Fusarium oxysporum lAMP detection primer, detection reagent kit and application thereof

Abstract

This invention belongs to the field of plant pathogenic microorganism detection technology, and discloses LAMP detection primers, detection kits and their applications for Fusarium oxysporum. The primers are targeted at Fusarium oxysporum. EF-1 alpha The gene design consists of four specific primers: F3-FO, B3-FO, FIP-FO, and BIP-FO. This invention establishes a loop-mediated isothermal amplification (LAMP) detection method using these primers. The detection results can be directly observed with the naked eye to determine the presence of *Fusarium oxysporum* in the sample. The primers of this invention have a sensitivity of up to 100 ag / μL and high specificity, enabling the detection of *Fusarium oxysporum* in tomato leaves at the early stage of infection, before obvious disease phenotypes are observed. This invention is suitable for rapid, sensitive, and visual detection of early-stage *Fusarium oxysporum* wilt in tomatoes.

Description

Technical Field

[0001] This invention belongs to the field of plant pathogenic microorganism detection technology, specifically to primers, detection kits and their applications for LAMP detection of Fusarium oxysporum. Background Technology

[0002] Fusarium wilt, caused by Fusarium oxysporum, is a highly prevalent fungal disease that can cause significant yield reductions. This pathogen is characterized by rapid spread, a wide host range, and a long disease cycle. In intensive greenhouse vegetable cultivation, large-scale outbreaks often result in irreparable economic losses. Therefore, early detection and precise intervention of the pathogen before or in the early stages of disease development are crucial for preventing disease outbreaks and ensuring the safe and green production of vegetables.

[0003] Currently, while pathogen identification techniques are constantly evolving, both traditional methods relying on morphological differences and molecular detection techniques based on temperature-dependent amplification of nucleic acid sequences have limitations in meeting the immediate testing needs of fields and grassroots stations. Traditional morphological identification methods are cumbersome, time-consuming, require a high level of professional experience from operators, and are susceptible to human error. In contrast, while conventional PCR technology significantly improves detection specificity, its operation is highly dependent on precise thermal cycling temperature control equipment, resulting in complex procedures and relatively long testing times, making it difficult to widely apply in rapid screening work outside of laboratory settings.

[0004] Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid isothermal amplification technique whose core advantage is the efficient amplification of target nucleic acid sequences within a constant temperature range. This technology integrates multiple advantages, including high sensitivity capture, high specificity recognition, short reaction time, colorimetric visualization, and relaxed hardware requirements, effectively overcoming the inherent technical shortcomings of variable-temperature amplification techniques. It has become a research hotspot in the field of plant disease detection, particularly suitable for emergency diagnosis at grassroots stations and in the field. However, existing LAMP detection methods for Fusarium oxysporum reported in the literature still have issues such as room for optimization of primer combination specificity and the fact that reaction system parameters have not yet reached optimal performance. Furthermore, in the early stages of pathogen colonization or when the spore suspension concentration is low, the minimum stable detection concentration cannot fully meet the needs for early warning at ultra-micro levels.

[0005] In summary, Fusarium wilt of tomatoes poses a serious threat to the greenhouse vegetable industry, and current detection technologies still have significant shortcomings in terms of detection limits and response efficiency. Therefore, it is practically necessary to develop a more sensitive and specific LAMP detection method for Fusarium oxysporum and to systematically optimize its reaction parameters for the very early and accurate identification of this pathogen. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention aims to provide LAMP detection primers, detection kits, and their applications for Fusarium oxysporum, in order to achieve specific and rapid detection of Fusarium oxysporum and solve the problem of insufficient sensitivity in the early detection of this pathogen in tomatoes.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A primer for detecting Fusarium oxysporum using LAMP consists of an outer primer and an inner primer. The outer primer is a forward outer primer F3-FO and a reverse outer primer B3-FO. The inner primer is a forward inner primer FIP-FO and a reverse inner primer BIP-FO.

