Primer probe combination for identifying wheat blast, product and application thereof

Abstract

The present invention relates to the biological field, and in particular to a primer-probe combination, product and application thereof for identifying wheat blast. The present invention provides a primer-probe combination for identifying wheat blast, the primer-probe combination comprising an upstream primer having a nucleotide sequence as shown in SEQ ID NO.4, a downstream primer having a nucleotide sequence as shown in SEQ ID NO.5 and a probe having a nucleotide sequence as shown in SEQ ID NO.6. The present invention designs a specifically labeled primer-probe combination based on the wheat blast fungus gene, and the primer-probe combination can accurately distinguish wheat blast fungus, gibberellosis and rice blast fungus. At the same time, based on the designed primer-probe combination, the present invention uses RPA amplification technology and nucleic acid colloidal gold detection test strips to detect wheat blast fungus, gibberellosis and rice blast fungus respectively, and the detection method can specifically detect wheat blast fungus.

Description

Technical Field

[0001] The invention relates to the biological field, and in particular to a primer-probe combination for identifying wheat blast, a product and an application thereof. Background Art

[0002] At present, nucleic acid detection technologies mainly include recombinase polymerase amplification technology (RPA) and lateral flow immunochromatography analysis technology. RPA technology is a technology that realizes exponential amplification of nucleic acids under constant temperature conditions with the participation of multiple enzymes and proteins. It is known as a nucleic acid detection technology that can replace PCR. At present, many companies have direct PRA kits available for purchase. You only need to synthesize the primers and probes of the target gene by yourself, and you can achieve room temperature amplification of nucleic acid products that do not rely on laboratory instruments. Only when the probe binds, the THF residue will be cut by the nfo enzyme. The 3'-OH group generated in this process is the target of polymerase extension and enables the 5'-label to be integrated into the amplified product; among them, the working principle of RPA technology is specifically as follows: the probe treated with the nof enzyme and the reverse primer produce a double-stranded amplification product, which jointly connects two antigen markers, and one of the markers is used to capture the product, and the other marker is used for visualization. In RPA technology, we often only need to design 2 pairs of primers and 1 probe, one of which is a common primer, and the other primer is labeled with biotin at the 5' end. The probe needs to have a FAM label, THF (tetrahydrofuran) group to replace any base in the probe base sequence, and the space group of C3. Lateral flow immunochromatography analysis technology is a classic rapid detection technology based on antigen-antibody immune reaction. This technology has colloidal gold antibodies on the binding pad, and its main function is color development; antibodies that can bind to FAM labels are fixed on the T line; secondary antibodies that can specifically bind to colloidal gold labels are fixed on the C line; after the synthesized probes and primers are added to the RPA reaction system, the RPA reaction is completed, and the amplified reaction solution (containing 2 labels, FAM label and biotin label) is diluted and dripped onto the sample pad, and the capillary action is used to move toward the water absorption pad. Because the amplification product at this time has both FAM labels and biotin labels and binds to the colloidal gold antibody, it can be colored on the T line and C line respectively. If there is no target template in the early stage and effective amplification cannot be achieved, then there are separate FAM labels and biotin labels. At this time, only the biotin label can bind to colloidal gold, so it can only be colored on the C line. That is, if the T line and the C line are colored at the same time, it means that the amplification of the target template is achieved. If only the C line is colored, it means that the amplification of the target template is not achieved.

[0003] The phenotype of wheat blast in the field is similar to the early symptoms of fusarium head blight. The results of genome analysis show that the genotype of wheat blast is highly similar to that of rice blast, with a consistency of 99.0%. At present, there is a lack of field detection methods that can accurately distinguish wheat blast from the other two diseases at the same time. The distinction between wheat blast and rice blast is mainly carried out in the laboratory internationally. The detection methods include: ① relying on PCR instrument and specific marker MoT3, ② relying on fluorescent quantitative PCR instrument and specific marker C17. Both of the above methods require expensive and bulky laboratory instruments and equipment, and the entire detection process is time-consuming. In addition, the field detection method that relies on CRISPR / Cas12 protein-mediated differentiation between wheat blast and rice blast requires the activation of CRISPR / Cas12 protein by gRNA. gRNA is unstable in the environment, so it has certain limitations for field detection. It can be seen that it is very necessary to find a field detection method that is suitable for field detection and can quickly and accurately distinguish wheat blast from fusarium head blight and rice blast. Summary of the invention

