RPA-LFD triple detection method and kit for rice blast, rice false smut and rhizoctonia solani and application of RPA-LFD triple detection method and kit

Abstract

The invention belongs to the field of biological detection, and particularly relates to an RPA-LFD triple detection method and kit for rice blast, rice false smut and rhizoctonia solani and application of the RPA-LFD triple detection method and kit. The detection method comprises the following steps: S.1, extracting DNA of a strain and a sample; s.2, designing primers and probes: designing corresponding RPA primers and probes according to specific target sequences of genome DNA of rice blast, rice false smut and rhizoctonia solani; and S.3, measuring the sensitivity of a multiple RPA-LFD reaction system. By adopting multiple RPA and designing multiple groups of specific primers and probes, synchronous detection of three pathogens can be completed in a single tube at the same time, so that the detection efficiency is improved; the detection flux of the multiple RPA-LFD technology is improved by more than 3 times, and the reagent consumption is reduced by 60%.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to the RPA-LFD triple detection method and kit for rice blast, rice false smut and sheath blight pathogens and their applications. Background Technology

[0002] Rice is one of the world's most important food crops. Rice blast, rice false smut, and rice sheath blight are the three major fungal diseases affecting rice yield, caused by the rice blast fungus (…). Magnaporthe oryzae ), rice false smut ( Ustilaginoidea virens ) and Rhizoctonia solani ( Rhizoctonia solani These three pathogens can overwinter in diseased plant debris or soil in the field. They have a long incubation period in the field and will cause irreversible damage to rice after infecting it. Therefore, early detection is of great significance for the prevention and control of rice diseases.

[0003] DNA-based molecular biology detection methods have become one of the mainstream technologies for pathogen detection due to their high sensitivity and specificity. Polymerase chain reaction (PCR), real-time fluorescence quantitative PCR (qPCR), and loop-mediated isothermal amplification (LAMP) are all important detection technologies in the field of molecular diagnostics. For rice blast fungus, the sensitivity of conventional PCR is 20 pg·μL. -1 qPCR, using TaqMan probe technology, can achieve 1 pg·μL -1 The sensitivity of LAMP technology is high, while its detection limit is 10 pg·μL. -1 In the detection of rice false smut, LAMP was used with HNB dye to achieve a colorimetric concentration of 100 pg·μL. -1 Its sensitivity is 10 times higher than that of conventional PCR, but lower than that of qPCR (50 fg·μL). -1 ).

[0004] Paper Quantitative Loop-Mediated Isothermal Amplification Detection of Ustilaginoidea virens In the study of Causing Rice False Smut, Zhang et al.'s qLAMP method, at an optimal reaction temperature of 63.4 °C, was able to detect a minimum spore concentration of 6.4 spores·mL within 60 min. -1In practical application, this method can detect 9 smut spores collected by polyester film tape. The paper Implementation of loop-mediated isothermal amplification methods in lateral flow devices for the detection of Rhizoctonia solani In the study of Jaimin et al. on rice sheath blight, the detection limit of qPCR was 2 fg·μL -1 However, the detection limit of the designed LAMP method was 5 times lower than that of qPCR. qPCR is suitable for precise quantification in the laboratory (such as resistance monitoring), while LAMP is more suitable for rapid screening in the field due to its constant temperature amplification characteristics (only a water bath is required). Currently, there are still few studies on multiple detection of field pathogens. When large-scale sample screening is required in the field or multiple pathogens need to be detected simultaneously, multiple detection becomes particularly important.

[0005] Recombinase polymerase amplification (RPA) is a new type of isothermal amplification technology that can complete DNA or RNA amplification within 30 min at a low temperature of 37-42℃, suitable for on-site detection and instant diagnosis. Multiple RPA detection technology can amplify multiple targets in one reaction system, significantly improving detection efficiency and reducing cost, and can be combined with lateral flow dipstick (LFD) technology for visual detection. The dipstick uses gold nanoparticle-labeled antibodies to specifically recognize certain antigens to detect RPA products, thereby eliminating the nucleotide purification step, and can visually display the results within 5-10 min. Currently, RPA-LFD has been reported in the detection of foodborne pathogens, parasites, viruses, and genetically modified organisms.

