Fusarium acuminatum LAMP (loop-mediated isothermal amplification) detection system, kit and detection method thereof

Abstract

The invention discloses an LAMP (Loop-Mediated Isothermal Amplification) detection system, a kit and a detection method of fusarium acuminatum. Comprising a forward outer primer F3 as shown in SEQ ID NO: 1, a reverse outer primer B3 as shown in SEQ ID NO: 2, a forward loop primer LF as shown in SEQ ID NO: 3, a reverse loop primer LB as shown in SEQ ID NO: 4, a forward inner primer FIP as shown in SEQ ID NO: 5 and a reverse inner primer BIP as shown in SEQ ID NO: 6. The invention establishes an LAMP (Loop-Mediated Isothermal Amplification) detection method for fusarium acuminatum The method has the advantages of high specificity and sensitivity, high repeatability and short detection time. The LAMP detection method disclosed by the invention can be used for preventing and treating fusarium root rot of medicago sativa, and has certain positive significance on production and protection of medicago sativa.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a LAMP detection system of Fusarium acuminatum, a kit and a detection method thereof. BACKGROUND

[0002] Medicago sativa is a perennial forage legume, and is known as the "king of forage" due to its rich nutrition, good palatability and strong stress resistance. It is the most common cultivated alfalfa in China. Root rot of alfalfa is one of the most common root diseases of Medicago sativa. The most harmful pathogenic fungus of the disease is Fusarium spp. The root rot caused by Fusarium is called Fusarium root rot, and Fusarium acuminatum is an important pathogenic fungus thereof.

[0003] Fusarium root rot is a soil-borne disease that occurs universally in alfalfa planting areas at home and abroad, and can cause plant disease during the whole growth period of alfalfa. The disease can cause the root of alfalfa to rot, leading to the gradual weakening of the function of the root system in absorbing water and nutrients until the death of the plant, and seriously harming the production of Medicago sativa. The existing agricultural and biological control measures can only control the disease preliminarily, and therefore, detecting whether the seedlings, soil and the like before planting contain Fusarium, and avoiding the use of materials contaminated by Fusarium, is an important means to reduce the risk of Medicago sativa being infected with Fusarium root rot.

[0004] Loop-mediated isothermal amplification (LAMP) is an isothermal amplification method mediated by loop primers, developed and published by Notomi et al. in 2000. It allows amplification to be completed under isothermal conditions and offers advantages over traditional PCR amplification techniques, including shorter reaction time, simpler reaction conditions, higher detection sensitivity, and lower cost. The LAMP detection system mainly consists of Bst DNA polymerase, dNTPs, magnesium ions, primers, and template DNA. Optimizing the concentration and ratio of these components can enhance specific amplification, reduce non-specific amplification, improve reaction efficiency, and shorten reaction time to achieve better amplification results. In the LAMP detection system, the reaction temperature directly affects the activity of DNA polymerase, influencing both amplification efficiency and specificity. The concentration of Bst DNA polymerase directly affects the reaction. Magnesium ions play a crucial role in the LAMP reaction; they are essential for the activity of Bst DNA polymerase and DNA chain elongation. Too low a magnesium ion concentration may lead to insufficient DNA polymerase activity, while too high a magnesium ion concentration may inhibit DNA polymerase activity. dNTPs, as essential nucleotide raw materials for DNA strand synthesis in isothermal amplification reactions, have a crucial impact on the reaction results. The appropriate addition of certain additives may promote the system's performance. Betaine can stabilize DNA-protein complexes and facilitate the smooth passage of DNA polymerase through some complex secondary structures of DNA, preventing DNA polymerase dissociation from the template DNA and thus promoting specific amplification of the sample. Dimethyl sulfoxide (DMSO) mainly affects the activity of DNA polymerase and DNA strand elongation by altering the viscosity and pH of the reaction mixture. Excessive DMSO may inhibit DNA polymerase activity, while insufficient DMSO may not be enough to reduce non-specific amplification. Furthermore, DMSO may also help reduce non-specific amplification by interacting with other reaction components such as magnesium ions, thus contributing to improved LAMP detection specificity and stability. Bovine serum albumin (BSA) can act as an enzyme stabilizer to prevent enzyme denaturation and non-specific adsorption, enhancing the reaction by protecting enzyme activity. It also plays a role in protecting the DNA template from high-temperature cleavage.

