Farmland soil free virus microscopic counting optimization method

Abstract

The invention discloses an optimization method for microscopic counting of free viruses in farmland soil, and belongs to the technical field of biological tests. According to the method, the test is carried out from the aspects of instrument selection, parameter setting, soil sample usage amount and microscopic observation key influence factors in the soil virus extraction test; analyzing the proportion of the farmland soil sample to the virus leaching solution, different oscillation instruments and parameter setting, and the influence of an anti-fluorescence quenching agent and an anti-fading solution in microscopic observation on the soil virus extraction efficiency and microscopic counting, and optimizing the farmland soil free virus microscopic counting process according to the analysis result. The invention provides an operation process suitable for the farmland soil virus fluorescence staining-microscopic counting method from instrument selection and parameter setting in soil virus extraction, soil sample usage amount and key influence factor control in microscopic observation. And a feasible technical reference can be provided for subsequent quantitative analysis and infection mode research of soil viruses.

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. They are composed of a protein shell and nucleic acids, are intracellular parasites, and can infect all organisms in the three-domain system of the biological kingdom (bacteria, archaea, and eukaryotes). As an important driver of the survival of soil microorganisms, viruses play an important role in regulating the soil ecological functions in which host microorganisms participate. When host cells are infected by viruses, the physiological metabolism, gene expression, and death rate of soil microorganisms change, causing changes in the number, population composition, and function of microorganisms. At the same time, a large amount of organic matter released by host cells becomes an effective substrate and energy for the circulation of elements such as carbon and nitrogen in the soil habitat, ultimately affecting the material cycle and energy flow of the soil.

[0003] With the rapid development of sequencing technology, soil virus research based on the gene level has been continuously reported. However, due to the influence of virus mutation rate, it is difficult to design universal primers, which limits the quantitative observation and detection of the number of viruses in the soil. Although the transmission electron microscope (TEM) observation and counting can quantify the number of viruses, the shortcomings of small detection area, high cost and complicated preparation work limit the counting research of soil viruses. Therefore, the use of fluorescent dyes to stain soil virus nucleic acids and count them with fluorescence microscopy has become the main method for quantitative analysis of soil viruses.

[0004] So far, there have been many reports at home and abroad on the use of fluorescent staining-microscopic counting to analyze the number of viruses in the environment, and the corresponding experimental process has been initially established. However, most of these research methods are for the study of viruses in marine ecosystems. The research methods for viruses in farmland soils are mostly different for different researchers and have a limited scope of application. The research results lack further verification and evaluation. 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 optimization 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 samples to virus extracts, different oscillation instruments and parameter settings, and the effects of anti-fluorescence quenching reagents on soil virus extraction efficiency and microscopic counting in microscopic observation, an operation process suitable for farmland soil virus fluorescence staining-microscopic counting method 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] The embodiment of the present invention provides a method for optimizing the microscopic counting of free viruses in farmland soil. The method conducts experiments on 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 in microscopic observation on soil virus extraction efficiency and microscopic counting, and optimizes the process of microscopic counting of free viruses in farmland soil according to 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 of the embodiments, 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.

[0014] In some embodiments, the virus extract is a potassium citrate phosphate buffer 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 fixing 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 microscopic counting of free viruses in farmland soil optimized according to the analysis results includes:

[0017] S1. Weigh 50 g of farmland soil sample, place it in 100 mL of 1% potassium citrate solution, stir with a handheld stirring rod for 3 min, and set the parameters to 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 and balance the filter pump pressure, then add S6 solution on the filter membrane for suction filtration and balance the filter pump pressure;

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

[0025] S9, take out the filter membrane and dry it in the dark;

[0026] S10, drip sterile HPLC water onto the slide, place a dried filter membrane on it, drip an anti-fluorescence quencher onto the filter membrane, and cover it 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 the embodiment of the present invention, the optimization method for microscopic counting of free viruses in farmland soil is provided, which uses a handheld stirring rod to break up the soil suspension to effectively increase the number of soil viruses extracted; compared with the traditional breaking 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 the embodiment of the present invention, the optimization method for microscopic counting of free viruses in farmland soil provided by the invention significantly improves the virus counting result by increasing the ratio of soil sample to extract; 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 fresh soil content is increased, that is, when the soil to extract ratio is 50:100 (50 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 one order of magnitude;

[0035] In an embodiment of the present invention, the provided optimization method for microscopic counting of free viruses in farmland soil effectively prolongs the observation time of soil viruses and increases the number of virus detections by using an anti-fluorescence quencher and / or an anti-fading liquid; when the anti-fading liquid and the anti-fluorescence quencher are not used, the virus is quenched in a short time, and 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 given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 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 crushing methods disclosed in one embodiment of the present invention are shown; wherein A is a vortex mixer; B is a water bath shaker; C is an ultrasonic cell crusher; and D is a handheld stirring rod;

[0039] Figure 3The 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 virus without using anti-fluorescence quenching agent disclosed in one embodiment of the present invention; wherein A is magnified 100 times; B is magnified 1000 times;

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

[0042] 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 part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are 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 in conjunction with 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. The method analyzes the influence of various factors on the microscopic counting of free viruses in soil from multiple aspects, and obtains a new microscopic counting method by combining various influencing factors, thereby achieving a good counting effect.

