Rapid enrichment and detection method of pathogens in soil

Abstract

The invention relates to a rapid enrichment and detection method for pathogens in soil, which comprises the following steps: uniformly mixing a soil sample with a gelatin solution, and standing the uniformly mixed soil solution; after standing is finished, a filter membrane is adopted for filtering supernate, the filter membrane is taken out after filtering is finished, and pathogen microorganisms are enriched on the obtained filter membrane, namely a pathogen enriched sample; performing low-temperature freezing on the filter membrane, taking out the filter membrane, immediately crushing, adding the crushed pathogen enrichment sample into a nucleic acid extraction buffer solution, and performing freeze thawing, so that cells are further crushed, thereby obtaining sample supernate; successively adding a magnetic bead solution and a sample supernatant, fully and uniformly mixing, and extracting pathogen nucleic acid by a magnetic bead method; and taking the extracted pathogen nucleic acid as a template, and carrying out nucleic acid detection by adopting an LAMP method. According to the method, rapid enrichment of pathogen cells in the soil without depending on complex instruments and equipment is realized by utilizing a gelatin-microporous filter membrane combination, and is combined with LAMP detection, so that the detection efficiency and sensitivity of soil-borne pathogens are improved, and the method can be used for prevention and control of agricultural diseases and has good practical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial enrichment and detection, and in particular to a method for rapid enrichment of pathogens in soil and detection thereof. Background Art

[0002] The facility planting of horticultural crops has improved the utilization rate of land, enriched the "vegetable basket" around people, and increased the economic benefits of agricultural land per unit. However, due to the single crop variety, high planting density, and high multiple cropping index in the production process of facility horticultural crops, soil-borne diseases, mainly wilt, often occur, resulting in huge economic losses.

[0003] Soil-borne pathogens often lurk in the soil, cultivation substrates, and weeds or diseased crop residues in the field, and spread and spread through the transportation of infected seedlings, irrigation of contaminated water sources, and agricultural operations. Pathogens invade plants from the root system or stem base, and there are usually no obvious symptoms in the early stages, but after symptoms become apparent, there is no effective control method. Therefore, the guiding principle for the prevention and control of soil-borne diseases has always been "prevention is better than cure", among which pathogen detection is an important part of disease prediction and prevention and control.

[0004] Soil-borne diseases have always restricted the stable and sustainable production of crops, often leading to serious agricultural economic losses, and the current production lacks corresponding rapid pathogen detection technology and early warning methods. Therefore, the development of accurate, sensitive and efficient detection methods for potential target pathogens in soil samples has become an important topic in current agricultural science research. Traditionally, the detection of soil-borne disease pathogens relies on culture methods, that is, the microorganisms in the soil are separated and cultured through specific culture media for identification. However, there are many types of soil microorganisms, and many pathogens are difficult to separate and identify under laboratory conditions in a short time. In recent years, with the rapid development of molecular biology technology, nucleic acid-based detection methods, such as polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP), have shown great potential in soil-borne disease detection. These methods have the advantages of high sensitivity, strong specificity and fast detection speed, but the premise is that high-quality nucleic acid templates need to be efficiently extracted from the soil.

[0005] However, the soil matrix is ​​complex and rich in organic matter, minerals and microbial residues. These components easily interfere with the purity and integrity of nucleic acids during nucleic acid extraction, resulting in low extraction efficiency and insufficient sensitivity. In order to solve the above problems, commercial soil DNA extraction kits are usually used to extract the DNA of soil microorganisms to complete the study of fungi and bacteria in the soil. However, due to factors such as the high price of the kit and the unclear composition of the reagents, more and more studies are devoted to designing a low-cost DNA extraction method. Subsequently, a scheme for extracting DNA from subsoil using phosphate lysis buffer and a rapid method for extracting high-quality DNA from soil for the preparation of metagenomic libraries were developed. However, these extraction methods have cumbersome operating steps and are not easy to use and study in practice.

[0006] Therefore, developing a rapid, efficient and highly sensitive method for extracting nucleic acid DNA of pathogens in soil is of great significance for improving the detection efficiency and accuracy of soil-borne diseases. Summary of the invention

[0007] In order to overcome at least one of the problems existing in the prior art: (1) although the previous methods for extracting DNA from soil can extract microbial DNA from the soil, they often require cumbersome experimental steps; (2) the experimental operations used in the existing detection methods also mostly rely on large-scale experimental equipment provided by the laboratory environment, and cannot be used to process soil samples under outdoor conditions; (3) the amount of samples that can be processed by the previous extraction methods is too small to achieve large-scale processing of samples. The present invention combines gelatin pretreatment with filter membrane enrichment technology to achieve rapid enrichment of Fusarium oxysporum and extraction of nucleic acid DNA, and uses the loop-mediated isothermal amplification (LAMP) method to achieve identification of pathogens. The above detection method is not only easy to use under the premise of limited experimental costs, but also improves the efficiency and sensitivity of detection.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] The first aspect of the present invention is to provide a method for enriching pathogens in soil, which comprises the steps of:

[0010] S1. Mixing a soil sample and a gelatin solution in a predetermined ratio, and leaving the mixed soil solution to stand for a predetermined time;

[0011] S2. After the standing is completed, the supernatant is taken out and filtered using a filter membrane. After the filtration is completed, the filter membrane is taken out; wherein, the filter membrane is enriched with pathogenic microorganisms, that is, the pathogen-enriched sample.

