of the invention

[0005] In view of the above problems, the purpose of the present invention is to provide a PCR enzyme cutting typing detection and identification method, kit and application of variegated rhododendron orchid, especially a PCR primer, detection method, kit and application suitable for rapid identification of variegated rhododendron orchid. The detection method can solve the technical problems that the gene sequence differences of similar species of variegated rhododendron orchid are small, the location of specific fragments of variegated rhododendron orchid is difficult, and false positive amplification is prone to occur, so that variegated rhododendron orchid can be detected, distinguished and identified quickly and accurately.

[0006] To this end, the present invention provides the following technical solutions:

[0007] Firstly, the present invention provides a PCR primer for rapid identification of variegated rhododendron orchid, comprising: an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2.

[0008] in:

[0009] The nucleotide sequence of the upstream primer SEQ ID NO.1 is: 5'-AGCCGTCCCAGTCGTTC-3';

[0010] The nucleotide sequence of the downstream primer SEQ ID NO. 2 is: 5'-AGCCGCCGCCTGTGTAT-3'.

[0011] Furthermore, the present invention provides a PCR enzyme digestion typing detection and identification method for variegated rhododendron orchid, which method comprises the following steps: using sample DNA as a template, performing PCR amplification using PCR primers, using restriction endonucleases to digest the PCR amplification products, subjecting the digestion products to agarose gel electrophoresis, and identifying variegated rhododendron orchid according to the number and size of specific electrophoresis bands. Wherein, the PCR primers include an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2; the restriction endonucleases are Afa I.

[0012] Preferably, the specific electrophoresis band includes two electrophoresis bands with sizes of 467 bp and 101 bp respectively.

[0013] Preferably, the total reaction system of the PCR amplification is calculated as 25 μL, including Mg 2+ 2.5 μL 10× PCR buffer, 2 μL 2.5 mmol / L dNTP mixture, 0.4 μL 5 U / μL Taq DNA polymerase, 1 μL 10 μmol / L upstream primer of the nucleotide sequence as shown in SEQ ID NO.1, 1 μL 10 μmol / L downstream primer of the nucleotide sequence as shown in SEQ ID NO.2, 1 μL DNA template, 17.1 μL ddH2O.

[0014] Preferably, the reaction conditions for the PCR amplification are: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 56°C for 45 s, extension at 72°C for 1 min, 40 cycles; extension at 72°C for 10 min.

[0015] Specifically, when using the PCR enzyme typing detection and identification method of the present invention, the following procedure can be used: extract sample DNA, use the sample DNA as a template, use a specific primer pair (SEQ ID NO.1, SEQ ID NO.2) for PCR amplification, and use a restriction endonuclease to amplify the amplified product. Afa I was used for enzyme digestion, and the digestion products were subjected to agarose gel electrophoresis. Before digestion, in the PCR amplification products, the variegated rhododendron orchid ( Cremastra unguiculata )、Azalea orchid( Cremastra appendiculate ) amplified a band of 568 bp, while other similar species had no specific electrophoresis bands. After enzyme digestion, the variegated azalea orchid showed specific electrophoresis bands of 467 bp and 101 bp, respectively, while other similar species did not have these two specific electrophoresis bands. Therefore, the variegated azalea orchid can be identified based on the number and size of electrophoresis bands, which improves the accuracy and efficiency of identification.

[0016] Then, the present invention provides a detection kit for identifying variegated azalea orchid, comprising an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2.

[0017] Preferably, the detection kit also contains a restriction endonuclease Afa I.

[0018] Finally, the present invention provides the use of the aforementioned PCR enzyme cleavage typing detection and identification method, primers and detection kit in identifying variegated azalea orchid. The variegated azalea orchid includes variegated azalea orchid plants or processed products. That is, the PCR enzyme cleavage typing detection and identification method and the detection kit can be used for the identification of variegated azalea orchid plants or processed products.

[0019] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects or advantages:

[0020] (1) The PCR enzyme cutting typing detection and identification method of the present invention has a wide range of applications, and is applicable to whole plants, incompletely developed plants, only partial plant materials or processed products, thereby increasing the scope of application of the identification method of variegated rhododendron orchid.

