Primer pair for detecting magnaporthe oryzae and application thereof

Abstract

The invention belongs to the technical field of crop disease detection, identification and prevention and treatment, and particularly relates to a primer pair for detecting magnaporthe oryzae and application of the primer pair, and base sequences of the primer pair are shown as SEQ ID NO.1 and SEQ ID NO.2. The rapid detection method for the magnaporthe oryzae is established on the basis of the MHP1-1 primer pair. According to the method, a magnaporthe oryzae MHP gene is taken as a target gene, the sensitivity reaches 10fg / uL after reaction is carried out for 20 minutes at the constant temperature of 38 DEG C, and the specificity is relatively good. The method does not depend on a complex thermal cycler, an experiment can be carried out only through a water bath kettle or a metal bath, the method is suitable for being used in grassroots units and field detection, a simpler and faster technical method is provided for rapid detection and early warning of magnaporthe oryzae, rice germplasm safety and the like, and the method can also be used for rapid detection of other plant diseases.

Description

Technical Field

[0001] This invention belongs to the field of crop disease detection, identification and control technology, specifically relating to a primer pair for detecting rice blast fungus and its application. Background Technology

[0002] Rice is an important food crop, widely cultivated globally. Rice blast fungus (… Pyricularia oryzae ) belongs to the genus Pyrethrum ( Pyricularia Rice blast is a fungus that infects the above-ground parts and roots of rice, causing rice blast disease, which leads to a significant reduction in rice yield and seriously threatens rice production. In my country, rice blast disease affects an average of about 5 million hectares annually, causing a loss of more than 3 billion kilograms of grain each year, seriously threatening food security. The wide range of occurrence, rapid spread, and variable physiological races of rice blast make its control extremely difficult. Therefore, early and rapid detection of the rice blast pathogen is crucial for the control of rice blast and reducing rice yield losses.

[0003] Traditional methods for detecting and identifying rice blast fungus include pathogen isolation, morphological observation, and physiological and biochemical identification. Since rice blast is an airborne disease, it is also possible to predict and warn of rice blast by capturing and counting airborne spores. However, these methods are inefficient and time-consuming. Therefore, there is an urgent need to provide a new strategy for detecting rice blast fungus. Summary of the Invention

[0004] The purpose of this invention is to provide a primer pair for detecting rice blast fungus, providing a simpler and faster technical method for rapid detection, early warning, and rice germplasm safety of rice blast fungus.

[0005] The technical solution adopted in this invention is: The present invention provides a primer pair for detecting rice blast fungus, the base sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0006] A second aspect of the present invention provides a kit for detecting rice blast fungus, the kit comprising the primer pair described above.

[0007] Preferably, the kit further includes at least one of the following: an enzyme system, a buffer, an activator, and proteinase K, required for the enzymatic recombination isothermal amplification reaction.

[0008] Preferably, the enzyme system comprises at least one of recombinase, single-stranded binding protein, DNA polymerase and reverse transcriptase; The buffer solution contains at least one of Mg(OAc)2, dNTPs and KOAc; The activator is Mg(OAc)2.

[0009] A third aspect of the present invention provides an application of the primer pair or the kit, wherein the application refers to any one of the following: 1) Detection of rice blast and / or rice blast fungus; 2) Identify rice blast disease and / or rice blast fungus.

[0010] Preferably, the method for detecting rice blast is as follows: DNA was extracted from the plants to be tested; Using DNA as a template, the kit was used for amplification to obtain amplification products; After digestion of the amplification products, agarose gel electrophoresis was performed. If bands appeared, the tested plants were infected with rice blast; if no bands appeared, the tested plants were not infected with rice blast.

[0011] Preferably, the amplification reaction conditions are as follows: React at 37℃~40℃ for 20min~25min.

[0012] Preferably, the amplification reaction conditions are as follows: React at 38℃ for 20 minutes.

[0013] Preferably, the digestion process of the amplification product is as follows: Add proteinase K to the amplification product and react at 55°C for 5 min.

[0014] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a primer pair for detecting rice blast fungus, the base sequences of which are shown in SEQ ID NO. 1 and SEQ ID NO. 2. This invention establishes a rapid detection method for rice blast fungus based on the MHP1-1 primer pair. This method uses rice blast fungus... MHP Using the target gene as the target gene, the sensitivity reached 10 fg / uL under a constant temperature of 38℃ for 20 min, with good specificity. This method does not rely on a complex thermal cycler; only a water bath or metal bath is needed for the experiment. It is suitable for use in grassroots units and field testing, providing a simpler and faster technical method for the rapid detection, early warning, and germplasm safety of rice blast fungus. It can also be used for the rapid detection of other plant diseases. Attached Figure Description

[0015] Figure 1 The results of the detection of rice blast fungus are from Examples 1 to 4.

