Primer probe combination, kit and detection method for corn small spot fungus LAMP-LFD detection

Abstract

The application provides a primer probe combination, a kit and a detection method for LAMP-LFD detection of corn small spot fungus, and belongs to the technical field of biology.The primer probe combination for LAMP-LFD detection of corn small spot fungus comprises an external upstream primer F3, an external downstream primer B3, an internal upstream primer FIP, an internal downstream primer BIP and a probe Bm-HP.The detection primer probe combination is used to take the genomic DNA of a sample to be detected as a template, and after LAMP constant-temperature amplification, LFD test paper is used for detection.If the quality control band and the detection band both show color, it indicates that the corn small spot fungus detection result of the sample is positive.The primer probe combination has good specificity, high accuracy, high sensitivity, and the detection limit is 1fg. ‑1 μL, is simple, fast and easy to operate, and has important significance for early warning and prevention and control of corn small spot, and prevention and control of the spread and spread of diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a primer probe combination for LAMP-LFD detection of corn small spot disease, a kit and a detection method thereof. BACKGROUND

[0002] Corn is an important food and feed crop in the world, and is also an indispensable raw material for the production of related industries such as industry, livestock, medicine and energy. High and stable yield of corn plays an extremely important role in ensuring China's food security. Corn small spot disease is a leaf fungal disease caused by Bipolaris maydis, which is widely distributed in major corn producing areas around the world. It can cause more than 10% yield reduction of corn, and the yield loss can reach 20% to 68% or even more than 40% in severe cases. Rapid and accurate detection of B. maydis at the early stage of disease occurrence and timely adoption of effective control measures are one of the effective measures to prevent the occurrence, spread and spread of the disease.

[0003] Traditional detection methods of corn disease pathogens mainly include disease tissue isolation, i.e. isolating target pathogens from diseased tissues, analyzing morphological characteristics or physiological and biochemical properties, or identifying with the aid of conserved gene sequences, pathogenicity determination, and then correctly classifying and identifying the pathogens by comprehensively analyzing the results, enzyme-linked immunoassay and ELIS analysis, etc. These identification and detection methods have the disadvantages of low sensitivity, poor specificity, complex operation, long detection period, etc., and are not suitable for large-scale sample and latent infection of plant tissue detection.

[0004] With the development of rapid molecular detection technology of plant pathogens, nucleic acid amplification techniques such as classical PCR assay, nested PCR, droplet digital PCR (ddPCR), real-time fluorescent quantitative PCR, recombinase-mediated isothermal amplification (PRA) and loop-mediated isothermal amplification (LAMP) have been successfully applied to rapid detection of corn pathogenic fungi. Compared with traditional identification and detection methods of corn pathogenic fungi, PCR detection technology has the advantages of strong specificity, high sensitivity, rapid and efficient, etc., which improves the accuracy of detection to a certain extent. However, PCR detection technology has high requirements for instruments and equipment, experimental environment and professional level of operators, and has the disadvantages of high cost, complex operation, low detection sensitivity, serious dependence on instruments and equipment, and the need for certain professional operation skills, easy to miss detection of trace pathogens and difficult to realize on-site detection, etc., which greatly limits its application and promotion. Loop-mediated isothermal amplification (LAMP) is a rapid nucleic acid amplification method. This method designs two pairs of primers for the conserved region of the target gene sequence, and uses a chain displacement active Bst DNA polymerase to amplify under isothermal conditions (about 63℃) for 30-60 min, which can realize the large-scale amplification of the target DNA. LAMP-LFD method has the advantages of short detection time, strong specificity, simple operation, no need for expensive and precise instruments, etc., and the results are visualized, which is convenient for observation. The patent with application number “201810482292.5” and the invention name “LAMP detection primer for corn small spot disease and visual detection method and application thereof” discloses a LAMP detection method for corn small spot disease, but the detection method has low sensitivity, and the detection limit is 100 fg. When the corn small spot disease occurs before and at the early stage, the concentration of corn small spot disease in the corn leaf tissue is low, and the above detection method cannot realize the detection of B. maydis. Therefore, it is particularly important to establish a rapid, simple, specific, sensitive and applicable B. maydis detection technology for the prevention of corn small spot disease. SUMMARY

[0005] In order to solve the above problems, the present application provides a primer probe combination with high sensitivity for LAMP-LFD detection of corn small spot disease, a kit and a detection method thereof.