[0012] Furthermore, in step S1, the mass volume ratio of the soil sample to the gelatin solution is 1:3-15 g / mL; preferably, the mass volume ratio is 1:4-10 g / mL, and more preferably, the mass volume ratio is 1:5 g / mL. Within the above mass volume ratio range, the soil sample and the gelatin solution can achieve the sedimentation of soil particles to a certain extent after mixing, and the microbial cells can be suspended in the gelatin solution as much as possible; in a specific embodiment of the present invention, the soil sample and the gelatin solution with a mass volume ratio of 1:5 g / mL are actually verified.

[0013] Furthermore, in step S1, the concentration of the gelatin solution is 0.1-3 w / v%; preferably, the concentration is 0.2-0.3 w / v%; more preferably, the concentration is 0.25 w / v%.

[0014] In a specific embodiment, in step S1, the mass volume ratio of the soil sample to the gelatin solution is 1:5 g / mL, and the concentration of the gelatin solution is 0.25 w / v%.

[0015] Furthermore, in step S1, the preparation step of the gelatin solution includes: weighing a predetermined amount of gelatin solid particles and mixing them with ultrapure water; melting the gelatin particles at a predetermined temperature to obtain a gelatin solution mother liquor of a predetermined concentration; and diluting the gelatin solution mother liquor with ultrapure water to prepare a gelatin solution of a predetermined concentration. It is understood that the preparation of the above gelatin solution can also adopt conventional preparation methods in the art.

[0016] Furthermore, the predetermined temperature is 60-70° C., preferably 65° C., and the constant temperature mode can be achieved by conventional means in the art, such as water bath heating.

[0017] In a specific embodiment, 0.3 g of gelatin solid particles are weighed and mixed with 10 mL of DDW (deuterium depleted water, ultrapure water), and the gelatin particles are melted at 65°C to obtain a 3% (w / v) gelatin solution mother liquor, which is then diluted with DDW to prepare a gelatin solution of a certain concentration (e.g., 0.25 w / v%, 0.1 w / v%, etc.). It is understood that the ratio and amount of gelatin solid particles to ultrapure water can be adaptively adjusted according to the different concentrations and volumes of the gelatin solution mother liquor to be prepared.

[0018] Furthermore, in step S1, the mixing operation is performed by conventional means in the art, for example, in the laboratory stage, vortex shaking is used for 5 seconds for mixing.

[0019] Furthermore, in step S1, the standing time of the soil solution is 5 to 40 minutes; preferably, the standing time is 15 to 35 minutes, more preferably, the standing time is 10 to 30 minutes; most preferably, the standing time is 20 to 30 minutes, for example, the standing time is 20 minutes, 22 minutes, 25 minutes, 28 minutes, or 30 minutes.

[0020] Further, in step S2, the filter membrane is a polyethersulfone microporous filter membrane, and the filtration is performed by a suction filtration device; preferably, the pore size of the polyethersulfone microporous filter membrane is 0.45 μm, and its diameter can be adjusted according to the amount of supernatant to be treated, for example, the diameter can be 25 mm, 65 mm, 70 mm, etc. It is understandable that the suction filtration device can be a conventional device in the art, for example, a Buchner funnel can be used in the laboratory stage, and its size is adjusted according to the diameter of the polyethersulfone microporous filter membrane.

[0021] Further, the pathogen includes bacteria and / or fungi; preferably, the pathogen includes Fusarium spp. and Verticillium dahliae, and more preferably, the pathogen is Fusarium oxysporum.

[0022] The second aspect of the present invention is to provide a method for extracting pathogen nucleic acid in soil, which comprises the steps of:

[0023] S31. Obtain a pathogen-enriched sample using any of the enrichment methods described in the first aspect;

[0024] S32, freezing the filter membrane containing the pathogen-enriched sample at low temperature, taking it out and crushing it, adding the crushed pathogen-enriched sample to a nucleic acid extraction buffer solution and performing freeze-thawing to further break the cells and obtain a sample supernatant;

[0025] S33, successively add the magnetic bead solution and the sample supernatant obtained in step S32, mix thoroughly, and extract the pathogen nucleic acid by the magnetic bead method.

[0026] Furthermore, the step S32 specifically includes the steps of: carefully transferring the filter membrane into a container, freezing it at a low temperature (-10°C or below, for example, -15°C, -20°C, -40°C, -60°C, -80°C, etc.), taking it out and immediately grinding it into powder, adding a nucleic acid extraction buffer solution (comprising Tris-HCl, NaCl, EDTA, SDS) and freezing and thawing it three times to further disrupt the cells and obtain a sample supernatant.

[0027] Furthermore, the step S33 specifically includes the steps of: taking another container, adding the magnetic bead solution and the sample supernatant obtained after freezing and thawing in a predetermined ratio (preferably a volume ratio of 6 to 15:5, more preferably a volume ratio of 8 to 12:5, and more preferably a volume ratio of 9:5), immediately mixing thoroughly, and completing the subsequent nucleic acid extraction step according to the instructions of the commercial magnetic beads (Novozyme Biotech Co., Ltd., catalog number N411-01).