[0004] The purpose of the present invention is to provide a primer-probe combination, product and application thereof for identifying wheat blast, so as to solve the problems existing in the above-mentioned prior art. The primer-probe combination provided by the present invention can effectively identify wheat blast and is suitable for field detection.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a primer-probe combination for identifying wheat blast, which comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO.4, a downstream primer with a nucleotide sequence as shown in SEQ ID NO.5 and a probe with a nucleotide sequence as shown in SEQ ID NO.6.

[0007] The primer-probe combination of the present invention is designed based on MoT6098, and its nucleotide sequence is shown in SEQ ID NO. 1. The results of the specific embodiment of the present invention show that the primer-probe combination can accurately distinguish wheat blast fungus, gibberellus and rice blast fungus. Therefore, the primer-probe combination can be used to prepare products for identifying rice blast, such as reagents, identification kits or chips.

[0008] The present invention provides application of the primer-probe combination in preparing a product for identifying rice blast.

[0009] Preferably, the product is a reagent, an identification kit or a chip.

[0010] The invention provides a product for identifying wheat blast, and the product comprises the primer-probe combination.

[0011] Preferably, the product further comprises a lysis solution; the lysis solution comprises 100 mM Tris-Cl, 10 mM EDTA and 1 M KCl.

[0012] Preferably, the product is a reagent, an identification kit or a chip.

[0013] The results of the specific examples of the present invention show that the primer-probe combination provided by the present invention or the product containing the primer-probe combination can accurately distinguish wheat blast fungus, Gibberella and rice blast fungus. It can be seen that the primer-probe combination and the product designed based on the primer-probe combination can be used to identify wheat blast.

[0014] The present invention provides the use of the primer-probe combination or the product in identifying wheat blast.

[0015] The present invention provides a method for identifying wheat blast, comprising the following steps:

[0016] The genome of the sample to be tested is used as template DNA, the template DNA is mixed with the above primer probe combination, and then recombinase polymerase amplification is performed, and the product obtained by the recombinase polymerase amplification is colorimetrically detected using a nucleic acid colloidal gold test strip;

[0017] If the T line and the C line are colored at the same time, it is judged that the sample to be tested is a sample infected with wheat blast.

[0018] Preferably, the recombinase polymerase amplification time is 10 minutes and the temperature is 39°C-42°C.

[0019] Preferably, the sample to be tested includes plant diseased tissue.

[0020] The present invention discloses the following technical effects:

[0021] The invention designs a primer-probe combination with specific markers based on the wheat blast fungus gene, and the primer-probe combination can accurately distinguish wheat blast fungus, Gibberella and rice blast fungus.

[0022] At the same time, based on the designed primer-probe combination, the present invention uses RPA amplification technology and nucleic acid colloidal gold test strips to detect wheat blast fungi, gibberellosis and rice blast fungi respectively, and the detection method can specifically detect wheat blast fungi. In a specific embodiment of the present invention, the detection method is used to detect the genomic DNA of wheat ears of the three pathogens after inoculation for 3 days, and the detection method can specifically detect wheat blast fungi. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 The results of rapid identification of wheat blast, Gibberella and rice blast after 3 days of inoculation, where + is the positive control wheat blast, - is the negative control water, MoT1 and MoT2 represent 2 different wheat blast, FHB1 and FHB2 represent 2 different Gibberella, MoO1 and MoO2 represent 2 different rice blast;

[0025] Figure 2 The sensitivity test results of the field rapid detection method; among them, 10 -2 , 10 -3 , 10 -4 and 10 -5 It is the multiple dilution concentration of wheat blast fungus mycelium. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031]