[0016] Figure 2 The results of the detection of rice blast fungus are from Examples 1, 5 to 11.

[0017] Figure 3 The results of the detection of rice blast fungus are from Example 1 and Comparative Examples 1 to 4.

[0018] Figure 4 The results are based on the specificity of different amplification templates.

[0019] Figure 5 The results show the sensitivity detection results for different primers.

[0020] Figure 6 To detect rice blast fungus in diseased rice tissues. Detailed Implementation

[0021] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0022] The inventive concept of this invention is as follows: With the development of molecular biology techniques, the detection methods for rice blast fungus based on PCR and Realtime PCR technologies are more sensitive and faster, but the required reagents and consumables are expensive, the requirements for operators are high, and complex instruments and equipment are needed.

[0023] Loop-mediated isothermal amplification (LAMP) is a PCR-based isothermal amplification technique. The reaction process is carried out at a constant temperature of 60℃~65℃, and it does not require the complex temperature-changing equipment used in PCR, making it more suitable for testing at the grassroots level and in frontline departments.

[0024] Enzymatic recombinase amplification (ERA) is a novel isothermal nucleic acid amplification technology developed by Suzhou Xianda Gene Technology Co., Ltd. Its main principle involves using a modified recombinase, endonuclease, DNA polymerase, and other multi-enzyme systems to establish an ERA amplification reaction system, enabling the amplification of specific segments of single-molecule DNA / RNA within 4-7 minutes. 9 The reaction time is 37℃~42℃ for 10min~20min, and the results can be detected by electrophoresis or fluorescence detection instruments. Currently, there are no reports in China regarding the application of ERA technology for the detection of rice blast fungus.

[0025] To ensure high specificity of the detection reaction and avoid cross-reactions with other microorganisms, pathogenicity-related genes are typically selected as ideal candidate target genes for detecting pathogens. Currently, the main target genes used for the detection of rice blast fungus include... 28S rRNA , Pot 2 , MHP 1 and Mif23 etc. Among them, 28S rRNA It is conserved within closely related species, making it prone to false positives during testing. Pot 2 The gene has 100 copies in the genome of *Oryza sativa* and is highly homologous to the transposon Fott in *Fusarium oxysporum*. MHP1 and Mif23 All of these factors are related to the pathogenicity of *Strombus oryzae* and exhibit high specificity for *Strombus oryzae*. Therefore, in this invention, MHP1 and Mif23 were selected as candidate target genes, and five pairs of primers were designed for ERA amplification. Primer screening showed that, under consistent reaction system and conditions, the MHP1-1 primer pair had the best amplification effect. The MHP gene encodes a class I hydrophobic protein of *Strombus oryzae*, which is closely related to the development of *Strombus oryzae* and its infection of rice, and is a single copy in the *Strombus oryzae* genome. The specificity test results in this invention also show that using the MHP gene as a target gene for ERA detection exhibits high specificity.

[0026] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0027] The materials and methods of this invention are as follows: 1. Materials.

[0028] The rice blast fungus used in this invention ( Pyricularia oryzae The strain was provided by the Institute of Plant Protection, Jilin Academy of Agricultural Sciences; other negative control bacteria were *Fusarium oxysporum* (…). Fusarium verticillioides Fusarium graminearum ( ), Fusarium graminearum Fusarium moniliforme ( Fusarium moniliforme ), rice false smut ( Ustilaginoidea virens ) and Rhizoctonia solani ( Rhizoctonia solani All of these are strains that have been preserved in our laboratory for a long time. The above pathogens are disclosed in the references:

[0029] 1) Chen Xing, MAZNAN Nur Atiqah Binti, Li Zhiqiang, et al. Establishment and application of LAMP detection method for rice seed carrying rice blast fungus [J]. Plant Protection, 2022, 48(03):204-210+224. DOI:10.16688 / j.zwbh.2021212.

[0030] 2) Zhang Yuanming. Research on physiological race identification and LAMP detection technology of rice blast fungus in Nanfan area [D]. South China Agricultural University, 2018.

[0031] 2. Methods.

[0032] (1) Culture of strains.

[0033] Rice blast fungus was inoculated onto PDA medium and cultured at 25°C for 7 days.

[0034] (2) Genomic DNA extraction.

[0035] Genomic DNA was extracted from *Oryza sativa* and other negative control bacteria using the Shanghai Sangon Fungal DNA Extraction Kit and stored at -20°C.

[0036] 3. Primer design.

[0037] According to ERA primer design requirements, MHP1 and mif23 Using the target gene as the primer, this invention designed a total of 5 pairs of primers, which were synthesized by Shanghai Sangon Biotech Co., Ltd. The primer sequences are shown in Table 1.

