High-throughput screening method for TMV (Tobacco Mosaic Virus) resistant microorganisms and application of high-throughput screening method
By using a high-throughput screening technology system based on 96-well plates, antiviral microorganisms can be rapidly screened, solving the problem of low efficiency in traditional methods. This enables the efficient screening of hundreds of strains, meeting the needs of agricultural disease prevention and control.
Patent Information
- Application Number
- CN202511600468.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-04
- Publication Date
- 2026-02-06
AI Technical Summary
Traditional methods for screening antiviral microorganisms are inefficient, time-consuming, and labor-intensive, making it difficult to quickly screen a large number of strains, and are limited by the amount of tobacco planted.
A high-throughput screening technology system based on 96-well plates was adopted, including the purification of tobacco mosaic virus, activation of test strains or compounds, co-incubation of virus and test substance, and detection of antiviral activity. The virus concentration changes were detected by ELISA, electron microscopy or RT-qPCR, which significantly improved the screening efficiency.
It can quickly and effectively screen hundreds of strains in a short period of time (3-5 days), significantly improving the screening efficiency of antiviral strains and meeting the needs of agricultural disease prevention and control.
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Figure CN121472365A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial application technology, specifically a high-throughput screening method for microorganisms resistant to the Tobacco Mosaic Virus genus and its application. Background Technology
[0002] Microorganisms have been proven to be an important source of natural antiviral agents. Currently, widely used antiviral agents in agriculture, such as ningnanmycin and pyrimethamine, are derived from microbial metabolites. The active ingredient of ningnanmycin is a cytosine nucleoside peptide compound, produced by the metabolism of *Streptomyces noursei* var. *xichangensis*. Ningnanmycin inhibits viral replication and proliferation by directly binding to the capsid protein and helicase of tobacco mosaic virus (TMV), disrupting the viral particle structure and causing the viral particles to break down. Its characteristics include high efficiency, low toxicity, and low residue, achieving up to 50% prevention and treatment effectiveness against TMV. Simultaneously, it can promote the production of trace elements and amino acids in crops, improving crop quality and yield, thus increasing production and income. In addition to ningnanmycin, which is derived from actinomycetes, bacteria and fungi, including Bacillus, Pseudomonas, Agrobacterium, Basidiomycetes, and Ascomycetes, have been found to have antiviral activity, demonstrating the rich diversity of antiviral microorganisms and their promising potential for application and development in biocontrol.
[0003] Efficiently identifying and utilizing antiviral microbial resources, and accelerating their screening and utilization, remains a key technical challenge. Traditional screening methods employ the half-leaf necrotic spot method, where strains and viruses are inoculated onto the heart leaves of tobacco, and the antiviral activity of the strains is assessed based on the number of necrotic spots produced. This method is time-consuming, labor-intensive, and inefficient, with a screening cycle exceeding one month from planting the heart leaves. Furthermore, the number of strains screened in a single batch is limited by the amount of heart leaves planted. This invention establishes a high-throughput screening technology system based on 96-well plates, which can rapidly and effectively screen hundreds of strains in a short time (3-5 days), significantly improving the screening efficiency of antiviral strains. Its applications include high-throughput rapid screening of antiviral microorganisms and antiviral compounds.
[0004] Traditional screening methods for antiviral microorganisms (including compounds) employ the half-leaf necrotic spot method, which involves inoculating the strain and virus onto the heart leaves of tobacco and assessing the antiviral activity of the strain based on the number of necrotic spots produced. This method is time-consuming, labor-intensive, and inefficient, with a screening cycle of more than a month from planting the heart leaves to completing the screening. Furthermore, the number of strains that can be screened in a single batch is limited by the amount of heart leaves planted. Summary of the Invention
[0005] The purpose of this invention is to provide a high-throughput screening method for microorganisms or compounds resistant to Tobacco Mosaic Virus (TMN) viruses and its application, which significantly improves screening efficiency, rapidly identifies microorganisms and compounds with TMN virus activity, and provides a new solution for agricultural disease control.
