A lAMP primer composition, kit and application for detecting fusarium equiseti

Abstract

Fusarium equiseti can cause root rot of many crops including red kidney beans, which seriously affects crop yield and quality. The present application selects CYP51C gene as a target to provide a LAMP primer composition for detecting Fusarium equiseti, a detection kit and application, and establishes a LAMP detection technology system for Fusarium equiseti. The technology can realize the rapid detection of Fusarium equiseti within 22 min, and the detection limit is 10 pg / μL. The method provides strong technical support for the field rapid diagnosis of red kidney bean root rot.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a LAMP primer composition, kit, and application for detecting Fusarium equisetifolium. Background Technology

[0002] Fusarium equisetifolium ( Fusarium equiseti Fusarium equisetifolium can cause root rot in various crops, including red kidney beans, severely affecting crop yield and quality. After infection, brown lesions appear on the base of the red kidney bean stem and the main root. These lesions gradually expand, leading to rotting of the affected area. Above-ground parts show poor growth, and in severe cases, the entire plant dies. Besides Fusarium equisetifolium, other fungi such as Fusarium oxysporum, Fusarium solani, Fusarium trifidum, and Rhizoctonia solani can also cause root rot in red kidney beans, and it is difficult to distinguish these pathogens based on symptoms alone.

[0003] Traditional pathogen identification techniques require steps such as isolation and purification, morphological identification, and molecular biological identification, which are cumbersome and time-consuming. PCR technology is highly dependent on equipment, complex to operate, and demands high levels of laboratory environment and operator skill. In the actual control of root rot, especially in cases of sudden outbreaks or when rapid on-site screening is required, there is an urgent need for a simpler, faster, and less equipment-dependent detection method. Loop-mediated isothermal amplification (LAMP) requires only simple heating equipment and has significant advantages such as simple operation, high speed and efficiency, high specificity and sensitivity, and visually perceptible results. Summary of the Invention

[0004] This invention selects CYP51C Using genes as targets, this study provides LAMP primer combinations, detection kits, and applications for the detection of *Fusarium equisetifolium*, establishing a LAMP detection technology system for *Fusarium equisetifolium*. This technology can achieve rapid detection of *Fusarium equisetifolium* within 22 minutes, which is shorter than the time required for previously reported LAMP detection technologies for *Fusarium equisetifolium*, providing strong technical support for rapid field diagnosis of root rot in red kidney beans.

[0005] This invention provides a LAMP primer composition for detecting Fusarium equisetifolium, characterized in that it comprises the forward outer primer Fe-F3 shown in SEQ ID NO.1, the reverse outer primer Fe-B3 shown in SEQ ID NO.2, the forward inner primer Fe-FIP shown in SEQ ID NO.3, the reverse inner primer Fe-BIP shown in SEQ ID NO.4, the forward loop primer Fe-LF shown in SEQ ID NO.5, and the reverse loop primer Fe-LB shown in SEQ ID NO.6. In the primer composition described above, the sequence of SEQ ID NO.1 is 5'-GTCAAGCAGCCTATGCGT-3'; the sequence of SEQ ID NO.2 is 5'-GCTGGTGACTGACTTGTTCA-3'; the sequence of SEQ ID NO.3 is 5'-TCCGAAGGTCGAGGGAAGAACTTAGTGCCTCCGTCCCATAC-3'; the sequence of SEQ ID NO.4 is 5'-TCCTCACCGCTGGGACAAGAAGCCATAATCCACGGTTTGT-3'; the sequence of SEQ ID NO.5 is 5'-TGGTGCCAGGAGACGCA-3'; and the sequence of SEQ ID NO.6 is 5'-TCGAACCTCTTGAGAAGAACGCT-3'.

[0006] This invention also provides the application of the above primer composition in the detection of Fusarium equisetifolium.

[0007] This invention also provides the application of the above primer composition in the preparation of a LAMP detection kit for Fusarium equisetifolium.

[0008] The present invention also provides a LAMP kit for detecting Fusarium equisetifolium, the LAMP kit comprising the above-described LAMP primer composition.

[0009] Furthermore, the reaction reagents in the above LAMP kit include 12.5 μL of 2×LAMP Master Mix, 0.5 μL of Fe-F3 (10 μmol / L), 0.5 μL of Fe-B3 (10 μmol / L), 2 μL of Fe-FIP (10 μmol / L), 2 μL of Fe-BIP (10 μmol / L), 1 μL of Fe-LF (10 μmol / L), 1 μL of Fe-LB (10 μmol / L), 1 μL of Template DNA, 0.5 μL of DNAPolymerase, and 4.0 μL of Sterilized ddH2O.

[0010] The present invention also provides a method for detecting Fusarium equisetifolium, the method comprising the following steps: extracting DNA from the sample to be tested, performing loop-mediated isothermal amplification of the DNA of the sample to be tested using the above-mentioned LAMP primer composition or the above-mentioned LAMP kit; and finally detecting the amplification product.

[0011] Furthermore, the above loop-mediated isothermal amplification conditions were 63℃ for 22 min.