[0006] In order to achieve the above-mentioned purposes, the present application provides the following technical solutions:

[0007] The present invention provides a primer-probe combination for detecting corn leaf blight fungus, comprising an external primer, an internal primer, and a probe Bm-HP, wherein the external primer comprises an external upstream primer F3 and an external downstream primer B3, and the internal primer comprises an internal upstream primer FIP and an internal downstream primer BIP;

[0008] The nucleotide sequence of the external upstream primer F3 is shown in SEQ NO.1;

[0009] The nucleotide sequence of the external downstream primer B3 is shown in SEQ NO.2;

[0010] The nucleotide sequence of the internal upstream primer FIP is shown in SEQ NO.3;

[0011] The nucleotide sequence of the internal downstream primer BIP is shown in SEQ NO.4;

[0012] The nucleotide sequence of the probe Bm-HP is shown in SEQ NO.5.

[0013] Preferably, the 5' end of the internal upstream primer FIP is labeled with biotin; and the 5' end of the probe Bm-HP is labeled with 6-FAM.

[0014] The present invention also provides application of the primer-probe combination in detecting Bipolaris maydis.

[0015] The present invention also provides a kit for detecting southern blight fungus, comprising the primer-probe combination.

[0016] Preferably, the molar ratio of the external upstream primer F3, the external downstream primer B3, the internal upstream primer FIP, and the internal downstream primer BIP in the kit is 0.8-1.2:0.8-1.2:7-9:7-9.

[0017] The present invention also provides a LAMP-LFD detection method for B. maydis, comprising the following steps:

[0018] (1) performing LAMP amplification on the sample to be tested using the primer-probe combination or the kit to obtain an amplified product;

[0019] (2) Detecting the amplified product with an LFD test strip and observing the test strip,

[0020] If both the test strip and the quality control strip show color, the sample to be tested is infected with the corn leaf blight pathogen;

[0021] If the quality control band develops color but the test band does not, the sample to be tested is not infected with corn leaf blight.

[0022] Preferably, the reaction system of the LAMP amplification is 25 μL, which uses water as a solvent and includes the following components at the following concentrations: external upstream primer F3 0.2 μmol / L, external downstream primer B3 0.2 μmol / L, internal upstream primer FIP 1.6 μmol / L, internal downstream primer BIP 1.6 μmol / L, dNTPs 0.96 mmol / L, Tris-HCl 20 mmol / L, KCl 10 mmol / L, MgSO4 5 mmol / L, (NH4)2SO4 10 mmol / L, 0.1% Triton X-100, 8 U Best DNA polymerase large fragment and 1 μL of sample DNA to be tested.

[0023] Preferably, the temperature of the LAMP amplification is 62-66° C., and the time of the LAMP amplification is 30-70 min.

[0024] By adopting the above technical solution, the present invention has the following beneficial effects: the primer-probe combination for LAMP-LFD detection of corn leaf blight of the present invention includes an external upstream primer F3, an external downstream primer B3, an internal upstream primer FIP, an internal downstream primer BIP and a probe Bm-HP. The detection primer-probe combination of the present invention is used to detect the genomic DNA of the sample to be tested as a template, and after LAMP constant temperature amplification, it is detected using an LFD test strip. If both the quality control band and the detection band are colored, it indicates that the corn leaf blight test result of the sample is positive. The present invention has good specificity, high accuracy, high sensitivity, and a detection limit of 1fg.μL -1 The operation is simple and fast, and the reaction results are easy to observe (visualize). It is of great significance for the early warning and prevention and control of corn leaf spot and the prevention and control of the spread of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of LAMP-LFD primer and probe design based on the reducease Brn1 gene of corn leaf blight fungus (Bipolaris maydis).

[0026] Figure 2 The results of the LAMP-LFD reaction temperature optimization experiment for Bipolaris maydis detection are shown in Figure 1. Among them, a is the detection result of adding fluorescent dye, b is the detection result under ultraviolet light irradiation at a wavelength of 365nm, c is the detection result of agarose gel electrophoresis, and d is the detection result of LFD test strips.

[0027] Figure 3The results of the LAMP-LFD reaction time optimization experiment for Bipolaris maydis detection are shown in Figure 1. Among them, a is the detection result of adding fluorescent dye, b is the detection result under ultraviolet light irradiation at a wavelength of 365nm, c is the detection result of agarose gel electrophoresis, and d is the detection result of LFD test strips.