[0009] The nucleotide sequences of each primer are as follows:

[0010] F3-FO: 5'-GAGCGACGGGGAGCGTTTGC-3';

[0011] B3-FO: 5'-CGGTGACATAGTAGCGAGGA-3';

[0012] FIP-FO: 5'-CGAGCTCAGCGGCTTCCTATTGCCCTCTTACCATTCTCACA-3';

[0013] BIP-FO: 5'-AGGGTTCCTTCAAGTACGCCTGATACCACGCTCACGCTCGGC-3'.

[0014] During the research, more than 10 sets of LAMP primers were designed using Fusarium oxysporum EF-1α and Tubulin genes as target genes. Unexpectedly, it was found that the LAMP-specific primers of this invention have outstanding advantages of high specificity and high sensitivity for early rapid detection of Fusarium oxysporum. In particular, the presence of the pathogen can be detected in tomatoes at the early stage of Fusarium oxysporum infection, when the bacterial load is extremely low and no disease phenotype is present.

[0015] The present invention also provides a tomato wilt LAMP detection kit prepared using the above primers, the components of which include: the primers, LAMP reaction buffer, thermophilic DNA polymerase for LAMP detection, and nucleic acid fluorescent dye;

[0016] The primers contain equal amounts of the two outer primers and equal amounts of the two inner primers.

[0017] The molar ratio of inner primer to outer primer is 3~1:1~3, preferably 2:1;

[0018] As a limitation of this invention, the LAMP reaction buffer contains 8 mM Mg 2+ 10×Bst II reaction buffer;

[0019] The thermophilic DNA polymerase used for LAMP detection is Bst II DNA polymerase.

[0020] As a further limitation of the present invention, the nucleic acid fluorescent dye is SYBR Green I.

[0021] Among them, SYBR Green I is used to stain the LAMP reaction product with the added nucleic acid fluorescent dye from orange (orange-yellow) to green (yellow-green).

[0022] Preferably, the nucleic acid fluorescent dye can also be replaced with hydroxynaphthol blue (HNB) or calcein / manganese ion.

[0023] This invention also provides a method for detecting tomato wilt using the above-mentioned tomato wilt LAMP detection kit, specifically: extracting DNA from the sample to be tested as a template, mixing the LAMP reaction system, performing the LAMP reaction, and adding nucleic acid fluorescent dye to the reaction product. If a positive color change occurs, the sample to be tested is a positive sample for tomato wilt.

[0024] As a limitation of the present invention, the LAMP reaction system has the following composition: 1×Bst II Buffer, 8 mg / mL MgSO₄. 2+ 0.2~1.0U / μL Bst II DNA polymerase, 0.5~1.5 mM dNTPs, 0.5 μM FIP-FO, 0.5 μM BIP-FO, 0.25 μM F3-FO, 0.25 μM B3-FO, 1 μL template, and sterile ultrapure water to bring the volume to 20 μL.

[0025] Preferably, it contains 0.6 U / μL Bst II DNA polymerase and 1 mM dNTPs.

[0026] As a further limitation of the present invention, the LAMP reaction conditions are 52~66℃ for 20~70 min, followed by inactivation at 85℃ for 5 min.

[0027] As a further limitation of the present invention, the LAMP reaction conditions are: reaction at 56°C for 30 min, followed by inactivation at 85°C for 5 min.

[0028] This invention also provides an application of the above-mentioned Fusarium oxysporum LAMP detection primers in the early identification of tomato wilt disease and the detection of Fusarium oxysporum.

[0029] This invention also provides the application of the above-mentioned tomato wilt LAMP detection kit in the early identification of tomato wilt and the detection of Fusarium oxysporum.

[0030] By adopting the above-described technical solution, the beneficial effects achieved by this invention compared to the prior art are as follows:

[0031] (1) The LAMP detection primers for Fusarium oxysporum of the present invention can achieve extremely early and ultra-high sensitivity detection of Fusarium oxysporum, providing a key window period for disease early warning.

[0032] (2) The detection kit prepared by the LAMP detection primers of Fusarium oxysporum of the present invention not only retains the advantages of visual visualization and convenience of the LAMP method, but also can complete the detection of Fusarium oxysporum within 30 min with a sensitivity of up to 100 ag / μL, which is significantly better than the LAMP method and conventional PCR method whose detection limit is still at the pg or fg level, thus achieving rapid and efficient detection.