[0005] Currently, molecular detection methods for Fusarium argentis include polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA). However, these methods suffer from long testing cycles, cumbersome procedures, and poor specificity, and remain unsatisfactory. Therefore, there is an urgent need to develop a simple, rapid, and accurate new method for detecting Fusarium argentis to meet the practical needs of large-scale field sample screening, early disease diagnosis, and precise control, thereby promoting the sustainable development of the alfalfa industry. Summary of the Invention

[0006] The technical problem to be solved by the present invention is how to detect Fusarium acuminatum rapidly, sensitively and / or specifically. The technical problem to be solved is not limited to the technical subject matter described herein. Other technical subject matter not mentioned herein can be clearly understood by those skilled in the art through the following description.

[0007] To address the aforementioned technical problems, the present invention first provides a primer composition for detecting Fusarium racifolium, the primer composition comprising the forward outer primer F3 shown in SEQ ID NO:1, the reverse outer primer B3 shown in SEQ ID NO:2, the forward loop primer LF shown in SEQ ID NO:3, the reverse loop primer LB shown in SEQ ID NO:4, the forward inner primer FIP shown in SEQ ID NO:5, and the reverse inner primer BIP shown in SEQ ID NO:6.

[0008] The present invention also provides a kit for detecting Fusarium racifolium, the kit comprising the primer composition described herein.

[0009] Furthermore, the kit may also include reagents required for LAMP detection. These reagents include, but are not limited to: reaction buffer, Mg... 2+ Solutions (such as MgSO4 solution or MgCl2 solution), Bst DNA polymerase, betaine, dNTPs (dATP, dTTP, dGTP, dCTP), colorimetric indicators (such as fluorescent dyes SYBR Green I, SYBR Gold, EvaGreen, SYTO9, etc.; colorimetric dyes phenol red, cresol red, neutral red, cresol purple, etc.), positive controls, negative controls, enzyme stabilizers (such as bovine serum albumin), and UDG enzyme.

[0010] LAMP assay buffers are well known to those skilled in the art and typically contain Tris-HCl, potassium chloride, ammonium sulfate, magnesium sulfate, surfactants (such as Tween 20), etc.

[0011] Furthermore, the kit may also include reaction buffer, Mg 2+ Solution, Bst DNA polymerase, betaine, and dNTPs.

[0012] Furthermore, the kit may also contain plasmid standards.

[0013] Furthermore, the plasmid standard can be a recombinant vector obtained by cloning the DNA molecule shown in SEQ ID NO:7 into a vector.

[0014] Furthermore, the kit may also include nucleic acid extraction reagents.

[0015] The nucleic acid extraction reagent is well known to those skilled in the art and is used to extract nucleic acids from a sample to be tested. For example, the nucleic acid extraction reagent may include lysis buffer (such as any one or more of guanidine isothiocyanate, guanidine hydrochloride, and trisodium citrate), lysozyme, proteinase K, washing buffer (such as ethanol and / or guanidine salt) and / or elution buffer (such as TE buffer), etc.

[0016] The positive control can refer to a set of controls that are guaranteed to be detected. For example, it can be a target gene (such as genomic DNA or cDNA), a target gene fragment (such as a specific conserved fragment), a plasmid containing the target gene or a target gene fragment, or a positive sample control (such as a sample known to be infected or a sample known to definitely yield the expected result). The purpose of the positive control is to prevent false negatives caused by problems such as target DNA loss or degradation, DNA polymerase inactivation, or PCR instrument malfunction.

[0017] The negative control can refer to a set of controls that are certain not to be detected. For example, it can be a template-free control (NTC, where water is used instead of nucleic acid in the PCR reaction, and other reagents are added normally in proportion), or a negative sample control (such as a sample that does not express the target gene). The purpose of the negative control is to prevent false positives caused by problems such as product contamination, reagent contamination, cross-contamination between samples, and contamination during sample collection and transportation.