[0021] (2) The PCR enzyme digestion typing detection and identification method of the present invention combines PCR with enzyme digestion, which not only overcomes the problem that simple PCR cannot effectively distinguish between variegated rhododendron orchid and similar species, but is also simple and fast. Compared with the traditional gene sequencing method, it saves more time and can obtain the test results within one day. It can realize the rapid and accurate identification and tracing of variegated rhododendron orchid in one reaction using a pair of primers and restriction endonucleases, and solves the technical problem of false positive amplification.

[0022] (3) The PCR enzyme digestion typing detection and identification method of the present invention has high PCR primer amplification efficiency and strong specificity. It can quickly identify the variegated azalea orchid from multiple similar species with only two electrophoresis bands combined with enzyme digestion and agarose gel electrophoresis. It solves the technical problem that the gene sequence differences of similar species of variegated azalea orchid are small and the specific fragments of variegated azalea orchid are difficult to locate, and can effectively distinguish the variegated azalea orchid and its similar species. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The specific primers SEQ ID NO.1, SEQ ID NO.2 and Afa Schematic diagram of the location of the I restriction site on the ribosomal gene sequence of Rhododendron variegatedefolium;

[0024] Figure 2 The PCR product amplification and identification results of Example 2;

[0025] Figure 3 The result of enzyme digestion identification of the PCR product in Example 2;

[0026] Among them, A is azalea orchid Cremastra unguiculata , B is azalea Cremastra appendiculate , C is mountain orchid Oreorchis patens , D is coral orchid Corallorhiza trifida , E is the tube distance blue Tipularia szechuanica , F is single flower orchid Amoeba changes , G is the bag orchid Calypso bulbosa , H is ddH2O. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The plant samples used in the following embodiments include variegated rhododendron orchid. Cremastra unguiculata , Rhododendron Cremastra appendiculata , Shanlan Oreorchis patens , Coral Orchid Corallorhiza trifida 、Tube distance blue Tipularia szechuanica , Orchidaceae Amoeba changes , Orchidaceae Calypso bulbosa All of them have been identified and confirmed by morphological characteristics.

[0028] Example 1 Establishment of specific primer pairs and restriction endonucleases

[0029] In this embodiment, a specific primer pair used in the detection method of the variegated rhododendron orchid is designed and synthesized.

[0030] By determining the ribosomal gene sequence of orchids and comparing it with the GenBank DNA sequence database, the difference sites of the variegated rhododendron orchid and its similar species were determined. According to the comparison results, the specific sites of the variegated rhododendron orchid were manually found and specific primers were designed upstream and downstream of the specific sites. The designed primers were checked for mismatches, dimers and hairpin structures, as well as for homologous sequences. Then, within the specific primer range of the variegated rhododendron orchid, the difference sites that can be distinguished from other species were manually found; in this way, specific enzyme cleavage sites were found. The enzyme cleavage sites must meet the specificity requirements, and their sites in the target sequence must be accurate and appropriate, so that the target sequence can be enzymatically cut into a band size that meets the identification requirements, so that the variegated rhododendron orchid can be quickly identified according to the number and size of electrophoretic bands.

[0031] After screening and comparison, a set of specific primer pairs and restriction endonucleaf primers for identifying Rhododendron variegatede enzyme Its position on the ribosomal gene sequence of Rhododendron variegatede Figure 1 The specific sequence is as follows:

[0032] The nucleotide sequence of the upstream primer SEQ ID NO.1 is: 5'-AGCCGTCCCAGTCGTTC-3';

[0033] The nucleotide sequence of the downstream primer SEQ ID NO.2 is: 5'-AGCCGCCGCCTGTGTAT-3';

[0034] Specific restriction endonucleases are: Afa I.

[0035] Example 2 Establishment of detection method

[0036] 1. PCR amplification and results

[0037] In this example, a PCR amplification detection method for identifying variegated rhododendron orchid was established. The total reaction system of PCR amplification was calculated as 25 μL, including 2.5 μL 10× PCR buffer (containing Mg 2+ ), 2 μL 2.5 mmol / L dNTP mixture, 0.4 μL 5U / μL Taq DNA polymerase, 1 μL 10 μmol / L upstream primer shown in SEQ ID NO.1, 1 μL 10 μmol / L downstream primer shown in SEQ ID NO.2, 1 μL DNA template, 17.1 μL ddH2O.

