Optimization method for microscopic counting of free viruses in farmland soil

Abstract

The present invention discloses an optimization method for microscopic counting of free viruses in farmland soil, which belongs to the field of biological test technology. The method conducts experiments from several aspects, including instrument selection, parameter setting, soil sample usage, and key influencing factors of microscopic observation in soil virus extraction experiments, analyzes the effects of the ratio of farmland soil samples to virus extracts, different oscillating instruments and parameter settings, and anti-fluorescence quenchers and anti-fading solutions on soil virus extraction efficiency and microscopic counting in microscopic observation, and optimizes the process of microscopic counting of free viruses in farmland soil based on the analysis results. The present invention can start from the instrument selection and parameter setting, soil sample usage, and key influencing factors in microscopic observation in soil virus extraction, and propose an operating procedure suitable for fluorescent staining-microscopic counting of farmland soil viruses, which can provide a feasible technical reference for subsequent quantitative analysis of soil viruses and research on infection modes.

Description

Technical Field

[0001] The invention belongs to the technical field of biological experiments, and in particular relates to an optimization method for microscopic counting of free viruses in farmland soil. Background Art

[0002] Viruses are the most abundant and diverse biological entities on Earth. Composed of a protein shell and nucleic acids, they are intracellular parasites and can infect all organisms in the three domains of the biological kingdom (Bacteria, Archaea, and Eukarya). As key drivers of the survival of soil microorganisms, viruses play a vital role in regulating the soil ecological functions in which their host microorganisms participate. When host cells are infected by viruses, changes occur in the physiological metabolism, gene expression, and death rates of soil microorganisms, leading to changes in microbial numbers, population composition, and function. Simultaneously, large amounts of organic matter released by host cells become effective substrates and energy sources for the cycling of elements such as carbon and nitrogen in the soil habitat, ultimately affecting the soil's material cycle and energy flow.

[0003] With the rapid development of sequencing technology, genetically-based soil virus research has been continuously reported. However, due to the high mutation rate of viruses, it is difficult to design universal primers, which in turn limits the quantitative observation and detection of viral populations in soil. Although transmission electron microscopy (TEM) can quantify viral populations, the small detection area, high cost, and complex preparations restrict soil virus enumeration studies. Therefore, staining soil viral nucleic acids with fluorescent dyes and counting them with fluorescence microscopy have become the main method for quantitative soil virus analysis.

[0004] To date, numerous reports have been published domestically and internationally on the use of fluorescent staining and microscopic counting to analyze viral populations in the environment, with preliminary experimental protocols established. However, most of these methods have been used to study viruses in marine ecosystems. Research methods for studying viruses in agricultural soils often vary widely among researchers and have limited applicability, leading to a lack of further validation and evaluation of the results. Summary of the Invention

[0005] The present invention aims to solve one of the technical problems in the above-mentioned related art at least to a certain extent.

[0006] To this end, the purpose of the present invention is to provide an optimized method for microscopic counting of free viruses in farmland soil. Starting from the instrument selection and parameter setting in soil virus extraction, the amount of soil sample used, and the control of key influencing factors in microscopic observation, by studying the ratio of farmland soil sample to virus extract, different oscillation instruments and parameter settings, and the effects of anti-fluorescence quenching reagents in microscopic observation on soil virus extraction efficiency and microscopic counting, an operational process suitable for the fluorescent staining-microscopic counting method of farmland soil viruses is proposed, which can provide a feasible technical reference for subsequent quantitative analysis of soil viruses and infection mode research.

[0007] In order to solve the above-mentioned technical problems, the present invention is achieved as follows:

[0008] Embodiments of the present invention provide a method for optimizing the microscopic counting of free viruses in farmland soil. The method conducts experiments on instrument selection, parameter settings, soil sample usage, and key influencing factors of microscopic observation in soil virus extraction experiments. The method analyzes the effects of the ratio of farmland soil sample to virus extract, different oscillating instruments and parameter settings, and anti-fluorescence quenchers and anti-fading solutions in microscopic observation on soil virus extraction efficiency and microscopic counting. The process of microscopic counting of free viruses in farmland soil is optimized based on the analysis results.