[0038] MHP1 GenBank: AF126872.1; mif23 GenBank: AF118227.1.

[0039] Table 1 Primers used for ERA detection of rice blast fungus Example 1 A primer pair for detecting rice blast fungus. In this example, the MHP1-1 primer pair was used for ERA reaction, as detailed below: Using 10 fg / µL of *Blastomyces oryzae* genomic DNA as a template, ERA amplification was performed using SEQ ID NO.1 and SEQ ID NO.2 listed in Table 1. The ERA amplification kit was purchased from Suzhou Xianda Gene Technology Co., Ltd., catalog number: KS101, and included lyophilized enzyme powder, solvent, and activator.

[0040] The ERA amplification process is as follows: 1) Preparation of ERA amplification system: Add 20 μL of dissolving agent, 4 μL of ddH2O, 2 μL each of upstream and downstream primers (final concentration 0.4 μmol / L), and 1 μL of template DNA to a 0.2 mL reaction tube containing lyophilized enzyme powder. Vortex, centrifuge to mix, add 2 μL of activator to the cap, and centrifuge rapidly.

[0041] 2) Immediately place the ERA amplification system in a 38℃ metal bath for 20 min. After the reaction, add 5 μL of loading buffer containing proteinase K to the reaction tube, mix quickly, and incubate at 55℃ for 5 min. Take 5 μL for 1% agarose gel electrophoresis and observe the electrophoresis results using a gel imaging system.

[0042] Examples 2 to 11 were conducted under different experimental conditions, as detailed in Table 2; unless otherwise specified, the remaining experimental conditions in Examples 2 to 11 were exactly the same as those in Example 1.

[0043] Table 2 Conditions for ERA reaction In Examples 1-4, the amplification products at reaction temperatures of 37°C, 38°C, 39°C, and 40°C were detected by agarose gel electrophoresis. The results are as follows: Figure 1 As shown, the electrophoretic bands of the amplified products were clearest when the ERA reaction temperature was 38°C, indicating that the amplification efficiency of ERA was highest at this temperature. Therefore, 38°C was chosen as the reaction temperature for the subsequent examples. Figure 1 In the diagram, M represents the DL2000 Marker; lanes 1 through 4 represent the amplification products of Examples 2, 1, 3, and 4, respectively.

[0044] The extracted genomic DNA of *Strombus haematous* was then diluted to 10 ng / µL, followed by a 10-fold serial dilution (7 dilutions in total), finally reducing the concentration to 1 fg / µL. The agarose gel electrophoresis results using serially diluted genomic DNA at different concentrations as templates in Examples 1, 5-11 are shown below. Figure 2 . Figure 2 The detection limit of ERA for rice blast fungus was 10 fg / µL. When the template concentration was 1 fg / µL, there was no obvious ERA amplification product band. Figure 2 In the text, M: DL2000 Marker; lanes 1 to 9 are respectively: Example 5, Example 6, Example 7, Example 8, Example 9, Example 10, Example 1, Example 11, and negative control ddH2O.

[0045] Comparative Example 1 A primer pair for detecting rice blast fungus This comparative example uses the MHP1-2 primer pair for ERA reaction. Except for the primer pair, the other conditions are exactly the same as in Example 1.

[0046] Comparative Example 2 A primer pair for detecting rice blast fungus This comparative example uses the MHP1-3 primer pair for ERA reaction. Except for the primer pair, the other conditions are exactly the same as in Example 1.

[0047] Comparative Example 3 A primer pair for detecting rice blast fungus This comparative example uses the mif23-1 primer pair for ERA reaction. Except for the primer pair, the other conditions are exactly the same as in Example 1.

[0048] Comparative Example 4 A primer pair for detecting rice blast fungus This comparative example uses the mif23-2 primer pair for ERA reaction. Except for the primer pair, the other conditions are exactly the same as in Example 1.

[0049] The products of Example 1 and Comparative Examples 1 to 4 were subjected to agarose gel electrophoresis, and the results are shown in the figure. Figure 3 Used to MHP1 or mif23 All five primer pairs targeting the gene amplified the desired sequence, and the fragment size was consistent with expectations. Among them, the ERA amplification product of primer MHP1-1 showed the clearest band.

[0050] Figure 3 In the text, M stands for DL2000 Marker; lanes 1 through 5 are respectively: Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4.

[0051] Example 12 An application of primer pairs for detecting rice blast fungus is as follows: 1. Specific detection.

[0052] Following the ERA amplification conditions in Example 1, different templates were used: rice blast fungus ( Pyricularia oryzae ), Fusarium pseudoverticum ( Fusarium verticillioides Fusarium graminearum ( ), Fusarium graminearum Fusarium moniliforme ( Fusarium moniliforme ), rice false smut ( Ustilaginoidea virens Rhizoctonia solani ( ), Rhizoctonia solani The genomic DNA of the sample was detected, and the specificity of the MHP1-1 primer pair was tested using ddH2O as a negative control.

