Detection kit and detection method for identifying Myeloidogyne and application of detection kit and detection method for identifying Myeloidogyne

Abstract

The invention belongs to the technical field of plant species identification, and particularly relates to a detection kit and a detection method for identifying Myeloidogyne spp. And application of the detection kit and the detection method. An upstream primer as shown in SEQ ID NO.1, a downstream primer as shown in SEQ ID NO.2 and a probe with a nucleotide sequence as shown in SEQ ID NO.3 are adopted, the 5'end of the probe is connected with a fluorescent reporter group, the 3 'end of the probe is connected with a quenching group, and the identification of the Myeloidogyne and the approximate species thereof is carried out by utilizing a real-time fluorescent quantitative PCR (Polymerase Chain Reaction) technology. The method is high in detection sensitivity and intuitive in identification result, effectively solves the technical problems that the gene sequence difference of the ametrychium coccineum and the approximate species of the ametrychium coccineum is small, and the positioning detection and identification of different fragments of the ametrychium coccineum are difficult, and realizes the rapid and accurate identification of the ametrychium coccineum.

Description

Technical Field

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

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

[0003] Real-time fluorescence quantitative PCR (Quantitative Real-Time Polymerase Chain Reaction) is a highly sensitive nucleic acid detection technology developed on the basis of PCR technology. Its basic principle is to use the characteristics of DNA polymerase to synthesize new DNA chains during the PCR process, combined with a fluorescently labeled probe or dye, to measure the progress of the PCR reaction by real-time monitoring of the increase in fluorescence signals. Real-time fluorescence quantitative PCR is used to detect and identify biological species and must meet specific primer and probe design requirements, especially to find specific primers and probes that are different from other similar species.

[0004] Due to the wide variety of orchids, the differences in the molecular level of species with close geographical distribution are small, 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 affects the amplification efficiency of the same probe. At present, there is no patent literature and non-patent literature related to the real-time fluorescence quantitative PCR detection and identification of Meizhulan. Even for the detection and identification of orchids, it is mainly through conventional PCR amplification sequencing, but PCR amplification sequencing has problems such as long detection cycle, low identification efficiency, and cumbersome operation. Therefore, how to establish a Meizhulan detection and identification method with high specificity, high sensitivity and non-morphology that reduces false positive amplification, non-PCR amplification sequencing is a technical problem that needs to be solved urgently. Summary of the invention

[0005] In view of the above problems, the purpose of the present invention is to provide a detection kit, a detection method and an application for identifying the scutellaria baicalensis, so as to effectively distinguish the scutellaria baicalensis and its similar species. To achieve the above purpose, the technical scheme of the present invention is as follows: 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. The 5'-end of the probe is linked to a fluorescent reporter group, and the 3'-end of the probe is linked to a quenching group.

[0006] Preferably, the fluorescent reporter group is any one of FAM, ROX, VIC, JOE, CY3, CY5, NED, and TEXAS RED, and the quenching group is any one of TAMRA, BHQ-1, and MGB. More preferably, the fluorescent reporter group is FAM, and the quenching group is MGB.

[0007] Preferably, the kit further contains Premix Ex Taq TM Probe qPCR. Wherein Premix Ex Taq TM Probe qPCR is a commonly used commercially available reagent.

[0008] Preferably, the kit further contains ROX reference dye (ROX Reference Dye). Wherein ROX reference dye (ROX Reference Dye) is a commonly used commercially available reagent.

[0009] The present invention further provides the use of the aforementioned kit as a detection kit for identifying Cymbidium erythraeum Lindl.

[0010] The present invention also provides a detection method for identifying Cymbidium erythraeum Lindl. The reaction system of the detection method, based on 25 μL, contains the following reagents: 12.5 μL of 2×Premix Ex Taq TM Probe qPCR, 0.5 μL of an upstream primer solution with a 10 μmol / L nucleotide sequence as shown in SEQID NO.1, 0.5 μL of a downstream primer solution with a 10 μmol / L nucleotide sequence as shown in SEQ ID NO.2, 0.5 μL of a probe solution with a 10 μmol / L nucleotide sequence as shown in SEQ ID NO.3, 0.5 μL of 50×ROX reference dye, 2 μL of DNA template, and ddH2O is added to make up to 25 μL. The upstream primer, downstream primer, and probe solution all use conventional ddH2O as the solvent.

