A detection kit, detection method and application for identifying melastoma

Abstract

The present invention belongs to the technical field of plant species identification, and in particular to a detection kit, detection method and application for identifying scutellaria baicalensis. The present invention adopts the upstream primer shown in SEQ ID NO.1, the downstream primer shown in SEQ ID NO.2, and the probe with the nucleotide sequence shown in SEQ ID NO.3, wherein the 5' end of the probe is connected to a fluorescent reporter group, and the 3' end is connected to a quenching group, and the identification of scutellaria baicalensis and its similar species is carried out by using real-time fluorescence quantitative PCR technology. The method has high detection sensitivity, and the identification result is intuitive, and effectively solves the technical problem that the gene sequence difference of scutellaria baicalensis and its similar species is small, and the positioning detection and identification of scutellaria baicalensis specific fragments are difficult, and realizes the rapid and accurate identification of scutellaria baicalensis.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant species identification, and in particular relates to a detection kit, a detection method and an application for identifying Stylophora japonica. Background Art

[0002] Meizhulan ( Callostylis rigida This species belongs to the genus Orchidaceae, ...

[0003] Real-time fluorescence quantitative PCR (qPCR) is a highly sensitive nucleic acid detection technique developed based on PCR. Its basic principle is to utilize the ability of DNA polymerase to synthesize new DNA chains during the PCR process. Combined with a fluorescently labeled probe or dye, the progress of the PCR reaction is measured by real-time monitoring of the increase in fluorescence signal. Real-time fluorescence quantitative PCR for the detection and identification of biological species requires specific primer and probe design requirements, particularly those that distinguish the species from those of similar species.

[0004] Because orchids are of various kinds, the difference of the molecular level of the species closer in geographical distribution is smaller, and it is very difficult to find specific primers and probe sites that are different from other similar species.The selection of different primers also has an impact on the amplification efficiency of the same probe.At present, there is no real-time fluorescence quantitative PCR detection and identification of the orchid orchid related patent literature and non-patent literature disclosed. Even if it is for the detection and identification of orchids, it is mainly also to be sequenced by conventional PCR amplification, but there is the problems such as long detection cycle, low identification efficiency, complicated operation in PCR amplification sequencing.Therefore, how to set up the non-morphology, non-PCR amplification sequencing with high specificity, high sensitivity and reducing false positive amplification of the orchid orchid detection and identification method is a technical problem to be solved urgently. Summary of the Invention

[0005] In view of the above problems, the present invention aims to provide a detection kit, detection method and application for identifying scutellaria baicalensis to effectively distinguish scutellaria baicalensis and its similar species. To achieve the above object, the technical solution of the present invention is as follows:

[0006] The present invention first provides a kit, which 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 probe with a nucleotide sequence as shown in SEQ ID NO.3, wherein the 5' end of the probe is connected to a fluorescent reporter group, and the 3' end of the probe is connected to a quencher group.

[0007] Preferably, the fluorescent reporter group is any one of FAM, ROX, VIC, JOE, CY3, CY5, NED and TEXAS RED, and the quencher group is any one of TAMRA, BHQ-1 and MGB. Further preferably, the fluorescent reporter group is FAM and the quencher group is MGB.

[0008] Preferably, the kit further contains Premix Ex Taq TM Probe qPCR. TM Probe qPCR is a commonly used commercially available reagent.

[0009] Preferably, the kit further contains ROX Reference Dye, which is a commonly used reagent available on the market.

[0010] The present invention further provides the use of the above-mentioned kit as a detection kit for identifying Melissa officinalis.

[0011] The present invention also provides a detection method for identifying meglitinidum. The reaction system of the detection method contains the following reagents per 25 μL: 2×Premix Ex Taq TM Probe qPCR: 12.5 μL, 0.5 μL of a 10 μmol / L upstream primer solution with the nucleotide sequence shown in SEQ ID NO. 1, 0.5 μL of a 10 μmol / L downstream primer solution with the nucleotide sequence shown in SEQ ID NO. 2, 0.5 μL of a 10 μmol / L probe solution with the nucleotide sequence shown in SEQ ID NO. 3, 0.5 μL of 50× ROX reference dye, 2 μL of DNA template, and ddH2O to 25 μL. The upstream primer, downstream primer, and probe solutions all used conventional ddH2O as the solvent.

[0012] Preferably, the 5' end of the probe is connected to a fluorescent reporter group FAM, and the 3' end of the probe is connected to a quencher group MGB.

[0013] Preferably, the reaction procedure of the detection includes: pre-denaturation at 94°C-96°C for 9 min-12 min, denaturation at 94°C-96°C for 12 sec-20 sec, annealing at 55°C-65°C for 0.5 min-2 min, and 40 cycles.