[0006] The technical solution adopted is:
[0007] A high-throughput screening method for resistance to Tobacco Mosaic Virus (TMV) viruses includes the following steps:
[0008] (1) Purification of Tobacco Mosaic Virus:
[0009] ① Take fresh or frozen infected leaves or fruits, remove the coarse main veins, add twice the volume of PB (pH=7.2), and homogenize thoroughly in a homogenizer.
[0010] ② After filtering the homogenate through three layers of gauze, add 6% (w / v) PEG6000 and 0.1M sodium chloride, stir thoroughly, and then let it stand overnight at 4℃.
[0011] ③ Centrifuge at 4℃, 10000g for 20 minutes, and retain the precipitate.
[0012] ④ Elute with a small amount of PB, centrifuge at 10000g for 20 minutes at 4℃, repeat the elution process several times, retain the supernatant and discard the precipitate.
[0013] ⑤ Combine all supernatants and centrifuge at 100,000g for 3 hours at 4°C, retaining the precipitate and discarding the supernatant.
[0014] ⑥ After suspending the precipitate with a small amount of PB, centrifuge at 10000g for 20 minutes at 4℃. Repeat the suspension several times and mix all the supernatants to make the virus concentration 5-10mg / mL.
[0015] (2) Activation of the test strain: The strain was inoculated on a solid culture medium and incubated upside down at 28°C for 24-48 hours. Then, a single colony was picked and cultured in a liquid culture medium at 28°C and 180 r / min on a constant temperature shaker for 24-48 hours. When the OD600 value was 0.6-1, the co-incubation experiment of bacterial solution and virus was carried out.
[0016] If the analyte is a compound, skip step (2) and directly incubate the analyte with the virus;
[0017] (3) Co-incubation of virus with test strain or test compound: During co-incubation, add liquid culture medium, test bacterial solution or compound and target virus in a ratio of 50:1:1, with a final virus concentration of 0.1 to 0.2 mg / mL;
[0018] The bacterial culture and virus were mixed and incubated on a constant temperature shaker at 28℃ and 180r / min for 0-3 days. The bacterial culture was taken every 24 hours for antiviral activity testing. A blank control was set up: only liquid culture medium was added; a negative control was set up: the target virus was added to the liquid culture medium; and a positive control was set up: the target virus and ningnanmycin were added to the liquid culture medium, with a final concentration of 0.16g / mL.
[0019] (4) Antiviral activity detection: The concentration of virus after co-incubation was detected by ELISA, electron microscopy or RT-qPCR. When the positive control can effectively inhibit the virus and the negative control is ineffective against the virus, the experimental group can be further calculated according to the P-vule value to see if the viral content can be significantly reduced compared with the negative control. This indicates that the microorganism or compound has an inhibitory effect on the virus.
[0020] Preferably, the Tobacco Mosaic Virus (TMV) viruses include, but are not limited to, Tobacco Mosaic Virus (TMV), Cucumber Green Mottle Virus (CGMMV), Tomato Brown Wrinkled Fruit Virus (ToBRFV), and Tomato Mosaic Virus (ToMV).
[0021] Preferably,
[0022] In step (1), the PB buffer solution for purifying tobacco mosaic virus was prepared as follows: 12.168 g of sodium dihydrogen phosphate and 43.69552 g of disodium hydrogen phosphate were weighed and dissolved in sterile water to a total volume of 1 L. After thorough mixing, a PB solution with a pH of 7.0 was obtained. The centrifugation conditions for purifying tobacco mosaic virus were as follows:
[0023] ①Preliminary purification: Centrifuge multiple times at 4℃, 10000g, 20min for 20min to remove precipitate;
[0024] ② Take the supernatant and centrifuge at 4°C, 100,000g for 3 hours;
[0025] ③ The centrifugation temperature should be controlled at around 4℃ throughout the process to avoid virus inactivation.
[0026] Preferably,
[0027] The method for preparing activated strains involves inoculating the strains onto solid culture plates in bacterial NA medium or fungal PDA medium, incubating them upside down at 28°C for 24–48 h, then picking single colonies and incubating them on liquid culture medium, bacterial NB medium, or fungal PDB medium at 28°C and 180 r / min on a constant temperature shaker for 24–48 h until the OD600 value is 0.6–1, and then conducting a co-incubation experiment between the strains and the virus.