[0053] The results of agarose gel electrophoresis are shown below. Figure 4 None of the negative control bacteria amplified the target band, indicating that the primer pair in Example 1 has good specificity.

[0054] Figure 4 In the middle, M: DL2000 Marker; Lane 1: Rice blast fungus ( Pyricularia oryzae Lane 2: Fusarium pseudovermiculosum ( Fusarium verticillioides Lane 3: Fusarium moniliformes ( Fusarium moniliforme Lane 4: Fusarium graminearum ( Fusarium graminearum Lane 5: Rice blast fungus ( Ustilaginoidea virens Lane 6: Rhizoctonia solani ( Rhizoctonia solani 7: ddH2O.

[0055] 2. Sensitivity detection.

[0056] The extracted genomic DNA of rice blast fungus was diluted to 1 fg / µl and used as a template, with ddH2O as a negative control. ERA amplification was performed using the five primer pairs listed in Table 1. The reaction was carried out at 38℃ for 20 min, and the sensitivity of the five primer pairs was compared.

[0057] The results of agarose gel electrophoresis are shown below. Figure 5 When the template concentration was 10 fg / µl, only primer pair MHP1-1 amplified the target band; primer pairs MHP1-2, MHP1-3, mif23-1, and mif23-2 failed to amplify the target band. This indicates that primer pair MHP1-1 has higher sensitivity than the other four primer pairs. Figure 5 In the diagram, M: DL2000 Marker; Lane 1: Primer pair MHP1-1; Lane 2: Primer pair MHP1-2; Lane 3: Primer pair MHP1-3; Lane 4: Primer pair mif23-1; Lane 5: Primer pair mif23-2.

[0058] 3. Detection of rice blast fungus in diseased rice tissues.

[0059] Germinated rice seeds were cultured at 25℃ until primary tillering. Mycelial blocks of *Oryza sativa*, cultured at 25℃ for 7 days, were inoculated onto rice hypocotyls and then cultured at 25℃ for 5 days. 200 mg of DNA was extracted from 8 diseased rice tissue samples using the Shanghai Sangon Biotech Ezup Column-Based Super Plant Genomic DNA Extraction Kit. ERA amplification was performed using this DNA as a template according to the protocol in Example 1 to detect *Oryza sativa*, with DNA from healthy rice tissue serving as a control.

[0060] The results of agarose gel electrophoresis are shown below. Figure 6All inoculated diseased tissue samples were positive, while healthy rice tissue and water were negative. These results indicate that the ERA method for detecting rice blast fungus based on the MHP1-1 method described in this invention can detect rice blast fungus in diseased rice samples and can be used for the detection of rice blast fungus. Figure 6 In the middle, M: DL2000 Marker; lanes 1 to 8: 8 different diseased tissue samples; 9: healthy rice tissue.

[0061] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0062] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A primer pair for detecting rice blast fungus, characterized in that, The base sequences of the primer pairs are shown in SEQ ID NO.1 and SEQ ID NO.

2.

2. A kit for detecting rice blast fungus, characterized in that, The kit contains the primer pair as described in claim 1.

3. The kit according to claim 2, characterized in that, The kit also includes at least one of the following: an enzyme system, a buffer, an activator, and proteinase K, required for the enzymatic recombination isothermal amplification reaction.

4. The kit according to claim 3, characterized in that, The enzyme system contains at least one of recombinase, single-stranded binding protein, DNA polymerase, and reverse transcriptase; The buffer solution contains at least one of Mg(OAc)2, dNTPs and KOAc; The activator is Mg(OAc)2.

5. The application of the primer pair as described in claim 1 or the kit as described in claim 2, characterized in that, The application refers to any one of the following: 1) Detection of rice blast and / or rice blast fungus; 2) Identify rice blast disease and / or rice blast fungus.

6. The application as described in claim 5, characterized in that, The methods for detecting rice blast are as follows: DNA was extracted from the plants to be tested; Using DNA as a template, the kit was used for amplification to obtain amplification products; After digesting the amplification product, perform agarose gel electrophoresis. If bands appear, the tested plant has been infected with rice blast. If no bands appear, the tested plant is not infected with rice blast.

7. The application as described in claim 6, characterized in that, The amplification reaction conditions are: React at 37℃~40℃ for 20min~25min.

8. The application as described in claim 7, characterized in that, The amplification reaction conditions are: React at 38℃ for 20 minutes.

9. The application as described in claim 6, characterized in that, The process of digesting the amplification products is as follows: Add proteinase K to the amplification product and react at 55°C for 5 min.

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