[0006] Traditional PCR technology is widely used in scientific research and clinical diagnosis due to its high specificity. qPCR technology can monitor the DNA amplification process in real time and quantitatively analyze the DNA concentration of pathogens by introducing fluorescent dyes or probes, greatly improving the accuracy and repeatability of detection. However, the equipment is expensive and takes a long time, which requires 2-3 h. LAMP technology breaks through the temperature cycle limitation and can complete amplification at a constant temperature of 60-65℃. The reaction time of 30-60 min and the visual detection results make it have great potential in on-site detection, but its multiple detection capability is limited, and the primer design is complex, which can easily cause non-specific amplification. Therefore, this invention uses multiple RPA to design multiple specific primers and probes to detect rice blast, smut, and sheath blight of rice. SUMMARY

[0007] In order to overcome the problems of long detection time, expensive detection equipment, complex primer design and low accuracy in the prior art, the present application provides a RPA-LFD triple detection method and kit for rice blast, rice sheath blight and sheath blight bacteria and its application by designing specific primers and probes for multiple RPA to amplify DNA and then synchronously detecting and judging the pathogens of rice blast, rice sheath blight and sheath blight bacteria.

[0008] The present application specifically adopts the following technical solutions to achieve the above purposes: In a first aspect, the present application provides a RPA-LFD triple detection method for rice blast, rice sheath blight and sheath blight bacteria, comprising the following steps: (S.1) Extraction of strains and sample DNA; (S.2) Design of primers and probes: design corresponding RPA primers and probes according to the target sequences of rice blast virus, rice sheath blight and sheath blight, wherein the primers include upstream primers and downstream primers, the upstream primers include M-TEF4-F1, U-1581-2-F2 and R-GD61-2-F2, and the nucleotide sequences thereof are shown in SEQ ID NO. 1, SEQ ID NO. 3 and SEQ ID NO. 5, respectively; the downstream primers include M-YEF4-R3B, U-1581-2-R3B and R-GD61-2-R2B, and the nucleotide sequences thereof are shown in SEQ ID NO. 2, SEQ ID NO. 4 and SEQ ID NO. 6, respectively; and the probes are M-TEF3-T1, U-1581-T1 and R-pg-GD61-T1, and the nucleotide sequences thereof are shown in SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9, respectively; (S.3) Multiple RPA-LFD determination: dilute the genomic DNA of rice blast, rice sheath blight and sheath blight bacteria to be used as a template, and perform amplification and observation of the results of the lateral flow chromatographic test strip under the RPA reaction system.

[0009] The probe has three modifications, including: base replacement with tetrahydrofuran at about 30 nt on the 5' end; 3' end label Spacer C3 modification; and 5' end modification label. The 5' end modification label of the probe M-TEF3-T1 is 6-carboxyfluorescein, the 5' end modification label of the probe U-1581-T1 is digoxin, and the 5' end modification label of the probe R-pg-GD61-T1 is 6-carboxytetramethylrhodamine.

[0010] Further, the reaction temperature of the multiple RPA-LFD is 35-39 DEG C.

[0011] Further, the reaction time of the multiple RPA-LFD is 15-25 min.

[0012] Further, the multiple RPA-LFD primer and probe concentrations are as follows: Magnaporthe oryzae and Ustilaginoidea virens primer concentration 200 nmol·L -1 , probe concentration 60 nmol·L -1 , Rhizoctonia solani primer concentration 300 nmol·L -1 , probe concentration 90 nmol·L -1 .

[0013] Further, in the multiple RPA system, the detection limit of Magnaporthe oryzae is 1 pg·μL -1 , the detection limit of Ustilaginoidea virens is 100 pg·μL -1 , and the detection limit of Rhizoctonia solani is 10 pg·μL -1 .

[0014] In a second aspect, the application provides a use of the RPA-LFD triple detection method for Magnaporthe oryzae, Ustilaginoidea virens and Rhizoctonia solani of rice as described above in the detection of rice leaves and soil.

