A method for efficient extraction and detection of red soil viruses

Abstract

This invention discloses a highly efficient method for the extraction and detection of typical red soil viruses, belonging to the field of soil biochemistry and environment. This invention overcomes the difficulty of extracting viruses due to their strong adsorption to red soil, and is of great significance for studying the abundance and diversity of viruses in red soil, the interaction between viruses and hosts, and the biogeochemical processes mediated by viruses.

Description

Technical Field

[0001] This invention relates to the fields of soil biochemistry and environment, and more specifically, to a method for the extraction and detection of a typical red soil virus. Background Technology

[0002] Viruses are the most numerous life forms in the biosphere, widely distributed in habitats such as rivers, oceans, lakes, deserts, and soil. It is estimated that there are over 10 million viruses on Earth. 31 Soil accounts for about 10% of these viruses. Although the abundance of viruses in soil is very high, the academic community has gradually realized that soil viruses may play an important role in regulating the structure and composition of microbial communities, influencing the recycling of soil elements, promoting biological evolution, and affecting plant and animal diseases, and even human health. However, research on the ecology of soil viruses lags far behind that on marine viruses.

[0003] The reason for this challenge lies in the high heterogeneity and diversity of soils, and the high adsorption of viruses onto soil particles (>90% of viruses tend to adsorb onto soil particles), making virus extraction from soil extremely difficult. Therefore, the key to soil virus research is establishing precise, specific, and efficient extraction methods to obtain virus particles adsorbed on and within soil particles. Although some virus extraction and counting methods exist, the highly variable properties of soil (such as mineral content and pH) affect virus adsorption and resuspension, thus different soil types require different soil virus extraction methods. Red soil is an important soil resource in my country, accounting for 22.7% of the country's total land area. Due to its high clay content, low pH, and high iron and aluminum oxide content, red soil has a stronger adsorption capacity for viruses than other soils. Therefore, establishing an efficient virus extraction method suitable for red soil is of great significance for studying virus abundance, virus diversity, virus-host interactions, and virus-mediated biogeochemical processes in red soil. Summary of the Invention

[0004] The problem to be solved by this invention is to establish a method for efficiently extracting and detecting viruses from red soil.

[0005] To address the above problems, this invention provides a method for efficiently extracting and detecting red soil viruses, comprising the following steps:

[0006] S1: Weigh a fresh soil sample after removing sand, gravel, and plant roots;

[0007] S2: Add buffer solution, sonicate at 0-4℃ for 1-10 minutes, shaking the soil suspension during sonication, and centrifuge at 4℃ after sonication.

[0008] S3: Take the supernatant after centrifugation in step S2, filter it, and obtain the virus extract;

[0009] S4: Treat the virus extract with DNAse and incubate at 37 degrees Celsius for 30-90 minutes;

[0010] S5: Perform PCR amplification on the 16S rRNA gene to detect whether the free DNA has been completely removed. If no band is found on agarose gel electrophoresis, it is considered a qualified viral solution.

[0011] S1: Weigh a fresh soil sample after removing sand, gravel, and plant roots;

[0012] S2: Add buffer solution, sonicate at 0-4℃ for 1-10 minutes, shaking the soil suspension during sonication, and centrifuge at 4℃ after sonication.

[0013] S3: Take the supernatant after centrifugation in step S2, filter and sterilize to obtain virus extract;

[0014] S4: Treat the virus extract with DNAse and incubate at 37 degrees Celsius for 30-90 minutes;

[0015] S5: Perform PCR amplification on the 16S rRNA gene to detect whether the free DNA has been completely removed. If no band is found on agarose gel electrophoresis, it is considered a qualified viral solution.

[0016] S6: Detect the viral morphology in the viral solution described in step S5 and calculate the viral abundance.

[0017] To ensure the activity of the extracted virus, the entire extraction experiment should be carried out at 0–4°C. In step S3, a filter with 0.22 μm pores should be selected for filtration to remove large soil particles, soil fungi, and most soil bacteria. The purpose of using DNase treatment is to remove free extracellular DNA.

[0018] Preferably, in step S2, the buffer solution is a potassium citrate buffer solution, which includes 10 g / L potassium citrate, 10% phosphate buffer solution, 5 mM ethylenediaminetetraacetic acid and 150 mM magnesium sulfate.

[0019] Preferably, in step S2, the parameters for ultrasound are: frequency 40kHz, duration 1-10min.

[0020] Preferably, in step S5, the primer sequences used for PCR amplification are 341F and 806R, and the sequences of 341F and 806R are as follows:

[0021] 341F:CCTACGGGNGGCWGCAG;

[0022] 806R:GGACTACHVGGGTATCTAAT.

[0023] Furthermore, the present invention provides a method for detecting the virus extracted by the aforementioned method, comprising the following steps:

[0024] a: Load the virus solution onto the filter membrane and dry the filter membrane;

[0025] b: Immerse the dried filter membrane from step a into the staining solution, stain, and then dry the filter membrane after staining.

