Rpa-lfd triple detection method and kit for rice blast, rice sheath blight and sheath blight of rice and application thereof

Abstract

The application belongs to the field of biological detection, and particularly relates to a RPA-LFD triple detection method and kit for rice blast, rice smut and sheath blight bacteria and application thereof. The detection method comprises the following steps: S.1 extraction of strain and sample DNA; S.2 design of primer and probe: corresponding RPA primers and probes are designed according to specific target sequences of rice blast, rice smut and sheath blight bacteria genomic DNA; S.3 sensitivity determination of multiplex RPA-LFD reaction system. The application can simultaneously complete synchronous detection of three kinds of pathogens in a single tube by designing multiple specific primers and probes through multiplex RPA, thereby improving detection efficiency; the detection throughput of multiplex RPA-LFD technology is improved by more than 3 times, and 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 applications, this method can detect nine rice false spores collected using polyester film tape. The paper, "Implementation of loop-mediated isothermal amplification methods in lateral flow devices for the detection of...", is titled "Implementation of loop-mediated isothermal amplification methods in lateral flow devices for the detection of...". Rhizoctonia solani In their study on rice sheath blight pathogens, Jaimin et al. found that the qPCR detection limit was 2 fg·μL. -1 However, the detection limit of the designed LAMP method is 5 times lower than that of qPCR. qPCR is suitable for precise quantification in the laboratory (such as antibiotic resistance monitoring), while LAMP, due to its isothermal amplification characteristics (requiring only a water bath), is more suitable for rapid field screening. Currently, there are relatively few studies on multiplex detection of pathogens in the field. Multiplex detection becomes particularly important when large-scale sample screening or simultaneous detection of multiple pathogens is required in the field.

[0005] Recombinase polymerase amplification (RPA) is a novel isothermal amplification technique that can amplify DNA or RNA within 30 minutes at low temperatures of 37-42°C, making it suitable for on-site detection and point-of-care diagnosis. Multiplex RPA detection technology can simultaneously amplify multiple targets in a single reaction system, significantly improving detection efficiency and reducing costs. Combined with lateral flow dipstick (LFD) technology, it enables visualized detection. The test strip utilizes gold nanoparticle-labeled antibodies to specifically recognize a particular antigen to detect RPA products, thus eliminating the need for nucleotide purification and providing results visually within 5-10 minutes. Currently, the application of RPA-LFD in the detection of foodborne pathogens, parasites, viruses, and genetically modified organisms has been reported.

[0006] Traditional PCR technology is widely used in scientific research and clinical diagnosis due to its high specificity. qPCR technology, by introducing fluorescent dyes or probes, can monitor the DNA amplification process in real time and quantitatively analyze the DNA concentration of pathogens, greatly improving the accuracy and repeatability of detection. However, the equipment is expensive and, like PCR, takes 2-3 hours. LAMP technology overcomes the limitations of temperature cycling, completing amplification under constant temperature conditions of 60-65℃. Its 30-60 minute reaction time and visually perceptible detection results make it highly promising for on-site detection. However, its multiplexing capability is limited, and primer design is complex, easily leading to non-specific amplification. Therefore, this invention utilizes multiplex RPA, designing multiple sets of specific primers and probes to detect rice blast, rice false smut, and sheath blight pathogens. Summary of the Invention

[0007] To overcome the problems of excessively long detection time, expensive detection equipment, complex primer design, and low accuracy in existing technologies, this invention provides a triple detection method and kit for rice blast, rice false smut, and rice sheath blight pathogens by designing specific primers and probes using multiple RPA, and then simultaneously detecting and interpreting these pathogens.

[0008] The present invention is specifically implemented using the following technical solutions:

[0009] In a first aspect, the present invention provides a triple detection method for rice blast, rice false smut, and sheath blight pathogens via RPA-LFD, comprising the following steps:

[0010] (S.1) Extraction of strain and sample DNA;

[0011] (S.2) Primer and probe design: Based on the target sequences of rice blast virus, rice false smut, and rice sheath blight, corresponding RPA primers and probes were designed. The primers include upstream primers and downstream primers. The upstream primers include M-TEF4-F1, U-1581-2-F2, and R-GD61-2-F2, with nucleotide sequences 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, with nucleotide sequences shown in SEQ ID NO.2, SEQ ID NO.4, and SEQ ID NO.6, respectively. The probes are M-TEF3-T1, U-1581-T1, and R-pg-GD61-T1, with nucleotide sequences shown in SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively.