[0028] Figure 4 The results of LAMP-LFD detection of Bipolaris maydis are specific, where a is the result of adding fluorescent dye, b is the result of detection under 365 nm ultraviolet light, c is the result of agarose gel electrophoresis, and d is the result of LFD test strip detection.

[0029] Figure 5 Figure 5 is the sensitivity test result of LAMP-LFD detection of Bipolaris maydis, where a is the detection result with fluorescent dye, b is the detection result under 365nm ultraviolet light, c is the detection result with agarose gel electrophoresis, and d is the detection result with LFD test strips.

[0030] Figure 6 The following are the results of LAMP-LFD detection of fungi on leaves infected with corn leaf spot; among them, a is the detection result of adding fluorescent dye, b is the detection result under ultraviolet light of 365nm wavelength, c is the detection result of agarose gel electrophoresis, and d is the detection result of LFD test strips. DETAILED DESCRIPTION

[0031] The present invention provides a primer-probe combination for detecting corn leaf blight fungus, comprising an external primer, an internal primer, and a probe Bm-HP, wherein the external primer comprises an external upstream primer F3 and an external downstream primer B3, and the internal primer comprises an internal upstream primer FIP and an internal downstream primer BIP;

[0032] The nucleotide sequence of the external upstream primer F3 is shown in SEQ NO.1;

[0033] The nucleotide sequence of the external downstream primer B3 is shown in SEQ NO.2;

[0034] The nucleotide sequence of the internal upstream primer FIP is shown in SEQ NO.3;

[0035] The nucleotide sequence of the internal downstream primer BIP is shown in SEQ NO.4;

[0036] The nucleotide sequence of the probe Bm-HP is shown in SEQ NO.5.

[0037] Preferably, the 5' end of the internal upstream primer FIP is labeled with biotin; and the 5' end of the probe Bm-HP is labeled with 6-FAM.

[0038] The present invention also provides application of the primer-probe combination in detecting Bipolaris maydis.

[0039] The present invention also provides a kit for detecting corn leaf blight pathogen, wherein the primer-probe combination; the molar ratio of the external upstream primer F3, the external downstream primer B3, the internal upstream primer FIP, and the internal downstream primer BIP in the kit is 0.8-1.2:0.8-1.2:7-9:7-9, further preferably 0.9-1.1:0.9-1.1:7.5-8.5:7.5-8.5, and more preferably 1:1:8:8.

[0040] The present invention also provides a LAMP-LFD detection method for B. maydis, comprising the following steps:

[0041] (1) performing LAMP amplification on the sample to be tested using the primer-probe combination or the kit to obtain an amplified product;

[0042] (2) Detecting the amplified product with an LFD test strip and observing the test strip,

[0043] If both the test strip and the quality control strip show color, the sample to be tested is infected with the corn leaf blight pathogen;

[0044] If the quality control band develops color but the test band does not, the sample to be tested is not infected with corn leaf blight.

[0045] The present invention first extracts genomic DNA of a sample to be tested. The present invention has no special limitation on the extraction of the sample DNA. The extraction can be performed using a method well known to those skilled in the art or a plant genomic DNA extraction kit. 1 μL of the genomic DNA of the sample to be tested is mixed with a kit for detecting corn leaf blight fungus, and LAMP amplification is performed to obtain an amplified product. The LAMP amplification reaction system is 25 μL and includes the following components at the following concentrations: an external upstream primer F3 0.2 μmol / L, an external downstream primer B3 0.2 μmol / L, an internal upstream primer FIP 1.6 μmol / L, an internal downstream primer BIP 1.6 μmol / L, dNTPs 0.96 mmol / L, Tris-HCl 20 mmol / L, KCl 10 mmol / L, MgSO4 5 mmol / L, (NH4)2SO4 10 mmol / L, 0.1% Triton X-100, and 8U Best DNA polymerase large fragment and 1 μL of the sample DNA to be tested, the balance is water; the temperature of the LAMP amplification is 62-66°C, more preferably 63-65°C, and more preferably 64°C; the time of the LAMP amplification is 30-70 minutes, more preferably 40-70 minutes, 50-70 minutes, 60-70 minutes; it can also be 30 minutes, 40 minutes, 50 minutes, 60 minutes or 70 minutes. After the amplification is completed, the amplification product is obtained. The amplification product is detected with a fluorescently labeled LFD test strip detection kit, and the test strip is observed. If both the test strip and the quality control strip are colored, the sample to be tested is infected with corn leaf blight; if the quality control strip is colored and the test strip is not colored, the sample to be tested is not infected with corn leaf blight.