[0018] Furthermore, the test samples for the kit include, but are not limited to, plant samples (such as plants, plant tissues or plant seeds), microbial samples (such as plant pathogenic fungi), and environmental samples (such as water, soil, air and objects).

[0019] Furthermore, the plant sample may be a sample of alfalfa (such as alfalfa).

[0020] The various reagent components of the kit may be present in separate containers, or may be pre-assembled, in whole or in part, into a reagent mixture.

[0021] The components of the kit may be provided in solution form, such as an aqueous solution. When present in aqueous solution, the concentration or content of these components can be readily determined by those skilled in the art according to different needs. For example, for storage purposes, the components may be present at a higher concentration, which can be reduced to the working concentration by diluting the higher concentration solution when in operation or for use.

[0022] Furthermore, the kit may also include a readable carrier describing a method for detecting Fusarium rashesii. The readable carrier may be a kit instruction manual (e.g., a printed manual) explaining how to practice the method of the present invention, or a computer-readable medium (e.g., a floppy disk, CD, etc.) on which information has been recorded.

[0023] The kit described in this article is a detection kit based on loop-mediated isothermal amplification (LAMP).

[0024] The present invention also provides the use of the primer composition in the preparation of products for detecting Fusarium argentis.

[0025] The products described in this article are selected from reagents, kits, chips, test strips, and test cards.

[0026] The present invention also provides the application of the primer composition or the kit in the detection of Fusarium racifolium.

[0027] The present invention also provides a method for detecting Fusarium racifolium, the method comprising using the primer composition or the kit to perform LAMP detection on the sample to be tested.

[0028] Furthermore, the method may include the following steps:

[0029] A1) Extract DNA from the sample to be tested;

[0030] A2) Using the DNA as a template, perform a LAMP reaction using the primer composition or the kit;

[0031] A3) Determine whether the sample to be tested contains Fusarium argentis based on the LAMP reaction results.

[0032] In the above method, the reaction system of the LAMP reaction includes the primer composition and Mg. 2+ Solution, Bst DNA polymerase, betaine, dNTPs, template DNA, and reaction buffer.

[0033] In the above method, in the reaction system of the LAMP reaction, Mg 2+ The concentration can be 2.5-3.5 mM, 2.6-3.4 mM, 2.7-3.3 mM, 2.8-3.2 mM, 2.9-3.1 mM or 3 mM, and / or the betaine concentration can be 0.5-0.7 mM or 0.6 mM, and / or the dNTPs concentration can be 0.7-1.3 mM, 0.8-1.2 mM, 0.9-1.1 mM or 1.0 mM.

[0034] Furthermore, in the primer composition, the molar ratio of the forward outer primer F3 (SEQ ID NO:1), the reverse outer primer B3 (SEQ ID NO:2), the forward loop primer LF (SEQ ID NO:3), the reverse loop primer LB (SEQ ID NO:4), the forward inner primer FIP (SEQ ID NO:5), and the reverse inner primer BIP (SEQ ID NO:6) can be 1:1:2:2:8:8.

[0035] In some embodiments, the LAMP reaction system (25 μL) comprises: 2.5 μL genomic DNA template, 2.5 μL 10× Reaction buffer, 0.75 μL MgSO4 (100 mM), 1 μL Hieff Bst Plus DNA Polymerase (40 U / μL), 3.5 μL dNTPs (10 mM), 4 μL primer 1-FIP-20 (10 μM), 4 μL primer 1-BIP-20 (10 μM), 1 μL primer 1-LF-20 (10 μM), 1 μL primer 1-LB-20 (10 μM), 0.5 μL primer 1-F3-20 (10 μM), 0.5 μL primer 1-B3-20 (10 μM), 0.5 μL LAMP Fluorescent Dye diluted 10-fold, and ddH2O to a final volume of 25 μL.

[0036] In the above method, the reaction temperature of the LAMP reaction can be 65°C.

[0037] Furthermore, the reaction conditions for the LAMP reaction can be 65°C for 30 min.

[0038] The A3) may include determining whether the sample to be tested contains Fusarium argentis based on the amplification curve and / or Ct value of the LAMP amplification product, or performing quantitative detection of Fusarium argentis in the sample to be tested.