[0015] In a third aspect, the application provides an RPA-LFD detection kit for Magnaporthe oryzae, Ustilaginoidea virens and Rhizoctonia solani of rice as described above, which comprises the upstream primers including M-TEF4-F1, U-1581-2-F2 and R-GD61-2-F2; the downstream primers including M-YEF4-R3B, U-1581-2-R3B and R-GD61-2-R2B; and the probes are M-TEF3-T1, U-1581-T1 and R-pg-GD61-T1, respectively.

[0016] In a fourth aspect, the application provides a use of the RPA-LFD detection kit for Magnaporthe oryzae, Ustilaginoidea virens and Rhizoctonia solani of rice in the simultaneous detection of Magnaporthe oryzae, Ustilaginoidea virens and Rhizoctonia solani of rice.

[0017] The application has the following beneficial effects: (1) The application uses multiple RPA to achieve efficient amplification of nucleic acids under constant temperature conditions of 37-42°C through the design of multiple specific primers and probes, and can simultaneously complete the synchronous detection of 3-5 kinds of pathogens in a single tube. Combined with a multi-target lateral flow chromatographic test strip, the results can be directly interpreted by the naked eye in only 15-30 minutes, greatly improving the detection reaction rate and saving the required time, and facilitating more timely and effective interpretation of the results.

[0018] (2) The detection flux of the multiple RPA-LFD technology used in the application is improved by more than 3 times, and the reagent consumption is reduced by 60%, which eliminates the dependence on professional laboratory equipment and only requires a simple constant temperature device to complete the entire detection process.

[0019] (3) The application is simple to operate, convenient for experiment, low in cost and easy to popularize, and is conducive to being widely used in production and practice. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Figure 3 is a sensitivity detection result diagram of a single RPA-LFD reaction system of rice blast fungus (A), rice sheath blight fungus (B) and rice sheath blight fungus (C), notes: 1-6: 100 pg·μL -1 , 10 pg·μL -1 , 1 pg·μL -1 , 100 fg·μL -1 , 10 fg·μL -1 , negative control group.

[0021] Figure 2 Figure 4 is a diagram of the establishment of a multiple RPA-LFD reaction system, notes: A: optimization of primer and probe concentration, 1-3 are grouped according to Table 2; B: optimization of reaction temperature, 1-6: 30℃, 33℃, 35℃, 37℃, 40℃, 43℃, 45℃; C: optimization of reaction time, 1-5: 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes.

[0022] Figure 3 Figure 5 is a sensitivity detection result diagram of a multiple RPA-LFD reaction system, notes: 1-8: 10 ng·μL -1 , 1 ng·μL -1 , 100 pg·μL -1 , 10 pg·μL -1 , 1 pg·μL -1 , 100 fg·μL -1 , 10 fg·μL -1 , negative control group.

[0023] Figure 4 Figure 6 is a specificity detection diagram of a multiple RPA-LFD reaction system, notes: 1: mixed rice blast fungus, rice sheath blight fungus and sheath blight fungus, 2: mixed rice blast fungus and rice sheath blight fungus, 3: mixed rice blast fungus and rice sheath blight fungus, 4: mixed rice sheath blight fungus and rice sheath blight fungus, 5-14: rice blast fungus, rice sheath blight fungus, rice sheath blight fungus, red fusarium, curvularia, flat pythium, bacterial leaf blight of rice, smut, negative control group.

[0024] Figure 5 Figure 7 is a result diagram of multiple RPA-LFD detection of rice blast, rice sheath blight and rice sheath blight from rice leaves and soil, notes: detection of rice leaves (A) and soil (B), 1: positive control group; 2-7: random samples; 8: negative control group. DETAILED DESCRIPTION

[0025] The present application is further explained by the following specific embodiments, which provide further advantages and features of the present application, and can be readily understood by those skilled in the art in view of the disclosure presented herein. The present application can also be practiced or carried out in various ways not specifically enumerated herein, with the understanding that the various embodiments and features of the present application can be modified or adapted in various ways based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that the following embodiments and features of the embodiments can be combined with each other without conflict. The methods used in the embodiments of the present application are conventional methods unless otherwise specified, and the reagents used are commercially available.