[0026] c: Add anti-fading solution to the glass slide, immerse the dried filter membrane from step b in the anti-fading solution, cover with a coverslip and add anti-fading solution to make an observation slide;

[0027] d: Observe the observation slide prepared in step c under a fluorescence microscope at 488nm, take fluorescence pictures, and count the fluorescence pictures;

[0028] e: Calculate viral abundance;

[0029] f: The virus solution was concentrated 10 to 1000 times using a 30-50K ultrafiltration tube to prepare a concentrated virus solution. The concentrated virus solution was then dropped onto a copper mesh, the mesh was negatively stained with 1% phosphotungstic acid, and the virus morphology was observed using a transmission electron microscope.

[0030] This method loads and enriches the virus onto the filter membrane, making the concentrated virus easier to observe. At the same time, loading the virus onto the filter membrane facilitates operations such as staining.

[0031] Preferably, in step a, the filter membrane is an Anodic Al2O3 filter membrane with a pore size of 0.02 μm.

[0032] Preferably, in step b, the staining solution is a SYBR GREENⅠ solution.

[0033] Preferably, in step c, the method for preparing the anti-fading liquid is as follows: adding 1% volume of 10% p-phenylenediamine solution to 99% volume of glycerol and / or PBS solution.

[0034] Furthermore, in step e, the formula for calculating viral abundance is:

[0035] Vt=a×K1÷K2×(V+m×ω)÷[m×(100%-ω)],

[0036] Where Vt: virus abundance in soil (virus count / g dry soil); a: average number of viruses per field of view under fluorescence microscopy; K1: area of ​​the Anodicel Al2O3 film (μm²). 2 K2: Area of ​​the field of view (μm)2 V: Buffer volume (mL); m: Soil mass (g); ω: Soil moisture content (%).

[0037] Preferably, in step f, the duration of the 1% phosphotungstic acid negative staining is 1 to 2 minutes.

[0038] This invention has the following technical advantages: 1. It uses a modified potassium citrate buffer (AKC). The phosphate and potassium citrate in the AKC buffer help maintain pH and osmotic balance, ethylenediaminetetraacetic acid helps prevent environmental interference and chelation, and magnesium sulfate is used to stabilize the viral capsid. Compared with other buffers, the number of viruses extracted by the AKC buffer is increased by 1 to 2 orders of magnitude, and the buffer is inexpensive and easy to prepare; 2. It uses sonication in an ice-water bath for 1 to 10 minutes. Sonication for 5 to 10 minutes has been verified as the most efficient physical dispersion method for virus extraction, which maintains the structure of the virus particles and yields the highest abundance of viruses extracted from red soil; 3. In addition, this invention overcomes the difficulty of virus extraction due to the strong adsorption of red soil, which is of great significance for studying the abundance and diversity of viruses in red soil, the interaction between viruses and hosts, and virus-mediated biogeochemical processes. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of the process for extracting viruses from red soil according to the present invention;

[0040] Figure 2 This is a statistical chart showing the viral abundance in the viral solution obtained by shaking and dispersing in a shaker in Example 12 of the present invention.

[0041] Figure 3 This is a comparison chart of virus abundance in virus solutions prepared by vortex dispersion, oscillation dispersion, and ultrasonic dispersion in Example 12 of the present invention.

[0042] Figure 4 This is a statistical chart showing the viral abundance in the viral solution obtained by ultrasonic dispersion in Example 12 of the present invention.

[0043] Figure 5 This is a statistical chart showing the viral abundance in the viral solution after the virus was extracted from red soil using 10 different chemical extractants in Example 14 of this invention.

[0044] Figure 6 This is a statistical comparison chart of virus abundance in virus solutions after extracting viruses from 10 types of red soil using three chemical extractants in Example 16 of this invention.

[0045] Figure 7 This is a transmission electron microscope image of the virus morphology in red soil solution in Example 17 of the present invention. Detailed Implementation

[0046] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the claims of the present invention.

[0047] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0048] As described in the background section, different soil types require different soil virus extraction methods. Red soil is an important soil resource in my country, accounting for 22.7% of the country's total land area. Due to its high clay content, low pH, and high iron and aluminum oxide content, red soil has a stronger adsorption capacity for viruses than other soils. Therefore, establishing an efficient virus extraction method suitable for red soil is of great significance for studying the abundance and diversity of viruses in red soil, the interaction between viruses and hosts, and virus-mediated biogeochemical processes.

[0049] Example 1

[0050] The formulation of potassium citrate (AKC) buffer in this embodiment is as follows: 10 g / L potassium citrate, 10% volume fraction phosphate buffer (Solepro, P1010), 5 mM ethylenediaminetetraacetic acid and 150 mM magnesium sulfate.