[0039] Furthermore, A3) may include any of the following:

[0040] (1) Determine whether the sample to be tested contains Fusarium argentis based on the amplification curve of the LAMP amplification product: if the LAMP amplification curve is an S-shaped curve, it is determined that the sample to be tested contains Fusarium argentis; if the LAMP amplification curve is not an S-shaped curve, it is determined that the sample to be tested does not contain Fusarium argentis.

[0041] (2) Determine whether the sample contains Fusarium argentis based on the Ct value of the LAMP amplification product: if the Ct value is <25, the sample contains Fusarium argentis; if the Ct value is ≥25, the sample does not contain Fusarium argentis.

[0042] Furthermore, quantitative analysis can be performed on the sample to be tested. For example, a standard with a known content can be diluted into samples of different concentrations, and these samples can be simultaneously subjected to real-time fluorescence LAMP amplification with the sample to be tested. A standard curve can be plotted based on the detection data (the horizontal axis represents the logarithm of the initial copy number of the standard, and the vertical axis represents the Ct value). When quantifying the sample to be tested, the initial copy number of the sample can be calculated from the standard curve equation based on the Ct value of the sample to be tested.

[0043] The samples to be tested described in this article include, but are not limited to, plant samples (such as plants, plant tissues or plant seeds), microbial samples (such as plant pathogenic fungi), and environmental samples (such as water, soil, air and objects).

[0044] Furthermore, the plant sample may be a sample of alfalfa (such as alfalfa).

[0045] The detection method for Fusarium argentis described in this invention can also be used for the identification or auxiliary identification of Fusarium argentis.

[0046] This invention is based on the principle of loop-mediated isothermal amplification (LAMP). A set of inner, outer, and loop primers for LAMP were designed and screened specifically for *Fusarium argentis*. Furthermore, by optimizing the reaction system and conditions, a reaction system capable of stably, accurately, and specifically detecting *Fusarium argentis* at a constant temperature of 65℃ for 30 minutes was obtained. This invention establishes a specific, sensitive, and rapid method for detecting *Fusarium argentis* (LAMP detection method). Experiments show that the LAMP detection method of this invention has high specificity and sensitivity, and high repeatability. Compared with the existing qPCR method, its sensitivity and specificity are higher, and the detection time is shortened by 40.12%, making it suitable for point-of-care testing (POCT). The LAMP detection method of this invention can be used for the prevention and control of *Fusarium argentis* root rot in alfalfa, which has certain positive significance for the production and protection of alfalfa. Attached Figure Description

[0047] Figure 1 The amplification curves of ten LAMP primer sets for different concentrations of Fusarium raffinosum DNA samples are shown. These include: a. 1 μg / ml template DNA; b. 0.1 μg / ml template DNA; c. 1 ng / ml template DNA; and d. ddH2O blank control group.

[0048] Figure 2 The amplification curves of primers 1-20 at different temperatures are shown.

[0049] Figure 3The effect of different temperatures on the LAMP reaction is investigated. This includes: a. Ct values ​​at different reaction temperatures; b. significance tests of Ct values ​​for positive samples at different temperatures.

[0050] Figure 4 The images show LAMP amplification curves at different magnesium ion concentrations.

[0051] Figure 5 The effect of magnesium ion concentration on the LAMP reaction was investigated. This included: a. Ct values ​​at different magnesium ion concentrations; b. significance test of Ct values ​​for positive samples at different magnesium ion concentrations.

[0052] Figure 6 The LAMP amplification curves are shown for different dNTP concentrations.

[0053] Figure 7 The effect of dNTP concentration on the LAMP response was investigated. This included: a. Ct values ​​at different dNTP concentrations; b. significance test of Ct values ​​for positive samples at different dNTP concentrations.

[0054] Figure 8 The LAMP amplification curves are shown for different betaine concentrations.

[0055] Figure 9 The effect of betaine concentration on the LAMP reaction was investigated. This included: a) Ct values ​​at different betaine concentrations; and b) a significance test of Ct values ​​for positive samples at different betaine concentrations.

[0056] Figure 10 The LAMP amplification curves are shown for different DMSO concentrations.

[0057] Figure 11 The effect of DMSO concentration on the LAMP reaction. This includes: a. Ct values ​​at different DMSO concentrations; b. significance test of Ct values ​​for positive samples at different DMSO concentrations.