[0028] Preferably,
[0029] In the co-incubation experiment, liquid culture medium, test bacterial solution or compound and target virus were added in a ratio of 50:1:1, with a final virus concentration of 0.1-0.2 mg / mL. The mixture was cultured on a constant temperature shaker at 28 ℃ and 180 r / min for 0-3 days, and samples were taken every 24 hours for antiviral activity detection.
[0030] Preferably,
[0031] The antiviral activity of the test strain was detected by ELISA, which was performed by adding 100 μL of samples from three control groups and three experimental groups to a 96-well plate and repeating the ELISA experiment three times.
[0032] The interpretation criteria are as follows: when the positive control can effectively inhibit the virus, while the negative control and blank control are ineffective against the virus, then calculate whether the P-vule value of the experimental group can significantly reduce the viral load compared with the negative control.
[0033] Electron microscopy detection features include: statistical comparison of viral particle count and length; calculation and measurement of viral particle count and length in three repeated fields of view to determine whether there is a significant difference between the experimental group and the negative control; RT-qPCR detection features include: RNA extracted from samples of the three control groups and the experimental group for absolute quantification RT-qPCR experiments; interpretation criteria are as follows:
[0034] When the positive control can effectively inhibit the virus, while the negative control and blank control are ineffective against the virus, the P-vule value of the experimental group is then calculated to determine whether it can significantly reduce the viral RNA content compared with the negative control.
[0035] Secondly, the present invention provides a high-throughput screening method for antiviral microorganisms of the Tobacco Mosaic Virus genus as described in the first aspect, including the screening and application of fungi, bacteria, artificially synthesized or naturally isolated compounds.
[0036] Compared with existing technologies, the advantages of this invention are: it establishes a high-throughput screening technology system based on 96-well plates, which can rapidly and effectively screen hundreds of strains in a short time (3-5 days), significantly improving the screening efficiency of antiviral strains. This is of great significance for accelerating the screening and application of antiviral microbial resources. Attached Figure Description
[0037] Figure 1 This is a negative staining image of the purified brown wrinkled fruit virus of the present invention;
[0038] Figure 2 The results of the enzyme-linked immunosorbent assay (ELISA) for screening antiviral microorganisms according to the present invention;
[0039] Figure 3This is an electron micrograph showing the effect of co-incubating the antiviral strain of the present invention with a virus;
[0040] Figure 4 This is a graph showing the RT-qPCR detection results of the strains screened in this invention after co-incubation with the virus;
[0041] Figure 5 The results of the enzyme-linked immunosorbent assay (ELISA) for screening antiviral compounds of the present invention are shown. Detailed Implementation
[0042] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0043] This high-throughput screening method mainly includes the following steps:
[0044] (1) Purification of Tobacco Mosaic Virus; (2) Activation of the test strain; (3) Co-incubation of the virus with the test strain (or compound); (4) Detection of the antiviral activity of the test strain;
[0045] In step (1), tobacco mosaic virus is separated and purified using ultracentrifugation:
[0046] ① Take fresh or frozen diseased leaves or fruits, remove the coarse main veins, and add twice the volume of PB (pH=7.2) to a homogenizer to homogenize thoroughly.
[0047] ② After filtering the homogenate through three layers of gauze, add 6% (W / V) PEG6000 and 0.1M sodium chloride, stir thoroughly, and then let it stand overnight at 4℃.
[0048] ③ Centrifuge at 10000g for 20 minutes at 4℃ and retain the precipitate.
[0049] ④ Elute with a small amount of PB, centrifuge at 10000g for 20 minutes at 4℃, repeat the elution process multiple times, retain the supernatant and discard the precipitate.
[0050] ⑤ Combine all supernatants and centrifuge at high speed (4℃, 100000g for 3h) to retain the precipitate and discard the supernatant.
[0051] ⑥ After suspending the precipitate with a small amount of PB, centrifuge at 10000g for 20 minutes at 4℃. Repeat the suspension several times and mix all the supernatants to make the virus concentration 5-10mg / mL.