[0028] The third aspect of the present invention is to provide a method for detecting pathogens in soil, which comprises the steps of: using the pathogen nucleic acid obtained by any extraction method described in the second aspect as a template, and adopting a specific method to perform nucleic acid detection.

[0029] Furthermore, the nucleic acid detection method is any suitable molecular biological detection method, including LAMP detection method, PCR amplification method, high-throughput sequencing and analysis, etc.

[0030] Furthermore, the nucleic acid detection method is a LAMP detection method, and the sensitivity of the detection method is 5×10 2 spores / g soil sample.

[0031] Further, in a specific embodiment, in the LAMP detection method, the LAMP reaction enzyme, reaction buffer, detection primers and reaction conditions required can all refer to the inventor's prior research: Sheng Deng et al. LAMP Assay for Distinguishing Fusarium oxysporum and Fusarium commune in Lotus (Nelumbonucifera) Rhizomes. Plant Dis. 2022 Jan; 106 (1): 231-246. It is understandable that the reaction system, detection primers and reaction conditions of the above-mentioned LAMP detection method can be optimized and designed according to conventional methods.

[0032] The fourth aspect of the present invention is to provide the application of the method for enriching pathogens in soil as described in any one of the first aspects, the application includes: application in the extraction and detection of pathogen nucleic acids in soil, and application in the prevention and control of crop diseases; wherein the nucleic acid extraction method refers to any one of the second aspects, and the nucleic acid detection method refers to any one of the third aspects. In a specific embodiment, the crop disease is tomato wilt caused by Fusarium oxysporum.

[0033] It is understandable that, based on the characteristics of pathogenic fungi and bacteria present in the soil, it can be reasonably inferred that, in addition to the Fusarium oxysporum verified in the embodiment, most of the other plant pathogenic fungi and bacteria present in the soil can be pretreated with gelatin and enriched by the filter membrane by the method of the present invention. Subsequently, it is only necessary to extract nucleic acid from the filter membrane enriched with pathogens, and then amplify it with the detection primers corresponding to the potential pathogens to achieve its detection. The above detection method is not limited to the LAMP detection method.

[0034] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0035] The present invention takes the detection of the important soil-borne pathogenic microorganism Fusarium oxysporum as the starting point, constructs a rapid enrichment, nucleic acid DNA extraction and detection technology system for crop pathogens in soil, and specifically adopts the loop-mediated isothermal amplification (LAMP) method to perform nucleic acid detection of pathogens.

[0036] The present invention firstly screens four kinds of microporous membrane materials and determines that PES polyethersulfone microporous membrane is most suitable for establishing the rapid fungal cell enrichment and nucleic acid extraction steps; then, through concentration gradient experiments, it is clarified that the best solution for the rapid separation of Fusarium oxysporum from sand and gravel in the soil is to mix the soil with 0.25% (w / v) gelatin solution and let it stand for 20-30 minutes; further, in artificially inoculated soil samples containing a specified conidia concentration, the 0.25% (w / v) gelatin pretreatment is integrated with the PES polyethersulfone microporous membrane enrichment technology, achieving a 10 2 spores / 0.2g soil sample (i.e. 5×10 2 spores / g soil sample); finally, relevant soil and plant samples were collected in the field where tomato wilt occurred to verify the simplified soil pathogen rapid detection method constructed by the present invention.

[0037] The pathogen enrichment and extraction detection method of the present invention does not require high-value instruments and equipment. The constructed detection method can quickly detect target pathogens in a large number of soil samples under field or simple laboratory conditions. It only takes about 3 hours from soil sample pretreatment to obtaining reaction results. The above detection method can provide accurate, efficient, sensitive and low-cost soil or cultivation matrix pathogen detection technical support and reference for grassroots plant protection departments and related service companies, and can also be further used for the prevention and control of crop diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0039] Figure 1Schematic diagram of LAMP test results of microporous filter membranes of four different materials in one embodiment of the present invention; wherein, part A represents PES polyethersulfone microporous filter membrane; part B represents CA-CN water-based mixed fiber microporous filter membrane; part C represents CA water-based cellulose acetate membrane; part D represents PP polypropylene microporous filter membrane, and the experiments were repeated twice;

[0040] Figure 2 It is a schematic diagram of the actual results of the membrane enrichment effect of different concentrations of gelatin solution mixed with soil and then subjected to static or centrifugal treatment in one embodiment of the present invention; wherein, part A is mixed; part B is static for 5 minutes; part C is static for 10 minutes; part D is static for 20 minutes; part E is static for 30 minutes; part F is static for 40 minutes; part G is centrifuged for 5 seconds;

[0041] Figure 3 It is a bar graph of the number of fluorescent spores at the same position in the supernatant after gelatin solutions of different concentrations are mixed with contaminated soil and then subjected to static or centrifugal treatment in one embodiment of the present invention.