[0058] Figure 12 The LAMP amplification curves are shown for different BSA concentrations.

[0059] Figure 13 The effect of BSA concentration on the LAMP response. This includes: a. Ct values ​​at different BSA concentrations; b. significance test of Ct values ​​for positive samples at different BSA concentrations.

[0060] Figure 14 This is the specificity verification result of the LAMP detection method for Fusarium racifolium in Example 4.

[0061] Figure 15 This is the sensitivity verification result of the LAMP detection method for Fusarium racifolium in Example 5.

[0062] Figure 16 This is the repeatability verification result of the LAMP detection method for Fusarium racifolium in Example 6. Specifically, it shows the amplification of high, medium, and low concentration plasmids in the LAMP detection system. Wherein: a. Amplification curves; b. Average Ct values ​​for each group of reactions.

[0063] Figure 17 The results show the detection results of the qPCR system on the serially diluted plasmids. Among them: a. qPCR amplification curve; b. qPCR melting peak curve.

[0064] Figure 18 This is the result of a specificity test for the qPCR system. Detailed Implementation

[0065] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0066] 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.

[0067] The 10×Reaction buffer used in the following examples is 10×Hieff Bst Plus DNA Polymerase Buffer, purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 14402ES97.

[0068] The LAMP Fluorescent Dye used in the following examples was purchased from NEB, catalog number B1700S.

[0069] Example 1: Design and Screening of LAMP Primers

[0070] Through extensive and in-depth research, the inventors of this application designed 10 sets of LAMP primers consisting of 6 primers each. Each primer set includes 2 outer primers (F3, B3), 2 inner primers (FIP, BIP), and 2 loop primers (LF, LB). The primer sequences are shown in Table 1.

[0071] Table 1. LAMP primer sequences

[0072]

[0073]

[0074] Example 2: Screening of LAMP primer sets and construction of positive plasmids

[0075] Ten designed LAMP primer sets were used as reaction primers. Fusarium raffinosum DNA (1 μg / ml, 0.1 μg / ml, and 1 ng / ml) was added as templates, ddH2O was used as a control, and 2.5 μL of 10×Reaction buffer, 0.75 μL of 100 mM MgSO4, 1 μL of Hieff Bst Plus DNA Polymerase (40 U / μL), 3.5 μL of 10 mM dNTPs, 4 μL of 10 μM M IP, 4 μL of 10 μM B IP, 1 μL of 10 μM LF, 1 μL of 10 μM LB, 0.5 μL of 10 μM F3, 0.5 μL of 10 μM B3, 0.5 μL of LAMP Fluorescent Dye diluted 10-fold, 2.5 μL of template DNA, and ddH2O was added to bring the total volume to 25 μL. React at 65℃ for 30 min, with fluorescence detected every minute, and then react at 85℃ for 5 min to inactivate.

[0076] The results are as follows Figure 1 As shown. The optimal primers for the LAMP detection system are primer set 1-20. Primer set 1-20 can successfully amplify 1 μg / ml and 0.1 μg / ml Fusarium raffinosum DNA templates, and has a smaller Ct value and a higher maximum fluorescence intensity Rn.

[0077] Based on the specific fragments of the selected LAMP primer sets 1-20, qPCR primers 1-2 (sequences shown in Table 2) were designed using Primer Premier 6 software. Plasmids were synthesized by extending the amplification fragments of the LAMP and qPCR primers 30 bp at both ends. The sequence of the target fragment used for plasmid synthesis is shown in Table 3.

[0078] Table 2. qPCR primer sequences

[0079]

[0080] LAMP plasmids are recombinant vectors obtained by cloning the DNA molecule shown in SEQ ID NO:7 into an E. coli plasmid.

[0081] qPCR plasmids are recombinant vectors obtained by cloning the DNA molecule shown in SEQ ID NO:8 into an E. coli plasmid.