[0009] In addition, the optimization method for microscopic counting of free viruses in farmland soil according to the present invention may also have the following additional technical features:

[0010] In some embodiments, the ratio of the farmland soil sample to the virus extract is 100 parts by volume of the virus extract with no less than 50 parts by mass of the farmland soil sample added.

[0011] In some embodiments, the oscillation instrument and parameters are set as follows: using a handheld stirring rod to oscillate at 650W and 50Hz for 3 minutes to break up the soil suspension.

[0012] In some embodiments, the anti-fluorescence quenching agent is a water-soluble anti-fluorescence quenching sealing agent.

[0013] In some embodiments, the anti-fading agent is prepared by mixing anhydrous ethanol and (S)-(-)-α-methylbenzylamine in a volume ratio of 1000:1 to form an anti-fading stock solution, mixing the anti-fading stock solution and glycerol-phosphate buffer in a volume ratio of 100:1, and filtering through a microporous filter membrane to obtain the anti-fading agent.

[0014] In some embodiments, the virus extract is a potassium citrate phosphate buffer solution with a pH of 7.0.

[0015] In some embodiments, the potassium citrate phosphate buffer is prepared by dissolving 1.44 parts of disodium hydrogen phosphate, 0.24 parts of dipotassium hydrogen phosphate, and 10.0 parts of potassium citrate in HPLC water, adding phosphoric acid to adjust the pH to 7.0 and then making up the volume, filtering the prepared buffer through a microporous filter membrane, and then sterilizing to obtain the potassium citrate phosphate buffer.

[0016] In some embodiments, the process of microscopically counting free viruses in farmland soil optimized according to the analysis results includes:

[0017] S1. Weigh 50 g of farmland soil sample and place it in 100 mL of 1% potassium citrate solution. Stir with a handheld stirring rod for 3 min at 650 W and 50 Hz.

[0018] S2, centrifuging the soil extract obtained in S1;

[0019] S3, obtaining the supernatant after centrifugation and filtering it through a pinhole filter membrane;

[0020] S4, adding the optimized solution and DNase1 to the filtered solution, and then performing a water bath reaction at a specific temperature;

[0021] S5, adding EDTA solution to the solution after water bath in S4, and then conducting water bath reaction at a specific temperature;

[0022] S6, cooling to room temperature;

[0023] S7, filter pump treatment: Place a filter membrane on the filter pump, add HPLC water for suction filtration, balance the filter pump pressure, then add the S6 solution on the filter membrane for suction filtration, balance the filter pump pressure;

[0024] S8. Add nucleic acid fluorescent dye to the filter membrane and stain in the dark; then filter, balance the filter pump pressure, and rinse with sterile HPLC water;

[0025] S9. Take out the filter membrane and dry it in the dark;

[0026] S10, add sterile HPLC water to the slide, place a dried filter membrane on it, add anti-fluorescence quenching agent to the filter membrane, and cover with a coverslip;

[0027] S11. Use a fluorescence microscope to observe and count free viruses.

[0028] In some embodiments, the temperature of the water bath reaction in step S4 is 37°C;

[0029] The temperature of the water bath reaction in step S5 is 65°C.

[0030] In some embodiments, the pinhole filter membrane in step S3 is a 0.22 μm pinhole filter membrane;

[0031] The filter membrane on the filter pump in step S7 is a 0.02 μm filter membrane.

[0032] Compared with the prior art, the present invention has at least the following beneficial effects:

[0033] In an embodiment of the present invention, an optimized method for microscopically counting free viruses in farmland soil is provided. The method of using a handheld stirring rod to disrupt the soil suspension can effectively increase the number of soil viruses extracted. Compared with the traditional disruption method, the handheld stirring rod method of the present invention can increase the number of soil viruses by at least one order of magnitude.

[0034] In an embodiment of the present invention, an optimized method for microscopic counting of free viruses in farmland soil is provided. By increasing the ratio of soil sample to extract, the virus count results are significantly improved. Compared with the traditional soil to extract ratio of 30:100 (30 parts by mass of soil are added to every 100 parts by volume of extract), the abundance of viruses per gram of soil can be significantly increased by an order of magnitude when the fresh soil content is increased, that is, the soil to extract ratio is 50:100 (50 parts by mass of soil are added to every 100 parts by volume of extract).