[0052] In step (2), the test strain is activated: the strain is inoculated onto a solid culture medium (bacterial NA medium, fungal PDA medium), incubated upside down at 28°C for 24-48 hours, and then a single colony is picked and incubated in a liquid culture medium (bacterial NB medium, fungal PDB medium) at 28°C and 180 r / min on a constant temperature shaker for 24-48 hours until its OD600 value is 0.6-1. Then, the bacterial solution is used for co-incubation with the virus. If the test substance is a compound, step (2) is skipped and the test compound is directly co-incubated with the virus.
[0053] In step (3), the virus is co-incubated with the test strain (or test compound): During co-incubation, liquid culture medium, test bacterial solution (or compound), and target virus (final virus concentration 0.1–0.2 mg / mL) are added at a ratio of 50:1:1. The bacterial solution and virus are mixed and incubated on a constant temperature shaker at 28°C and 180 r / min for 0–3 days, and the bacterial solution is taken every 24 hours for antiviral activity detection. At the same time, a blank control is set up: only liquid culture medium is added; a negative control: target virus is added to the liquid culture medium; a positive control: target virus and ningnanmycin (final concentration 0.16 g / mL) are added to the liquid culture medium.
[0054] In step (4), antiviral activity was detected using ELISA, electron microscopy, or RT-qPCR to determine the viral concentration after co-incubation. Interpretation criteria: When the positive control effectively inhibited the virus, while the negative control was ineffective, the P-value was used to further calculate whether the experimental group significantly reduced the viral load compared to the negative control.
[0055] Example 1
[0056] Purification of Tomato Brown Wrinkle Fruit Virus:
[0057] ① Take fresh or frozen diseased leaves or fruits, remove the coarse main veins, and add twice the volume of PB (pH=7.2) to a homogenizer to homogenize thoroughly.
[0058] ② After filtering the homogenate through three layers of gauze, add 6% (W / V) PEG6000 and 0.1M sodium chloride, stir thoroughly, and then let it stand overnight at 4℃.
[0059] ③ Centrifuge at 4℃, 10000g for 20 minutes, and retain the precipitate.
[0060] ④ Elute with a small amount of PB, centrifuge at 10000g for 20 minutes at 4℃, repeat the elution process multiple times, retain the supernatant and discard the precipitate.
[0061] ⑤ Combine all supernatants and centrifuge at high speed (4℃, 100000g for 3h) to retain the precipitate and discard the supernatant.
[0062] ⑥ After suspending the precipitate with a small amount of PB, centrifuge at 10000g for 20 minutes at 4℃. Repeat the suspension several times and store all supernatants at -20℃. Discard the precipitate (to keep the virus concentration at 5-10 mg / mL).
[0063] The virus concentration was calculated using the ultraviolet spectrophotometer reading at 280 nm and the Lambert-Beer law formula C=A / (ε×L). As shown in Figure 1, after purifying the tomato brown wrinkle virus, negative staining was used for electron microscopy observation of the virus. It was found that the purified virus had a clean background, no impurities, and the virus particles had an intact structure, approximately 300 nm in length and 18 nm in diameter.
[0064] Example 2
[0065] Activation of test strain
[0066] Several bacterial strains preserved in glycerol at -80℃ were selected and inoculated onto NA solid medium plates. After incubation at 28℃ for 24 hours, single colonies were picked and cultured in 5 mL NB liquid medium on a constant temperature shaker at 28℃ and 180 r / min for 24 hours. When the OD600 value was 0.6-1, the bacterial culture was used for co-incubation with the virus.
[0067] In Example 2, the NA solid culture medium consisted of the following components: 10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, 15 g / L agar, diluted to 1 L of distilled water, with a pH of 7.3 ± 0.1, and sterilized at 121 °C for 15 min.
[0068] like Figure 2-4 As shown:
[0069] Example 3
[0070] Co-incubation experiment of antiviral microorganisms and viruses
[0071] Three control groups were set up as follows:
[0072] Blank control: 5 mL of NB liquid culture medium;
[0073] Negative control: Add 100 μL of purified brown wrinkled fruit virus (final virus concentration 0.1-0.2 mg / mL) to 5 mL of NB liquid culture medium.