[0033] (3) The LAMP detection primers for Fusarium oxysporum of the present invention exhibit high specificity. In the detection of Fusarium oxysporum, Corynebacterium multiflorum, Pseudomonas syringae, Staphylococcus aureus, Pectinobacillus carotenoides, Fusarium graminearum, Fusarium graminearum, and Rhizoctonia solani, Fusarium oxysporum is specifically detected only for Fusarium oxysporum, and the results are accurate and reliable.

[0034] This invention is applicable to the early identification of tomato wilt and the detection of Fusarium oxysporum, and can be used for early monitoring and prevention of vegetable diseases. Attached Figure Description

[0035] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0036] Figure 1 This is a schematic diagram of the target sequence in Embodiment 1 of the present invention;

[0037] Figure 2 The graph shows the detection results of LAMP reaction systems with different enzyme amounts in this invention.

[0038] Figure 3 The graph shows the detection results of LAMP reaction systems with different dNTP contents in this invention.

[0039] Figure 4 The graph shows the detection results of LAMP reaction systems with different inner and outer primer ratios in this invention.

[0040] Figure 5 The graph shows the detection results at different LAMP reaction temperatures in this invention.

[0041] Figure 6 The graph shows the detection results for different LAMP reaction times in this invention.

[0042] Figure 7 This is a graph showing the detection results of LAMP reactions with different template concentrations in this invention;

[0043] Figure 8 This is a graph showing the PCR reaction detection results of different template concentrations in Comparative Example 1 of the present invention;

[0044] Figure 9 This is a graph showing the results of targeted monitoring of Fusarium wilt pathogen in tomato grown in protected cultivation areas using the kit in Example 3 of this invention.

[0045] Figure 10 This is a graph showing the specific detection results in Comparative Example 2 of the present invention;

[0046] Figure 11 This is a graph showing the specificity detection results of different primers in Comparative Example 3 of this invention. Detailed Implementation

[0047] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustrative and understanding purposes only and are not intended to limit the scope of the invention.

[0048] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available. Experimental methods not specifically described in the embodiments are generally performed under standard conditions or as recommended by the manufacturer.

[0049] In this example, 1.5% agarose gel electrophoresis and SYBR Green I fluorescence staining were used to determine the LAMP reaction results.

[0050] The strains involved in the examples—Corynespora cassiicola, Botryosphaeria dothidea, Pseudomonas syringae DC3000, Pectobacterium carotovorum, and Gibberella zeae—were all derived from the Institute of Biology, Hebei Academy of Sciences; Fusarium pseudograminearum was derived from Hebei University of Science and Technology; and Fusarium oxysporum and Rhizoctonia solani were derived from Hebei Agricultural University. All fungal strains were cultured on PDA solid medium, and bacteria on LB medium at 28°C.

[0051] BstDNA polymerase, Bst Buffer, and dNTP Mixture were purchased from Bomeide Biotechnology; the genomic DNA extraction kit was purchased from Beijing Polymer Biotechnology Co., Ltd.; SYBR Green I was purchased from Shanghai Ruichu Biotechnology Co., Ltd. The clean bench was purchased from Suzhou Antai Air Technology Co., Ltd., the benchtop high-speed refrigerated centrifuge was purchased from Hunan Xiangyi Laboratory Instrument Development Co., Ltd., the constant temperature metal bath was purchased from CoYoTebio, and the biochemical incubator was purchased from Shanghai Yiheng Scientific Instruments Co., Ltd.

[0052] Example 1

[0053] This embodiment describes a LAMP detection primer for Fusarium oxysporum, consisting of an outer primer and an inner primer. The outer primers are a forward outer primer F3-FO and a reverse outer primer B3-FO; the inner primers are a forward inner primer FIP-FO and a reverse inner primer BIP-FO.

[0054] The nucleotide sequences of each primer are as follows:

[0055] F3-FO: 5'-GAGCGACGGGAGCGTTTGC-3', denoted as SEQ ID NO.1;

[0056] B3-FO: 5'-CGGTGACATAGTAGCGAGGA-3', denoted as SEQ ID NO.2;

[0057] FIP-FO: 5'-CGAGCTCAGCGGCTTCCTATTGCCCTCTTACCATTCTCACA-3', recorded as SEQ IDNO.3;

[0058] BIP-FO: 5'-AGGGTTCCTTCAAGTACGCCTGATACCACGCTCACGCTCGGC-3', recorded as SEQ IDNO.4.