[0012] The LAMP detection system for Fusarium equisetifolium established in this invention can be used for rapid and visual detection of root rot in red kidney bean samples. This technology can achieve rapid detection of Fusarium equisetifolium within 22 minutes, providing strong technical support for rapid field diagnosis of root rot in red kidney bean. Attached Figure Description

[0013] Figure 1 Figure 1 shows the optimized reaction conditions for the LAMP detection system of *Fusarium equisetifolium*. (A) Real-time fluorescence amplification curves under different temperature conditions; (B) Gel electrophoresis image; (C) Visualization results under natural light; (D) Visualization results under ultraviolet light. M: DL 2000 molecular weight standard; 1: 15 min; 2: 30 min; 3: 45 min; 4: 60 min; 5: 75 min; Figure 2 This image shows the specificity detection of *Fusarium equisetifolium* using the LAMP assay system. (A) Gel electrophoresis image; (B) Visualization results under natural light; (C) Visualization results under ultraviolet light. M: DL 2000 molecular weight standard; 1-2: *Fusarium equisetifolium*; 3-4: *Fusarium oxysporum*; 5-6: *Fusarium trifidum*; 7-8: *Fusarium solani*; 9-10: *Fusarium latifolium*; 11-12: *Fusarium aromaticum*; 13: *Fusarium verticillatum*; 14: *Fusarium chrysogenum*; 15-16: *Fusarium graminearum*; 17: *Alternaria alternata*; 18: *Botrytis cinerea*; 19: *Phytophthora*; 20: *Helicobacter convexum*; 21: *Colletotrichum gloeosporioides*; 22: Negative control; Figure 3 The sensitivity detection results of the LAMP detection system for Fusarium equisetifolium are shown in Figure 1. (A) Gel electrophoresis image; (B) Visualized detection results under natural light; (C) Visualized detection results under ultraviolet light.

[0014] Figure 4 LAMP assay results for field samples. (A) Visualization results under natural light; (B) Visualization results under ultraviolet light. 1-18: Diseased plants in the field; 19: Negative control; 20: Positive control; Figure 5LAMP assay for Fusarium equisetifolium in red kidney bean seeds. (A) 8 red kidney bean seed samples; (B) Visualization results under natural light; (C) Visualization results under ultraviolet light. 1-8: Seed samples from different plots; 9: Positive control; 10: Negative control; Detailed Implementation

[0015] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to examples. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained in the art without creative effort should fall within the scope of protection of the present invention.

[0016] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Example 1: LAMP Primer Design and Synthesis

[0017] Select CYP51C Genes were used as target regions. Fusarium-related pathogens were downloaded from the GenBank database. CYP51C Gene sequences were analyzed and aligned using MEGA11 software to identify specific regions. Once identified, the sequence information for these regions was uploaded to the NEB LAMP Primer Design Tool online primer design platform. Based on the alignment results, four LAMP primer combinations were designed for specific regions of *Fusarium equisetifolium*. The specificity and sensitivity of each primer combination were compared systematically, and the primer combination with superior overall performance was ultimately selected.

[0018] Experimental results: The primer combinations shown in Table 1 below can be used for LAMP detection of Fusarium equisetifolium.

[0019] Table 1 Primer combinations obtained in this study Example 2: Genomic DNA extraction from test strains and samples

[0020] Genomic DNA extraction of the tested strains: The representative strains in Table 1 (all isolated and identified by the Plant Pathology Laboratory of Shanxi Agricultural University) were inoculated on PDA plates and cultured in the dark at 25°C for 7 days. The mycelia were scraped into a mortar, and liquid nitrogen was added to grind the strains into dry powder. The DNA was extracted using a rapid fungal genomic DNA extraction kit (Sangon Biotech Shanghai Co., Ltd.) and stored at -20°C for later use.

[0021] Extraction of genomic DNA from plant samples: The plant roots and stems were ground into powder using liquid nitrogen and transferred to 2.0 mL EP tubes. The plant genomic DNA was extracted using a plant genomic DNA rapid extraction kit (Sangon Biotech Shanghai Co., Ltd.). The DNA concentration was measured and stored at -20℃ for later use.

[0022] Table 2. Sources of tested bacterial strains Example 3: Optimization of reaction conditions for the LAMP detection system of Fusarium equisetifolium

[0023] To optimize reaction conditions, the reaction temperature was set to 60.0, 60.5, 61.5, 63.0, 65.0, 66.5, 67.5, and 68.0 °C, and the real-time amplification curves at different temperatures were displayed using a real-time quantitative PCR instrument to determine the optimal reaction temperature. At the optimal reaction temperature, the reaction time was set to 15, 30, 45, 60, and 75 min, respectively, and the reaction was carried out in a constant temperature water bath. Before the reaction, 0.25 μL of SYBR Green I was pre-added to the inner wall of the tube cap. After the reaction was completed, the reaction system was mixed with SYBR Green I in a centrifuge tube, and the color change under natural light, the fluorescence reaction under 365 nm UV light, the results of 2.5% agarose gel electrophoresis, and the real-time amplification curves were observed to determine the optimal reaction time. Each experiment was repeated three times. The reaction system is shown in Table 3.