[0032] The amino acid sequence of the protein encoded by MoT6098 is shown in SEQ ID NO.3, specifically: MRLLVLAAASVC*。

[0033] Example 1

[0034] 1. Determination of specific primers and probes for wheat blast fungus

[0035] Primers and probes were designed according to MoT6098. The sequences of primers and probes are as follows:

[0036] Primer probe set 1:

[0037] MoT6098-F: 5'-TGTAAGCTGGTATCTGGAGTGTTTTGGTTGGT-3', SEQ ID NO.4;

[0038] B-MoT6098-R: 5'-Biotin-GACTTGACTGTTGGCGTTTGCCCCTTTTTTG-3', SEQ ID NO.5;

[0039] F-MoT6098-probe: 5'-FAM-CTGGCTGAACGACGTCTGGCAGCTGTGAAACT (THF)GCCATCTCCAGTGAACA-C3 Spacer-3', SEQ ID NO.6;

[0040] Primer probe set 2:

[0041] MoT6098-F1: 5'-AACGCAGCCGTCGGAATGACTCGGAAGGGT-3', SEQ ID NO.7;

[0042] B-MoT6098-R1: 5'-TAAAGCGGACTCGGTGTCCAGCTTGTCAATAAT-3', SEQ ID NO.8;

[0043] F-MoT6098-probe: 5'-FAM-CTGGCTGAACGACGTCTGGCAGCTGTGAAACT (THF)GCCATCTCCAGTGAACA-C3 Spacer-3', SEQ ID NO.6;

[0044] Primer probe set 3:

[0045] MoT6098-F2: 5'-GTGCCTCACTTTACCGATTTGCTGG-3', SEQ ID NO.9;

[0046] B-MoT6098-R2: 5'-GACTGTTGGCGTTTGCCCCTTTTTT-3', SEQ ID NO.10;

[0047] F-MoT6098-probe: 5'-FAM-CTGGCTGAACGACGTCTGGCAGCTGTGAAACT (THF)GCCATCTCCAGTGAACA-C3 Spacer-3', SEQ ID NO.6.

[0048] 2. Rapid extraction of genomic DNA from samples to be tested

[0049] Wheat seedlings were grown in an incubator for 10 days, and spore suspensions of two wheat blast fungus strains (PR01-37.V.1 (MoT1) and PR01-37.V.3 (MoT2), the strains were provided by the applicant's cooperative laboratory) and spore suspensions of two Gibberella strains (PH-1 (FHB1) and 3ADON (FHB2)) were cultured on oat medium in a light incubator for 10 days. Then, spore suspensions of the four strains were prepared with 0.05% (v / v) Tween-20 aqueous solution, and the concentrations of the spore suspensions were adjusted to 1×10 5 After the rice seedlings were grown in the incubator for 14 days, two strains of rice blast fungus (RB22 (MoO1) and Guy11 (MoO2), which were cultured in the light incubator for 10 days on oatmeal medium, were taken. Then, spore suspensions were prepared with 0.05% (v / v) Tween-20 aqueous solution, and the concentrations of the spore suspensions of the two strains were adjusted to 1×10 5 / mL, and rice was spray-inoculated.

[0050] 3 days after inoculation, take an appropriate amount of wheat leaf disease tissue or rice leaf disease tissue and place it in a 1.5mL centrifuge tube, add 100μL of lysis solution (100mM Tris-Cl (pH 8.0), 10mM EDTA, 1M KCl) to the centrifuge tube to cover the plant tissue, crush it as much as possible with the pipette tip, heat at 65℃ for 30-45min, let it stand for 2min, take the supernatant, add 900μL ddH2O to dilute it 10 times for subsequent detection.

[0051] 3. RPA amplification

[0052] The primers and probes synthesized by Qingke Biotechnology Co., Ltd. were entrusted to Anpu Future (Changzhou) Biotechnology Co., Ltd. to prepare a customized kit for microspheres (WLN8203KIT). The concentration of the upstream primer (SEQ ID NO.3) in the kit is 10μM, the concentration of the downstream primer (SEQ ID NO.4) is 10μM, and the concentration of the probe (SEQ ID NO.5) is 10 μM. During detection, only the diluted template needs to be added, and the amount used is 50μL.