[0059] The design and synthesis process of the above primers is as follows:

[0060] Using the EF-1α gene sequence of *Fusarium oxysporum* publicly available on the NCBI website as the target gene sequence, sequence alignment was used to identify gene fragments with low homology to other pathogenic fungi as target sequences. Figure 1 Access the PrimerExplorerV5 website (http: / / primerexplorer.jp) and design a set of LAMP primers based on the B1c, B2, B3, F1c, F2, and F3 regions of the target sequence: forward outer primer F3-FO and reverse outer primer B3-FO; the inner primers are forward inner primer FIP-FO and reverse inner primer BIP-FO. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0061] Example 2

[0062] This embodiment describes a LAMP detection kit for tomato Fusarium wilt prepared using the primers from Example 1. The kit includes: forward outer primer F3-FO, reverse outer primer B3-FO; inner primers are forward inner primer FIP-FO and reverse inner primer BIP-FO, containing 8 mM Mg... 2+ The reaction mixture contained 10×Bst II reaction buffer, Bst II DNA polymerase, and the nucleic acid fluorescent dye SYBR Green I, as a positive control, an EF-1α synthesized DNA fragment (target sequence), and nuclease-free water as a negative control.

[0063] The usage method of this embodiment is as follows:

[0064] S1. Template extraction: Genomic DNA of Fusarium oxysporum was extracted using a DNA extraction kit and diluted with sterile ultrapure water to 1 ng / μL as a template for later use.

[0065] S2. Mixing LAMP reaction system: Take the reagents from the tomato wilt LAMP detection kit and mix them according to the following original concentrations and volumes: 10×Bst II Buffer 2 μL (final concentration containing 8 mM Mg). 2+), 8 U / μL Bst II DNA polymerase 1.5 μL, 10 mM dNTPs 2 μL, 10 μM FIP-FO 1 μL, 10 μM BIP-FO 1 μL, 10 μM F3-FO 0.5 μL, 10 μM B3-FO 0.5 μL, template 1 μL, sterile ultrapure water 10.5 μL.

[0066] The final concentrations of each substance in the system are: 1×Bst II Buffer, 8 mM Mg 2+ 0.6 U / μL Bst II DNA polymerase, 1 mM dNTPs, 0.5 μM FIP-FO, 0.5 μM BIP-FO, 0.25 μM F3-FO, 0.25 μM B3-FO, 1 μL template, and sterile ultrapure water were added to bring the volume to 20 μL to obtain the LAMP reaction system.

[0067] S3. Perform the LAMP reaction: Incubate the LAMP reaction system at 56℃ for 30 min, and then inactivate it at 85℃ for 5 min.

[0068] S4. Reading the test results: After the LAMP reaction is complete, add 0.5 μL of SYBR Green I to the reaction product and visually observe the color development results. If the reaction product is observed to be green fluorescent, it is considered positive, indicating that Fusarium oxysporum is present in the sample. If orange is observed, it is considered negative, indicating that Fusarium oxysporum is not present.

[0069] The results showed that the reaction product was green fluorescent, indicating a positive result, which means that Fusarium oxysporum was present in the sample to be tested. That is, the primers and kit of the present invention can successfully detect Fusarium oxysporum.

[0070] In other embodiments, the amount of SYBR Green I used in the 20 μL LAMP reaction system can be any integer value from 0.5 to 1 μL.