[0026] Example 1: RPA-LFD triple detection method of Magnaporthe grisea, Ustilaginoidea virens and Rhizoctonia solani 1. Experimental materials 1.1 Strains for testing Magnaporthe grisea, Ustilaginoidea virens, Rhizoctonia solani, Fusarium oxysporum f. sp. ( Fusarium proliferatum ), Curvularia ( Curvularia sp. ), Pyricularia grisea ( Nigrospora oryzar ), Pyricularia grisea ( Biopolaris oryzae ) and Xanthomonas oryzae pv. oryzae ( Xanthomonas oryzae pv. oryzicola ) were provided by the Plant Protection and Microbial Institute of the Zhejiang Academy of Agricultural Sciences.

[0027] 1.2 Main reagents RPA nucleic acid amplification kit and nucleic acid detection test strip were purchased from Ampure Future Biotechnology Co., Ltd. Soil DNA extraction kit was purchased from MP Biomedicals LLC. Centrifuge, CTAB extraction solution, shaker.

[0028] 2. Experimental methods 2.1 Extraction of strains and sample DNA The genomic DNA of the test strain was extracted by the CTAB method: a small amount of bacterial body was taken into a 2 ml centrifuge tube, 10 small steel balls with a diameter of 1 mm and 800 μL of preheated CTAB extraction solution were added, and the mixture was shaken in a shaker at 70 Hz for 150 s. After 1 h of water bath at 65 °C, an equal volume of phenol: chloroform: isopropyl alcohol (25:24:1) was added, mixed well, and then left to stand at room temperature for 15 min. After centrifugation at 5180 x g for 10 min, the supernatant was transferred to a new centrifuge tube. An equal volume of isopropyl alcohol was added to the supernatant, and the mixture was mixed gently by inversion. Centrifugation was performed at 4 °C and 5180 x g for 10 min. The supernatant was discarded, 1 mL of 75% ethanol was added, and the centrifuge tube was gently inverted. Centrifugation was performed at 4 °C and 5180 x g for 10 min. The supernatant was discarded, and the tube was inverted on a paper towel for 5-10 min. 20-50 μL of sterile water was added, and the mixture was incubated at 50 °C for 20 min. The resulting solution was the solution containing the genomic DNA of the strain. The genomic DNA of the soil sample was extracted using an MP soil DNA extraction kit.

[0029] 2.2 Design of primers and probes The target sequence of Magnaporthe grisea was the translation elongation factor 1-alpha gene (accession number: MGG_03641), the target sequence of Ustilaginoidea virens was G1581 (accession number: KY617824.1), and the target sequence of Rhizoctonia solani was the endopolygalacturonase gene (accession number: HQ197944.1). The RPA primers M-TEF4-F1 / R3B, U-1581-2-F2 / R3B, and R-GD61-2-F2 / R2B corresponding to the target were designed using Primer Premier 5.0 software. The 5' end of the downstream primer was labeled with biotin modification. The specificity of the primers was checked using the primer BLAST tool in NCBI. A sequence of 46-52 nt in length, M-TEF3-T1, U-1581-T1, and R-pg-GD61-T1, was designed between the respective upstream and downstream primers as a colloidal gold probe. This sequence was the same as or complementary to the target fragment amplified by the primer pair. The probe had three modifications: the base at about 30 nt on the 5' end was replaced with tetrahydrofuran (THF); the 3' end was labeled with Spacer C3 modification; and the 5' end of the three probes was labeled differently, with 6-carboxyfluorescein (6-FAM), digoxigenin (Digoxigenin), and 6-carboxytetramethylrhodamine (TAMRA), respectively. The detailed information of the primers and probes is shown in Table 1.

[0030] Table 1 Nucleotide sequences of the primers and probes designed and used for RPA-LFD assay

[0031] Note: idSp refers to the absence of tetrahydrofuran.