[0046] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0047] The main reagents of the embodiment of the present invention: Bst DNA polymerase large fragment (containing 10× buffer) was purchased from the official website of NewEngland Biolabs, dNATs was purchased from the official website of TaKaRa, LFD lateral flow test strips were purchased from Weibaiao (Beijing) Biotechnology Co., Ltd., Taq DNA polymerase and 2000DL DNA Marker were purchased from Bao Bioengineering (Dalian) Co., Ltd., and SYBRgreen I was purchased from the official website of Invitrogen.

[0048] Escherichia coli strain DH5α was maintained by the Plant Pathology Laboratory, Institute of Plant Protection, Fujian Academy of Agricultural Sciences. pMD19-T Vector, rTaq DNA polymerase, and T4 DNA ligase were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd., the plasmid Mini kit was purchased from Guangzhou Omega Biotechnology Co., Ltd., the Gel Extraction kit was purchased from Qiagen-Shanghai Bojing Biotechnology Co., Ltd., and the plant genomic DNA extraction kit (spin column type, catalog number DP302-02) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0049] Example 1 Cloning and sequence analysis of the reducease Brn1 gene of Bipolaris maydis

[0050] In 2022, the pathogenic strain SM-1 was isolated from the diseased leaves of corn leaf blight at the fresh corn planting base in Shangjie Village, Yangxi Town, Sanyuan District, Sanming City, Fujian Province, and was identified as Bipolaris maydis.

[0051] Amplification, cloning, and sequencing of the B. maydis reductaseBrn1 gene: Based on the nucleotide sequence of the B. maydis reductaseBrn1 gene from GenBank, a pair of amplification primers, BmF: 5'-TTCGGCCACTTCAAGGATGT-3' (SEQ NO. 6) and BmR: 5'-CCGAGAATCCAAAGCCTGAAG-3' (SEQ NO. 7), were designed. PCR was used to amplify the B. maydis reductaseBrn1 gene fragment. The PCR amplification reaction system was 25 μL, including 10 μmol / L outer primers BmF and BmR, 1.0 μL each, and 10× Buffer (containing MgCl2). 2+ ) 5.0 μL, 4.0 μL of 10 mM dNTPs, 1.0 μL of Taq enzyme (5 U / μL), 1.0 μL of DNA template, and sterile double-distilled water (ddH2O) were added to 25 μL. The PCR amplification procedure (reaction conditions) included 94°C pre-denaturation for 4 minutes, denaturation at 94°C for 1 minute, annealing (annealing) at 55°C for 45 seconds, and extension at 72°C for 1 minute, for 35 cycles, followed by a final extension at 72°C for 10 minutes. The B. maydis reductaseBrn1 gene fragment was amplified by PCR and cloned into pMD19-T to generate a recombinant plasmid. The sequencing results were analyzed to facilitate specific primer design.

[0052] Example 2 Design of LAMP primers and probes

[0053] Based on the six sites in the conserved region of the reductase gene (reductaseBrn1) sequence of Bipolaris maydis (accession number: MT130208.1), two pairs of specific primers and a probe Bm-HP (one pair of external primers F3 and B3, one pair of internal primers FIP and BIP) were designed. The schematic diagram of the primer and probe design is shown in the figure. Figure 1 As shown, the primer and probe sites are shown by straight lines, the reverse complementary sequences of F2 and F1c constitute the upstream inner primer FIP, the B2 and B1c sequences constitute the downstream inner primer BIP, Bm-HP is the probe for LFD detection, and the nucleotide sequences of the primers and probes are shown in Table 1.