[0051] Weigh 5g of red soil (after removing sand and plant roots) and mix with 15mL of AKC buffer. Place the mixture in a 50mL sterile centrifuge tube and shake on a shaker for 0.5h at 4℃. Centrifuge the shaken solution at 5000×g for 15min at 4℃. Use a 20mL sterile syringe to aspirate the supernatant and pass it through a 0.45μm pore size needle filter and a 0.22μm pore size needle filter to remove large soil particles, soil fungi, and most soil bacteria. Treat the filtered solution with DNase and incubate at 37℃ for 1h. The final concentration of DNase is 100U / mL to remove free extracellular DNA. PCR amplification of the 16S rRNA gene was performed using universal primers 341F / 806R to detect whether free DNA was completely removed. Agarose gel electrophoresis showed no bands, indicating a successful result. 341F: CCTACGGGNGGCWGCAG; 806R: GGACTACHVGGGTATCTAAT.

[0052] Example 2

[0053] The formulation of AKC buffer in this embodiment is: 10 g / L potassium citrate, 10% volume fraction phosphate buffer (Solepro, P1010), 5 mM ethylenediaminetetraacetic acid and 150 mM magnesium sulfate.

[0054] Weigh 5g of red soil (after removing sand and plant roots) and mix with 15mL of AKC buffer. Place the mixture in a 50mL sterile centrifuge tube and shake on a shaker for 1h at 4℃. Centrifuge the shaken solution at 5000×g for 15min at 4℃. Use a 20mL sterile syringe to aspirate the supernatant and pass it through a 0.45μm pore size needle filter and a 0.22μm pore size needle filter to remove large soil particles, soil fungi, and most soil bacteria. Treat the filtered solution with DNase and incubate at 37℃ for 1h. The final concentration of DNase is 100U / mL to remove free extracellular DNA. PCR amplification of the 16S rRNA gene was performed using universal primers 341F / 806R to detect whether free DNA was completely removed. Agarose gel electrophoresis showed no bands, indicating a successful result. 341F: CCTACGGGNGGCWGCAG; 806R: GGACTACHVGGGTATCTAAT.

[0055] Example 3

[0056] The formulation of AKC buffer in this embodiment is: 10 g / L potassium citrate, 10% volume fraction phosphate buffer (Solepro, P1010), 5 mM ethylenediaminetetraacetic acid and 150 mM magnesium sulfate.

[0057] Weigh 5g of red soil (after removing sand and plant roots) and mix with 15mL of AKC buffer. Place the mixture in a 50mL sterile centrifuge tube and shake on a shaker for 2 hours at 4℃. Centrifuge the shaken solution at 5000×g for 15 minutes at 4℃. Use a 20mL sterile syringe to aspirate the supernatant and pass it through a 0.45μm pore size needle filter and a 0.22μm pore size needle filter to remove large soil particles, soil fungi, and most soil bacteria. Treat the filtered solution with DNase and incubate at 37℃ for 1 hour. The final concentration of DNase is 100U / mL to remove free extracellular DNA. PCR amplification of the 16S rRNA gene was performed using universal primers 341F / 806R to detect whether free DNA was completely removed. Agarose gel electrophoresis showed no bands, indicating a successful result. 341F: CCTACGGGNGGCWGCAG; 806R: GGACTACHVGGGTATCTAAT.

[0058] Example 4

[0059] The formulation of AKC buffer in this embodiment is: 10 g / L potassium citrate, 10% volume fraction phosphate buffer (Solepro, P1010), 5 mM ethylenediaminetetraacetic acid and 150 mM magnesium sulfate.

[0060] Weigh 5g of red soil (after removing sand and plant roots) and mix with 15mL of AKC buffer. Place the mixture in a 50mL sterile centrifuge tube and shake on a shaker at 4℃ for 4h. Centrifuge the shaken solution at 5000×g for 15min at 4℃. Use a 20mL sterile syringe to aspirate the supernatant and pass it through a 0.45μm pore size needle filter and a 0.22μm pore size needle filter to remove large soil particles, soil fungi, and most soil bacteria. Treat the filtered solution with DNase and incubate at 37℃ for 1h. The final concentration of DNase is 100U / mL to remove free extracellular DNA. PCR amplification of the 16S rRNA gene was performed using universal primers 341F / 806R to detect whether free DNA was completely removed. Agarose gel electrophoresis showed no bands, indicating a successful result. 341F: CCTACGGGNGGCWGCAG; 806R: GGACTACHVGGGTATCTAAT.

[0061] Example 5

[0062] The formulation of AKC buffer in this embodiment is: 10 g / L potassium citrate, 10% volume fraction phosphate buffer (Solepro, P1010), 5 mM ethylenediaminetetraacetic acid and 150 mM magnesium sulfate.