[0024] Table 3 LAMP detection reaction system for Fusarium equisetifolium

[0025] Experimental results: Real-time amplification curves at eight temperature gradients within the range of 60.0℃ to 68.0℃ were analyzed. Figure 1 A) determined 63.0℃ as the optimal reaction temperature. Under this temperature condition, different reaction times were set for testing. Gel electrophoresis and colorimetric results showed that effective amplification occurred within the range of 15–30 min ( Figure 1 (B, C, and D). Further analysis using real-time fluorescence amplification curves revealed that the system entered a plateau phase at 22 min. Based on these results, the optimal reaction conditions for this system were determined to be 63.0 °C for 22 min. Example 4 Specificity analysis of the LAMP detection system for Fusarium equisetifolium

[0026] Using the DNA of representative strains in Table 2 as templates, LAMP amplification was performed under the optimized reaction conditions of Example 3. The established detection system was then subjected to specific detection. After the reaction, positive results showed a yellow-green color under natural light and green fluorescence under ultraviolet light, while negative results showed an orange color and no fluorescence. The products were then analyzed by electrophoresis; positive results showed a trapezoidal band, while negative results did not exhibit this characteristic band.

[0027] Experimental results: The results showed that, except for *Fusarium equisetifolium*, none of the other tested strains showed electrophoretic bands after amplification. Figure 2 -A), and it appears orange under natural light and shows no fluorescence under ultraviolet light (Figure 2-B, C). Example 5 Sensitivity Analysis of the LAMP Detection System for Fusarium equisetifolium

[0028] The genomic DNA of *Fusarium equisetifolium* was serially diluted 10-fold (10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, and 100 fg / μL) and then amplified using LAMP. ddH2O was used as a negative control, and the method for determining the amplification products was the same as in Example 4.

[0029] Test results: such as Figure 3 As shown, the results were obtained by agarose gel electrophoresis ( Figure 3 -A) Color change under visible light (turns to yellowish-green, Figure 3 -B) and fluorescence signal under ultraviolet light (producing green fluorescence, Figure 3 -C) The three methods are combined for determination, and the detection limit of this method is 10 pg / μL. Example 6: LAMP detection of field samples

[0030] Eighteen diseased plants from the field were tested according to the established testing system, with ddH2O as a negative control. Each sample was tested in triplicate.

[0031] Test results: such as Figure 4 As shown, a clear colorimetric reaction was observed in all samples, with green fluorescence under ultraviolet light, indicating a positive result. The negative control showed no color change and no fluorescence under ultraviolet light. These results demonstrate that the detection system established in this study can be used for rapid and visual detection of *Fusarium equisetifolium* in field samples. Example 7: LAMP detection of Fusarium equisetifolium in red kidney bean seeds

[0032] Eight samples of red kidney bean seeds were tested according to the established testing system, with ddH2O as a negative control. Each sample was tested in triplicate.

[0033] Test results: such as Figure 5As shown, the established detection system was used to test eight red kidney bean seed samples, with YG-1 and YX-2 showing positive results. This demonstrates that the proposed method can be used for the detection of red kidney bean seed samples.

Claims

1. A LAMP primer composition for detecting Fusarium equisetifolium, characterized in that: This includes the forward outer primer Fe-F3 as shown in SEQ ID NO.1, the reverse outer primer Fe-B3 as shown in SEQ ID NO.2, the forward inner primer Fe-FIP as shown in SEQ ID NO.3, the reverse inner primer Fe-BIP as shown in SEQ ID NO.4, the forward loop primer Fe-LF as shown in SEQ ID NO.5, and the reverse loop primer Fe-LB as shown in SEQ ID NO.

6.

2. The application of the primer composition as described in claim 1 in the detection of Fusarium equisetifolium.

3. The application of the primer composition as described in claim 1 in the preparation of the Fusarium equisetifolium LAMP detection kit.

4. A LAMP kit for detecting Fusarium equisetifolium, characterized in that: The composition comprises the LAMP primers of claim 1.

5. The LAMP kit as described in claim 4, characterized in that: The reaction reagents in the LAMP kit include 12.5 μL 2×LAMP Master Mix, 0.5 μL 10 μmol / L Fe-F3, 0.5 μL 10 μmol / LFe-B3, 2 μL 10 μmol / L Fe-FIP, 2 μL 10 μmol / LFe-BIP, 1 μL 10 μmol / LFe-LF, 1 μL 10 μmol / LFe-LB, 1 μL Template DNA, 0.5 μL DNA Polymerase, and 4.0 μL Sterilized ddH2O.

6. A method for detecting Fusarium equisetifolium, characterized in that: The procedure includes the following steps: extracting DNA from the sample to be tested, performing loop-mediated isothermal amplification of the DNA using the primer composition described in claim 1 or the LAMP kit described in claim 4 or 5, and finally detecting the amplification products.

7. The method as described in claim 6, characterized in that: The loop-mediated isothermal amplification conditions were 63°C for 22 min.

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