[0054] Table 1 Primer and probe sequences for LAMP-LFD detection of B. maydis

[0055]

[0056] The primer FIP was labeled with biotin at its 5' end, and the probe HP was labeled with 6-carboxyfluorescein (6-FAM) at its 5' end. Both primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0057] Example 3 Optimization of LAMP amplification conditions

[0058] Genomic DNA from the test strain of Bipolaris maydis was extracted using conventional nucleic acid extraction methods or commercially available kits and used as a template for LAMP amplification. A 25 μL LAMP reaction system was prepared in a sterile 200 μL Sppendorf tube. The reaction system contained 0.2 μmol / L each of outer primers F3 and B3, 1.6 μmol / L each of inner primers FIP and BIP, 0.96 mmol / L dNTPs, 20 mmol / L Tris-HCl (pH 8.8), 10 mmol / L KCl, 5 mmol / L MgSO₄, 10 mmol / L (NH₄)₂SO₄, 0.1% Triton X-100, 8 U of Best DNA Polymerase Large Fragment, and 1 μL of sample template. Double-distilled water was added to bring the total reaction volume to 25 μL.

[0059] To obtain the optimal LAMP reaction conditions, the prepared LAMP reaction solution was first incubated in a water bath at different temperatures (60, 61, 62, 63, 64, 65, 66, 67, and 68°C) for 60 min, and the appropriate reaction temperature was determined based on the amplification results.

[0060] The LAMP amplification results were detected by three methods: visual observation, 2% agarose gel electrophoresis and lateral flow cytometry (LFD).

[0061] Colorimetric naked eye observation method: SYBR Green I nucleic acid dye is added to the LAMP amplification product, if the color of the amplification product becomes yellow-green, it indicates that the detection result is positive, and the sample contains Bipolaris maydis, if the amplification product is orange, it indicates that the detection result is negative, and the sample does not contain Bipolaris maydis, or the LAMP amplification product is placed under a wavelength of 365 nm ultraviolet light, if the amplification product appears turbid white precipitate, it indicates that the detection is positive, and the sample contains Bipolaris maydis, if the amplification product is transparent and colorless, it indicates that the detection result is negative, and the sample does not contain Bipolaris maydis.

[0062] Agarose gel electrophoresis method: 5.0 μL of LAMP amplification product is taken for 2% agarose gel electrophoresis, if ladder bands appear in the gel, it indicates that the detection is positive, and the sample contains Bipolaris maydis, if there are no ladder bands in the gel, it indicates that the detection result is negative, and the sample does not contain Bipolaris maydis.

[0063] LFD test strip detection method: After LAMP amplification, 20 pmol of Bm-HP probe is added to the reaction solution without termination reaction, and incubation is continued at 64°C for 5 min, 10 μL of hybridization solution is mixed with 90 μL of Buffer, and the LFD test strip is immersed in it, and whether the detection band develops color is observed. If the quality control band and the detection band develop color, it indicates that the detection result of Bipolaris maydis in the sample is positive, if only the quality control band C develops color and the detection band does not develop color, it indicates that the detection result of Bipolaris maydis in the sample is negative.

[0064] The LAMP-LFD reaction temperature and time optimization results are shown in Table 1. Figure 2 Figure 2 ​Figures 1-8 (left to right) show the amplification results using Bipolaris maydis as a template at 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, and 67°C, respectively. Figure 9: Negative control (sterile water). M: DL2000 DNA marker. The results show that when the reaction time is 60 minutes, the color of the LAMP amplification reaction solution changes from orange to yellow-green at 62°C, 63°C, 64°C, 65°C, and 66°C, producing a white, turbid precipitate. Typical ladder-like DNA bands were observed during electrophoresis, and all results were positive on the LFD test strip. The DNA band at 64°C was the brightest, and the positive band on the LFD test strip was most obvious at 64°C.

[0065] At the optimal reaction temperature, the reaction time optimization results are as follows Figure 3 As shown, Figure 3 Center: 1, 8, and 9 are negative controls (sterile water). 2-7 (from left to right) show the amplification results using Bipolaris maydis as a template at reaction times of 20, 30, 40, 50, 60, and 70 minutes, respectively. M: DL 2000 DNA marker. The results show that positive LAMP amplification results were observed at 30, 40, 50, 60, and 70 minutes, with the DNA electrophoresis bands and LFD test strip detection lines being most pronounced at 60 and 70 minutes.

[0066] By comprehensively comparing the amplification effects of different reaction temperatures and reaction times, the final reaction system of the Bipolaris maydis LAMP-LFD detection method was determined to be: 25 μL, including 12.5 μL of 2× reaction buffer (RM), 1.0 μL each of 0.2 μM outer primers (F3, B3) and 1.6 μM inner primers (FIP, BIP), and Bst DNA polymerase (8 U. -1 ) 1.0 μL, nucleic acid fluorescent dye (FD) 1.0 μL, genomic DNA template 1.0 μL, and sterile ddH2O to 25 μL. The optimal reaction conditions were 64°C for 60 minutes. The results of visual color change observation, agarose gel electrophoresis, and LFD test strips were consistent.