[0038] PCR reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 56°C for 45 s, and extension at 72°C for 1 min, 40 cycles; extension at 72°C for 10 min.

[0039] Therefore, this embodiment also establishes a detection kit for rapid identification of variegated leaf azalea orchid, and the detection kit comprises: an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1, and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2. The detection kit for rapid identification of variegated leaf azalea orchid may further comprise a restriction endonuclease Afa I.

[0040] When performing sample identification, sample DNA is extracted and used as a template. PCR amplification is performed using the primer pair obtained in Example 1 (the nucleotide sequences of the upstream and downstream primers are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively), and the amplified product is subjected to agarose gel electrophoresis.

[0041] like Figure 2 The PCR product amplification results showed that the variegated rhododendron orchid ( Cremastra unguiculata )、Azalea orchid( Cremastra appendiculata ) amplified a band of 568 bp. Other similar species had no specific electrophoresis bands. It can be seen that the PCR amplification product cannot distinguish between the variegated rhododendron orchid and the rhododendron orchid.

[0042] 2. PCR amplification product digestion, electrophoresis and results

[0043] Restriction endonucleases Afa I digested the PCR amplification product, and the digested product was subjected to agarose gel electrophoresis. The digested product identification result of the PCR product is as follows: Figure 3 As shown, due to the presence of Afa I restriction site, two bands appeared after restriction electrophoresis, the sizes of which were 467 bp and 101 bp respectively, while Cremastra appendiculate , Shanlan Oreorchis patens , Coral Orchid Corallorhiza trifida 、Tube distance blue Typical szechuan , Orchidaceae Amoeba changes , Orchidaceae Calypso bulbosa There are no these two bands in either sample, so the variegated azalea orchid can be identified based on the number and size of the electrophoresis bands.

[0044] in Figure 2 , Figure 3 A in the middle is azalea orchid Cremastra unguiculata , B is azalea Cremastra appendiculate , C is mountain orchid Oreorchis patens , D is coral orchid Corallorhiza trifida , E is the tube distance blue Tipularia szechuanica , F is single flower orchid Amoeba changes , G is the bag orchid Calypso bulbosa, H is ddH2O. This method can quickly and efficiently distinguish and identify variegated rhododendron orchids.

[0045] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A PCR enzyme typing detection and identification method for variegated rhododendron orchid, characterized in that: The PCR enzyme digestion typing detection and identification method for variegated azalea orchid comprises the following steps: using sample DNA as a template, performing PCR amplification using PCR primers, performing enzyme digestion on the PCR amplification product using restriction endonucleases, subjecting the enzyme digestion product to agarose gel electrophoresis, and identifying the variegated azalea orchid according to the number and size of specific electrophoresis bands; the PCR primers include an upstream primer having a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.2; the restriction endonucleases are Afa I.

2. The PCR enzyme typing detection and identification method of Rhododendron variegatedefolium according to claim 1, characterized in that: The specific electrophoresis bands include two electrophoresis bands with sizes of 467 bp and 101 bp respectively.

3. The PCR enzyme typing detection and identification method of Rhododendron variegatedefolium according to claim 1, characterized in that: The total reaction system of the PCR amplification is calculated as 25 μL, including Mg 2+ 2.5 μL 10× PCR buffer, 2 μL 2.5 mmol / L dNTP mixture, 0.4 μL 5 U / μL Taq DNA polymerase, 1 μL 10 μmol / L upstream primer of the nucleotide sequence shown in SEQ ID NO.1, 1 μL 10 μmol / L downstream primer of the nucleotide sequence shown in SEQ ID NO.2, 1 μL DNA template, 17.1 μL ddH2O.

4. The PCR enzyme typing detection and identification method of Rhododendron variegatedefolium according to claim 1, characterized in that: The reaction conditions of the PCR amplification are: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 56°C for 45 s, extension at 72°C for 1 min, 40 cycles; extension at 72°C for 10 min.

5. A detection kit for identifying variegated rhododendron orchid, characterized in that: The detection kit contains an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1, a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2, and a restriction endonuclease Afa I.

6. Use of the PCR enzyme typing detection and identification method of variegated rhododendron orchid according to any one of claims 1 to 4 or the detection kit according to claim 5 in identifying variegated rhododendron orchid.

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