[0042] Figure 4 The present invention is a microscope photo of fluorescent spores in which contaminated soil is treated with a 0.25 w / v% gelatin solution for different time periods;

[0043] Figure 5 is a graph showing the results of LAMP detection in soil contaminated by Fusarium oxysporum using a gelatin-membrane enrichment method in one embodiment of the present invention; wherein Negative: negative control, Positive: positive control, 10 2 :The concentration of spore solution is 1×10 2 / mL, the obtained filter membrane sample, 10 3 :The concentration of spore solution is 1×10 3 / mL, the obtained filter membrane sample; 10 4 :The concentration of spore solution is 1×10 4 The filter membrane sample obtained under the condition of 100 / mL; H2O: The filter membrane sample obtained under the condition of water treatment;

[0044] Figure 6 Schematic diagram of sampling and detection results of tomato plants in one embodiment of the present invention; wherein, part A shows the disease situation of tomatoes in a greenhouse; part B shows the sample tissues of healthy and diseased tomato plants taken from the greenhouse; part C shows the results of pathogen isolation of diseased tomato plant tissue samples and NCBI comparison of sequencing results; part D shows the LAMP detection results of tomato tissue samples; S1 to S6 are diseased tomato samples, and H1 to H3 are healthy tomato samples;

[0045] Figure 7It is a schematic diagram of the LAMP test results of tomato soil in one embodiment of the present invention; wherein, Part A is the LAMP test results of diseased tomato greenhouse soil, Negative: negative control, Positive: positive control, A1~A3 represent soil samples taken from the seedling root position, seedling spacing position and tomato seedling planting aisle position of tomato seedlings, and so on; Part B is the LAMP test results of healthy tomato greenhouse soil, Negative: negative control, Positive: positive control, D1 and D2 represent soil samples taken from the seedling spacing position and tomato seedling planting aisle position, and so on.

[0046] Figure 8 The present invention is an experimental flow chart of a rapid enrichment, nucleic acid DNA extraction and detection technology system for crop pathogens in soil in one embodiment of the present invention. DETAILED DESCRIPTION

[0047] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source indicated in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0048] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0049] In the following examples, the test strains used: Fusarium oxysporum LYG 9-1 strain (isolated from a tomato plant collected in Lianyungang, Jiangsu) and its green fluorescent protein (GFP) labeled strain were used in this study. The strains were stored in a low temperature refrigerator for a long time and activated with potato culture medium (PDB) or potato agar medium (PDA) before use.

[0050] In the following examples, the collection of test soil and plant materials: (1) Preparation of sterilized soil: Soil samples were collected from the corn test field of Jiangsu Academy of Agricultural Sciences in Nanjing, Jiangsu. (2) For soil containing suspected soil-borne pathogens, soil samples were collected from a plantation suspected of tomato wilt disease in Hushu Street, Jiangning District, Nanjing. The sampling rule for plant samples is as follows: two tomato seedlings were randomly selected from the diseased greenhouse and the healthy greenhouse, and the root base was taken as tomato tissue samples. The sampling rule for soil samples is as follows: according to the planting area of ​​the greenhouse, the greenhouse is divided into three parts, A, B, and C, in equal proportion. In each part, three holes were drilled at the root position of the tomato seedlings, the seedling spacing position, and the planting aisle position of the tomato seedlings using a soil sampler for sampling. The drilling depth of the soil sampler is about 20 cm. The soil samples taken from each position were mixed and put into a sampling bag as soil samples.

[0051] In the following examples, the main reagents used were: gelatin (McLean Biochemical Technology Co., Ltd.), nucleic acid extraction buffer solution (20 mM Tris [pH 8.0], 25 mM NaCl, 50 mM EDTA, 0.05% SDS), 80% ethanol, and magnetic beads (Novozyme Biotech Co., Ltd., catalog number N411-01).

[0052] In the following embodiments, the main instruments and tools used are: 1.5mL centrifuge tubes, 5mL centrifuge tubes, high-speed tissue grinders, vortexers, magnetic racks, small filters, microporous filter membranes (diameters of 25mm and 70mm respectively (can be adjusted or repaired according to the inner diameter of the funnel), pore size of 0.45μm, produced by Haining Delv New Material Technology Co., Ltd., corresponding to different materials and diameters, the product numbers are PESLM-25 / 70, XHCALM-25 / 70, PPLM-25 / 70, MECLM-25 / 70), Buchner funnel, 20mL syringe needle, glass jar with a volume of 200mL, soil sampler, and fluorescence microscope (Zeiss Axioscope 5).

[0053] In certain specific embodiments of the present invention, a method for rapid enrichment and detection of pathogens in soil is provided, which is as follows: Figure 8 As shown, the method comprises the following steps: (1) enriching pathogens in soil to obtain a pathogen-enriched sample; (2) extracting pathogen nucleic acid from the pathogen-enriched sample by a magnetic bead method; (3) using the obtained pathogen nucleic acid as a template and performing nucleic acid detection by a LAMP method; wherein the pathogens include bacteria and / or fungi, such as Fusarium and Verticillium, and specifically may be Fusarium oxysporum.

[0054] In certain preferred embodiments, the method for enriching pathogens in soil in step (1) comprises the steps of: mixing a soil sample with a gelatin solution (concentration of 0.1 to 3 w / v%) at a mass volume ratio of 1:3 to 15 g / mL, and allowing the mixed soil solution to stand for 5 to 40 minutes; after the standing period is completed, taking out the supernatant, and filtering the supernatant with a polyethersulfone microporous filter membrane, and after the filtration is completed, taking out the filter membrane, and the obtained filter membrane is the pathogen enrichment sample.