[0063] Weigh 5g of red soil (after removing sand and plant roots) and mix it with 15mL of AKC buffer. Place the mixture in a 50mL sterile centrifuge tube and vortex for 5min at 4℃. Centrifuge the vortexed solution at 5000×g for 15min at 4℃. Use a 20mL sterile syringe to aspirate the supernatant and pass it through a 0.45μm pore size needle filter and a 0.22μm pore size needle filter to remove large soil particles, soil fungi, and most soil bacteria. Treat the filtered solution with DNase and incubate at 37℃ for 1h. The final concentration of DNase is 100U / mL to remove free extracellular DNA. The 16S rRNA gene was amplified by PCR using universal primers 341F / 806R. The removal of free DNA was checked, and agarose gel electrophoresis showed no bands, indicating that the gene was qualified. The results were as follows: 341F: CCTACGGGNGGCWGCAG; 806R: GGACTACHVGGGTATCTAAT.

[0064] Example 6

[0065] The formulation of AKC buffer in this embodiment is: 10 g / L potassium citrate, 10% volume fraction phosphate buffer (Solepro, P1010), 5 mM ethylenediaminetetraacetic acid and 150 mM magnesium sulfate.

[0066] Weigh 5g of red soil (after removing sand and plant roots) and mix it with 15mL of AKC buffer. Place the mixture in a 50mL sterile centrifuge tube and vortex for 20min at 4℃. Centrifuge the vortexed solution at 5000×g for 15min at 4℃. Use a 20mL sterile syringe to aspirate the supernatant and pass it through a 0.45μm pore size needle filter and a 0.22μm pore size needle filter to remove large soil particles, soil fungi, and most soil bacteria. Treat the filtered solution with DNase and incubate at 37℃ for 1h. The final concentration of DNase is 100U / mL to remove free extracellular DNA. The 16S rRNA gene was amplified by PCR using universal primers 341F / 806R. The removal of free DNA was checked, and agarose gel electrophoresis showed no bands, indicating that the gene was qualified. The results were as follows: 341F: CCTACGGGNGGCWGCAG; 806R: GGACTACHVGGGTATCTAAT.

[0067] Example 7

[0068] The formulation of AKC buffer in this embodiment is: 10 g / L potassium citrate, 10% volume fraction phosphate buffer (Solepro, P1010), 5 mM ethylenediaminetetraacetic acid and 150 mM magnesium sulfate.

[0069] Weigh 5g of red soil (after removing sand and plant roots) and mix it with 15mL of AKC buffer. Place the mixture in a 50mL sterile centrifuge tube and sonicate it at 40kHz for 0min at 4℃. Centrifuge the sonicated solution at 5000×g for 15min at 4℃. Use a 20mL sterile syringe to aspirate the supernatant and pass it through a 0.45μm pore size needle filter and a 0.22μm pore size needle filter to remove large soil particles, soil fungi, and most soil bacteria. Treat the filtered solution with DNase and incubate it at 37℃ for 1h. The final concentration of DNase is 100U / ml to remove free extracellular DNA. The 16S rRNA gene was amplified by PCR using universal primers 341F / 806R. The removal of free DNA was checked, and agarose gel electrophoresis showed no bands, indicating that the gene was qualified. The results were as follows: 341F: CCTACGGGNGGCWGCAG; 806R: GGACTACHVGGGTATCTAAT.

[0070] Example 8

[0071] The formulation of AKC buffer in this embodiment is: 10 g / L potassium citrate, 10% volume fraction phosphate buffer (Solepro, P1010), 5 mM ethylenediaminetetraacetic acid and 150 mM magnesium sulfate.

[0072] Weigh 5g of red soil (after removing sand and plant roots) and mix it with 15mL of AKC buffer. Place the mixture in a 50mL sterile centrifuge tube and sonicate it at 40kHz for 1min in an ultrasonic cleaner at 4℃. Centrifuge the sonicated solution at 5000×g for 15min at 4℃. Use a 20mL sterile syringe to aspirate the supernatant and pass it through a 0.45μm pore size needle filter and a 0.22μm pore size needle filter to remove large soil particles, soil fungi, and most soil bacteria. Treat the filtered solution with DNase and incubate it at 37℃ for 1h. The final concentration of DNase is 100U / mL to remove free extracellular DNA. The 16S rRNA gene was amplified by PCR using universal primers 341F / 806R. The removal of free DNA was checked, and agarose gel electrophoresis showed no bands, indicating that the gene was qualified. The results were as follows: 341F: CCTACGGGNGGCWGCAG; 806R: GGACTACHVGGGTATCTAAT.

[0073] Example 9

[0074] The formulation of AKC buffer in this embodiment is: 10 g / L potassium citrate, 10% volume fraction phosphate buffer (Solepro, P1010), 5 mM ethylenediaminetetraacetic acid and 150 mM magnesium sulfate.