[0035] In an embodiment of the present invention, an optimized method for microscopic counting of free viruses in farmland soil is provided. By using an anti-fluorescence quencher and / or an anti-fading liquid, the soil virus observation time is effectively extended, and the number of virus detections is increased. When the anti-fading liquid and the anti-fluorescence quencher are not used, the virus is quenched in a short time. When the magnification is 1000 times, the presence of soil virus particles cannot be observed in the field of view. The use of both anti-fading liquid and anti-fluorescence quencher can effectively prevent fluorescence quenching, but the number of soil virus particles after using the anti-fluorescence quencher can be one order of magnitude higher than that after using the anti-fading agent.

[0036] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a flow chart of an optimization method for microscopic counting of free viruses in farmland soil disclosed in one embodiment of the present invention;

[0038] Figure 2 The microscopic observation results of soil viruses under different disruption methods disclosed in one embodiment of the present invention are shown; A is a vortex mixer; B is a water bath shaker; C is an ultrasonic cell disruptor; and D is a handheld stirring rod.

[0039] Figure 3The following are the microscopic observation results of soil viruses under different soil-liquid ratios disclosed in one embodiment of the present invention; wherein, A is a soil-liquid ratio of 30:100 (meaning: 30 parts by mass of soil are added to every 100 parts by volume of the extract); B is a soil-liquid ratio of 50:100 (meaning: 50 parts by mass of soil are added to every 100 parts by volume of the extract);

[0040] Figure 4 This is the microscopic observation result of soil viruses without using an anti-fluorescence quencher disclosed in one embodiment of the present invention; wherein A is a 100-fold magnification; B is a 1000-fold magnification;

[0041] Figure 5 The present invention discloses microscopic observation results of soil viruses using an anti-fading agent and an anti-fluorescence quenching agent, wherein A is an anti-fading agent and B is an anti-fluorescence quenching agent. DETAILED DESCRIPTION

[0042] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0043] The embodiments of the present invention are described in detail below through specific embodiments and application scenarios with reference to the accompanying drawings.

[0044] In some embodiments of the present invention, a method for optimizing the microscopic counting of free viruses in farmland soil is provided. This method analyzes the influence of various factors on the microscopic counting of free viruses in soil from multiple aspects, and combines various influencing factors to obtain a new microscopic counting method, which achieves good counting results.

[0045] 1 Materials and Methods

[0046] 1.1 Test materials and instruments

[0047] The test soils were soil from the campus of Shenyang Agricultural University, soil with long-term fertilization, and soil from farmland around Shenyang City. After collection, the soils were stored at -80℃ for future use.

[0048] Experimental consumables and reagents: glass slides (1-1.2 mm, 25.4 mm × 76.2 mm); cover slips (Nr. 1.0, 25 mm × 25 mm); Anodisc 25 filter membrane (0.02 μm pore size, 25 mm diameter; Cat. 6809-6002, Cytiva, USA); 0.22 μm pore size syringe filter (Cat. SLGV033RB, Millipore, USA); HPLC water (Cat. W820536, MacLean, China); molecular-grade tris (Cat. ZZ02531, NOVON, UK); SYBR Green I nucleic acid dye (10000×) (Cat.SY1020, Solebao, China); Fluoromount-G anti-fluorescence fading mounting medium (Cat.36307ES08, Yeasen, China); cedar oil (Cas.8000-27-9, Hualing, China).

[0049] The potassium citrate phosphate buffer solution was prepared as follows: 1.44 g disodium hydrogen phosphate, 0.24 g dipotassium hydrogen phosphate, and 10.0 g potassium citrate were dissolved in HPLC water, the pH was adjusted to 7.0 with phosphoric acid, and the volume was made up to 1.0 L. The prepared buffer solution was passed through a 0.1 μm microporous filter membrane, sterilized at 121°C for 30 minutes, cooled, and stored at 4.0°C for later use.

[0050] The anti-fading agent was prepared by mixing anhydrous ethanol and (S)-(-)-α-methylbenzylamine in a ratio of 1000:1 to prepare an anti-fading stock solution. 100.0 ml of the anti-fading stock solution was mixed evenly with 1.0 ml of the glycerol-phosphate buffer stock solution, filtered through a 0.1 μm microporous membrane, and stored at room temperature for later use.