[0074] Positive control: 100 μL of purified brown wrinkled fruit virus and ningnanmycin (final concentration 0.16 g / mL) were added to 5 mL of NB liquid culture medium.
[0075] The experimental group was prepared by adding 100 μL of purified brown wrinkled fruit virus and 100 μL of activated test bacterial solution to 5 mL of NB liquid culture medium.
[0076] Each group had three replicates. The samples were cultured on a constant temperature shaker at 28 ℃ and 180 r / min for 0–3 days. Samples were taken every 24 hours and frozen at -80℃. After all samples were collected, the samples were tested for antiviral activity.
[0077] Example 4
[0078] Co-incubation experiment of antiviral compounds with viruses:
[0079] Compound A was isolated and purified from NA25. The original solution of compound A was diluted 5 times to obtain 6 concentration gradients: 1X, 5X, 25X, 125X, 625X, and 3125X.
[0080] Four control groups were set up: blank control: 1 mL of sterile water; negative control: 1 mL of sterile water with 20 μL of purified brown wrinkled fruit virus (final virus concentration 0.1–0.2 mg / mL); positive control: 1 mL of sterile water with 20 μL of purified brown wrinkled fruit virus and 200 μL of ningnanmycin (final concentration 0.16 g / mL); experimental group: 1 mL of sterile water with 20 μL of purified brown wrinkled fruit virus and 200 μL of compound A at different gradient concentrations.
[0081] In the co-incubation experiment, each group was repeated in triplicate. Samples were incubated on a constant-temperature shaker at 28 ℃ and 180 r / min for 0–3 days. Samples were collected every 24 hours and frozen at -80℃. After all samples were collected, their antiviral activity was detected. ELISA results after co-incubation are shown below. Figure 5 As shown in the figure, the results indicate that the antiviral activity of compound A decreases with increasing concentration, and the original solution at concentration 1X exhibits the greatest antiviral activity.
[0082] Example 5
[0083] like Figure 2 As shown, the ELISA assay for antiviral microbial (or compound) activity follows these steps:
[0084] Add blank, negative, positive, and experimental control samples to the ELISA plate (avoid touching the well walls). Each sample must be on the same plate for 0-3 days. Each plate must include both positive and negative samples. Set up 3 replicates, 100 μL / well, incubate at 37℃ for 3 hours or 4℃ overnight. Wash with PBST and drain (vertically invert) using a fast 3-times-slow 3-times-3-times cycle (slow 3 times, 3 minutes per wash). Wash once more in the side wells.
[0085] Add 5% skim milk powder or 2% bovine serum albumin in PBS×1 blocking buffer, 200uL / well, incubate at 37℃ for 30min, drain without washing the plate.
[0086] Add polyclonal antibody (mouse polyclonal antibody) at a ratio of 1:5000 (1 is mouse polyclonal antibody, 5000 is PBS×1), 100 μL / well, incubate at 37℃ / 3h or 4℃ / overnight, wash with PBST.
[0087] Add enzyme-labeled secondary antibody (mouse secondary antibody) at a ratio of 1:5000 (1 is mouse polyclonal antibody, 5000 is PBS×1), 100 μL / well, incubate at 37℃ / 3h or 4℃ / overnight, and wash with PBST.
[0088] Add 100 μL of substrate development solution per well and incubate at room temperature for 15–30 minutes to develop the color (protect from light after adding the development solution).
[0089] PBS solution formula: NaCl 40g, Na2HPO4∙12H2O 7.2g, KH2PO4 1g, KCl 1g, add 1L of distilled water to make PBS×5, and finally add distilled water to dilute to make PBS×1.
[0090] PBST solution formulation: PBS + Tween (1L PBS × 1 + 0.5mL Tween).
[0091] ELISA results of antiviral microorganisms showed that co-incubation of microorganisms in experimental group 1 with ToBRFV significantly reduced viral load, while experimental groups 2, 3, 4, and 5 did not show significant viral inhibition compared to the positive control, as shown in Tables 1 and 2 below.