[0075] Weigh 5g of red soil (after removing sand and plant roots) and mix it with 15mL of AKC buffer. Place the mixture in a 50mL sterile centrifuge tube and sonicate it at 40kHz for 3min at 4℃. Centrifuge the sonicated solution at 5000×g for 15min at 4℃. Use a 20mL sterile syringe to aspirate the supernatant and pass it through a 0.45μm pore size needle filter and a 0.22μm pore size needle filter to remove large soil particles, soil fungi, and most soil bacteria. Treat the filtered solution with DNase and incubate it at 37℃ for 1h. The final concentration of DNase is 100U / mL to remove free extracellular DNA. The 16S rRNA gene was amplified by PCR using universal primers 341F / 806R. The removal of free DNA was checked, and agarose gel electrophoresis showed no bands, indicating that the gene was qualified. The results were as follows: 341F: CCTACGGGNGGCWGCAG; 806R: GGACTACHVGGGTATCTAAT.

[0076] Example 10

[0077] The formulation of AKC buffer in this embodiment is: 10 g / L potassium citrate, 10% volume fraction phosphate buffer (Solepro, P1010), 5 mM ethylenediaminetetraacetic acid and 150 mM magnesium sulfate.

[0078] Weigh 5g of red soil (after removing sand and plant roots) and mix it with 15mL of AKC buffer. Place the mixture in a 50mL sterile centrifuge tube and sonicate it at 40kHz for 5min at 4℃. Centrifuge the sonicated solution at 5000×g for 15min at 4℃. Use a 20mL sterile syringe to aspirate the supernatant and pass it through a 0.45μm pore size needle filter and a 0.22μm pore size needle filter to remove large soil particles, soil fungi, and most soil bacteria. Treat the filtered solution with DNase and incubate it at 37℃ for 1h. The final concentration of DNase is 100U / mL to remove free extracellular DNA. The 16S rRNA gene was amplified by PCR using universal primers 341F / 806R. The removal of free DNA was checked, and agarose gel electrophoresis showed no bands, indicating that the gene was qualified. The results were as follows: 341F: CCTACGGGNGGCWGCAG; 806R: GGACTACHVGGGTATCTAAT.

[0079] Example 11

[0080] The formulation of AKC buffer in this embodiment is: 10 g / L potassium citrate, 10% volume fraction phosphate buffer (Solepro, P1010), 5 mM ethylenediaminetetraacetic acid and 150 mM magnesium sulfate.

[0081] Weigh 5g of red soil (after removing sand and plant roots) and mix it with 15mL of AKC buffer. Place the mixture in a 50mL sterile centrifuge tube and sonicate it at 40kHz for 10min at 4℃. Centrifuge the sonicated solution at 5000×g for 15min at 4℃. Use a 20mL sterile syringe to aspirate the supernatant and pass it through a 0.45μm pore size needle filter and a 0.22μm pore size needle filter to remove large soil particles, soil fungi, and most soil bacteria. Treat the filtered solution with DNase and incubate it at 37℃ for 1h. The final concentration of DNase is 100U / mL to remove free extracellular DNA. The 16S rRNA gene was amplified by PCR using universal primers 341F / 806R. The removal of free DNA was checked, and agarose gel electrophoresis showed no bands, indicating that the gene was qualified. The results were as follows: 341F: CCTACGGGNGGCWGCAG; 806R: GGACTACHVGGGTATCTAAT.

[0082] Example 12

[0083] Virus solutions prepared by different physical dispersion methods in Examples 1 to 11 were used as experimental materials. The filter core of a vacuum filter was moistened with sterile water. A 0.22-micron pore size filter membrane and a 0.02-micron pore size Anodices Al2O3 filter membrane were placed sequentially on the filter stage, ensuring both membranes were moistened. The filter cup was fixed in place, and 400 μL of virus solution was added to the filter cup. 2 mL of sterile water was slowly added along the filter cup. The vacuum pump was turned on to load virus particles onto the Al2O3 filter membrane. After filtration, vacuum filtration continued for 1 minute. The filter cup was removed, and the Al2O3 filter membrane was removed with tweezers. The bottom of the filter membrane was wiped dry with low-dust paper and placed on new low-dust paper to air dry in the dark. 150 μL of 2.5× SYBR water was added to a sterile petri dish. Green I solution was used, and the filter membrane was immersed in the staining solution and stained in the dark for 15 minutes. The bottom of the filter membrane was wiped dry with dust-free paper, placed on new dust-free paper, and air-dried in the dark. 15 μL of anti-fading solution (10 μL of 10% p-phenylenediamine solution added to 990 μL of glycerol / PBS solution) was added to a glass slide, and the filter membrane was immersed in the anti-fading solution. 15 μL of anti-fading solution was added to a coverslip, and the coverslip was placed on the filter membrane. The coverslip was gently squeezed to remove air bubbles. The fluorescence signal was observed at a wavelength of about 488 nm under a fluorescence microscope (EFM) at 1000× magnification. Fluorescence images were taken, and the fluorescence images were counted using ImageJ software. Open the image to be processed (File-Open, or drag the image directly to the menu bar); convert the image to 8-bit (Image-Type-8-bit); adjust the threshold, remove the background, and select the cell nucleus (Image-Adjust-Threshold); automatically analyze and count particles (Analyze-AnalyzeParticles), and calculate the viral abundance using formula (1):