[0067] Example 4 Determination of specificity of LAMP-LFD detection of Bipolaris maydis

[0068] To verify the specificity of LAMP-LFD for Bipolaris maydis, specificity assays were performed using Bipolaris maydis and test strains from the same genus and other genera (Table 2).

[0069] Table 2 Test strains and test results

[0070]

[0071] Note: “+” indicates that Bipolarismaydis was detected positive, and (-) indicates that it was not detected.

[0072] All test strains were inoculated on PDA solid medium plates and cultured at 28°C for 5-7 days. Genomic DNA of each test strain was extracted using a plant genomic DNA extraction kit and dissolved in 60 μL ddH2O. The DNA concentration was measured using a spectrophotometer and diluted to 50 ng.μL. -1 , store at -20℃ for future use.

[0073] Specificity was verified using the optimized LAMP-LFD assay from Example 3, using genomic DNA from the test strain as a template. Bipolaris maydis genomic DNA served as a positive control, and sterile ddH₂O served as a negative control. LAMP amplification products were detected by visual inspection, 2% agarose gel electrophoresis, and LFD strips. The experiment was repeated three times.

[0074] Amplification results such as Figure 4 As shown, Figure 4 The samples in the figure are: 1-3: Bipolaris maydis; 4-11: from left to right: Psoralea maydis, Helminthosporium oryzae, Helminthosporium maydis, Helminthosporium spp., Psoralea maydis, Psoralea spp., Psoralea spp., Psoralea spp., Psoralea spp., Psoralea spp., Psoralea spp., Psoralea spp., Psoralea spp., Psoralea spp., Psoralea spp.; 12: negative control (sterile water); M: DL2000 DNA marker. The results showed that only Psoralea maydis showed positive amplification, with the amplified product exhibiting a yellow-green color and a white turbid precipitate. Typical ladder-like bands appeared on agarose gel electrophoresis, and the LFD test strip had clear control and detection lines. No amplification product was produced for other species of the genus and sterile water, all showing negative results. This demonstrates that the established LAMP-LFD assay is highly specific for Bipolaris maydis, capable of separating Bipolaris maydis from closely related species of the same genus and strains of other genera, and specifically detecting Bipolaris maydis.

[0075] Example 5 LAMP-LFD sensitivity detection of Bipolaris maydis

[0076] Sterile ddH2O was used to perform 10-fold serial dilution of Bipolaris maydis genomic DNA, and 1 ng.μL -1 , 100μg.μL -1 , 10μg.μL-1 , 1pg.μL -1 , 100fg.μL -1 , 10fg.μL -1 ,

[0077] 1fg.μL -1 , 100ag.μL -1 , 10ag.μL -1 and 1ag.μL -1 A total of 10 concentrations of genomic DNA were used as templates for the LAMP-LFD reaction, and sterile water was used as a negative control. The sensitivity of Bipolaris maydis genomic DNA was detected using the LAMP-LFD optimized in Example 3. The sensitivity of the detection was evaluated based on the test results. The test was repeated 3 times under the same conditions. The test results are shown in Figure 2. Figure 5 shown. Figure 5 The template concentrations from left to right in 1-8 are: 10μg.μL -1 , 1μg.μL -1 , 1pg.μL -1 、10fg.μL -1 , 1fg.μL -1 、100ag.μL -1 、10ag.μL -1 and 1ag.μL -1 ,9: negative control (sterile water), M: DL2000 DNA marker.

[0078] The concentration of Bipolaris maydis genomic DNA was higher than 1 fg.μL -1 The test results of the above treatments were all positive, that is, the LAMP product showed yellow-green, a white turbid precipitate was observed under ultraviolet light, a typical ladder-like band appeared in gel electrophoresis, and the LFD test strip had clearly visible quality control lines and detection lines, indicating that the established LAMP-LFD method has a detection sensitivity of 1 fg.μL for Bipolaris maydis genomic DNA. -1 .