[0051] The main instruments included a 100% electronic balance (CP224S, Sartorius, Germany), a vortex mixer (Vortex-Genie 2, Scientific Industries, USA); a reciprocating water bath constant temperature shaker (ZWY-110X50, Zhicheng, China), an ultrasonic cell disruptor (SCIENTZ-ⅡD, Xinzhi, China), a handheld stirring rod (HR2534, Philips, China), an ultracentrifuge (CR21N, HITACHI, Japan), a vacuum pump (SCJ-15, Supo, China), and a fluorescence microscope (DM4B, Leica, Germany). The microscopic counting software was Leica Application Suite (Leica Microsystems, LASV4.12, Germany).

[0052] 1.2 Test methods and result analysis

[0053] 1.2.1 Effect of soil suspension fragmentation method on virus count

[0054] Four different disruption methods were used to treat the soil suspension, and their effects on virus extraction efficiency were compared. These included: 1) vortex mixer at maximum speed (3200 rpm) for 3 minutes; 2) water bath shaker at 120 rpm for 60 minutes; 3) ultrasonic cell disruptor (100 W, 40 kHz) for 19 seconds, followed by 3 seconds of rest, for a total of 3 minutes; and 4) handheld stir bar (650 W, 50 Hz) for 3 minutes. Images captured by fluorescence microscopy using the Leica Application Suite (Leica Microsystems, LAS V4.12, Germany) were counted, and data were processed and graphed using Excel 2019.

[0055] The experimental results of different soil suspension crushing methods showed that after crushing with an ultrasonic cell crusher, the presence of soil viruses could not be observed under a microscope, but more virus particles could be observed using a handheld stirring rod. Figure 2 Although soil virus particles were observed using both a vortex mixer and a water bath shaker, the extraction efficiencies were 9.67% and 19.34% of those using a handheld stirrer, respectively, as shown in Table 1. These results indicate that during soil virus extraction, the instrument used for disruption, as well as its speed and power settings, significantly affect the extraction efficiency of the soil suspension (P < 0.05). Among the four disruption methods, 3 minutes of oscillation with a handheld stirrer (650W, 50Hz) resulted in the highest extraction efficiency.

[0056] Table 1 Soil virus count results under different treatments

[0057]

[0058] 1.2.2 Effect of soil sample to extract ratio on virus count

[0059] Based on the optimal soil suspension fragmentation method, 30.0 g and 50.0 g of fresh soil were added to 100.0 ml of potassium citrate phosphate buffer (pH = 7.0), respectively, and the effects of different soil-liquid ratios (30:100 and 50:100) on soil virus counts were analyzed.

[0060] The potassium citrate phosphate buffer solution was prepared as follows: 1.44 g disodium hydrogen phosphate, 0.24 g dipotassium hydrogen phosphate, and 10.0 g potassium citrate were dissolved in HPLC water, the pH was adjusted to 7.0 with phosphoric acid, and the volume was made up to 1.0 L. The prepared buffer solution was passed through a 0.1 μm microporous filter membrane, sterilized at 121°C for 30 minutes, cooled, and stored at 4.0°C for later use.

[0061] Images collected by fluorescence microscope were counted using Leica Application Suite (Leica Microsystems, LAS V4.12, Germany), and data were processed and graphs were drawn using Excel 2019.

[0062] When the soil-liquid ratio is 30:100 (30 parts by mass of soil are added to every 100 parts by volume of the extract), relatively obvious virus particles can be observed under a microscope. Figure 3 As shown in middle A, the abundance reaches 1.56×10 8 VPL -1 As shown in Table 1. When the soil-liquid ratio increased to 50:100 (50 parts by mass of soil was added to every 100 parts by volume of the extract), the abundance of soil viruses increased by 810.26%, as shown in Table 1. Figure 3 As shown in B. The above results indicate that increasing the amount of soil samples (soil-liquid ratio of 50:100) compared with the soil-liquid ratio of 30:100 can help better reflect the amount of virus particles in the soil.

[0063] 1.2.3 Effect of anti-fluorescence quenching agents on virus counts

[0064] After determining the most suitable soil suspension crushing method and the ratio of soil sample to extract, the effects of anti-fading agents and anti-fluorescence quenching agents on virus counts were compared.