[0012] (S.3) Multiplex RPA-LFD assay: Diluted genomic DNA of rice blast, rice false smut and sheath blight pathogens were used as templates for amplification in the RPA reaction system and the results of the side-flow chromatography test strips were observed.

[0013] The probes of this invention have three modifications, including: replacement of the base at approximately 30 nt at the 5' end with tetrahydrofuran; a Spacer C3 modification at the 3' end; and a 5' end modification marker. Specifically, the 5' end modification marker for probe M-TEF3-T1 is 6-carboxyfluorescein, the 5' end modification marker for probe U-1581-T1 is digoxigenin, and the 5' end modification marker for R-pg-GD61-T1 is 6-carboxytetramethylrhodamine.

[0014] Furthermore, the reaction temperature of the multiple RPA-LFD is 35℃-39℃.

[0015] Furthermore, the reaction time of the multiple RPA-LFD is 15 min-25 min.

[0016] Furthermore, the concentrations of the multiplex RPA-LFD primers and probes are as follows: 200 nmol·L⁻¹ for the primers of *Strombus oryzae* and *Strombus oryzae*. -1 Probe concentration 60 nmol·L -1 Primer concentration for *Rhizoctonia solani* was 300 nmol·L⁻¹ -1 Probe concentration 90 nmol·L -1 .

[0017] Furthermore, in the multiplex RPA system, the detection limit for rice blast fungus was 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 .

[0018] Secondly, the present invention provides an application of the RPA-LFD triple detection method for rice blast, rice false smut and sheath blight pathogens as described above in the detection of rice leaves and soil.

[0019] Thirdly, the present invention provides an RPA-LFD detection kit for rice blast, rice false smut, and sheath blight pathogens as described above, comprising 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 being M-TEF3-T1, U-1581-T1, and R-pg-GD61-T1, respectively.

[0020] Fourthly, the present invention provides an RPA-LFD detection kit for rice blast, rice false smut and sheath blight pathogens, and its application in the simultaneous detection of rice blast, rice false smut and sheath blight pathogens.

[0021] The present invention has the following beneficial effects:

[0022] (1) This invention uses multiple RPA to design multiple sets of specific primers and probes to achieve efficient amplification of nucleic acids under constant temperature conditions of 37-42℃. It can simultaneously detect 3-5 pathogens in a single tube. With the multi-target side-flow chromatography test strip, the results can be directly interpreted by the naked eye in just 15-30 minutes, which greatly improves the detection reaction rate, saves the required time, and facilitates obtaining interpretation results more timely and effectively.

[0023] (2) The detection throughput of the multiple RPA-LFD technology used in this invention is increased by more than 3 times, and the reagent consumption is reduced by 60%. It eliminates the dependence on professional laboratory equipment and only requires a simple constant temperature device to complete the entire detection process.

[0024] (3) The present invention is simple to operate, convenient to experiment, low in cost and easy to promote, which is conducive to its widespread use in production and practice. Attached Figure Description

[0025] Figure 1 The graph shows the sensitivity detection results of single RPA-LFD reaction systems for rice blast fungus (A), rice false smut fungus (B), and rice sheath blight fungus (C). Note: 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.

[0026] Figure 2 The diagram shows the establishment of the multiplex RPA-LFD reaction system. Note: A: Optimization of primer and probe concentrations, 1-3 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.

[0027] Figure 3 The graph shows the sensitivity detection results in the multiple RPA-LFD reaction system. Note: 1-8: 10 ng·μL -1 1ng·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.

