Primer probe combination, kit and detection method for corn bipolaris maydis LAMP-LFD (loop-mediated isothermal amplification-lateral flow dipstick) detection

Abstract

The invention provides a primer probe combination, a kit and a detection method for corn bipolaris maydis LAMP-LFD (loop-mediated isothermal amplification-lateral flow dipstick) detection, and belongs to the technical field of biology. The primer and probe combination for LAMP-LFD detection of the bipolaris maydis 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. According to the detection primer probe combination, genome DNA of a to-be-detected sample is used as a template, LAMP isothermal amplification is performed, then the LFD test strip is used for detection, and if a quality control band and a detection band both develop colors, it is indicated that the detection result of the corn bipolaris maydis of the sample is positive. The primer probe combination is good in specificity, high in accuracy and high in sensitivity, the detection limit is 1 fg.mu L <-1 >, operation is easy, convenient and rapid, and the primer probe combination has important significance on early warning, prevention and control of corn southern leaf blight and prevention and control of disease diffusion and spreading.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a primer-probe combination, a kit and a detection method thereof for detecting southern blight pathogen LAMP-LFD. Background Art

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

[0003] Traditional detection methods for maize pathogens mainly include diseased tissue separation, that is, separation of target pathogens from diseased tissues, analysis of morphological characteristics or physiological and biochemical characteristics of pathogens, or identification with the aid of conservative gene sequences, pathogenicity determination, and then correct classification and identification of pathogens based on comprehensive results, enzyme-linked immunosorbent assay and ELIS analysis, etc. These identification and detection methods have the disadvantages of low sensitivity, poor specificity, complex operation, and long detection cycle, so they are not suitable for the detection of large quantities of samples and latently infected plant tissues.

[0004] With the development of rapid molecular detection technology for plant pathogens, nucleic acid amplification technologies such as conventional PCR assay, nested PCR, droplet digital PCR (ddPCR), real-time fluorescence quantitative PCR, recombinase-mediated isothermal amplification (PRA) and loop-mediated constant temperature amplification (LAMP) have been successfully applied to the rapid detection of maize pathogens. Compared with traditional identification and detection methods for maize pathogens, PCR detection technology has the advantages of strong specificity, high sensitivity, rapidity and high efficiency, which improves the accuracy of detection to a certain extent. However, PCR detection technology has high requirements for instruments and equipment, experimental environment, and the professional level of operators. It has the disadvantages of high cost, complex operation, low detection sensitivity, heavy dependence on instruments and equipment, and the need for certain professional operating skills, easy to cause missed detection of trace pathogens, and difficulty in achieving on-site detection, 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 six sites in the conservative region of the target gene sequence, and uses a strand displacement active Bst DNA polymerase to amplify for 30 to 60 minutes under isothermal conditions (about 63°C), so as to achieve a large amount of target DNA amplification. The LAMP-LFD method has the advantages of short detection time, strong specificity, simple operation, no need for expensive precision instruments, and the results are visualized for easy observation. The patent with application number "201810482292.5" and invention name "LAMP detection primers for corn leaf blight and its visualization detection method and application" discloses a LAMP detection method for corn leaf blight, but the sensitivity of the detection method is low, with a detection limit of 100fg. Before and at the early stage of corn leaf blight, the concentration of corn leaf blight in corn leaf tissue is low, and the above detection method can be used to infinitely 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 leaf blight. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a primer-probe combination, a kit and a detection method thereof for LAMP-LFD detection of Xanthomonas maydis with high sensitivity.

[0006] In order to achieve the above-mentioned object of the invention, the present invention 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 external 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 corn leaf blight pathogen.

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

[0016] Preferably, the molar ratio of the external upstream primer F3, the external downstream primer B3, the external upstream primer FIP, and the external 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 corn leaf blight pathogen, 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 amplification product;

[0019] (2) using an LFD test strip to detect the amplification product and observing the test strip,

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

[0021] If the quality control band is colored but the test band is not colored, 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 scheme, 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 take the genomic DNA of the sample to be detected as a template, and after LAMP isothermal amplification, it is detected with an LFD test strip. If both the quality control band and the detection band are colored, it means that the corn leaf blight detection 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 blight and the prevention and control of the spread of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the design of LAMP-LFD primers and probes based on the reductase Brn1 gene of corn leaf blight pathogen (Bipolaris maydis).