[0045] 1 Materials and methods

[0046] 1.1 Test materials and instruments

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

[0048] Experimental consumables and reagents: glass slide (1-1.2 mm, 25.4 mm × 76.2 mm); cover glass (Nr.1.0, 25 mm × 25 mm); Anodisc25 filter (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, McLean, China); molecular grade tris(hydroxymethylaminomethane) (Cat.ZZ02531, NOVON, UK); SYBR Green I Nucleic acid dye (10000×) (Cat.SY1020, Solebao, China); Fluoromount-G anti-fluorescence quenching mounting medium (Cat.36307ES08, Yeasen, China); cedar oil (Cas.8000-27-9, Hualing, China).

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

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

[0051] The main instruments included: 1% electronic balance (CP224S, Sartorius, Germany), vortex mixer (Vortex-Genie 2, Scientific Industries, USA); reciprocating water bath constant temperature shaker (ZWY-110X50, Zhicheng, China), ultrasonic cell crusher (SCIENTZ-ⅡD, Xinzhi, China), handheld stirring rod (HR2534, Philips China, China), ultracentrifuge (CR21N, HITACHI, Japan), vacuum pump (SCJ-15, Supo, China) and fluorescence microscope (DM4B, Leica, Germany), and 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 crushing methods were used to treat the soil suspension, and the effects of different methods on virus extraction efficiency were compared. Specifically, they included: 1) the vortex mixer was turned to the maximum speed (3200 rpm) and oscillated for 3 minutes; 2) the water bath shaker was oscillated at 120 rpm for 60 minutes; 3) the ultrasonic cell crusher (100w, 40kHz) was oscillated for 19 seconds each, and then stood for 3 seconds, for a total of 3 minutes; 4) the handheld stirring rod (650w, 50Hz) was oscillated for 3 minutes. Images collected by fluorescence microscope using Leica Application Suite (Leica Microsystems, LAS V4.12, Germany) were counted, and Excel 2019 was used to process data and draw charts.

[0055] The experimental results of different soil suspension crushing methods showed that after crushing with an ultrasonic cell crusher, no soil virus could be observed under a microscope, but more virus particles could be observed using a handheld stirring rod. Figure 2 As shown. Although the presence of soil virus particles can be observed using a vortex mixer and a water bath shaker, the extraction efficiencies of the two are 9.67% and 19.34% of that using a handheld stirring rod, respectively, as shown in Table 1. From the above results, it can be seen that during the soil virus extraction process, the instrument used for crushing and its speed and power settings will significantly affect the extraction efficiency of the soil suspension (P<0.05). Among the four crushing methods, the extraction efficiency is the highest after 3 minutes of oscillation with a handheld stirring rod (650w, 50Hz).

[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 preparation method of potassium citrate phosphate buffer is as follows: 1.44g disodium hydrogen phosphate, 0.24g dipotassium hydrogen phosphate, and 10.0g potassium citrate are dissolved in HPLC water, and the pH is adjusted to 7.0 with phosphoric acid and the volume is made up to 1.0L. The prepared buffer is passed through a 0.1μm microporous filter membrane, sterilized at 121℃ for 30 minutes, cooled, and stored at 4.0℃ for use.

[0061] Images collected by fluorescence microscope were counted using Leica Application Suite (Leica Microsystems, LAS V4.12, Germany), and Excel 2019 was used to process the data and draw charts.

[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 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 number of soil samples (soil-to-liquid ratio of 50:100) helps to better reflect the number of virus particles in the soil compared with the soil-to-liquid ratio of 30:100.

[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 preparation method is as follows: anhydrous ethanol and (S)-(-)-α-methylbenzylamine are prepared in a ratio of 1000:1 to prepare an anti-fading stock solution, 100.0 ml of the anti-fading stock solution is mixed evenly with 1.0 ml of glycerol-phosphate buffer stock solution, passed through a 0.1 μm microporous filter 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 Excel 2019 was used to process the data and draw charts.