[0084] Vt=a×K1÷K2×(V+m×ω)÷[m×(100%-ω)]···········(Formula 1)

[0085] Vt: Virus abundance in soil (virus count / g dry soil); a: Average number of viruses per field of view under fluorescence microscopy; K1: Area of ​​the Anodicel Al2O3 film (μm²) 2 K2: Area of ​​the field of view (μm) 2 V: Buffer volume (mL); m: Soil mass (g); ω: Soil moisture content (%).

[0086] Virus abundance in virus solutions prepared by different physical dispersion methods is as follows: Figures 2-4 As shown, ultrasound extraction of red soil virus for 5 minutes is the best physical dispersion method.

[0087] Example 13

[0088] In this embodiment, the inventors used 10 different chemical extractants to extract the virus from a type of red soil. The 10 chemical extractants and their parameters are as follows:

[0089] PPBS1: 10% PBS, 10 g / L potassium citrate, 150 mM MgSO4, 2% BSA, pH = 4.8;

[0090] PPBS2: 10% PBS, 10 g / L potassium citrate, 150 mM MgSO4, 2% BSA, 5 g / L sodium hexametaphosphate, pH=4.7;

[0091] PPBS3: 10% PBS, 10 g / L potassium citrate, 150 mM MgSO4, 2% BSA, pH = 5.1;

[0092] AK: 10 g / L potassium citrate, 1.44 g / L Na2HPO4·7H2O, 0.24 g / L KH2PO4, pH=7;

[0093] Gly: 250mM glycine solution, pH=8.0;

[0094] SM: 5.8g NaCl, 2g MgSO4·7H2O, 50mL 1M Tris-HCl, pH=7.5;

[0095] AKC+CER: 10% PBS, 10 g / L potassium citrate, 150 mM MgSO4, 5 mM EDTA, 33.3% cation exchange resin, pH = 4.6;

[0096] Tris-HCl: 0.1M Tris-HCl, 1M NaCl, pH=7.0;

[0097] D-MEM: 1×DMEM culture medium, pH=7.2;

[0098] AKC: 10% PBS, 10 g / L potassium citrate, 150 mM MgSO4, 5 mM EDTA, pH = 4.6.

[0099] Weigh 5g of red soil (after removing sand and plant roots) and mix it with 15mL of chemical extraction agent. Place the mixture in a 50mL sterile centrifuge tube and sonicate it at 40kHz for 5min at 4℃. Centrifuge the sonicated solution at 5000×g for 15min at 4℃. Use a 20mL sterile syringe to aspirate the supernatant and pass it through a 0.45μm pore size needle filter and a 0.22μm pore size needle filter to remove large soil particles, soil fungi, and most soil bacteria. Treat the filtered solution with DNase and incubate it at 37℃ for 1h. The final concentration of DNase is 100U / mL to remove free extracellular DNA. The 16S rRNA gene was amplified by PCR using universal primers 341F / 806R. The removal of free DNA was checked, and agarose gel electrophoresis showed no bands, indicating that the gene was qualified. The results were as follows: 341F: CCTACGGGNGGCWGCAG; 806R: GGACTACHVGGGTATCTAAT.

[0100] Example 14

[0101] Using virus solutions prepared with 10 different chemical extractants as experimental materials in Example 13, the filter core of the vacuum filter was moistened with sterile water. A 0.22-micron pore size filter membrane and a 0.02-micron pore size Anodices Al2O3 filter membrane were placed sequentially on the filter stage, ensuring both membranes were moistened. The filter cup was then fixed in place. 400 μL of virus solution was added to the filter cup, followed by 2 mL of sterile water slowly added along the filter cup. The vacuum pump was turned on to load virus particles onto the Al2O3 filter membrane. After filtration, vacuum filtration continued for 1 minute. The filter cup was removed, and the Al2O3 filter membrane was removed with tweezers. The bottom of the filter membrane was wiped dry with low-dust paper and placed on new low-dust paper to air dry in the dark. 150 μL of 2.5× SYBR water was then added to a sterile petri dish. Green I solution was used, and the filter membrane was immersed in the staining solution and stained in the dark for 15 minutes. The bottom of the filter membrane was wiped dry with dust-free paper, placed on new dust-free paper, and air-dried in the dark. 15 μL of anti-fading solution (10 μL of 10% p-phenylenediamine solution added to 990 μL of glycerol / PBS solution) was added to a glass slide, and the filter membrane was immersed in the anti-fading solution. 15 μL of anti-fading solution was added to a coverslip, and the coverslip was placed on the filter membrane. The coverslip was gently squeezed to remove air bubbles. The fluorescence signal was observed at a wavelength of about 488 nm under a fluorescence microscope (EFM) at 1000× magnification. Fluorescence images were taken, and the fluorescence images were counted using ImageJ software. Open the image to be processed (File-Open, or drag the image directly to the menu bar); convert the image to 8-bit (Image-Type-8-bit); adjust the threshold, remove the background, and select the cell nucleus (Image-Adjust-Threshold); automatically analyze and count particles (Analyze-Analyze Particles), and calculate the viral abundance using formula (1):