[0011] Preferably, the 5'-end of the probe is linked to the fluorescent reporter group FAM, and the 3'-end of the probe is linked to the quenching group MGB.

[0012] Preferably, the reaction procedure for 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, with 40 cycles.

[0013] The technical solution of the present invention has the following beneficial effects: (1) The kit and detection method of the present invention can achieve the detection and identification of Callostylis rigida under the condition of relatively low nucleic acid concentration, and the lowest detectable DNA concentration can reach 1x10 ﹣2 ng / μL. Moreover, the kit and detection method of the present invention can achieve specific identification and differentiation of Callostylis rigida and its similar species without the need for complex gene sequencing and internal reference genes.

[0014] (2) The kit and detection method of the present invention only require a pair of primers and a probe in one reaction system to quickly and accurately identify Callostylis rigida and its similar species. Moreover, the detection sensitivity is high, the identification result is intuitive, effectively solving the technical problem of difficult detection and identification of the specific fragment location of Callostylis rigida due to the small difference in gene sequences between Callostylis rigida and its similar species, and achieving the rapid and accurate identification of Callostylis rigida. Description of the Drawings

[0015] Figure 1 It is the specific detection result of real-time fluorescence quantitative PCR for the combination of primers SEQ ID NO.1 / SEQ ID NO.2 and probe SEQ ID NO.3 for Callostylis rigida; Figure 2 It is the sensitivity detection result of the real-time fluorescence quantitative PCR identification method for Callostylis rigida; 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, H is ddH2O.

[0016] Among them, the DNA template concentrations corresponding to 1, 2, 3, 4, 5, and 6 are 100 ng / μL, 10 ng / μL, 1 ng / μL, 1×10 - 1 ng / μL, 1×10 -2 ng / μL, 1×10 -3 ng / μL respectively, and the DNA template concentration of 7 is 0 (i.e., ddH2O is used instead). Detailed Embodiments

[0017] The technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and embodiments. 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 of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention. Unless otherwise specified, the reagents used in the following embodiments 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 embodiments of the present invention are conventional methods and techniques in the art, such as the technique of connecting a fluorescent reporter group and a quenching group to the probe.

[0018] Example 1 Specific Primers, Probes and Identification Methods for Real-Time Fluorescent Quantitative PCR Detection of Cymbidium erythraeum Lindl. 1. In this example, specific primers and probes for detecting and identifying Cymbidium erythraeum Lindl. by real-time fluorescent quantitative PCR were designed and prepared. The nucleotide sequences of the primers and probes are as follows: The nucleotide sequence of the upstream primer is (SEQ ID NO.1): 5'-ACATCGCTCCGTGGCAACTTC-3' (artificial sequence); The nucleotide sequence of the downstream primer is (SEQ ID NO.2): 5'-TTGGGATCACCTCTCGGGCTTAT-3' (artificial sequence); The nucleotide sequence of the probe primer is (SEQ ID NO.3): 5'-CGTCGAAGGGCGTGTC-3' (artificial sequence); Among them, the 5' end of the probe primer is connected to the fluorescent reporter group FAM (carboxyfluorescein), and the 3' end is connected to the quenching group MGB (Minor Groove Binder).

[0019] 2. In this example, a real-time fluorescence quantitative PCR detection and identification method for Callostylis rigida was established. The reaction system includes (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 as shown in SEQ ID NO.1, 10 μmol / L), 0.5 μL of downstream primer solution (nucleotide sequence as shown in SEQ ID NO.2, 10 μmol / L), 0.5 μL of probe solution (nucleotide sequence as 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. Among them, the 5' end of the probe primer is linked to the fluorescent group FAM (carboxyfluorescein), and the 3' end is linked to the quenching group MGB (Minor Groove Binder). The upstream primer, downstream primer, and probe solution all use routinely used ddH2O as the solvent.