[0092] Table 1 ELISA test results
[0093]
[0094] Table 2 Sample loading sequence
[0095]
[0096] Example 6
[0097] Electron microscopic observation of antiviral microbial (or compound) activity
[0098] The suspension of the target antagonistic virus strain A within 0-3 days of incubation is diluted to an appropriate concentration with buffer (such as PBS). Prepare a clean electron microscope screen, avoiding touching the membrane surface when handling it with tweezers to prevent contamination. Use a pipette to add 5-10 μL of the sample suspension to the supporting membrane surface of the screen, and let it stand at room temperature for 2 minutes to allow the sample to fully adhere to the membrane. Gently touch the edge of the screen (not the membrane surface) with the edge of clean filter paper to aspirate excess sample solution (leaving only a thin film on the membrane surface). Immediately add 5-10 μL of negative staining solution (1%–2% sodium phosphotungstenate solution) to the sample-adsorbed surface of the screen, and stain at room temperature for 1 minute and 30 seconds. Gently aspirate excess negative staining solution again with filter paper, then place the screen on a clean petri dish or filter paper and place it under an infrared lamp to accelerate drying, avoiding direct sunlight. The dried screen can be directly placed on the electron microscope stage for observation.
[0099] The experimental control group for 0-3 days was subjected to counterstaining electron microscopy. The results were further evaluated by observing the number of virus particles and the number of virus fragments. Electron microscopy showed that the target antagonistic virus strain A had a significant inhibitory effect on virus particles within 0-3 days of treatment: firstly, the number of virus particles was effectively reduced, and secondly, more virus particles were fragmented. This preliminarily verified the high antiviral activity of the strain.
[0100] Example 7
[0101] RT-qPCR detection of antiviral microbial activity (or test compound)
[0102] Absolute quantitative RT-qPCR was performed on the blank, negative, positive, and experimental control samples after co-incubation experiments. RNA was extracted from each sample and then subjected to reverse transcription and qPCR detection. For qPCR detection, a plasmid containing the ToBRFV CP gene sequence was constructed. The plasmid was serially diluted to establish a standard curve, and the viral load was calculated based on the Ct value of the target sample. The results are shown below. Figure 4 As shown, both the test strain and the positive control were able to significantly identify the viral load compared to the negative control.
[0103] The above-described embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application, and should all be included within the protection scope of this invention.
Claims
1. A high-throughput screening method for microorganisms resistant to Tobacco Mosaic Virus (TMV), characterized in that... Specifically, it includes the following steps: Tobacco mosaic virus purification: ① Take fresh or frozen infected leaves or fruits, remove the coarse main veins, add twice the volume of PB (pH=7.2), and homogenize thoroughly in a homogenizer. ② After filtering the homogenate through three layers of gauze, add 6% (w / v) PEG6000 and 0.1M sodium chloride, stir thoroughly, and then let it stand overnight at 4℃. ③ Centrifuge at 4℃, 10000g for 20 minutes, and retain the precipitate. ④ Elute with a small amount of PB, centrifuge at 10000g for 20 minutes at 4℃, repeat the elution process several times, retain the supernatant and discard the precipitate. ⑤ Combine all supernatants and centrifuge at 100,000g for 3 hours at 4°C, retaining the precipitate and discarding the supernatant. ⑥ After suspending the precipitate with a small amount of PB, centrifuge at 10000g for 20 minutes at 4℃. Repeat the suspension several times and mix all the supernatants to make the virus concentration 5-10mg / mL. Activation of the test strain: The strain was inoculated on a solid culture medium and incubated upside down at 28°C for 24-48 hours. Then, a single colony was picked and incubated in a liquid culture medium at 28°C and 180 r / min on a constant temperature shaker for 24-48 hours. When the OD600 value was 0.6-1, the bacterial solution and virus co-incubation experiment was carried out. If the analyte is a compound, skip step (2) and directly incubate the analyte with the virus; Co-incubation of the virus with the test strain or test compound: During co-incubation, add liquid culture medium, test bacterial solution or compound and target virus at a ratio of 50:1:1, with a final virus concentration of 0.1–0.2 mg / mL; The bacterial culture and virus were mixed and incubated on a constant temperature shaker at 28℃ and 180r / min for 0-3 days. The bacterial culture was taken every 24 hours for antiviral activity testing. A blank control was set up: only liquid culture medium was added; a negative control was set up: the target virus was added to the liquid culture medium; and a positive control was set up: the target virus and ningnanmycin were added to the liquid culture medium, with a final concentration of 0.16g / mL. (4) Antiviral activity detection: The concentration of virus after co-incubation was detected by ELISA, electron microscopy or RT-qPCR. When the positive control can effectively inhibit the virus and the negative control is ineffective against the virus, the experimental group can be further calculated according to the P-vule value to see if the viral content can be significantly reduced compared with the negative control. This indicates that the microorganism or compound has an inhibitory effect on the virus.