[0082] Example 3: Optimization of the LAMP detection system

[0083] 1. Screening of the optimal reaction temperature for the LAMP system

[0084] The selected primer sets 1-20 were used in a 1×10⁻⁶ ratio. -4 Using μg / ml plasmid (LAMP plasmid) as DNA template and ddH2O as blank control, the reaction was carried out at 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, 66℃ and 67℃ for 30 min, and then inactivated at 85℃ for 5 min. Based on the reaction results, the optimal reaction temperature was determined.

[0085] The results are as follows Figure 2 and Figure 3 As shown, the optimal reaction temperature for the LAMP system is 65℃.

[0086] 2. LAMP system Mg 2+ Concentration screening

[0087] Primers 1-20 were reacted at 65℃ for 30 min, with fluorescence detected every minute, and then inactivated at 85℃ for 5 min. Nine different Mg2+ primers at concentrations of 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, and 10 mM were used. 2+ Concentrations were determined, with each concentration replicated three times to screen for the optimal Mg concentration within the range of 2-10 mM. 2+ Concentration (due to the presence of Mg in the 10×Reaction buffer itself) 2+ Therefore, the minimum initial concentration is 2 mM.

[0088] The results are as follows Figure 4 and Figure 5 As shown. The optimal Mg content in the LAMP system. 2+ The concentration is 3 mM.

[0089] 3. Screening of dNTP concentrations in LAMP systems

[0090] Primer sets 1-20 in 3mM Mg 2+ The reaction was carried out at 65℃ for 30 min, with fluorescence detected every minute, followed by inactivation at 85℃ for 5 min. Six different dNTP concentration gradients were set up: 0.2 mM, 0.6 mM, 1.0 mM, 1.4 mM, 1.8 mM, and 2.2 mM. Each concentration was repeated three times to screen for the optimal dNTP concentration.

[0091] The results are as follows Figure 6 and Figure 7 As shown, the optimal dNTP concentration for the LAMP system is 1.0 mM.

[0092] 4. Screening of betaine concentration in LAMP system

[0093] Primer sets 1-20 in 3mM Mg 2+1.0 mM dNTPs were added, and the reaction was carried out at 65℃ for 30 min, with fluorescence detected every minute. The reaction was then carried out at 85℃ for 5 min to inactivate the betaine. Seven final concentrations of betaine were set: 0 mol / L, 0.2 mol / L, 0.4 mol / L, 0.6 mol / L, 0.8 mol / L, 1.0 mol / L, and 1.2 mol / L. Each concentration was repeated three times to determine the optimal concentration of betaine.

[0094] The results are as follows Figure 8 and Figure 9 As shown, the optimal betaine concentration for the LAMP system is 0.6 M.

[0095] 5. Screening of DMSO concentration in LAMP system

[0096] Primer sets 1-20 in 3mM Mg 2+ 1.0 mM dNTPs, 0.6 M betaine, reaction temperature 65℃ for 30 min, fluorescence detected every minute, inactivated at 85℃ for 5 min. 0%, 2.5%, 7.5%, and 10% DMSO (dimethyl sulfoxide) were added respectively, with each concentration repeated three times to observe the effect of DMSO addition on the reaction.

[0097] The results are as follows Figure 10 and Figure 11 As shown, the optimal DMSO concentration for the LAMP system is 0.

[0098] 6. Screening of BSA concentration in LAMP system

[0099] Primer sets 1-20 in 3mM Mg 2+ 1.0 mM dNTPs, 0.6 M betaine, reaction temperature 65℃ for 30 min, fluorescence detected every minute, inactivated at 85℃ for 5 min. Five different BSA (bovine serum albumin) concentration gradients of 0 μg / ml, 50 μg / ml, 100 μg / ml, 150 μg / ml, and 200 μg / ml were set up, with each concentration performed in triplicate to observe the effect of different concentrations of BSA in the range of 0-200 μg / ml on the reaction.

[0100] The results are as follows Figure 12 and Figure 13 As shown, the addition of BSA has no significant effect on the LAMP system.

[0101] The optimized LAMP reaction system and reaction conditions are as follows:

[0102] The reaction system includes: primer set 1-20, template DNA, reaction buffer, and Mg. 2+The primers consist of Bst DNA polymerase, betaine, and dNTPs. Primer set 1-20 comprises primers 1-F3-20 (SEQ ID NO:1), 1-B3-20 (SEQ ID NO:2), 1-LF-20 (SEQ ID NO:3), 1-LB-20 (SEQ ID NO:4), 1-FIP-20 (SEQ ID NO:5), and 1-BIP-20 (SEQ ID NO:6).