[0028] Figure 4 This is a specific detection diagram of the multiplex RPA-LFD reaction system. Note: 1: Mixed strains of rice blast fungus, rice false smut fungus, and rice sheath blight fungus; 2: Mixed strains of rice blast fungus and rice false smut fungus; 3: Mixed strains of rice blast fungus and rice sheath blight fungus; 4: Mixed strains of rice false smut fungus and rice sheath blight fungus; 5-14: Rice blast fungus, rice false smut fungus, rice sheath blight fungus, Fusarium oxysporum, Curvularia, Helicobacter pylori, Bacterial leaf streak of rice, Ustilago maydis, negative control group.

[0029] Figure 5Figure 1 shows the results of multiplex RPA-LFD detection of rice blast, rice false smut, and rice sheath blight pathogens in rice leaves and soil. Note: Detection of rice leaves (A) and soil (B), 1: positive control group; 2-7: random samples; 8: negative control group. Detailed Implementation

[0030] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0031] Example 1: RPA-LFD triple detection method for rice blast, rice false smut, and rice sheath blight pathogens

[0032] 1. Experimental Materials

[0033] 1.1 Test strains

[0034] Rice blast fungus, rice false smut fungus, rice sheath blight fungus, Fusarium graminearum (… Fusarium proliferatum ), Curvularia ( Curvularia sp. ), *Ustilago maydis* ( Nigrospora oryzar ), *Helicobacter pylori* ( Biopolaris oryzae ) and rice bacterial leaf streak ( Xanthomonas oryzae pv. oryzicola All of these were provided by the Crop Disease Control Team of the Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences.

[0035] 1.2 Main Reagents

[0036] The RPA nucleic acid amplification kit and nucleic acid test strips were purchased from Anpu Future Biotechnology Co., Ltd. The soil DNA extraction kit was purchased from MP Biopharmaceuticals, Inc. (USA). Centrifuge, CTAB extraction buffer, and shaker were also used.

[0037] 2. Experimental Methods

[0038] 2.1 Extraction of DNA from Strains and Samples

[0039] Genomic DNA was extracted from the tested strain using the CTAB method: A small amount of bacterial cells was placed in a 2 ml centrifuge tube, along with 10 1 mm diameter steel beads and 800 μL of preheated CTAB extraction buffer. The mixture was shaken at 70 Hz for 150 s. After incubating at 65 °C for 1 h, an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added and mixed thoroughly. After standing at room temperature for 15 min, the mixture was centrifuged at 5180 × g for 10 min, and the supernatant was transferred to a new centrifuge tube. An equal volume of isopropanol was added to the supernatant, and the mixture was gently inverted to mix. The mixture was centrifuged at 5180 × g for 10 min at 4 °C. The supernatant was discarded, and 1 mL of 75% ethanol was added. The centrifuge tube was gently inverted. The mixture was centrifuged at 5180 × g for 10 min at 4 °C. The supernatant was discarded, and the tube was inverted onto a paper towel for 5-10 min. Add 20-50 μL of sterile water, incubate at 50 ℃ for 20 min to remove volatiles, and the resulting solution contains the genomic DNA of this strain. Genomic DNA from soil samples was extracted using the MP Soil DNA Extraction Kit.

[0040] 2.2 Primer and probe design

[0041] The target sequences for *Bacillus oryzae* are the translation elongation factor 1-alpha gene (accession number: MGG_03641), the target sequence for *Syngonium oryzae* is G1581 (accession number: KY617824.1), and the target sequence for *Rhizoctonia solani* is the endoplasmic polygalacturonase gene (accession number: HQ197944.1). RPA primers M-TEF4-F1 / R3B, U-1581-2-F2 / R3B, and R-GD61-2-F2 / R2B were designed using Primer Premier 5.0 software, with biotin-modified 5' ends of all downstream primers. Primer specificity was checked using the primer BLAST tool in NCBI. A 46-52 nt sequence, M-TEF3-T1, U-1581-T1, and R-pg-GD61-T1, was designed as a colloidal gold probe between each primer pair and its upstream and downstream primers. This sequence must be identical or complementary to the target fragment amplified by the primer pair. The probes underwent three modifications: the base at approximately 30 nt at the 5' end was replaced with tetrahydrofuran (THF); the 3' end was labeled with Spacer C3; and the 5' end of the three probes was labeled differently: 6-carboxyfluorescein (6-FAM), digoxigenin, and 6-carboxytetramethylrhodamine (TAMRA), respectively. Detailed information on the primers and probes is shown in Table 1.