[0071] This invention verified the optimal implementation method by adjusting the composition of the LAMP reaction system, the LAMP reaction conditions, and the template concentration in Example 2, as detailed below:

[0072] (I) Different LAMP reaction system compositions and their implementation effects

[0073] Based on Example 2, this invention adjusts the amounts of the main components Bst II DNA polymerase and dNTPs, as well as the ratio of inner and outer primers in the reaction system. The adjustments for each single factor are as follows: the final concentration of Bst II DNA polymerase is 0.2 U / μL, 0.4 U / μL, 0.6 U / μL, 0.8 U / μL, or 1.0 U / μL; the final concentration of dNTPs is 0.5 mmol / L, 0.75 mmol / L, 1 mmol / L, 1.25 mmol / L, or 1.5 mmol / L; and the ratio of inner and outer primers is 3:1, 2:1, 1:1, 1:2, or 1:3. With other component amounts and conditions remaining the same as in Example 2, the single-factor conditions are varied to conduct the LAMP reaction. Sterile ultrapure water is used as a negative control. After the reaction, the optimal LAMP reaction system for *Fusarium oxysporum* is determined by gel electrophoresis and fluorescent dye detection.

[0074] The results are as follows Figures 2-4 As shown, Figure 2 The results showed that LAMP reactions using different concentrations of Bst II DNA polymerase yielded positive amplification results when the Bst II DNA polymerase concentration was between 0.2 and 1.0 U / μL, with specific ladder-like bands observed on electrophoresis. SYBR Green I staining indicated a green band, while the negative control (0 U / μL) showed no amplification band and stained orange. LAMP reactions using different concentrations of dNTPs also yielded the following results: Figure 3 As shown, when dNTPs are used at amounts of 0.5-1.5 mM, the amplification result is positive, and electrophoresis shows specific ladder-shaped bands. SYBR Green I staining is green. The negative control (0 mM) shows no amplification band and is stained orange. LAMP reactions were performed using different ratios of inner and outer primers, and the results are as follows. Figure 4 As shown, both the inner and outer primer ratios at the indicated ratios amplified positive results, with specific ladder-like bands observed on electrophoresis and staining green with SYBR Green I. The negative control (CK) showed no amplification bands and stained orange. Considering both amplification efficiency and cost, the optimal amounts of Bst II DNA polymerase, dNTPs, and the inner-outer primer ratio were determined to be 0.6 U / μL, 1 mM, and 2:1.

[0075] Therefore, the LAMP reaction system for Fusarium oxysporum was established as follows: a final concentration of 1×Bst II Buffer and 8 mM Mg. 2 +0.6 U / μL Bst II DNA polymerase, 1 mM dNTPs, 0.5 μM FIP-FO, 0.5 μM BIP-FO, 0.25 μM MF3-FO, 0.25 μM B3-FO, 1 μL DNA template, and sterile ultrapure water to bring the volume to 20 μL.

[0076] (II) Different LAMP reaction conditions and their implementation effects

[0077] Based on Example 2, this invention adjusts the reaction conditions as follows: reaction temperatures are 52℃, 54℃, 56℃, 58℃, 60℃, 62℃, 64℃, 66℃, or 68℃; reaction times are 10, 20, 30, 40 min, 50 min, 60 min, or 70 min. With other component amounts and conditions remaining the same as in Example 2, the single-factor conditions are varied to conduct the LAMP reaction. Sterile ultrapure water is used as a negative control. After the reaction, the optimal LAMP reaction conditions for *Fusarium oxysporum* are determined by gel electrophoresis and fluorescent dye detection.

[0078] The results are as follows Figure 5 As shown, amplification at 52-66℃ yielded a positive result, with electrophoresis showing specific ladder-shaped bands. SYBR Green I staining indicated a green band, while the negative control (CK) showed no amplification band and stained orange. Figure 6 Amplification results were positive between 20 and 70 min, showing specific ladder-like bands on electrophoresis. SYBR Green I staining was green. The negative control (CK) and the 10-min control showed no amplification bands and stained orange. Amplification at 68℃ and for 10 min yielded poor results. Therefore, the optimal LAMP reaction conditions for Fusarium oxysporum are: 56℃ for 30 min, followed by inactivation at 85℃ for 5 min.