[0014] The technical solution of the present invention has the following beneficial effects:

[0015] (1) The kit and detection method of the present invention can detect and identify Melanophora in the case of low nucleic acid concentration, and the minimum DNA concentration detected can reach 1x10 ﹣2 ng / μL. The kit and detection method of the present invention can realize the specific identification and differentiation of Melanocyma and its similar species without the need for complex gene sequencing and internal reference genes.

[0016] (2) The kit and detection method of the present invention only require a pair of primers and a probe in a reaction system to quickly and accurately identify Melanocymidae and its similar species, and the detection sensitivity is high, and the identification results are intuitive. It effectively solves the technical problem that the gene sequence differences between Melanocymidae and its similar species are small, and the positioning, detection and identification of Melanocymidae-specific fragments are difficult, thereby achieving rapid and accurate identification of Melanocymidae. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The specific detection results of the real-time fluorescence quantitative PCR of the combination of primers SEQ ID NO.1 / SEQ ID NO.2 and probe SEQ ID NO.3 of the mesitylene;

[0018] Figure 2 This is the sensitivity test result of the real-time fluorescence quantitative PCR identification method for Messylophyllum;

[0019] In the figure, A is Callostylis rigida, B is Callostylis bambusifolia, C is Cryptochilus luteus, D is Porpax ustulata, E is Ceratostylis caespitosa, F is Eria corneri, G is Pinalia pachyphylla, and H is ddH2O.

[0020] The DNA template concentrations corresponding to 1, 2, 3, 4, 5, and 6 were 100 ng / μL, 10 ng / μL, 1 ng / μL, and 1×10 - 1 ng / μL, 1×10 -2 ng / μL, 1×10 -3ng / μL, and the DNA template concentration of 7 was 0 (i.e., ddH2O was used instead). DETAILED DESCRIPTION

[0021] The technical solutions of the present invention are described clearly and completely below in conjunction with the accompanying drawings and examples. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Unless otherwise specified, the reagents used in the following examples of the present invention are all commercially available common reagents. Unless otherwise specified, the preparation techniques of the primers and probes used in the following examples of the present invention are conventional methods and techniques in the art, such as the technology of connecting the probe to a fluorescent reporter group and a quencher group.

[0022] Example 1 Real-time fluorescence quantitative PCR detection primers, probes and identification methods for Melaleuca alternifolia

[0023] 1. In this example, specific primers and probes for real-time fluorescence quantitative PCR were designed and prepared for the detection and identification of Melilotrichia. The nucleotide sequences of the primers and probes are as follows:

[0024] The nucleotide sequence of the upstream primer is (SEQ ID NO.1):

[0025] 5′-ACATCGCTCCGTGGCAACTTC-3′ (artificial sequence);

[0026] The nucleotide sequence of the downstream primer is (SEQ ID NO.2):

[0027] 5′-TTGGGATCACCTCTCGGGCTTAT-3′ (artificial sequence);

[0028] The nucleotide sequence of the probe primer is (SEQ ID NO.3):

[0029] 5′-CGTCGAAGGGCGTGTC-3′ (artificial sequence);

[0030] The 5' end of the probe primer is connected to the fluorescent reporter group FAM (carboxyfluorescein), and the 3' end is connected to the quencher group MGB (Minor Groove Binder).

[0031] 2. This example establishes a real-time fluorescence quantitative PCR detection and identification method for melaleuca alternifolia. The reaction system comprises (based on a 25 μL reaction system): 12.5 μL of 2× Premix Ex Taq Probe qPCR, 0.5 μL of upstream primer solution (nucleotide sequence shown in SEQ ID NO. 1, 10 μmol / L), 0.5 μL of downstream primer solution (nucleotide sequence shown in SEQ ID NO. 2, 10 μmol / L), 0.5 μL of probe solution (nucleotide sequence shown in SEQ ID NO. 3, 10 μmol / L), 0.5 μL of 50× ROX Reference Dye, 2 μL of DNA template, and 8.5 μL of ddH₂O. The probe primer is linked to a fluorescent group (FAM) (carboxyfluorescein) at its 5' end and a quencher (MGB) (minor groove binder) at its 3' end. Conventionally available ddH₂O is used as the solvent for the upstream primer, downstream primer, and probe solution.

[0032] The reaction procedure includes: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 sec, annealing at 60°C for 1 min, and 40 cycles.

[0033] Example 2 Specificity test of the real-time fluorescence quantitative PCR identification method of Melaleuca alternifolia

[0034] The primer and probe combination and the real-time fluorescence quantitative PCR reaction system and method of Example 1 were used to perform real-time fluorescence quantitative PCR detection on Melissa officinalis and its similar species. The specific detection results of the obtained real-time fluorescence quantitative PCR were as follows: Figure 1 A is Callostylis rigida, B is Callostylis bambusifolia, C is Cryptochilus luteus, D is Porpax ustulata, E is Ceratostyliscaespitosa, F is Eria corneri, G is Pinalia pachyphylla, and H is ddH2O.