[0055] In certain preferred embodiments, the method for extracting pathogen nucleic acids from soil in step (2) comprises the steps of: freezing the pathogen enriched sample (filter membrane) at low temperature (-10°C or below), taking it out and immediately crushing it, adding the crushed pathogen enriched sample to a nucleic acid extraction buffer solution for freeze-thawing to further break the cells, and obtaining a sample supernatant; adding a magnetic bead solution and a sample supernatant in a volume ratio of 6 to 15:5, mixing them immediately and thoroughly, and extracting the pathogen nucleic acid by a magnetic bead method.

[0056] In certain preferred embodiments, in step (3), in the LAMP detection method, the LAMP reaction enzyme, reaction buffer, detection primers and conditions required for the reaction can be referred to the paper published by Deng et al. (2022) (https: / / doi.org / 10.1094 / PDIS-06-21-1223-RE).

[0057] The present invention is illustrated by way of examples below.

[0058] Example 1 - Microporous membrane selection and screening

[0059] In this embodiment, four common microporous filter membranes are selected to screen filter membranes suitable for enrichment of soil pathogens. The types and characteristics of the four microporous filter membranes are shown in Table 1 below:

[0060] Table 1- Microporous membrane materials and their functions

[0061]

[0062] The experimental steps used in this example include: (1) thawing a sample of a liquid cryopreservation tube of Fusarium oxysporum LYG 9-1 strain stored in a refrigerator in the laboratory and activating the sample in a PDA medium. After mycelium grows, the mycelium is picked up and transferred to a PDB medium, and the mixture is cultured in a shaking incubator at 28°C and 180 rpm for 3-5 days to collect conidia; (2) the conidia are diluted with sterile water to the following concentration: 1×10 2 , 1×10 3 , 1×10 4spores / mL. Take 5mL (or 10mL) of the above three different concentrations of spore solution with a 20mL needleless syringe and pass it through the above four different materials and 25mm diameter filter membranes; (3) Carefully transfer the filter membrane to a 1.5mL sterile centrifuge tube, put it into a low-temperature refrigerator, take it out and immediately grind it with a pestle until it is crushed, then add nucleic acid extraction buffer solution and mix well, freeze and thaw three times to further break the cells and obtain the supernatant after freezing and thawing; (4) Take a new 1.5mL centrifuge tube, add 36μL of magnetic bead solution and 20μL of supernatant after freezing and thawing, mix well immediately, and complete the subsequent nucleic acid extraction steps according to the magnetic bead instructions; (5) The extracted DNA is used for subsequent LAMP detection (refer to Sheng Deng et al. LAMP Assay for Distinguishing Fusarium oxysporum and Fusariumcommune in Lotus (Nelumbo nucifera) Rhizomes. Plant Dis. 2022 Jan; 106(1): 231-246.), LAMP detection was performed with a 25 μL reaction system to obtain the final detection results.

[0063] In this embodiment, the DNA reaction template in the solution is obtained by using the above-mentioned membrane filtration method. The conidia solutions of different dosages and concentrations are filtered by the membrane. The nucleic acid DNA of the conidia intercepted by the membrane is extracted and used as the template for the LAMP detection reaction. The detection results of the four different materials of the membrane used for the test are shown in FIG. Figure 1 As shown in the figure, the PES polyethersulfone microporous membrane is relatively stable and can detect 5mL and 10mL of conidia suspension (concentration of 1×10 4 spores / mL), while other filter membranes could not obtain detection results at this concentration and dosage. Therefore, this example determined that PES polyethersulfone microporous filter membrane is preferred as the filter membrane material used for pathogen enrichment.

[0064] Example 2 - Gelatin solution preparation and screening method

[0065] In order to ensure the enrichment effect of the filter membrane, based on the adsorption and affinity of gelatin to cells, this example verifies the adsorption of different concentrations of gelatin solutions on Fusarium oxysporum spores in the soil and their suspension effects on the spores, thereby screening out the optimal gelatin solution concentration and its operating parameters. The above verification experiment specifically includes the following steps:

[0066] (1) Preparation of gelatin solution: Weigh 0.3 g of gelatin solid particles and place them into a 15 mL centrifuge tube filled with 10 mL DDW. Place the centrifuge tube in a 65°C water bath to melt the gelatin particles to obtain a 3% (w / v) gelatin solution mother liquor. Use this mother liquor to prepare gelatin solutions with concentrations of 0.1%, 0.25%, 0.5%, 1%, 1.5%, and 2%.

[0067] (2) First, by observing the clarity of the solution under different treatments, the degree of separation of soil and spores in the solution was simply judged: water and 0.1%, 0.25%, 0.5%, 1%, 1.5%, 2% and 3% gelatin solutions were mixed evenly with soil at a ratio of 1:10 (g / mL), and then allowed to stand for 0 min (after mixing), 5 min, 10 min, 20 min, 30 min and 40 min, or centrifuged for 5 s, and then the stratification of the soil mixture in the centrifuge tube was observed. The experimental results are as follows: Figure 2 As shown in the figure, the gelatin suspension of the soil-gelatin mixed solution gradually became clear as the standing time increased, indicating that most of the soil particles had settled; the suspension of the soil-water mixture also gradually became clear as the standing time increased, but the clarification speed was very slow, indicating that the solution still contained a lot of soil particles. The suspension of the soil gelatin solution or soil-water mixture after centrifugation was relatively clear, but due to the centrifugal force, most of the conidia were likely to gather at the bottom of the centrifuge tube together with the soil particles, which was not conducive to the separation of microbial cells and soil particles.