[0079] (III) Different LAMP reaction template concentrations and their implementation effects

[0080] Genomic DNA of *Fusarium oxysporum* was diluted to 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL, 100 ag / μL, 10 ag / μL, and 1 ag / μL, respectively. Using these different concentrations of DNA as templates, LAMP amplification was performed using the LAMP detection method established in Example 2. The results are as follows: Figure 7As shown, the lowest template concentration for a positive reaction is 100 ag / μL, indicating that the detection sensitivity for Fusarium oxysporum is 100 ag / μL. In contrast, when the conventional PCR method amplifies the above templates at different concentrations, the lowest template concentration for a positive reaction is 1 ng / μL, indicating that the LAMP detection using the primers of this invention has extremely high sensitivity.

[0081] Comparative Example 1

[0082] This comparative example used conventional PCR technology, with different concentrations of *Fusarium oxysporum* genomic DNA from (III) as templates. The outer primers F3-FO and B3-FO from the LAMP primer set were used as the upstream and downstream primers for conventional PCR. The reaction system was adaptively adjusted based on the *Fusarium oxysporum* LAMP reaction system obtained in Example 2, maintaining the same volume. The results of conventional PCR amplification are shown below. Figure 8 The results showed that the minimum template concentration for a positive PCR reaction was 1 ng / μL, which was less sensitive than the LAMP reaction of this invention.

[0083] In this comparative example, the reaction system for conventional PCR technology was as follows: 10 μL of Permix Taq (TaKaRa Taq Versin 2.0plus dye), 1 μL each of 10 μM F3-FO, 10 μM B3-FO and template, and 7 μL of sterile ultrapure water; the reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 53℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 30 cycles; and 72℃ extension for 10 min.

[0084] Example 3

[0085] In this embodiment, the kit from Example 2 is used for targeted monitoring of Fusarium wilt pathogen in greenhouse-grown tomatoes. The specific detection method is as follows:

[0086] Five pathogen monitoring points were set up in the greenhouse cultivation area. LAMP tests were conducted on air, tomato leaves, and soil samples from the tomato root colonization area during the tomato seedling stage, flowering and fruit setting stage, fruiting stage, and maturity stage.

[0087] The sampling method for air samples in the protected cultivation area is as follows: Reaction tubes containing template-free LAMP reaction solution were placed between the rows at the monitoring points, with the caps open for 30-60 minutes to allow air near the monitoring points to diffuse into the reaction tubes, serving as air templates for the LAMP reaction. Phenotypic healthy young leaves from the middle of tomato plants on both sides of the air sampling points were selected as leaf samples, and soil from the root colonization area of ​​the tomato plants was selected as soil samples. Genomic DNA was extracted from both leaves and soil samples and used as templates for the LAMP reaction. Two replicates were set up at each sampling point, resulting in a total of 10 samples, numbered 1-10. The 10 samples, along with positive and negative control samples, were subjected to LAMP reactions under the optimal LAMP reaction conditions and system described above for *Fusarium oxysporum*.

[0088] The results are as follows Figure 9 As shown in Table 1.

[0089] Table 1. Correspondence between Fusarium oxysporum pathogen detection and various factors during tomato growth period.

[0090]

[0091] The results showed that all tomato seedlings in the greenhouse exhibited healthy phenotypes, with no Fusarium oxysporum pathogen detected in the air or leaves. However, the pathogen was detected in the soil of the root colonization area, indicating that Fusarium oxysporum was present in the greenhouse soil during the seedling stage, but it had not spread into the air or infected the plant leaves. During the flowering and fruiting stage, Fusarium oxysporum was not detected in the air or leaves, but it was detected in the soil. At this time, a small number of plants in the greenhouse showed yellowing and wilting of the lower, older leaves. During the fruiting stage, Fusarium oxysporum was widely detected in both the air and soil, and it was also detected in some leaves that did not show any symptoms of infection. A small number of fruits showed symptoms of infection such as early reddening and mold, indicating that Fusarium oxysporum contamination was relatively severe in the greenhouse during this period. Fusarium oxysporum was also detected in the air and soil during the ripening period. The number of leaves with the pathogen detected decreased, possibly because the removal of diseased fruits, leaves, and susceptible old leaves at the bottom of the plant reduced the source of infection.