[0032] 2.3 Single RPA-LFD reaction system and sensitivity determination In each reaction tube containing dry powder, 29.4 μL A buffer, 200 nmol·L -1 Upstream and downstream primers, 60 nmol·L -1 Probe, 5 μL template DNA, 2.5 μL B buffer, and ddH2O to 50 μL. After mixing, centrifuge quickly to make the mixed reaction liquid at the bottom of the reaction tube, and then immediately place it in 37 ℃ for 20 min. After the completion of the amplification reaction, take 5 μL of the amplification product, dilute it 20 times with sterile ddH2O, mix well, take 80 μL of the diluted product, and drop it on the sample port of the test strip. After 5 min, observe the results.

[0033] Dilute the genomic DNA of rice blast, rice smut, and sheath blight to 1 ng·μL -1 , and sequentially dilute to 100 pg·μL -1 , 10 pg·μL -1 , 1 pg·μL -1 , 100 fg·μL -1 , and 10 fg·μL -1 , respectively, as templates. Observe the results of the lateral flow chromatographic test strip to evaluate the sensitivity of the multiplex RPA-LFD detection.

[0034] 3. Results analysis Using the gradient-diluted genomic DNA of rice blast, rice smut, and sheath blight as templates, and ddH2O as a negative control template, perform RPA reaction and test strip detection according to the method described in 2.3. The results show that all the control lines of the reactions have blue bands, and the negative control group does not have a dark red detection line. The color of the detection line of the test strip gradually weakens as the concentration of the template DNA decreases. When the concentration of the template DNA is 100 fg·μL -1 , the single RPA reaction results of the test strip detection of rice blast and rice smut show weak bands; when the concentration of the template DNA is 10 fg·μL -1 , the single RPA reaction results of the test strip detection of rice blast and rice smut show no bands, so the sensitivity of the single RPA detection of rice blast and rice smut is 100 fg·μL -1 ( Figure 1 A, B). When the concentration of the template DNA is 1 pg·μL -1 , the single RPA reaction results of the test strip detection of sheath blight show weak bands; when the concentration of the template DNA is 100 fg·μL -1The single RPA reaction result of the test strip showed no band, so the sensitivity of the single RPA detection of the sheath blight fungus was 1 pg·μL -1 . Figure 1 C).

[0035] Example 2: Optimization of multiplex RPA-LFD reaction system 1. Experimental method On the basis of the single RPA-LFD reaction, the concentration ratio of the three groups of primers in the RPA reaction, the reaction temperature and the reaction time were optimized. Different combinations of the addition amount of primers of rice blast, rice smut and sheath blight fungus were set up, as shown in Table 2. The RPA reaction temperature was set to 6 groups, which were set to 30 ℃, 33 ℃, 35 ℃, 37 ℃, 40 ℃, 43 ℃, 45 ℃ in turn. The RPA reaction time was set to 5 groups, which were set to 5 min, 10 min, 15 min, 20 min, 25 min in turn. After the RPA reaction, the bands on the lateral flow chromatographic test strip were observed and the results were analyzed.

[0036] Table 2 Concentration combinations of primers and probes for system screening

[0037] 2. Results analysis The primers and probes concentration, reaction temperature and reaction time in the multiplex RPA system were optimized and screened. Referring to the single RPA reaction condition, the reaction temperature was set to 37 ℃ and the reaction time was set to 20 min. It was found in the pre-experiment that when the concentrations of the three groups of primers or probes were the same, the amplification efficiency of the sheath blight fungus was weak, so the concentrations of the primers and probes of the sheath blight fungus were increased. The concentration combinations of the primers and probes were set up according to Table 2, and the results showed that the three positive bands on the test strip in the first group and the third group were obvious, and the positive band for detecting the sheath blight fungus in the first group was darker than that in the third group (A in Figure 2 , so the optimal primer and probe concentration of the multiplex RPA-LFD was 200 nmol·L -1 for the primers of the rice blast and the rice smut, 60 nmol·L -1 for the probe, 300 nmol·L -1 for the primers of the sheath blight fungus and 90 nmol·L -1 for the probe. With the selected optimal primer and probe combination, the reaction time was set to 20 min, and the temperature gradient of 30 ℃, 33 ℃, 35 ℃, 37 ℃, 40 ℃, 43 ℃ and 45 ℃ was set for the reaction optimal temperature screening. The results showed that when the reaction temperature was 37 ℃, the detection bands of the three kinds of bacteria on the test strip were the most obvious (B in Figure 2B), so 37 ℃ was selected as the RPA reaction temperature. With the selected optimal primer probe combination and optimal reaction temperature 37 ℃, reaction time gradients of 5 min, 10 min, 15 min, 20 min, and 25 min were set to screen the optimal reaction time. The results showed that when the reaction time was 5 min, there was no obvious band on the test strip; when the reaction time was 10 min or more, obvious bands appeared on the test strip; when the reaction time was 20 min, the band color reached the most obvious state, and with the increase of time, the band color did not deepen obviously Figure 2 Therefore, the optimal RPA reaction time was selected as 20 min.