[0103] In the reaction system, the optimal Mg 2+ The optimal concentration was 3 mM; the optimal dNTP concentration was 1.0 mM; the optimal betaine concentration was 0.6 M. The optimal reaction temperature was 65 °C.

[0104] Example 4: Specificity validation of the LAMP detection method for Fusarium racifolium

[0105] Genomic DNA from six Fusarium species—*Fusarium roseum*, *Fusarium improliferatum*, *Fusarium redolens*, *Fusarium solani*, *Fusarium oxysporum*, and *Fusarium cuminatum*—was used as samples, with ddH2O as a blank control. Primers 1-20 were prepared in 3 mM Mg... 2+ 1.0 mM dNTPs, 0.6 M betaine, reaction temperature 65℃ for 30 min, fluorescence detected once per minute, inactivated at 85℃ for 5 min, and amplification results were statistically analyzed.

[0106] The specific reaction system (25 μL) is as follows: 2.5 μL genomic DNA template, 2.5 μL 10× Reaction buffer, 0.75 μL MgSO4 (100 mM), 1 μL Hieff Bst Plus DNA Polymerase (40 U / μL), 3.5 μL dNTPs (10 mM), 4 μL primer 1-FIP-20 (10 μM), 4 μL primer 1-BIP-20 (10 μM), 1 μL primer 1-LF-20 (10 μM), 1 μL primer 1-LB-20 (10 μM), 0.5 μL primer 1-F3-20 (10 μM), 0.5 μL primer 1-B3-20 (10 μM), 0.5 μL LAMP Fluorescent Dye diluted 10-fold, and ddH2O to a final volume of 25 μL.

[0107] The results are as follows Figure 14As shown, only Fusarium argentis showed a positive result, indicating that the LAMP detection method for Fusarium argentis of this invention has good specificity.

[0108] Example 5: Sensitivity verification of the LAMP detection method for Fusarium racifolium

[0109] The synthesized target fragment plasmid (LAMP plasmid in Example 2) was diluted to eight different concentrations: 100 ng / ml, 10 ng / ml, 1 ng / ml, 100 pg / ml, 10 pg / ml, 1 pg / ml, 0.1 pg / ml, and 0.01 pg / ml. Each concentration was diluted three times and mixed thoroughly. This was used as template DNA, with ddH2O as a blank control. Primers 1-20 were prepared in 3 mM Mg... 2+ 1.0 mM dNTPs, 0.6 M betaine, reaction temperature 65℃ for 30 min, fluorescence detected once per minute, inactivated at 85℃ for 5 min, and amplification results were statistically analyzed.

[0110] Copy number (copies / ul) = (6.02 × N) A ×concentration (ng / μL) × 10 -9 ) / (DNA length×660).

[0111] The specific reaction system (25 μL) is the same as the specific reaction system in Example 4, except that the DNA template is replaced with LAMP plasmid templates of different concentrations.

[0112] The results are as follows Figure 15 As shown. The LAMP detection method for Fusarium raffins of the present invention can detect plasmid samples as low as 0.01 pg / ml, exhibiting high sensitivity.

[0113] Example 6: Repeatability Validation of the LAMP Detection Method for Fusarium racifolium

[0114] LAMP plasmids at concentrations of 100 ng / ml, 100 pg / ml, and 0.1 pg / ml were used as high, medium, and low concentrations of template DNA, respectively. ddH2O was used as a blank control. Primers 1-20 were prepared in 3 mM Mg... 2+ 1.0 mM dNTPs, 0.6 M betaine, reaction temperature 65℃ for 30 min, fluorescence detected once per minute, inactivated at 85℃ for 5 min, repeated 6 times for each concentration, according to formula Calculate the coefficient of variation for each concentration to determine the repeatability of the system.

[0115] The specific reaction system (25 μL) is the same as the specific reaction system in Example 4, except that the DNA template is replaced with LAMP plasmid templates of different concentrations.