[0065] The anti-fading agent was prepared by mixing anhydrous ethanol and (S)-(-)-α-methylbenzylamine in a ratio of 1000:1 to prepare an anti-fading stock solution. 100.0 ml of the anti-fading stock solution was mixed evenly with 1.0 ml of the glycerol-phosphate buffer stock solution, filtered through a 0.1 μm microporous membrane, and stored at room temperature for later use.

[0066] Fluoromount-G anti-fluorescence quenching mounting medium (water-soluble) was used as the anti-fluorescence quenching agent.

[0067] Images collected by fluorescence microscope were counted using Leica Application Suite (Leica Microsystems, LAS V4.12, Germany), and data were processed and graphs were drawn using Excel 2019.

[0068] from Figure 4 It can be seen that when no anti-fading solution and anti-fluorescence quenching agent are used, a certain number of fluorescent particles can be observed under a microscope at a magnification of 100 times. Figure 4 As shown in Figure A, however, when the magnification is 1000 times, no soil virus particles can be observed in the field of view. Figure 4 As shown in B, it shows that the quenching phenomenon of virus particles in a short time affects the observation results.

[0069] When using anti-fading solution or anti-fluorescence quenching agent, both can effectively prevent fluorescence quenching, see Figure 4 Among them, the number of virus particles in the soil after using the anti-fading agent was 8.78×10 7 VPL -1 The number of soil virus particles after using the anti-fluorescence quencher was one order of magnitude higher than that after using the anti-fading agent, reaching 3.75×10 8 VPL -1 , see Table 1 for details.

[0070] 2 Conclusion

[0071] Farmland soil virus particles are often adsorbed on the surface of soil particles, making efficient extraction a key step in soil virus research. Commonly used soil virus extractants include 10% beef extract (10g beef extract diluted to 100ml), 250mmol / L glycine solution, 10mmol / L sodium pyrophosphate, or 1% potassium citrate solution. Relatively speaking, 1% potassium citrate solution is more suitable for farmland soil virus extraction. The stirring method determines whether the virus can be fully separated and released from the soil.

[0072] In conjunction with the above research results, please refer to Figure 1 As shown, the steps for detecting farmland soil viruses of the present invention are as follows:

[0073] Step 1. Weigh 50 g of farmland soil sample and place it in 100 mL of 1% potassium citrate solution. Use a handheld stirring rod (650W, 50Hz) to stir for 3 minutes. The farmland soil sample is measured by mass, and the virus extract is measured by volume. This is the current common usage in the field.

[0074] Step 2: The stirred soil extract was divided into two 50 ml sterile centrifuge tubes and centrifuged at 6000 g, 4 °C for 20 min;

[0075] Step 3: Filter the supernatant obtained after centrifugation through a 0.22 μm pinhole filter membrane, and transfer 1.8 ml of the filtrate to a 2 ml sterile centrifuge tube;

[0076] Step 4: Add 100 μL of 10× optimized solution and 2 μL of DNase1 (deoxyribonuclease I) to the centrifuge tubes, and react in a water bath at 37°C for 30 min.

[0077] The optimized solution is a mixture of 100mM Tris solution, 25mM MgCl2 solution, and 1mM CaCl2 solution at pH 7.5, and its purpose is to remove free DNA;

[0078] Step 5: Add 35 μL of EDTA solution (0.5 M) and react in a 65°C water bath for 10 min.

[0079] Step 6: Take out the reaction solution and place it at room temperature;

[0080] Step 7: Use 75% by volume anhydrous ethanol to wipe the filter pump for surface disinfection, place a 0.02 μm filter membrane on the filter pump, add 1 ml of sterile HPLC water (make the membrane in close contact with the filter pump), filter, balance the filter pump pressure, then add the cooled reaction solution from step 6 to the filter membrane, filter, balance the filter pump pressure;

[0081] Step 8: Add 500 μL of SYBR Green nucleic acid fluorescent dye to the filter membrane and stain for 20 minutes in the dark; filter, balance the filter pump pressure, and rinse with sterile HPLC water 2-3 times;

[0082] Step 9: Take out the filter membrane and dry it in the dark;