[0092] Fusarium wilt, caused by Fusarium oxysporum, is a soil-borne disease that can occur throughout the entire tomato growth cycle, but is most prevalent from transplanting to fruiting. Continuous cropping exacerbates the disease, leading to earlier and more severe outbreaks. High temperature and humidity, along with a soil pH below 7.0, easily induce the disease. LAMP assays of Fusarium oxysporum in greenhouse air, leaves, and soil at different tomato growth stages revealed that soil is the primary source of pathogen contamination. Therefore, soil sterilization and disinfection should be prioritized before transplanting. Infected plants begin to appear during the flowering and fruiting stages, with the fruiting period being the peak period for disease incidence. The frequency of LAMP assays should be increased during the flowering and fruiting stages to detect the pathogen promptly and implement targeted prevention measures.

[0093] The above results indicate that the kit in Example 2 can detect pathogens significantly earlier than the appearance of the disease phenotype in tomatoes. It can be used for early detection of pathogens. Combined with plant disease phenotype, environmental factors and agricultural operations, it can assess the risk level of wilt disease and thus achieve early warning of wilt disease.

[0094] Comparative Example 2

[0095] In this comparative example, the LAMP detection primers for *Fusarium oxysporum* from Example 1 were used to detect different strains or negative controls to verify the specificity of the primers of the present invention. The specific detection method was the same as in Example 2, wherein the detection objects were numbered sequentially as follows: 1: negative control; 2: *Fusarium oxysporum*; 3: *Corynebacterium multiflorum*; 4: *Pseudomonas syringae*; 5: *Rhizoctonia solani*; 6: *Pectinobacterium soft rot*; 7: *Fusarium graminearum*; 8: *Fusarium pseudograecum*; 9: *Staphylococcus aureus*.

[0096] The results are as follows Figure 10 As shown, only *Fusarium oxysporum* strain 2 showed a positive amplification result, exhibiting a specific ladder-like band on electrophoresis and turning yellow with SYBR Green I staining. The negative control and other non-target pathogens showed no amplification bands and stained orange. These results indicate that the primers of this invention have high specificity, specifically detecting *Fusarium oxysporum* without detecting other non-target pathogens, and can be used for LAMP detection of *Fusarium oxysporum*.

[0097] The LAMP reaction results were further evaluated using 1.5% agarose gel electrophoresis, and the results were consistent with those of SYBR GreenI staining.

[0098] Comparative Example 3

[0099] In this comparative example, three sets of LAMP primers were designed targeting the EF-1α and Tubulin gene sequences of Fusarium oxysporum. The sequences of each primer are shown in Table 2. Each primer was used to perform LAMP amplification with Fusarium oxysporum genomic DNA as a template, following the method in Example 2. Positive results were obtained in all cases.

[0100] Further verification of specificity revealed that only the LAMP-FO primer combination of this invention has the effect of specifically detecting Fusarium oxysporum, as detailed below:

[0101] Following the method described in Example 2, LAMP amplification was performed using genomic DNA from *Fusarium oxysporum*, *Corynebacterium multiflorum*, *Pseudomonas syringae*, *Rhizoctonia solani*, *Pectinobacter softrot*, *Fusarium graminearum*, and *Staphylococcus aureus* as templates. The LAMP reaction results were evaluated using SYBR Green I fluorescence staining. The results are as follows: Figure 11 .

[0102] Table 2 Primer sequences and target genes

[0103]

[0104] Figure 11 A represents the detection result of primer LAMP-FO, compared with control group 2.

[0105] Figure 11 B represents the detection results of primer LAMP-FOEF1. The detection objects are numbered sequentially as follows: 1: negative control; 2: Fusarium oxysporum; 3: Corynebacterium multiflorum; 4: Pseudomonas syringae; 5: Rhizoctonia solani; 6: Pectinobacterium soft rot; 7: Fusarium graminearum; 8: Fusarium pseudograss; 9: Staphylococcus aureus.

[0106] Figure 11 C represents the detection result of primer LAMP-FOTUB. The detection objects are numbered sequentially as follows: 1: negative control; 2: Fusarium oxysporum; 3: Corynebacterium multiflorum; 4: Pseudomonas syringae; 5: Staphylococcus aureus; 6: Pectinobacter soft rot; 7: Fusarium graminearum; 8: Fusarium pseudograss; 9: Rhizoctonia solani.