[0042] Table 1. Nucleotide sequences of primers and probes designed for RPA-LFD assay.

[0043]

[0044] Note: idSp refers to tetrahydrofuran deletion.

[0045] 2.3 Single RPA-LFD reaction system and sensitivity determination

[0046] Add 29.4 μL of buffer A and 200 nmol·L⁻¹ to each reaction tube containing the dry powder. -1 Upstream and downstream primers, 60 nmol·L -1 The sample consisted of a probe, 5 μL template DNA, 2.5 μL B buffer, and ddH2O to a final volume of 50 μL. After mixing, the mixture was rapidly centrifuged to ensure all the reaction mixture reached the bottom of the tube. The tube was then immediately incubated at 37 °C for 20 min. After the amplification reaction was complete, 5 μL of the amplification product was diluted 20-fold with sterile ddH2O and mixed thoroughly. 80 μL of the diluted product was then dropped into the sample application port of the test strip. The results were observed after 5 min.

[0047] Genomic DNA from rice blast, rice false smut, and sheath blight was diluted to 1 ng / μL. -1 Diluted sequentially to 100 pg·μL -1 10 pg·μL -1 1 pg·μL -1 100 fg·μL -1 10 fg·μL -1 Used as a template. Observe the results of the side-flow chromatography test strip to evaluate the sensitivity of the analysis of multiplex RPA-LFD.

[0048] 3. Results Analysis

[0049] Using serially diluted genomic DNA from *Oryza sativa*, *Oryza sativa* blast fungus, and *Sheath blight* as templates, and ddH2O as a negative control template, RPA reactions and test strip detection were performed according to the method described in section 2.3. The results showed that all control lines had blue bands, while no dark red test lines appeared in the negative control groups. The color of the test lines in the experimental groups gradually decreased with decreasing template DNA concentration. When the template DNA concentration was 100 fg·μL... -1 At that time, the single RPA reaction results of rice blast and rice false smut fungus showed weak bands in the test strip detection; when the template DNA concentration was 10 fg·μL -1 At that time, the single RPA reaction results for rice blast and rice false smut showed no bands on the test strips; therefore, the sensitivity of single RPA detection for rice blast and rice false smut is 100 fg·μL. -1 ( Figure 1 (A and B in the original text). When the template DNA concentration is 1 pg·μL -1 At that time, the single RPA reaction result of *Rhizoctonia solani* showed a weak band in the test strip; when the template DNA concentration was 100 fg·μL-1 At that time, the single RPA reaction result of *Rhizoctonia solani* showed no band in the test strip, therefore the sensitivity of the single RPA detection of *Rhizoctonia solani* was 1 pg·μL. -1 ( Figure 1 (C in the middle).

[0050] Example 2: Optimization of Multiple RPA-LFD Reaction Systems

[0051] 1. Experimental Methods

[0052] Based on the single RPA-LFD reaction, the concentration ratios of the three primer groups, reaction temperature, and reaction time in the RPA reaction were optimized. Three combinations of primer additions for rice blast, rice false smut, and sheath blight were set up (see Table 2). Six RPA reaction temperatures were set at 30 ℃, 33 ℃, 35 ℃, 37 ℃, 40 ℃, 43 ℃, and 45 ℃. Five RPA reaction times were set at 5 min, 10 min, 15 min, 20 min, and 25 min. After the RPA reaction, the bands on the lateral flow chromatography strips were observed and the results were analyzed.