2. The high-throughput screening method for antiviral microorganisms of the Tobacco Mosaic Virus genus according to claim 1, characterized in that, The viruses of the Tobacco Mosaic Virus genus include, but are not limited to, Tobacco Mosaic Virus. TMV Cucumber green mottle virus CGMMV Tomato brown wrinkled fruit virus ToBRFV Tomato mosaic virus ToMV .
3. The high-throughput screening method for antiviral microorganisms of the Tobacco Mosaic Virus genus according to claim 1, characterized in that: In step (1), the PB buffer solution for purifying tobacco mosaic virus was prepared as follows: 12.168 g of sodium dihydrogen phosphate and 43.69552 g of disodium hydrogen phosphate were weighed and dissolved in sterile water to a total volume of 1 L. After thorough mixing, a PB solution with a pH of 7.0 was obtained. The centrifugation conditions for purifying tobacco mosaic virus were as follows: ①Preliminary purification: Centrifuge multiple times at 4℃, 10000g, 20min for 20min to remove precipitate; ② Take the supernatant and centrifuge at 4°C, 100,000g for 3 hours; ③ The centrifugation temperature should be controlled at around 4℃ throughout the process to avoid virus inactivation.
4. The high-throughput screening method for antiviral microorganisms of the Tobacco Mosaic Virus genus according to claim 1, characterized in that, The method for preparing activated strains involves inoculating the strains onto solid culture plates in bacterial NA medium or fungal PDA medium, incubating them upside down at 28°C for 24–48 h, then picking single colonies and incubating them on liquid culture medium, bacterial NB medium, or fungal PDB medium at 28°C and 180 r / min on a constant temperature shaker for 24–48 h until the OD600 value is 0.6–1, and then conducting a co-incubation experiment between the strains and the virus.
5. The high-throughput screening method for antiviral microorganisms of the Tobacco Mosaic Virus genus according to claim 1, characterized in that, In the co-incubation experiment, liquid culture medium, test bacterial solution or compound and target virus were added in a ratio of 50:1:1, with a final virus concentration of 0.1-0.2 mg / mL. The mixture was cultured on a constant temperature shaker at 28 ℃ and 180 r / min for 0-3 days, and samples were taken every 24 hours for antiviral activity detection.
6. The high-throughput screening method for antiviral microorganisms of the Tobacco Mosaic Virus genus according to claim 1, characterized in that, The antiviral activity of the test strain was detected by ELISA, which was performed by adding 100 μL of samples from three control groups and three experimental groups to a 96-well plate and repeating the ELISA experiment three times. The interpretation criteria are as follows: when the positive control can effectively inhibit the virus, while the negative control and blank control are ineffective against the virus, then calculate whether the P-vule value of the experimental group can significantly reduce the viral load compared with the negative control. Electron microscopy detection features include: statistical comparison of viral particle count and length; calculation and measurement of viral particle count and length in three repeated fields of view to determine whether there is a significant difference between the experimental group and the negative control; RT-qPCR detection features include: RNA extracted from samples of the three control groups and the experimental group for absolute quantification RT-qPCR experiments; interpretation criteria are as follows: When the positive control can effectively inhibit the virus, while the negative control and blank control are ineffective against the virus, the P-vule value of the experimental group is then calculated to determine whether it can significantly reduce the viral RNA content compared with the negative control.
7. A high-throughput screening method for antiviral microorganisms of the Tobacco Mosaic Virus genus as described in claim 1, characterized in that... It is applicable to screening of fungi, bacteria, synthetic compounds and naturally isolated compounds.