[0053] The specific steps are as follows:

[0054] (1) Add 50 μL of the DNA extraction lysis solution diluted 10 times with water to the reaction tube of the customized kit, quickly cover the reaction tube (cover it immediately after adding a sample to prevent cross contamination), and mix it upside down 8-10 times. A positive control (mycelium of wheat blast fungus) and a negative control (water) need to be prepared for each test.

[0055] (2) Place the reaction tube at 39-42°C for 10 min, remove it, and obtain the RPA reaction product.

[0056] 4. Visual detection of nucleic acid colloidal gold test strips

[0057] Take 50 μL of the RPA reaction obtained in the above steps, dilute it 10 times with water, mix it, and use the nucleic acid colloidal gold test strip (Anpu Future (Changzhou) Biotechnology Co., Ltd.) for color development. The results were read within 5 minutes. It was found that only primer probe set 1 could specifically detect wheat blast fungus. Among them, the detection results of primer probe set 1 are as follows Figure 1 As shown. MoT1 and MoT2 showed obvious positive bands. In summary, the primer-probe combination provided by the present invention can specifically detect wheat blast fungus.

[0058] Example 2 Sensitivity Experiment

[0059] Take wheat blast fungus and inoculate it into oat culture medium, culture it in a light incubator for 10 days, scrape an appropriate amount of mycelium, and extract wheat blast fungus DNA by 2% CTAB lysis method (this step is completed by the Brazilian laboratory that the applicant cooperates with and mailed to the applicant). Determine the concentration of the extracted DNA and dilute it to 10 -2 ng / μL, 10 -3 ng / μL, 10 -4 ng / μL, 10 -5 ng / μL, 10 -6 ng / μL.

[0060] Add different gradient dilutions of wheat blast fungus mycelium DNA to the custom tube, place the reaction tube at 39-42℃ for 10 minutes, take it out, and get the RPA reactant. Repeat the above nucleic acid colloidal gold test strip visual detection. The detection sensitivity is measured by wheat blast fungus mycelium concentration. The results are as follows Figure 2 The results showed that the minimum detection threshold of the primer-probe combination for wheat blast fungus was 10 -5 ng / μL.

[0061] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer-probe combination for identifying wheat blast, characterized in that: The primer-probe combination comprises an upstream primer whose nucleotide sequence is shown as SEQ ID NO.4, a downstream primer whose nucleotide sequence is shown as SEQ ID NO.5, and a probe whose nucleotide sequence is shown as SEQ ID NO.

6.

2. Use of the primer-probe combination according to claim 1 in the preparation of a product for identifying rice blast.

3. The use according to claim 2, characterized in that: The product is a reagent, an identification kit or a chip.

4. A product for identifying wheat blast, characterized in that: The product comprises the primer-probe combination according to claim 1.

5. The product according to claim 4, characterized in that The product also includes a lysis solution; the lysis solution includes 100mM Tris-Cl, 10mM EDTA and 1M KCl.

6. The product according to claim 4, characterized in that The product is a reagent, an identification kit or a chip.

7. Use of the primer-probe combination of claim 1 or the product of any one of claims 4 to 6 in identifying wheat blast.

8. A method for identifying wheat blast, characterized in that: The following steps are involved: The genome of the sample to be tested is used as a template DNA, the template DNA is mixed with the primer-probe combination of claim 1, and then recombinase polymerase amplification is performed, and the product obtained by the recombinase polymerase amplification is colorimetrically detected using a nucleic acid colloidal gold test strip; If the T line and the C line are colored at the same time, it is judged that the sample to be tested is a sample infected with wheat blast.

9. The method according to claim 8, characterized in that The amplification time of the recombinase polymerase is 10 minutes, and the temperature is 39°C-42°C.

10. The method according to claim 8, characterized in that The sample to be tested includes plant diseased tissue.

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