[0035] Depend on Figure 1 It can be seen that only the amplification curve of Melissa officinalis showed specific amplification, with a Ct value of 15.86, that is, the fluorescence signal increased exponentially from the 15th cycle, while other similar species and ddH2O showed no fluorescence growth signal before the end of all cycle reactions, indicating that this probe and primer combination is species-specific only for Melissa officinalis.

[0036] Example 3 Sensitivity of the Real-time Fluorescence Quantitative PCR Identification Method for Melaleuca alternifolia

[0037] The concentrations of DNA templates of Melissa officinalis were 100ng / μL, 10ng / μL, 1ng / μL, and 1×10 -1 ng / μL, 1×10 -2 ng / μL, 1×10 -3 ng / μL and 0 ng / μL (i.e., ddH2O was used instead), and the primer, probe combination, and real-time fluorescence quantitative PCR reaction system and method obtained in Example 1 were used for detection. The sensitivity detection results of the obtained real-time fluorescence quantitative PCR identification method are as follows Figure 2 The DNA template concentrations corresponding to 1, 2, 3, 4, 5, and 6 are 100 ng / μL, 10 ng / μL, 1 ng / μL, and 1×10 - 1 ng / μL, 1×10 -2 ng / μL, 1×10 -3 ng / μL, and the DNA template concentration of 7 was 0 (i.e., ddH2O was used instead).

[0038] Depend on Figure 2 It can be seen that the Ct values ​​of the detection were different for DNA templates of different concentrations. When the template concentration was 100ng / μL, 10ng / μL, 1ng / μL, 1×10 -1 ng / μL, 1×10 -2 ng / μL, 1×10 -3 ng / μL, the Ct values ​​were 16.12, 20.39, 24.08, 27.91, 31.96, and 36.05, respectively. According to the principle of real-time fluorescence quantitative PCR detection results, when the concentration was 1×10 -3 ng / μL, the Ct value was 36.05, and it was impossible to determine whether it was positive. Therefore, the detection limit DNA concentration of the mesitylene-specific probe SEQ ID NO.3 was 1×10 -2 ng / μL.

[0039] The present invention has been disclosed above with preferred embodiments, which are not intended to limit the present invention. Any technical solutions obtained by adopting equivalent replacement or equivalent transformation solutions fall within the protection scope of the present invention.

Claims

1. A real-time fluorescence quantitative PCR detection kit for identifying Melanophora spp., characterized in that: The 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 probe with a nucleotide sequence as shown in SEQ ID NO.3, wherein the 5' end of the probe is connected to a fluorescent reporter group, and the 3' end of the probe is connected to a quencher group.

2. The real-time fluorescence quantitative PCR detection kit according to claim 1, characterized in that The fluorescent reporter group is any one of FAM, ROX, VIC, JOE, CY3, CY5, NED and TEXAS RED, and the quencher group is any one of TAMRA, BHQ-1 and MGB.

3. The real-time fluorescence quantitative PCR detection kit according to claim 2, characterized in that The fluorescent reporter group is FAM, and the quencher group is MGB.

4. The real-time fluorescence quantitative PCR detection kit according to claim 1, characterized in that The kit also contains Premix Ex Taq TM Probe qPCR reagents.

5. The real-time fluorescence quantitative PCR detection kit according to claim 1, characterized in that The kit also contains ROX reference dye.

6. Use of the real-time fluorescence quantitative PCR kit according to any one of claims 1 to 5 as a detection kit for identifying Melanocarpa.

7. A real-time fluorescence quantitative PCR detection method for identifying Melanophora spp., characterized in that: The reaction system of the real-time fluorescence quantitative PCR detection method contains the following reagents per 25 μL: 2×Premix Ex Taq TM Probe qPCR 12.5 μL, 10 μmol / L upstream primer solution with the nucleotide sequence shown in SEQ ID NO.1 0.5 μL, 10 μmol / L downstream primer solution with the nucleotide sequence shown in SEQ ID NO.2 0.5 μL, 10 μmol / L probe solution with the nucleotide sequence shown in SEQ ID NO.3 0.5 μL, 50× ROX reference dye 0.5 μL, DNA template 2 μL, and ddH2O to 25 μL.

8. The real-time fluorescence quantitative PCR detection method for identifying Mycophyllum according to claim 7, characterized in that: The 5' end of the probe is connected to a fluorescent reporter group FAM, and the 3' end of the probe is connected to a quencher group MGB.

9. The real-time fluorescence quantitative PCR detection method for identifying Mycophyllum according to claim 7, characterized in that: The reaction procedure of the detection includes: pre-denaturation at 94°C-96°C for 9 minutes-12 minutes, denaturation at 94°C-96°C for 12 seconds-20 seconds, annealing at 55°C-65°C for 0.5 minutes-2 minutes, and 40 cycles.

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