[0068] (3) Subsequently, in order to determine whether the spore concentration in the supernatant at the same location changed after the static treatment, an experiment with different concentrations of gelatin solution and a range of static time was conducted: 0.2 g of soil, 100 μL of 1×10 8spores / mL of GFP-labeled conidia of Fusarium oxysporum (LYG 9-1 strain), 900 μL of the specified concentration was added to the gelatin solution, inverted several times, and vortexed for 5 seconds to mix. Seven groups of experiments were set up, and the centrifuge tube was placed on the centrifuge rack to stand (standing for 0 minutes (after mixing), 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes) or centrifuged (centrifuged at 4000 rpm for 5 seconds). These two treatment methods were used to determine the number of fluorescent spores in the supernatant. The specific operation is as follows: After standing, 16 μL of the supernatant solution was taken and placed under a microscope, and the fluorescent spores were observed with a 10× microscope and photographed and recorded. After the solution was vortexed again, it was immediately placed on a small centrifuge for centrifugation for 5 seconds. After sampling at the same position, the fluorescent spores were observed with a 10× microscope and photographed and recorded. After the photos were taken, three areas in the field of view were randomly selected (each area was 2.2 mm*1.6 mm in size) and the number of spores was counted using Image J software. Specifically, the gelatin solutions containing GFP fluorescently labeled conidia with different treatments were observed under a fluorescence microscope, and the fluorescent spores observed under a 10× microscope were photographed and counted using Image J software. The above statistical results are shown in Tables 2 and Figure 3 .

[0069] Table 2 - Number of fluorescent spores in three random areas under different solutions

[0070]

[0071] From Table 2 and Figure 3 The experimental results show that during the filtration operation, it was found that the gelatin solution with a concentration of more than 0.25% had a high concentration. Even after centrifugation or long-term standing, fine soil particles were still suspended in the solution, resulting in the inability of the filter to pass a large volume of solution during processing (the larger the volume of filtration, the more potential pathogen cells are enriched), affecting the enrichment effect of the filter membrane. The above experimental results show that in gelatin solutions of various concentrations, the number of conidia is the highest just after mixing and when it is left standing for 5 minutes. With the extension of the standing time, the number of conidia in the gelatin solution gradually decreases, among which the aqueous solution and 0.1% gelatin solution decrease the most. When the standing time exceeds 20 minutes, the number of conidia in the solution shows a negative growth trend with the extension of time. However, during the 5-40 minutes of standing of gelatin solutions with a concentration of 0.25% and above, the number of conidia did not decrease significantly, and basically remained in the range of 200-350. For gelatin solutions with a concentration below 0.25%, although centrifugation helps to separate soil from conidia, the amount of conidia in the supernatant is too low due to the centrifugal effect, with the number being around 50.

[0072] (4) Finally, considering the uncontrollable factors such as the large amount of soil and the instability of experimental time in the subsequent actual application in the soil field, further experimental attempts were made on the 0.25% gelatin solution with a relatively stable spore concentration after standing for a long time. Figure 4 The following are microscope photos of fluorescent spores treated with 0.25% gelatin solution for different time periods. Figure 3 and Figure 4 As shown in the results, it is obvious that after the standing time reaches 20 minutes, the number of fluorescent spores is still relatively stable. It can be seen that the standing time range can be selected after 20 minutes. In order to optimize the experimental efficiency, the final choice is to stand for 20 to 30 minutes.

[0073] Therefore, the above experimental results show that when enriching pathogens, the concentration of the gelatin solution is preferably 0.25% (w / v), and after mixing with the pathogen sample, it is preferably left to stand for 20 to 30 minutes.

[0074] Example 3 - Gelatin-microporous membrane combination test

[0075] Based on the experimental results of Example 1 and Example 2, in order to explore whether the mixed solution can smoothly pass through the filter membrane to achieve the effect of enriching spores under different treatments, this example reconfigured the above-mentioned soil gelatin mixed solution, absorbed the supernatant of the solution under different treatments, and filtered it using the filter membrane to verify the effect of the gelatin-microporous filter membrane combination. The specific experimental steps include:

[0076] According to the final selected filter membrane material (Example 1) and gelatin solution concentration (Example 2), this example combined them for a combined experiment, and added 0.2 g sterilized soil, 100 μL target pathogen spore liquid (Fusarium oxysporum LYG 9-1 strain (spore liquid concentrations were 1×10 3 , 1×10 4 , 1×10 5 spores / mL) and 900 μL gelatin solution to obtain soil preparation solutions containing different conidia concentrations (1×10 2 , 1×10 3 , 1×10 4spores / mL). At the same time, a solution obtained by mixing 0.2 g sterilized soil, 100 μL sterilized water and 900 μL gelatin solution was used as the experimental control group. After standing for 20 to 30 minutes (specifically 30 minutes), the supernatant was filtered with a polyethersulfone microporous filter membrane (diameter 25 mm) with a pore size of 0.45 μm; after the filtration, the filter membrane was placed in a 1.5 mL centrifuge tube, frozen in a -80 ° C refrigerator, taken out and quickly crushed with a grinding pestle, 200 μL of nucleic acid extraction buffer solution was added, and the mixture was frozen and thawed repeatedly for 3 times, and then the nucleic acid was extracted by the magnetic bead method described in Example 1 to prepare a subsequent LAMP detection reaction template; the extracted DNA was used for subsequent LAMP detection (refer to Sheng Deng et al. LAMP Assay for Distinguishing Fusarium oxysporum and Fusariumcommunein Lotus (Nelumbo nucifera) Rhizomes. Plant Dis. 2022 Jan; 106 (1): 231-246.), and the final detection result of the LAMP detection was obtained with a 25 μL reaction system.

[0077] The above test results are as follows Figure 5 As shown in the figure, it can be clearly observed that according to the above optimized gelatin-microporous filter membrane combination method, the detection concentration limit of soil-borne disease genome in soil has successfully reached 1×10 2 This significant progress not only improves the detection efficiency, but also simplifies the reaction template preparation steps, thus bringing great convenience and benefits to current research work.

[0078] Through the above test results, the optimal process conditions of the pathogen enrichment method of gelatin-microporous filter membrane combination were finally determined as follows: the soil sample and gelatin solution were mixed at a ratio of 1:5g / mL, and after standing for 20 to 30 minutes, a 0.45μm polyethersulfone microporous filter membrane was used to filter to obtain a pathogen enriched sample (i.e., a filter membrane for enriching spores). The extraction steps of the above pathogen enriched sample include: freezing the filter membrane and then quickly breaking it, adding nucleic acid extraction buffer, and repeatedly freezing and thawing for 3 times to obtain a sample supernatant; mixing the magnetic bead solution and the sample supernatant, and extracting the nucleic acid sample using the magnetic bead method as a template for the LAMP reaction.

[0079] Example 4 - Verification of collected field soil samples using gelatin-microporous membrane combination method

[0080] To verify the effectiveness of the gelatin-microporous membrane combination method, this example was conducted on a tomato plantation suspected of being infected by Fusarium oxysporum in Hushu Street, Jiangning District, Nanjing ( Figure 6As shown in Part A of the present invention, plant and soil sampling was carried out: for plant samples, this embodiment uses three methods to identify pathogens, (1) traditional culture plate separation method; (2) DNA extraction of diseased tissue combined with loop-mediated isothermal amplification technology (LAMP) for rapid molecular detection; (3) soil gelatin-microporous filter combination DNA extraction combined with LAMP for rapid molecular detection; among them, LAMP detection refers to Sheng Deng et al. LAMP Assay for Distinguishing Fusarium oxysporum and Fusariium commune in Lotus (Nelumbo nucifera) Rhizomes. Plant Dis.. 2022 Jan; 106 (1): 231-246. The above experimental results are shown in the figure. Figure 6 and Figure 7 shown.

[0081] The experimental steps of the soil gelatin-microporous filter membrane combination DNA extraction combined with LAMP detection method include: referring to Example 3, the proportion of soil gelatin is proportionally enlarged, 30g of soil is mixed with 150mL of gelatin solution, put into a glass jar and mix well, and then stand for 20-30min; after the soil solution is allowed to stand, cover the bottle mouth with sterilized gauze, pour out 20-30mL of supernatant into a new glass jar, and then transfer 15mL of supernatant to a Buchner funnel filtration device for filtration. After the filtration is completed, the filter membrane (diameter 65mm) is taken out, divided into four equal parts, and respectively loaded into 1.5mL centrifuge tubes, frozen in a -80℃ refrigerator, taken out and quickly crushed with a grinding pestle, 200μL of nucleic acid extraction buffer solution is added, and repeated freezing and thawing 3 times, and then the magnetic bead method described in Example 1 is referred to extract nucleic acid to prepare a subsequent LAMP detection reaction template; the extracted DNA is used for subsequent LAMP detection, and the LAMP detection obtains the final detection result with a 25μL reaction system.

[0082] like Figure 6 As shown in Part B of the conventional identification method (conventional culture plate separation method), the cross section of the diseased plant was observed, and the vascular tissue was found to have obvious browning phenomenon; the surface of the diseased tissue was cleaned, then disinfected with 10% sodium hypochlorite solution, and placed on a potato dextrose agar (PDA) plate and cultured at 28°C for 3 days, and white hyphae growth was observed ( Figure 6 A trace amount of DNA was extracted from the growing mycelium for PCR amplification of the transcribed spacer region of the genome, and the amplified product was sequenced and compared, and it was determined to be Fusarium oxysporum.

[0083] like Figure 6As shown in Part D, for the diseased tissue DNA extraction combined with the LAMP detection method, the vascular infected tissue was taken, and the DNA of a small amount of tissue was quickly obtained by freeze grinding and magnetic bead method, and then used as a template for LAMP detection. The test results showed that all samples from diseased plants showed positive reaction to Fusarium oxysporum.