[0102] Vt=a×K1÷K2×(V+m×ω)÷[m×(100%-ω)]···········(Formula 1)

[0103] Vt: Virus abundance in soil (virus count / g dry soil); a: Average number of viruses per field of view under fluorescence microscopy; K1: Area of ​​the Anodicel Al2O3 film (μm²) 2 K2: Area of ​​the field of view (μm) 2 V: Buffer volume (mL); m: Soil mass (g); ω: Soil moisture content (%).

[0104] like Figure 5 As shown, the viral abundance extracted by AKC and DMED was the highest, followed by the viral abundance extracted by Tris-HCl.

[0105] Example 15

[0106] This embodiment verifies the universality of AKC, DMED, and Tris-HCl in extracting viruses from 10 different red soils. The specific implementation method is as follows:

[0107] Weigh 5g of red soil (after removing sand and plant roots) and mix it with 15mL of chemical extraction agent. Place the mixture in a 50mL sterile centrifuge tube and sonicate it at 40kHz for 5min at 4℃. Centrifuge the sonicated solution at 5000×g for 15min at 4℃. Use a 20mL sterile syringe to aspirate the supernatant and pass it through a 0.45μm pore size needle filter and a 0.22μm pore size needle filter to remove large soil particles, soil fungi, and most soil bacteria. Treat the filtered solution with DNase and incubate it at 37℃ for 1h. The final concentration of DNase is 100U / mL to remove free extracellular DNA. The 16S rRNA gene was amplified by PCR using universal primers 341F / 806R. The removal of free DNA was checked, and agarose gel electrophoresis showed no bands, indicating that the gene was qualified. The results were as follows: 341F: CCTACGGGNGGCWGCAG; 806R: GGACTACHVGGGTATCTAAT.

[0108] Example 16

[0109] Using virus solutions prepared from 10 different red soils and 3 different chemical extractants as experimental materials in Example 15, the filter core of the vacuum filter was moistened with sterile water. A 0.22-micron pore size filter membrane and a 0.02-micron pore size Anodices Al2O3 filter membrane were placed sequentially on the filter platform, ensuring both membranes were moistened. The filter cup was then fixed in place. 400 μL of virus solution was added to the filter cup, followed by 2 mL of sterile water slowly added along the filter cup. The vacuum pump was turned on to load virus particles onto the Al2O3 filter membrane. After filtration, vacuum filtration continued for 1 minute. The filter cup was removed, and the Al2O3 filter membrane was removed with tweezers. The bottom of the filter membrane was wiped dry with low-dust paper and placed on new low-dust paper to air dry in the dark. 150 μL of 2.5× SYBR water was then added to a sterile petri dish. Green I solution was used, and the filter membrane was immersed in the staining solution and stained in the dark for 15 minutes. The bottom of the filter membrane was wiped dry with dust-free paper, placed on new dust-free paper, and air-dried in the dark. 15 μL of anti-fading solution (10 μL of 10% p-phenylenediamine solution added to 990 μL of glycerol / PBS solution) was added to a glass slide, and the filter membrane was immersed in the anti-fading solution. 15 μL of anti-fading solution was added to a coverslip, and the coverslip was placed on the filter membrane. The coverslip was gently squeezed to remove air bubbles. The fluorescence signal was observed at a wavelength of about 488 nm under a fluorescence microscope (EFM) at 1000× magnification. Fluorescence images were taken, and the fluorescence images were counted using ImageJ software. Open the image to be processed (File-Open, or drag the image directly to the menu bar); convert the image to 8-bit (Image-Type-8-bit); adjust the threshold, remove the background, and select the cell nucleus (Image-Adjust-Threshold); automatically analyze and count particles (Analyze-AnalyzeParticles), and calculate the viral abundance using formula (1):

[0110] Vt=a×K1÷K2×(V+m×ω)÷[m×(100%-ω)]···········(Formula 1)

[0111] Vt: Virus abundance in soil (virus count / g dry soil); a: Average number of viruses per field of view under fluorescence microscopy; K1: Area of ​​the Anodicel Al2O3 film (μm²) 2 K2: Area of ​​the field of view (μm) 2 V: Buffer volume (mL); m: Soil mass (g); ω: Soil moisture content (%).