[0053] Table 2 Primer and probe concentration combinations used for system screening

[0054]

[0055] 2. Results Analysis

[0056] The primer and probe concentrations, reaction temperature, and reaction time in the multiplex RPA system were optimized and screened. Referring to the single-RPA reaction conditions, the reaction temperature was set to 37 ℃ and the reaction time to 20 min. Preliminary experiments showed that when the concentrations of the three primers or probes in the multiplex RPA were the same, the amplification efficiency of *Rhizoctonia solani* was weak. Therefore, the primer and probe concentrations for *Rhizoctonia solani* were increased. The primer and probe concentration combinations are shown in Table 2. The results showed that all three positive bands on the test strips in groups 1 and 3 were obvious, and the positive band for *Rhizoctonia solani* in group 1 was darker than that in group 3. Figure 2 Therefore, the optimal primer and probe concentrations for multiplex RPA-LFD are 200 nmol·L⁻¹ for both rice blast fungus and rice false smut fungus. -1 Probe concentration 60 nmol·L -1 Primer concentration for *Rhizoctonia solani* was 300 nmol·L⁻¹ -1 Probe concentration 90 nmol·L -1The optimal reaction temperature was determined by screening the best primer-probe combination, setting a reaction time of 20 min, and establishing temperature gradients of 30 ℃, 33 ℃, 35 ℃, 37 ℃, 40 ℃, 43 ℃, and 45 ℃. The results showed that the detection bands for the three bacteria were most prominent on the test strip at a reaction temperature of 37 ℃. Figure 2 Therefore, 37 ℃ was chosen as the RPA reaction temperature (based on the optimal primer-probe combination and optimal reaction temperature of 37 ℃). Optimal reaction times were selected by setting reaction time gradients of 5 min, 10 min, 15 min, 20 min, and 25 min. The results showed that no obvious bands appeared on the test strip at a reaction time of 5 min; obvious bands appeared on the test strip at reaction times of 10 min and above; and the band color reached its most obvious state at 20 min, with no significant deepening of the band color over time. Figure 2 (C in the text); therefore, the optimal reaction time for RPA is 20 min.

[0057] Example 3: Sensitivity detection of multiple RPA-LFD reaction systems

[0058] 1. Experimental Methods

[0059] The genomic DNA of rice blast, rice false smut, and sheath blight pathogens were serially 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 Using the optimized RPA reaction system as a template, the results of the side-flow chromatography test strip were amplified and observed to evaluate the sensitivity of the multiplex RPA-LFD assay.

[0060] 2. Results Analysis

[0061] Based on the optimized multiple RPA reaction conditions, the concentrations of rice blast, rice false smut, and sheath blight pathogen DNA in the system were set at 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 -1The results showed that all three positive bands on the test strips became lighter in color as the concentration of template DNA decreased; when the concentrations of genomic DNA of the three pathogens in the reaction system were 100 pg·μL, the color change was more pronounced. -1 The test strips all showed three distinct positive bands; when the concentrations of the genomic DNA of the three pathogens in the reaction system were 100 fg·μL, respectively... -1 There was no positive band on the test strip, only the control line. Figure 3 The lowest concentration of rice blast fungus genomic DNA in the multiplex reaction system was 1 pg·μL. -1 At 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 .

[0062] Example 4: Specificity detection of multiple RPA-LFD reaction systems

[0063] 1. Experimental Methods

[0064] 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.

[0065] 2. Results Analysis

[0066] 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.

[0067] Example 5: Application of a Multiple RPA-LFD Reaction System

[0068] 1. Experimental Methods

[0069] 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.

[0070] 2. Results Analysis

[0071] Multiplex RPA-LFD was performed on rice leaf and soil samples collected in the field. The results showed that multiplex RPA-LFD could detect rice blast, rice false smut, and rice sheath blight pathogens from rice leaves and soil. These pathogens were not detected in healthy rice or soil. Figure 5 This indicates that the RPA-LFD can be used for the detection of rice leaves and soil, and has strong practicality.