[0083] Step 10: Add 35 μL of sterile HPLC water to a clean glass slide, place a dried filter membrane on it, add 35 μL of anti-fluorescence quencher on the filter membrane, and cover with a coverslip (try to remove bubbles to avoid affecting observation);

[0084] Step 11: After the preparation is completed, the free viruses are observed and counted using a fluorescence microscope under a 100× objective lens;

[0085] Step 12: Counting formula

[0086]

[0087] Where:

[0088] N is the number of virus particles per gram dry weight of the assay sample (VLP / g);

[0089] N t The number of virus particles (VLP) counted under a microscope;

[0090] A t is the membrane area (mm 2 ), with a diameter of 25 mm;

[0091] V0 is the volume of soil extract (mL);

[0092] C is the CCD coefficient of 2.0408, which is used to correct the microscope image;

[0093] A m is the microscope field of view area (mm 2 ), diameter 22mm, eyepiece 10×, objective lens 100×;

[0094] V t To determine the sample volume, that is, the volume of the sample reaction solution that finally passes through the filter membrane (mL);

[0095] T0 is the dry weight of the sample (g).

[0096] For parts of the present invention that are not described in detail, reference may be made to the prior art in the art or to technologies known to those skilled in the art.

[0097] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.

Claims

1. An optimization method for microscopic counting of free viruses in farmland soil, characterized in that: The steps of the method include: S1. Weigh 50 parts by mass of farmland soil sample and place it in 100 parts by volume of 1% potassium citrate solution. Stir with a handheld stirring rod for 3 minutes at 650W and 50Hz. S2, centrifuging the soil extract obtained in S1; S3, obtaining the supernatant after centrifugation and filtering it through a pinhole filter membrane; S4, adding an optimized solution and DNase1 to the filtered solution, and then performing a water bath reaction at a specific temperature; the optimized solution is a solution that can remove free DNA; S5, adding EDTA solution to the solution after water bath in S4, and then conducting water bath reaction at a specific temperature; S6, cooling to room temperature; S7, filter pump treatment: Place a filter membrane on the filter pump, add HPLC water for suction filtration, balance the filter pump pressure, then add the S6 solution on the filter membrane for suction filtration, balance the filter pump pressure; S8. Add nucleic acid fluorescent dye to the filter membrane and stain in the dark; then filter, balance the filter pump pressure, and rinse with sterile HPLC water; S9. Take out the filter membrane and dry it in the dark; S10, add sterile HPLC water to the slide, place a dried filter membrane on it, add anti-fluorescence quencher and anti-fading agent to the filter membrane, and cover with a coverslip; S11. Observe and count free viruses using a fluorescence microscope; The anti-fluorescence quenching agent is a water-soluble anti-fluorescence quenching sealing agent; The anti-fading agent is prepared by mixing anhydrous ethanol and (S)-(-)-α-methylbenzylamine in a volume ratio of 1000:1 to prepare an anti-fading stock solution, mixing the anti-fading stock solution and glycerol-phosphate buffer in a volume ratio of 100:1, and filtering through a microporous filter membrane to obtain the anti-fading agent.

2. The optimization method for microscopic counting of free viruses in farmland soil according to claim 1, characterized in that: The virus extract is a potassium citrate phosphate buffer solution with a pH of 7.

0.

3. The optimization method for microscopic counting of free viruses in farmland soil according to claim 2, characterized in that, The potassium citrate phosphate buffer solution is prepared by dissolving 1.44 parts of disodium hydrogen phosphate, 0.24 parts of dipotassium hydrogen phosphate, and 10.0 parts of potassium citrate in HPLC water, adding phosphoric acid to adjust the pH to 7.0 and then making up the volume, filtering the prepared buffer solution through a microporous filter membrane, and then sterilizing the solution to obtain the potassium citrate phosphate buffer solution.

4. The optimization method for microscopic counting of free viruses in farmland soil according to claim 1, characterized in that: The temperature of the water bath reaction in step S4 is 37°C; The temperature of the water bath reaction in step S5 is 65°C.

5. The optimization method for microscopic counting of free viruses in farmland soil according to claim 1, characterized in that: The pinhole filter membrane in step S3 is a 0.22 μm pinhole filter membrane; The filter membrane on the filter pump in step S7 is a 0.02 μm filter membrane.

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