[0026] Figure 2 The figure is the experimental result of optimizing the reaction temperature of LAMP-LFD detection for Bipolaris maydis; wherein, a is the detection result of adding fluorescent dye, b is the detection result under ultraviolet light irradiation with a wavelength of 365nm, c is the detection result of agarose gel electrophoresis, and d is the detection result of LFD test strip.

[0027] Figure 3The figure is the experimental result of optimizing the reaction time of LAMP-LFD detection for Bipolaris maydis; wherein, a is the detection result of adding fluorescent dye, b is the detection result under ultraviolet light irradiation with a wavelength of 365nm, c is the detection result of agarose gel electrophoresis, and d is the detection result of LFD test strip.

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

[0029] Figure 5 The results of sensitivity determination of LAMP-LFD detection of Bipolaris maydis are shown in Figure 1, where a is the detection result of adding fluorescent dye, b is the detection result under ultraviolet light irradiation with a wavelength of 365 nm, c is the detection result of agarose gel electrophoresis, and d is the detection result of LFD test strips.

[0030] Figure 6 The figure is the LAMP-LFD detection result of corn leaf blight infected leaves; among them, a is the detection result of adding fluorescent dye, b is the detection result under ultraviolet light of wavelength 365nm, c is the agarose gel electrophoresis detection result, and d is the LFD test strip detection result. 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 external 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 corn leaf blight pathogen.

[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 external 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 corn leaf blight pathogen, 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 amplification product;

[0042] (2) using an LFD test strip to detect the amplification product and observing the test strip,

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

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

[0045] The invention first extracts genomic DNA of a sample to be tested. The invention has no special limitation on the extraction of sample DNA. The method well known to those skilled in the art or a plant genomic DNA extraction kit can be used for extraction. 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 reaction system of the LAMP amplification is 25 μL, and includes components with 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, 8U Best DNA polymerase large fragment and 1 μL of 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 30-70min, more preferably 40-70min, 50-70min, 60-70min; it can also be 30min, 40min, 50min, 60min or 70min. 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 the detection band and the quality control band are colored, the sample to be tested is infected with corn leaf blight; if the quality control band is colored and the detection band 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 in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0047] The main reagents of the embodiments 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] The 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., plasmidMini kit was purchased from Guangzhou Omega Biotechnology Co., Ltd., Gel Extraction kit was purchased from Qiagen-Shanghai Bojing Biotechnology Co., Ltd., and the plant genomic DNA extraction kit was a centrifugal column type, catalog number DP302-02, purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0049] Example 1 Cloning and sequence analysis of the reductase 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 corn leaf blight pathogen (Bipolaris maydis).

[0051] Amplification, cloning and sequencing of the reductaseBrn1 gene of Bipolaris maydis: Based on the nucleotide sequence of the reductase Brn1 gene of B. maydis in GenBank, a pair of amplification primers BmF: 5'-TTCGGCCACTTCAAGGATGT-3' (SEQNO.6) and BmR: 5'-CCGAGAATCCAAAGCCTGAAG-3' (SEQNO.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, 10× Buffer (containing Mg 2+ ) 5.0μL, 10mM dNTPs 4.0μL, Taq enzyme (5U / μL) 1.0μL, DNA template 1.0μL, sterile double distilled water (ddH2O) to 25μL; PCR amplification program (reaction conditions) is 94℃ pre-denaturation 4min, 94℃ denaturation 1min, 55℃ annealing (renaturation) 45s, 72℃ extension 1min, 35 cycles, and finally 72℃ extension 10min. PCR amplification obtained the reductaseBrn1 gene fragment of B. maydis, cloned it into pMD19-T to obtain the recombinant plasmid and sequenced and analyzed the sequencing results for specific primer design.

[0052] Example 2 Design of LAMP primers and probes

[0053] According to the 6 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 primer and probe design is shown in 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 X. maydis

[0055]

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

[0057] Example 3 Optimization of LAMP amplification conditions

[0058] The genomic DNA of the test strain of Bipolarismaydis was extracted by conventional nucleic acid extraction methods or commercial kits as a template for LAMP amplification. The LAMP reaction system was prepared in a sterile 200 μL Leppendorf tube, and the 25 μL reaction system included 0.2 μmol / L of outer primers F3 and B3, 1.6 μmol / L of inner primers FIP and BIP, 0.96 mmol / L of dNTPs, 20 mmol / L of Tris-HCl (pH 8.8), 10 mmol / L of KCl, 5 mmol / L of MgSO4, 10 mmol / L of (NH4)2SO4, 0.1% Triton X-100, 8 U of Best DNA polymerase large fragment and 1 μL of sample template, and double distilled water was added to make the total volume of the reaction system 25 μL.