[0107] Depend on Figure 11 It is known that although all primers can amplify the genome of Fusarium oxysporum, compared with the primer LAMP-FO of this invention, the other primers have poor specificity and cannot distinguish Fusarium oxysporum from Pseudomonas syringae, Pectinobacter soft rot, Fusarium pseudograss, Rhizoctonia solani, and Staphylococcus aureus, and cannot be used to accurately identify Fusarium oxysporum.

[0108] The above results demonstrate that the loop-mediated isothermal amplification detection method using primers LAMP-FO of this invention allows for direct visual observation to determine the presence of Fusarium oxysporum in the sample. The primers of this invention exhibit a sensitivity of up to 100 ag / μL and high specificity. When used for tomato disease detection, they can detect the disease in tomato leaves before obvious disease phenotypes appear. This invention is suitable for early, rapid, sensitive, and visual detection of tomato wilt disease.

[0109] It should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments or make equivalent substitutions for some of the technical features. Any equivalent substitutions should be included within the protection scope of the present invention.

Claims

1. A primer for detecting Fusarium oxysporum using LAMP, comprising an outer primer and an inner primer, characterized in that, The outer primers are the forward outer primer F3-FO and the reverse outer primer B3-FO; the inner primers are the forward inner primer FIP-FO and the reverse inner primer BIP-FO. The nucleotide sequences of each primer are as follows: F3-FO: 5'-GAGCGACGGGGAGCGTTTGC-3'; B3-FO: 5'-CGGTGACATAGTAGCGAGGA-3'; FIP-FO: 5'-CGAGCTCAGCGGCTTCCTATTGCCCTCTTACCATTCTCACA-3'; BIP-FO: 5'-AGGGTTCCTTCAAGTACGCCTGATACCACGCTCACGCTCGGC-3'.

2. A LAMP detection kit for tomato Fusarium wilt prepared using the primers described in claim 1, characterized in that, Its components include: the primers, LAMP reaction buffer, thermophilic DNA polymerase for LAMP detection, and nucleic acid fluorescent dye; The primers contain equal amounts of the two outer primers and equal amounts of the two inner primers. The molar ratio of inner primer to outer primer is 3~1:1~3.

3. The tomato wilt LAMP detection kit according to claim 2, characterized in that, The LAMP reaction buffer contains 8 mM Mg 2+ 10×Bst II reaction buffer; The thermophilic DNA polymerase used for LAMP detection is Bst II DNA polymerase.

4. The tomato wilt LAMP detection kit according to claim 3, characterized in that, The nucleic acid fluorescent dye is SYBR Green I.

5. A method for detecting tomato wilt disease using the tomato wilt LAMP detection kit according to any one of claims 2 to 4, characterized in that, DNA from the sample to be tested is extracted as a template, mixed into a LAMP reaction system, and subjected to a LAMP reaction. If a positive color change occurs after adding a nucleic acid fluorescent dye to the reaction product, the sample to be tested is a positive sample for tomato wilt disease.

6. The method for detecting tomato wilt according to claim 5, characterized in that, The LAMP reaction system consisted of the following components: 1×Bst II Buffer, 8 mM Mg 2+ 0.2~1.0U / μL Bst II DNA polymerase, 0.5~1.5mM dNTPs, 0.5 μM FIP-FO, 0.5 μM BIP-FO, 0.25 μM F3-FO, 0.25 μM B3-FO, 1 μL template, and sterile ultrapure water to bring the volume to 20 μL.

7. The method for detecting tomato wilt according to claim 6, characterized in that, The LAMP reaction conditions are 52-66℃ for 20-70 min, followed by inactivation at 85℃ for 5 min.

8. The method for detecting Fusarium wilt in tomatoes according to claim 7, characterized in that, The LAMP reaction conditions were 56°C for 30 min, followed by inactivation at 85°C for 5 min.

9. The application of the LAMP detection primers for Fusarium oxysporum as described in claim 1 in the early identification of Fusarium wilt in tomato and the detection of Fusarium oxysporum.

10. The application of the tomato wilt LAMP detection kit according to any one of claims 2 to 4 in the early identification of tomato wilt and the detection of Fusarium oxysporum.

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