[0038] Example 3: Sensitivity detection of multiplex RPA-LFD reaction system 1. Experimental method The genomic DNA of rice blast, rice sheath blight and sheath blight was diluted to 10 ng·μL -1 , 1 ng·μL -1 , 100 pg·μL -1 , 10 pg·μL -1 , 1 pg·μL -1 , 100 fg·μL -1 , and 10 fg·μL -1 , respectively, and used as templates. The optimized RPA reaction system was used for amplification and observation of the results of lateral flow chromatographic test strips, so as to evaluate and analyze the sensitivity detection of multiplex RPA-LFD.

[0039] 2. Results analysis According to the optimized multiplex RPA reaction conditions, the DNA concentrations of rice blast, rice sheath blight and sheath blight in the system were set to 10 ng·μL -1 , 1 ng·μL -1 , 100 pg·μL -1 , 10 pg·μL -1 , 1 pg·μL -1 , 100 fg·μL -1 , and 10 fg·μL -1 , respectively. The results showed that the color of the three positive bands in the test strip became lighter as the concentration of the template DNA decreased; when the genomic DNA concentrations of the three pathogens in the reaction system were 100 pg·μL -1 , there were three obvious positive bands on the test strip; when the genomic DNA concentrations of the three pathogens in the reaction system were 100 fg·μL -1 , there was no positive band detected on the test strip, only the quality control line Figure 3 . Among them, the minimum concentration of rice blast genomic DNA in the multiplex reaction system was 1 pg·μL -1At that time, positive bands were still detected; the lowest concentration of rice false smut genomic DNA was 100 pg·μL. -1 At that time, positive bands were still detected; the lowest concentration of genomic DNA of *Rhizoctonia solani* was 10 pg·μL. -1 At that time, a positive band was still detected. Therefore, the detection limit for *Strombus rice* in this multiplex RPA system is 1 pg·μL. -1 The detection limit for rice false smut is 100 pg·μL. -1 The detection limit for rice sheath blight pathogen is 10 pg·μL. -1 .

[0040] Example 4: Specificity detection of multiple RPA-LFD reaction systems 1. Experimental Methods Genomic DNA was extracted from the tested strains using the CTAB method. This genomic DNA, along with ddH2O, served as a negative control, while genomic DNA from rice blast, rice false smut, and sheath blight pathogens served as positive controls. The results of the lateral flow chromatography test strips were observed to evaluate the specificity of the multiplex RPA-LFD assay.

[0041] 2. Results Analysis To verify the specificity of this multiple RPA reaction system, *Helicobacter pylori* (a type of fungus) was selected. Biopolaris oryzae ), Curvularia ( Curvularia sp. Rice streak pathogen ( Xanthomonas oryzae pv.oryzicola, Xoc Specific detection of common pathogenic microorganisms in rice fields, such as rice blast, rice false smut, and rice sheath blight pathogens, was performed. Results showed that no positive bands were detected in the detection systems using non-target strain DNA and ddH2O as templates; however, in triple, double, and single detection systems using rice blast, rice false smut, and rice sheath blight pathogen DNA as templates, the corresponding strains' bands were accurately detected. Figure 4 This demonstrates that the reaction system has good specificity.