[0059] To obtain the optimal reaction conditions for LAMP, 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 results of LAMP amplification were detected by three methods: colorimetric observation, 2% agarose gel electrophoresis and lateral flow test strips (LFD).

[0061] Colorimetric naked eye observation method: Add SYBR Green I nucleic acid dye to the LAMP amplification product. If the color of the amplification product changes to yellow-green, it means that the test result is positive and the sample contains corn leaf blight (Bipolaris maydis). If the amplification product is orange-yellow, it means that the test result is negative and the sample does not contain corn leaf blight (Bipolaris maydis). Alternatively, place the LAMP amplification product under a 365nm ultraviolet lamp for observation. If a turbid white precipitate appears in the amplification product, it means that the test is positive and the sample contains corn leaf blight (Bipolaris maydis). If the amplification product is transparent and colorless, it means that the test result is negative and the sample does not contain corn leaf blight (Bipolaris maydis).

[0062] Agarose gel electrophoresis: Take 5.0 μL LAMP amplification product and perform 2% agarose gel electrophoresis. If a ladder-like band appears in the gel, it means the test result is positive and the sample contains Bipolaris maydis. If there is no ladder-like band in the gel, it means the test result is negative and the sample does not contain Bipolaris maydis.

[0063] LFD test strip detection method: After LAMP amplification, without stopping the reaction, add 20pmol of Bm-HP probe to the reaction solution, continue incubation at 64℃ for 5min, take 10μL hybridization solution and mix with 90μL Buffer, immerse the LFD test strip in it, and observe whether the detection band is colored. If both the quality control band and the detection band are colored, it means that the test result of the corn leaf blight (Bipolaris maydis) of the sample is positive. If the quality control band is colored, only the quality control band C is colored and the detection band is not colored, it means that the test result of the corn leaf blight (Bipolaris maydis) of the sample is negative.

[0064] The optimization results of LAMP-LFD reaction temperature and time are shown in Figure 2 shown. Figure 2In the figure, 1-8: from left to right, the amplification results of corn leaf blight pathogen at 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, 66℃ and 67℃, 9: negative control (sterile water), M: DL2000 DNA marker. The results showed that when the reaction time was 60min, the color of the LAMP amplification reaction solution changed from orange to yellow-green at 62℃, 63℃, 64℃, 65℃ and 66℃, and a white turbid precipitate was produced. Typical ladder-shaped DNA bands were observed by electrophoresis, and the LFD test strip test results were all positive, among which the electrophoresis DNA band at 64℃ had the highest brightness, and the positive band of the LFD test strip test line at 64℃ was the most obvious.

[0065] At the optimal reaction temperature, the reaction time optimization results are as follows Figure 3 As shown, Figure 3 Middle: 1, 8 and 9 are negative controls (sterile water), 2-7 from left to right are the amplification results of using corn leaf blight as template, and the reaction time is 20min, 30min, 40min, 50min, 60min and 70min respectively; M: DL 2000DNA marker. The results showed that positive results were observed at 30min, 40min, 50min, 60min and 70min of LAMP amplification reaction, and the DNA electrophoresis bands and LFD test strip detection lines at 60min and 70min were the most obvious.

[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 of each of 0.2 μM outer primers (F3, B3) and 1.6 μM inner primers (FIP, BIP), and 8 U. μL of Bst DNA polymerase. -1 ) 1.0μL, nucleic acid fluorescent dye (FD) 1.0μL, genomic DNA template 1.0μL, sterile ddH2O to 25μL, the best reaction conditions are 64℃, 60min. The results of the three observations of color change by naked eye, agarose electrophoresis and LFD test strips are consistent.