[0020] 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, for 40 cycles.

[0021] Example 2 Specificity test of the real-time fluorescence quantitative PCR identification method for Callostylis rigida Using the primer and probe combination, real-time fluorescence quantitative PCR reaction system and method of Example 1, real-time fluorescence quantitative PCR detection was performed on Callostylis rigida and its similar species. The specific detection results of real-time fluorescence quantitative PCR are as Figure 1 shown. Among them, 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.

[0022] It can be Figure 1 seen that only the amplification curve of Callostylis rigida has specific amplification, and the Ct value is 15.86, that is, the fluorescence signal starts to increase exponentially from the 15th cycle, while there is no fluorescence growth signal in other similar species and ddH2O before the end of all cycle reactions, indicating that this probe and primer combination is species-specific only for Callostylis rigida.

[0023] Example 3 Sensitivity of the real-time fluorescence quantitative PCR identification method for Callostylis rigida The DNA template concentrations of Cymbidium erythraeum Lindl. were 100 ng / μL, 10 ng / μL, 1 ng / μL, 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). The primers, probe combination, real-time fluorescence quantitative PCR reaction system and method obtained in Example 1 were used for detection. The detection results of the sensitivity of the real-time fluorescence quantitative PCR identification method are as shown in Figure 2 . Among them, the DNA template concentrations corresponding to 1, 2, 3, 4, 5, and 6 were 100 ng / μL, 10 ng / μL, 1 ng / μL, 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).

[0024] It can be seen from Figure 2 that for DNA templates with different concentrations, the detected Ct values are different. When the template concentrations were 100 ng / μL, 10 ng / μL, 1 ng / 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 determination principle of the real-time fluorescence quantitative PCR detection results, when the concentration was 1×10 -3 ng / μL, its Ct value was 36.05, and it was impossible to determine whether it was positive. Therefore, the detection limit of the specific probe SEQ ID NO.3 of Cymbidium erythraeum Lindl. was 1×10 -2 ng / μL.

[0025] The present invention has been disclosed in the above preferred embodiments, but it is not intended to limit the present invention. Any technical solutions obtained by adopting equivalent replacement or equivalent transformation shall fall within the protection scope of the present invention.

Claims

1. A detection kit for identifying Melaleuca alternifolia, 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 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 detection kit according to claim 2, characterized in that The fluorescent reporter group is FAM, and the quencher group is MGB.

4. The detection kit according to claim 1, characterized in that The kit also contains Premix Ex Taq TM Probe qPCR reagents.

5. The detection kit according to claim 1, characterized in that The kit also contains ROX reference dye.

6. Use of the kit according to any one of claims 1 to 5 as a detection kit for identifying Melaleuca alternifolia.

7. A detection method for identifying Melaleuca alternifolia, characterized in that: The reaction system of the detection method contains the following reagents per 25 μL: 2×Premix Ex Taq TM Probe qPCR 12.5μL, 10μmol / L upstream primer solution with a nucleotide sequence as shown in SEQ ID NO.1 0.5μL, 10μmol / L downstream primer solution with a nucleotide sequence as shown in SEQ ID NO.2 0.5μL, 10μmol / L probe solution with a nucleotide sequence as shown in SEQ ID NO.3 0.5μL, 50×ROX reference dye 0.5μL, DNA template 2μL, ddH2O is supplemented to 25μL.

8. A method for identifying Melaleuca 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. A method for identifying Melaleuca according to claim 7, characterized in that: The reaction procedure of the detection includes: pre-denaturation at 94°C-96°C for 9min-12min, denaturation at 94°C-96°C for 12sec-20sec, annealing at 55°C-65°C for 0.5min-2min, and 40 cycles.

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