[0112] like Figure 6 As shown, overall, the highest viral abundance was obtained in most red soils using modified potassium citrate buffer (AKC).

[0113] Example 17

[0114] The virus solution prepared in Example 10 was used to observe the virus morphology using transmission electron microscopy (TEM). 10 mL of the concentrated virus solution was transferred to a 50 mL 30 K ultrafiltration tube and centrifuged at 3500 g for 10 minutes at 4°C to concentrate the virus particles to approximately 200 μL. This process was repeated three times. 20 μL of the concentrated virus solution was dropped onto a copper mesh. The mesh was negatively stained with 1% phosphotungstic acid for 1 minute and 30 seconds, and the morphology and structure of the virus were observed under 85,000x magnification using a transmission electron microscope.

[0115] Virus structure such as Figure 7 As shown, the results indicate that the extracted viruses exhibit diverse morphologies, primarily tailed bacteriophages, along with other virus types such as baculoviruses and filoviruses. This demonstrates that the virus extraction was relatively successful.

[0116] While the disclosure is as stated above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of this invention.

Claims

1. A method for efficient extraction and detection of red soil viruses, characterized by, The method comprises the following steps: S1: weighing fresh red soil samples without sand and plant roots; S2: adding a buffer solution, ultrasonicating for 1-10 minutes at 0-4°C, oscillating the soil suspension during ultrasonication, and centrifuging at 4°C after ultrasonication, wherein the buffer solution is a potassium citrate buffer solution with pH=4.6, the potassium citrate buffer solution comprises 10 g / L of potassium citrate, 10% by volume of a phosphate buffer solution, 5 mM of ethylenediaminetetraacetic acid, and 150 mM of magnesium sulfate, and the ultrasonication parameters are a frequency of 40 kHz and a time length of 1-10 minutes; S3: aspirating the supernatant after centrifugation in step S2, filtering and sterilizing to obtain a virus extraction solution; S4: treating the virus extraction solution with DNAase, and incubating at 37°C for 30-90 minutes; S5: performing PCR amplification on a 16S rRNA gene to detect whether free DNA is removed completely, and the result is qualified if no band is shown in agarose gel electrophoresis, that is, the virus solution is obtained; S6: detecting the virus morphology in the virus solution in step S5 and calculating the virus abundance, and the specific steps are as follows: a: loading the virus solution onto a filter membrane and drying the filter membrane; b: immersing the dried filter membrane in step a in a staining solution for staining, and drying the filter membrane after staining; c: adding a fade-proof solution onto a glass slide, immersing the dried filter membrane in step b in the fade-proof solution, covering a cover glass and adding the fade-proof solution, and preparing an observation slide; d: observing the observation slide prepared in step c under a fluorescence microscope at 488 nm, taking a fluorescence photo, and counting the fluorescence photo; e: calculating the virus abundance, and the formula for calculating the virus abundance is as follows: Vt=a×K1÷K2×(V+m×ω)÷[m×(100%-ω)], wherein, Vt: virus abundance in soil (number of viruses / g of dry soil); a: average number of viruses per field of view under fluorescence microscope; K1: area of Anodisc Al203 membrane (pm 2 ); K2: area of field of view (pm 2 ); V: volume of buffer (mL); m: mass of soil (g); ω: water content of soil (%); f: concentrating the virus solution by 10-1000 times by using a 30-50K ultrafiltration tube to prepare a virus concentrated solution, adding the virus concentrated solution to a copper mesh grid, negatively staining the grid with 1% phosphotungstic acid, and observing the virus morphology by using a transmission electron microscope.

2. The method of efficient extraction and detection of red loam viruses as claimed in claim 1, wherein, In step S5, the primer sequences used in PCR amplification are 341F and 806R, and the sequences of 341F and 806R are as follows: 341F: CCTACGGGNGGCWGCAG; 806R: GGACTACHVGGGTATCTAAT.

3. The method of efficient extraction and detection of red loam viruses as claimed in claim 1 wherein, In step a, the filter membrane is an Anodisc Al2O3 filter membrane with a pore size of 0.02 μm.

4. The method of efficient extraction and detection of red loam viruses as claimed in claim 1 wherein, In step b, the staining solution is a SYBR GREEN I solution.

5. The method of efficient extraction and detection of red loam viruses as claimed in claim 1 wherein, In step c, the preparation method of the fade-proof solution is as follows: adding 1% by volume of a 10% p-phenylenediamine solution to 99% by volume of a glycerol and / or PBS solution.

6. The method of efficient extraction and detection of red loam viruses as claimed in claim 1 wherein, In step f, the negative staining time of the 1% phosphotungstic acid is 1-2 minutes.

Images