[0084] like Figure 7 As shown, for the soil gelatin-microporous filter combination DNA extraction combined with LAMP detection method, the collected soil samples were processed according to the gelatin-filter method determined in Example 3 and then subjected to LAMP detection. The experimental results showed that the Fusarium oxysporum test in the diseased tomato soil was positive, while the root soil of healthy tomatoes was negative. The above experimental results further verified the effectiveness of the experimental method.

[0085] As can be seen from the above examples, the present invention successfully improves the detection efficiency and sensitivity of soil-borne diseases by constructing a rapid enrichment, nucleic acid DNA extraction and detection technology system for crop pathogens in soil. The present invention first screened out PES polyethersulfone microporous filter membrane as a material for cell enrichment and nucleic acid extraction, and determined through a concentration gradient test that the best solution for the rapid separation of Fusarium oxysporum from sand and gravel in the soil is to mix the soil with 0.25% (w / v) gelatin solution and let it stand for 20 to 30 minutes. Subsequently, the 0.25% (w / v) gelatin pretreatment was combined with the PES polyethersulfone microporous filter membrane enrichment method to achieve 10 2 The detection sensitivity is 10 spores / 0.2 g soil sample. In addition, the present invention also verifies the feasibility of the above detection method for rapid detection of target pathogens in a large number of soil samples under field or simple laboratory conditions.

[0086] The present invention utilizes the adsorption and affinity of gelatin to cells, and configures a gelatin solution of corresponding concentration to achieve the effect of adsorption of Fusarium oxysporum spores in the soil and suspension of gelatin molecules in the solution after the spores are combined, and the mixed solution after standing by filtering with a filter membrane is enriched to achieve spores. At the same time, by combining with LAMP detection, rapid detection of soil pathogens is achieved. In the above detection method, the experimental materials used are low in price, and the early soil treatment method is relatively simple and easy to operate.

[0087] In summary, the present invention not only improves the detection efficiency and sensitivity of soil-borne diseases by optimizing the enrichment step of pathogens in soil and the rapid preparation step of nucleic acid DNA, but also contributes new strength to the innovative development of agricultural disease prevention and control technology. It is expected to be promoted and applied in a wider range of agricultural production fields.

[0088] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A method for enriching pathogens in soil, characterized in that: The method for enriching pathogens in soil comprises the following steps: S1. Mixing a soil sample and a gelatin solution in a predetermined ratio, and leaving the mixed soil solution to stand for a predetermined time; S2. After the standing is completed, the supernatant is taken out, and the supernatant is filtered using a filter membrane. After the filtration is completed, the filter membrane is taken out; wherein the pathogenic microorganisms are enriched on the filter membrane, that is, the pathogen-enriched sample.

2. The method for enriching pathogens in soil according to claim 1, characterized in that: In the step S1, the mass volume ratio of the soil sample to the gelatin solution is 1:3-15 g / mL, and the concentration of the gelatin solution is 0.1-3 w / v%; and / or, in the step S1, the standing time of the soil solution is 5-40 min; And / or, in step S2, the filter membrane is a polyethersulfone microporous filter membrane, and the filtration is performed using a suction filtration device.

3. The method for enriching pathogens in soil according to claim 2, characterized in that: In the step S1, the mass volume ratio of the soil sample to the gelatin solution is 1:5 g / mL, and the concentration of the gelatin solution is 0.25 w / v%; And / or, in step S1, the soil solution is allowed to stand for 20 to 30 minutes.

4. The method for enriching pathogens in soil according to claim 1, characterized in that: In step S1, the preparation steps of the gelatin solution include: weighing a predetermined amount of gelatin solid particles and mixing them with ultrapure water; melting the gelatin particles at a predetermined temperature to obtain a gelatin solution mother liquor of a predetermined concentration; and diluting the gelatin solution mother liquor with ultrapure water to prepare a gelatin solution of a predetermined concentration.

5. The method for enriching pathogens in soil according to claim 1, characterized in that: The pathogens include bacteria and / or fungi.

6. The method for enriching pathogens in soil according to claim 5, characterized in that: The pathogen is Fusarium oxysporum.

7. A method for extracting pathogen nucleic acid in soil, characterized in that: Includes steps: S31. Obtaining a pathogen-enriched sample by using the enrichment method described in any one of claims 1 to 6; S32, freezing the filter membrane containing the pathogen-enriched sample at low temperature, taking it out and crushing it, adding the crushed pathogen-enriched sample to a nucleic acid extraction buffer solution and performing freeze-thawing to further break the cells and obtain a sample supernatant; S33, successively add the magnetic bead solution and the sample supernatant obtained in step S32, mix thoroughly, and extract the pathogen nucleic acid by the magnetic bead method.

8. A method for detecting pathogens in soil, characterized in that: The method comprises the following steps: using the pathogen nucleic acid obtained by the extraction method according to claim 7 as a template and adopting a specific method to perform nucleic acid detection.

9. The method for detecting pathogens in soil according to claim 8, characterized in that: The nucleic acid detection method is a LAMP detection method, and the sensitivity of the detection method is 5×10 2 spores / g soil sample.

10. An application of the method for enriching pathogens in soil as claimed in any one of claims 1 to 6, characterized in that: The applications include: extraction and detection of pathogen nucleic acids in soil, and prevention and treatment of crop diseases.

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