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

[0068] To verify the specificity of LAMP-LFD to 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. The 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] The genomic DNA of the strain to be tested was used as a template, and the LAMP-LFD optimized in Example 3 was used for specificity verification, with Bipolaris maydis genomic DNA as a positive control and sterilized ddH2O as a negative control. The LAMP amplification product was detected by naked eye observation, 2% agarose gel electrophoresis and LFD test strips. The experiment was repeated 3 times.

[0074] The amplification results are as follows Figure 4 As shown, Figure 4 The samples to be tested in the figure are: 1-3: corn leaf spot pathogen; 4-11: from left to right, corn round spot pathogen, rice flat umbilical worm spore, corn flat umbilical worm spore, wheat root rot flat umbilical worm spore, corn large leaf spot pathogen, corn curved spore leaf spot pathogen, corn alternaria leaf spot pathogen, corn verticillium fusarium; 12: negative control (sterile sterile water), M: DL2000DNA marker. The results showed that only corn leaf spot pathogen showed positive amplification results, the amplification product showed yellow-green and white turbid precipitation, agarose electrophoresis showed typical ladder bands, LFD test strips had obvious quality control lines and detection lines, while other species under the genus and sterile water had no amplification products, all showing negative results, indicating that the established LAMP-LFD has a strong specificity for Bipolaris maydis, can separate Bipolaris maydis from closely related species of the same genus and strains of other genera, and can specifically detect Bipolaris maydis.

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

[0076] Use sterile ddH2O to dilute Bipolaris maydis genomic DNA 10-fold, and select 1ng.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 LAMP-LFD reaction templates, and sterile water was used as a negative control. The optimized LAMP-LFD in Example 3 was used to perform sensitivity detection of Bipolaris maydis genomic DNA, and the detection sensitivity was evaluated based on the detection results. The test was repeated 3 times under the same conditions, and the detection results were as follows: 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: DL2000DNA marker.

[0078] Bipolaris maydis genomic DNA concentration is higher than 1fg.μL -1 The test results of the above treatments were all positive, that is, the LAMP product showed yellow-green color, white turbid precipitate was observed under ultraviolet light, typical ladder-like bands appeared in gel electrophoresis, and the LFD test strip had clearly visible quality control line and detection line, indicating that the established LAMP-LFD method had 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 corn plants with 4 leaves (corn variety "Xuetian 232"). Five days after inoculation, the symptomatic part of the diseased leaves was cut off as simulated diseased leaf tissue. At the same time, the diseased leaves suspected of corn leaf spot in the field were collected as natural disease samples in the field.

[0081] Leaf infection detection: The collected artificial simulated diseased leaf tissues and field-derived corn leaf blight leaf tissues were placed in a mortar, ground into powder with liquid nitrogen, and genomic DNA was extracted using a plant genomic DNA extraction kit. The optimized LAMP-LFD method in Example 3 was used for detection. 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 location Collection time variety Sample size Detection rate (%) ZT1~ZT15 Zhangzhou Changtai 2024-5-12 Top grade 15 93.33 SY1~SY20 Sanming Sanyuan 2024-6-21 Snow sweet 232 20 100.00 NX1~NX23 Ningde Xiapu 2024-5-30 unknown 23 95.65

[0084] The LAMP-LFD test results showed that the LAMP-LFD test results of all simulated bacteria-carrying leaves were positive, while the healthy corn leaves that were not inoculated with Bipolaris maydis were negative. Among the 15 leaf samples collected from Changtai, Zhangzhou, 14 tested positive and 1 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 were positive. This shows that the established LAMP-LFD can be used for the detection of 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 blight. 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 It is easy and quick to operate, and is of great significance for the early warning and prevention and control of corn leaf blight, and for preventing 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A primer-probe combination for detecting corn leaf blight, 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 external 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 Blight of Corn.

4. A kit for detecting corn leaf blight, characterized in that: The invention 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 external upstream primer FIP, and the external 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 corn leaf blight pathogen, characterized in that: The steps include: (1) performing LAMP amplification on a sample to be tested using the primer-probe combination of claim 1 or the kit of claim 4 to obtain an amplification product; (2) using an LFD test strip to detect the amplification product and observing the test strip, 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 band is colored but the test band is not colored, 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 reaction system of the LAMP amplification is 25 μL, which uses water as a solvent and includes the following components in concentration: 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% TritonX-100, 8 U Best DNA polymerase large fragment and 1 μL of sample DNA to be tested.

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

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