Claims

1. A triple detection method for rice blast, rice false smut, and sheath blight pathogens via RPA-LFD, characterized in that, Includes the following steps: (S.1) Extraction of strain and sample DNA; (S.2) Primer and probe design: Based on the target sequences of rice blast, rice false smut, and sheath blight pathogens, corresponding RPA primers and probes were designed. The primers include upstream and downstream primers. The upstream primer designed based on the rice blast target sequence is M-TEF4-F1, with the nucleotide sequence SEQ ID NO.1; the downstream primer is M-YEF4-R3B, with the nucleotide sequence SEQ ID NO.2; and the probe is M-TEF3-T1, with the nucleotide sequence SEQ ID NO.

7. The upstream primer designed based on the rice blast target sequence is U-1581-2-F2, with the nucleotide sequence SEQ ID NO.3; the downstream primer is U-1581-2-R3B, with the nucleotide sequence SEQ ID NO.4; and the probe is U-1581-T1, with the nucleotide sequence SEQ ID NO.

8. The upstream primer designed based on the target sequence of *Rhizoctonia solani* is R-GD61-2-F2, with the nucleotide sequence SEQ ID NO.5; the downstream primer is R-GD61-2-R2B, with the nucleotide sequences SEQ ID NO.6; and the probe is R-pg-GD61-T1, with the nucleotide sequence SEQ ID NO.

9. (S.3) Multiplex RPA-LFD assay: Diluted genomic DNA of rice blast, rice false smut and sheath blight pathogens were used as templates for amplification in the RPA reaction system and the results of the side-flow chromatography test strips were observed.

2. The RPA-LFD triple detection method for rice blast, rice false smut, and sheath blight pathogens as described in claim 1, characterized in that, The reaction temperature of the multiple RPA-LFD is 35℃-39℃.

3. The RPA-LFD triple detection method for rice blast, rice false smut, and sheath blight pathogens as described in claim 1, characterized in that, The reaction time of the multiple RPA-LFD is 15 min-25 min.

4. The RPA-LFD triple detection method for rice blast, rice false smut, and sheath blight pathogens as described in claim 1, characterized in that, In the aforementioned multiplex RPA reaction system, the concentration of the detection primers for rice blast fungus and rice false smut fungus was 150-250 nmol·L⁻¹. -1 Probe concentration 55-65 nmol·L -1 Primer concentration for *Rhizoctonia solani* was 250-350 nmol·L⁻¹ -1 Probe concentration 85-95 nmol·L -1 .

5. The RPA-LFD triple detection method for rice blast, rice false smut, and sheath blight pathogens as described in claim 4, characterized in that, In the multiplex RPA system, the detection limit for rice blast fungus was 1 pg·μL. -1 The detection limit for rice false smut is 100 pg·μL. -1 The detection limit for *Rhizoctonia solani* is 10 pg·μL. -1 .

6. The application of the RPA-LFD triple detection method for rice blast, rice false smut and sheath blight as described in any one of claims 1-5 in the detection of rice leaves and soil.

7. An RPA-LFD detection kit for rice blast, rice false smut, and rice sheath blight pathogens, used for the simultaneous detection of rice blast, rice false smut, and rice sheath blight pathogens, characterized in that... The kit includes RPA primers and probes designed based on the target sequences of rice blast, rice false smut, and sheath blight pathogens. The primers include upstream and downstream primers. The upstream primer designed based on the rice blast target sequence is M-TEF4-F1, with the nucleotide sequence SEQ ID NO.1; the downstream primer is M-YEF4-R3B, with the nucleotide sequence SEQ ID NO.2; and the probe is M-TEF3-T1, with the nucleotide sequence SEQ ID NO.

7. The upstream primer designed based on the rice blast target sequence is U-1581-2-F2, with the nucleotide sequence SEQ ID NO.3; the downstream primer is U-1581-2-R3B, with the nucleotide sequence SEQ ID NO.4; and the probe is U-1581-T1, with the nucleotide sequence SEQ ID NO.

8. The upstream primer designed based on the target sequence of *Rhizoctonia solani* is R-GD61-2-F2, with the nucleotide sequence SEQ ID NO.5; the downstream primer is R-GD61-2-R2B, with the nucleotide sequences SEQ ID NO.6; and the probe is R-pg-GD61-T1, with the nucleotide sequence SEQ ID NO.9.

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