[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 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, this indicates that the quenching phenomenon of virus particles in a short period of time affects the observed 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 mostly adsorbed on the surface of soil particles, and their efficient extraction is a key link in the study of soil viruses. Commonly used soil virus extracts are 10% beef extract (10g beef extract is diluted to 100ml of 10% beef extract solution), 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 of detecting farmland soil viruses of the present invention are as follows:

[0073] Step 1, weigh 50g of farmland soil sample, place it in 100mL of 1% potassium citrate solution, use a handheld stirring rod (650W, 50Hz), stir for 3min; the farmland soil sample is in mass unit, and the virus extract is in volume unit, which is the current conventional usage in this 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 into a 2 ml sterile centrifuge tube;

[0076] Step 4, add 100 μL of 10× optimization solution and 2 μL of DNase1 (deoxyribonuclease I) to the centrifuge tube, 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 LEDA solution (0.5 M), place in a 65°C water bath, and react for 10 min;

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

[0080] Step 7, wipe the filter pump with 75% by volume anhydrous ethanol to disinfect the surface, 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 reaction solution cooled in step 6 to the filter membrane, filter, balance the filter pump pressure;

[0081] Step 8, add 500 μL 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 away from light;

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

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

[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 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 modes, which are merely illustrative rather than 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 within the protection of the present invention.

Claims

1. An optimization method for microscopic counting of free viruses in farmland soil, characterized in that: The method conducts experiments on instrument selection, parameter settings, 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 in microscopic observation on soil virus extraction efficiency and microscopic counting, and optimizes the process of microscopic counting of free viruses in farmland soil according to the analysis results.

2. The optimization method for microscopic counting of free viruses in farmland soil according to claim 1, characterized in that: The ratio of the farmland soil sample to the virus extract is that no less than 50 parts by mass of the farmland soil sample is added to 100 parts by volume of the virus extract.

3. The optimization method for microscopic counting of free viruses in farmland soil according to claim 1, characterized in that: The oscillation instrument and parameters were set as follows: the soil suspension was broken up by oscillating at 650 W and 50 Hz for 3 minutes using a hand-held stirring rod.

4. The optimization method for microscopic counting of free viruses in farmland soil according to claim 1, characterized in that: The anti-fluorescence quenching agent is a water-soluble anti-fluorescence quenching sealing agent.

5. The optimization method for microscopic counting of free viruses in farmland soil according to claim 1, characterized in that: The preparation method of the anti-fading agent is: anhydrous ethanol and (S)-(-)-α-methylbenzylamine are mixed evenly at a volume ratio of 1000:1 to prepare an anti-fading stock solution, the anti-fading stock solution and glycerol-phosphate buffer are mixed evenly at a volume ratio of 100:1, and filtered through a microporous filter membrane to obtain the anti-fading agent.

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

0.

7. The optimization method for microscopic counting of free viruses in farmland soil according to claim 6, 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 fixing the volume, filtering the prepared buffer solution through a microporous filter membrane, and then sterilizing the buffer solution to obtain the potassium citrate phosphate buffer solution.

8. The optimization method for microscopic counting of free viruses in farmland soil according to claim 1, characterized in that: The process of microscopic counting of free viruses in farmland soil optimized according to the analysis results includes: S1. Weigh 50 g of farmland soil sample, place it in 100 mL of 1% potassium citrate solution, stir it with a handheld stirring rod for 3 min, and set the parameters to 650 W and 50 Hz; S2, centrifuging the soil extract obtained in S1; S3, obtaining the supernatant after centrifugation and filtering it through a pinhole filter membrane; S4, adding the optimized solution and DNase1 to the filtered solution, and then performing a water bath reaction at a specific temperature; S5, adding EDTA solution to the solution after water bath in S4, and then conducting water bath reaction at a specific temperature; S6, cooling at room temperature; S7, filter pump treatment: place a filter membrane on the filter pump, add HPLC water for suction filtration and balance the filter pump pressure, then add S6 solution on the filter membrane for suction filtration and balance the filter pump pressure; S8, add nucleic acid fluorescent dye to the filter membrane, and stain it in the dark; then filter, balance the filter pump pressure, and then rinse with sterile HPLC water; S9, take out the filter membrane and dry it in the dark; S10, drip sterile HPLC water onto the slide, place a dried filter membrane on it, drip an anti-fluorescence quencher onto the filter membrane, and cover it with a coverslip; S11. Use a fluorescence microscope to observe and count free viruses.

9. The optimization method for microscopic counting of free viruses in farmland soil according to claim 8, 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.

10. The optimization method for microscopic counting of free viruses in farmland soil according to claim 8, characterized in that: In step S3, the pinhole filter membrane 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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