[0042] Example 5: Application of a Multiple RPA-LFD Reaction System 1. Experimental Methods Six samples each of rice leaves and soil around the roots were collected from the field. Genomic DNA was extracted from the leaves and soil for RPA analysis. The positive control was a mixture of genomic DNA from rice blast, rice false smut, and rice sheath blight pathogens, while the negative control consisted of genomic DNA from healthy rice leaves and soil.

[0043] 2. Results Analysis The multiple RPA-LFD practicality detection was carried out on the rice leaf and soil samples collected in the field. The results showed that the multiple RPA-LFD could detect Magnaporthe oryzae, Ustilaginoidea virens and Rhizoctonia solani from the rice leaf and soil, and no Magnaporthe oryzae, Ustilaginoidea virens and Rhizoctonia solani were detected in the healthy rice and soil Figure 5 ), indicating that the RPA-LFD can be used for the detection of rice leaf and soil, and has strong practicability.

Claims

1. A triple detection method of Magnaporthe oryzae, Ustilaginoidea virens and Rhizoctonia solani by RPA-LFD, characterized by, Comprising the following steps: (S.1) Extraction of strains and sample DNA; (S.2) Design of primers and probes: design corresponding RPA primers and probes according to the target sequences of rice blast, rice smut and sheath blight, the primers include upstream primers and downstream primers, the upstream primers include: M-TEF4-F1, U-1581-2-F2, R-GD61-2-F2, the nucleotide sequences are shown in SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5 respectively; the downstream primers include M-YEF4-R3B, U-1581-2-R3B, R-GD61-2-R2B, the nucleotide sequences are shown in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6 respectively; the probes are M-TEF3-T1, U-1581-T1, R-pg-GD61-T1, the nucleotide sequences are shown in SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 respectively; (S.3) Multiple RPA-LFD assay: dilute the genomic DNA of rice blast, rice smut and sheath blight as a template, and perform amplification under RPA reaction system and observe the results of lateral flow chromatographic test strip.

2. The RPA-LFD triplex detection method for Magnaporthe oryzae, Ustilaginoidea virens and Rhizoctonia solani of rice according to claim 1, wherein, The multiple RPA-LFD reaction temperature is 35℃-39℃.

3. The RPA-LFD triplex detection method for Magnaporthe oryzae, Ustilaginoidea virens and Rhizoctonia solani of claim 1, wherein, The multiple RPA-LFD reaction time is 15min-25min.

4. The RPA-LFD triplex detection method for Magnaporthe oryzae, Ustilaginoidea virens and Rhizoctonia solani of claim 1, wherein, The detection primer concentration of Magnaporthe grisea and Ustilaginoidea virens in the multiplex RPA reaction system is 150-250 nmol / L -1 , the probe concentration is 55-65 nmol / L -1 , the primer concentration of Rhizoctonia solani is 250-350 nmol / L -1 , and the probe concentration is 85-95 nmol / L -1 .

5. The RPA-LFD triplex detection method for Magnaporthe oryzae, Ustilaginoidea virens and Rhizoctonia solani of rice according to claim 4, wherein, In the multiplex RPA system, the detection limit of M. grisea was 1 pg·μL -1 , that of M. graminicola was 100 pg·μL -1 , and that of R. solani was 10 pg·μL -1 .

6. The application of a rice blast, rice smut and sheath blight RPA-LFD triple detection method according to any one of claims 1-5 in the detection of rice leaves and soil.

7. A RPA-LFD detection kit for Magnaporthe oryzae, Ustilaginoidea virens and Rhizoctonia solani of rice, characterized by, Comprising upstream primers, downstream primers and probes, the upstream primers include: M-TEF4-F1, U-1581-2-F2 and R-GD61-2-F2, the nucleotide sequences are shown in SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5 respectively; the downstream primers include: M-YEF4-R3B, U-1581-2-R3B and R-GD61-2-R2B, the nucleotide sequences are shown in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6 respectively; the probes include: M-TEF3-T1, U-1581-T1 and R-P8-GD61-T1, the nucleotide sequences are shown in SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 respectively.

8. The application of a rice blast, rice smut and sheath blight RPA-LFD detection kit according to claim 7 in the simultaneous detection of rice blast, rice smut and rice sheath blight.

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