[0079] Example 6 Detection of simulated diseased leaves and naturally diseased leaves in the field

[0080] Collection of simulated diseased leaves and naturally diseased leaves in the field: Bipolaris maydis conidia suspension (1×10 6 CFU.mL -1) was spray-inoculated into the true leaves of four-leaf corn plants (corn variety "Xuetian 232"). Five days after inoculation, symptomatic areas of diseased leaves were excised to serve as simulated diseased leaf tissue. Simultaneously, leaves suspected of being infected with Southern Blight were collected from the field as naturally diseased samples.

[0081] Leaf infection detection: The collected artificially simulated diseased leaf tissue and field-derived corn leaf blight tissue were placed in a mortar and ground into powder with liquid nitrogen. Genomic DNA was extracted using a plant genomic DNA extraction kit and detected using the optimized LAMP-LFD method in Example 3. Sterile ddH2O was used as a negative control, and 1 ng.μL - 1 Bipolaris maydis genomic DNA was used as a positive control. Figure 6 and as shown in Table 3.

[0082] Table 3 LAMP-LFD detection results of corn leaf spot pathogen

[0083] Sample code Collection site Collection time Variety Sample number Detection rate (%) ZT1~ZT15 Changtai, Zhangzhou 2024-5-12 Shangpin 15 93.33 SY1~SY20 Sanyuan, Sanming 2024-6-21 Xuetian 232 20 100.00 NX1~NX23 Xiapu, Ningde 2024-5-30 Unknown 23 95.65

[0084] The LAMP-LFD test results showed that all simulated bacteria-infected leaves were positive, while healthy corn leaves that were not inoculated with Bipolaris maydis were negative. Fourteen of the 15 leaf samples collected from Changtai, Zhangzhou, tested positive and one was negative. The positive rate of 20 leaf samples collected from Sanyuan District, Sanming was 100%, and 22 of the 23 samples collected from Xiapu, Ningde, tested positive. This indicates that the established LAMP-LFD can be used to detect Bipolaris maydis in plant leaves.

[0085] From the above examples, it can be seen that the present invention provides a primer-probe combination, a kit and a detection method for LAMP-LFD detection of corn leaf spot fungus. The primer-probe combination of the present invention has good specificity, high accuracy and high sensitivity, and the detection limit is 1 fg.μL -1 The operation is simple and quick, and it is of great significance for the early warning and prevention of corn leaf spot and the prevention and control of the spread of the disease.

[0086] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A primer-probe combination for detecting corn leaf spot pathogen, characterized in that: It comprises external primers, internal primers and a probe Bm-HP, wherein the external primers comprise an external upstream primer F3 and an external downstream primer B3, and the internal primers comprise an internal upstream primer FIP and an internal downstream primer BIP; The nucleotide sequence of the external upstream primer F3 is shown in SEQ NO.1; The nucleotide sequence of the external downstream primer B3 is shown in SEQ NO.2; The nucleotide sequence of the internal upstream primer FIP is shown in SEQ NO.3; The nucleotide sequence of the internal downstream primer BIP is shown in SEQ NO.4; The nucleotide sequence of the probe Bm-HP is shown in SEQ NO.

5.

2. The primer-probe combination according to claim 1, characterized in that The 5' end of the internal upstream primer FIP is labeled with biotin; the 5' end of the probe Bm-HP is labeled with 6-FAM.

3. Use of the primer-probe combination according to claim 1 or 2 in detecting Bacterial leaf spot pathogenicity of corn.

4. A kit for detecting corn leaf spot pathogen, characterized in that: The method comprises the primer-probe combination according to claim 1.

5. The kit according to claim 4, characterized in that The molar ratio of the external upstream primer F3, the external downstream primer B3, the internal upstream primer FIP, and the internal downstream primer BIP in the kit is 0.8-1.2:0.8-1.2:7-9:7-9.

6. A LAMP-LFD detection method for Bacteroides maydis, characterized in that: The steps include: (1) performing LAMP amplification on the sample to be tested using the primer-probe combination of claim 1 or the kit of claim 4 to obtain an amplified product; (2) Detect the amplified product using an LFD test strip and observe the test strip. If both the test strip and the quality control strip show color, the sample to be tested is infected with the corn leaf blight pathogen; If the quality control band develops color but the test band does not, the sample to be tested is not infected with corn leaf blight.

7. The LAMP-LFD detection method according to claim 6, characterized in that The temperature of the LAMP amplification is 62-66° C., and the time of the LAMP amplification is 30-70 min.

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