[0116] The results are as follows Figure 16 As shown in Table 4, the coefficient of variation of the LAMP detection method for Fusarium racifolium of this invention is less than 5%, and the repeatability is good.

[0117] Table 4. Coefficients of variation for LAMP amplification with high, medium, and low concentration plasmids.

[0118]

[0119] Example 7: Comparison of LAMP and qPCR detection methods for Fusarium argentis

[0120] The qPCR specific detection reaction system was as follows: genomic DNA from six Fusarium species (Fusarium moniliforme, Fusarium latifolium, Fusarium aromaticum, Fusarium solani, Fusarium oxysporum, and Fusarium scutellarioides) was used as samples, with ddH2O as a blank control. 10 μL of ArtiCanCEO SYBR qPCR Mix, 0.4 μL of 10 μM upstream primer F1-2, 0.4 μL of 10 μM downstream primer R1-2, 2 μL of sample DNA, and ddH2O was added to a final volume of 20 μL. The qPCR instrument was programmed to react at 95℃ for 10 min, with cycles of 95℃ for 10 s and 60℃ for 30 s, for a total of 40 cycles, followed by the melting curve program.

[0121] The qPCR sensitivity detection reaction system consisted of qPCR plasmids at concentrations of 100 ng / ml, 10 ng / ml, 1 ng / ml, 100 pg / ml, 10 pg / ml, 1 pg / ml, 0.1 pg / ml, and 0.01 pg / ml, respectively. The following were added: 10 μL of ArtiCanCEO SYBRqPCR Mix, 0.4 μL of 10 μM upstream primer F1-2, 0.4 μL of 10 μM downstream primer R1-2, 2 μL of sample DNA, and ddH2O to a final volume of 20 μL. The qPCR instrument was programmed to react at 95℃ for 10 min, with 40 cycles of 95℃ for 10 s and 60℃ for 30 s, followed by the melting curve program.

[0122] The comparison results showed that the qPCR system used as a control could detect plasmid samples as low as 0.1 pg / ml. Figure 17 The qPCR system is specific for the detection of Fusarium raffins. Figure 18 The Ct value of qPCR for a 0.1 pg / ml plasmid sample was 35.15, and the detection time was 33.43 minutes. The Ct value of LAMP detection was 20.02, and the detection time was 20.02 minutes. The detection time was shortened by 40.12% compared with qPCR.

[0123] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

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

6.

2. A kit for detecting Fusarium raffins, characterized in that, The kit contains the primer composition of claim 1.

3. The reagent kit according to claim 2, characterized in that, The kit also includes reaction buffer, Mg 2+ Solution, Bst DNA polymerase, betaine, and dNTPs.

4. The use of the primer composition of claim 1 in the preparation of a product for detecting Fusarium argentis.

5. The use of the primer composition of claim 1 or the kit of claim 2 or 3 in the detection of Fusarium racifolium.

6. A method for detecting Fusarium argentis, characterized in that, The method includes performing LAMP detection on the sample to be tested using the primer composition of claim 1 or the kit of claim 2 or 3.

7. The method according to claim 6, characterized in that, The method includes the following steps: A1) Extract DNA from the sample to be tested; A2) Using the DNA as a template, perform a LAMP reaction using the primer composition of claim 1, or the kit of claim 2 or 3; A3) Determine whether the sample to be tested contains Fusarium argentis based on the LAMP reaction results.

8. The method according to claim 7, characterized in that, The LAMP reaction system includes the primer composition described in claim 1, Mg 2+ Solution, Bst DNA polymerase, betaine, dNTPs, template DNA, and reaction buffer.

9. The method according to claim 8, characterized in that, In the LAMP reaction system, Mg 2+ The concentrations are 2.5-3.5 mM, 2.6-3.4 mM, 2.7-3.3 mM, 2.8-3.2 mM, 2.9-3.1 mM or 3 mM, and / or the betaine concentrations are 0.5-0.7 mM or 0.6 mM, and / or the dNTPs concentrations are 0.7-1.3 mM, 0.8-1.2 mM, 0.9-1.1 mM or 1.0 mM.

10. The method according to claim 8 or 9, characterized in that, The reaction temperature for the LAMP reaction is 65°C.

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