A method for improving the efficiency of virus extraction from sludge

Abstract

The present application provides a method for improving the efficiency of virus extraction in sludge, which belongs to the technical field of safe sludge treatment, and includes the following steps: before extracting the virus from the sludge, adding ethylenediaminetetraacetate and lysozyme to the original sludge sample to pre-treat the original sludge; extracting the virus from the pre-treated sludge. The present application adds ethylenediaminetetraacetate and lysozyme to the original sludge sample to pre-treat the original sludge. Ethylenediaminetetraacetate can promote the shedding of viruses in the sludge from the surface of the sludge solid particles; lysozyme can inhibit the denaturation of viral cells, thereby improving the efficiency of virus enrichment in the sludge; and the residue of lysozyme can stabilize the viral cells in the sludge, overcoming the defect that the virus in the sludge is easily denatured after extraction, resulting in inaccurate analysis of the virus types in the sludge, and can improve the amplification efficiency of the viral genes in the sludge in the qPCR amplification system, so as to improve the detection limit of the viral genes in the sludge.

Description

Technical Field

[0001] The present application belongs to the technical field of safe sludge treatment, and more specifically, to a method for improving the efficiency of virus extraction in sludge. Background Art

[0002] Sludge not only has a high water content, is easy to rot, and has a strong odor, but is also rich in a variety of nutrients and is a good nutrient base for pathogens. Municipal sewage treatment plants receive a large amount of wastewater rich in organic pollutants discharged by urban residents. About 90% of pathogenic microorganisms can be enriched from sewage to sludge through the sewage treatment process. If not properly treated, the subsequent sludge transportation, landfill, land use and other processes are very likely to cause the risk of spreading pathogenic microorganisms.

[0003] However, sludge contains a large amount of solid particles and gel-like structure EPS, and has a complex microbial community structure, which can firmly bind to viruses in sludge. As a result, traditional sewage virus extraction methods are unable to effectively extract viruses from sludge, and the extracted total viral genes cannot accurately reflect the actual occurrence characteristics of viruses in sludge. Summary of the invention

[0004] The purpose of this application is to provide a method for improving the efficiency of virus extraction in sludge based on an EDTA-coupled lysozyme pretreatment process. This method effectively improves the subsequent QPCR amplification efficiency by destroying the binding force between sludge and virus and shielding the amplification factors that affect the sludge virus gene, thereby improving the concentration and detection efficiency of viruses in sludge, that is, improving the extraction efficiency of viruses.

[0005] To achieve the above object, the present application provides a method for improving the efficiency of virus extraction in sludge, comprising the following steps:

[0006] Before extracting the virus from the sludge, ethylenediaminetetraacetate and lysozyme are added to the original sludge sample to pre-treat the original sludge;

[0007] The pretreated sludge is subjected to virus extraction.

[0008] Furthermore, the preprocessing comprises the following steps:

[0009] Adding the ethylenediaminetetraacetate to the original sludge sample and stirring evenly to perform a first reaction;

[0010] After the first reaction is completed, lysozyme is added to carry out the second reaction.

[0011] Furthermore, based on dry sludge weight (TSS), 0.1 to 0.3 g of the ethylenediaminetetraacetic acid salt is added per gram of dry sludge.

[0012] Furthermore, based on the dry weight of the sludge, 10 to 30% of lysozyme is added to each gram of dry sludge.

[0013] Furthermore, the temperature of the first reaction is -4±0.5°C and the time is 1 to 1.5 hours.

[0014] Furthermore, the temperature of the second reaction is -4±0.5°C and the time is 2 to 2.5 hours.

[0015] Furthermore, the ethylenediaminetetraacetate is any one of EDTA-Na2, EDTA-Na3, Na4EDTA·4H2O, and EDTA-K2.

[0016] Furthermore, the virus extraction comprises the following steps:

[0017] Adding beef extract buffer to the pretreated sludge, mixing and performing a third reaction, then ultrasonicating and centrifuging, and collecting the supernatant;

[0018] The supernatant is filtered, and an iron flocculant is added to the filtrate to precipitate the sludge virus;

[0019] The settled sludge virus is resuspended with oxalic acid buffer, and RNA is extracted and reverse transcribed after resuspension; the type and content of the sludge virus are determined by QPCR amplification system.

[0020] Furthermore, the temperature of the third reaction is 4±0.5° C., and the time is 2 to 2.5 h; and / or the filtration is performed using 0.45 um and 0.22 um water filter membranes, respectively.

[0021] Furthermore, the iron flocculant is any one of ferric chloride (FeCl3), potassium iron oxalate (K3[Fe(C2O4)3]), and ferric sulfate (Fe2(SO4)3).

[0022] Compared with the prior art, this application has the following technical effects:

[0023] The present invention discloses a method for improving the efficiency of virus extraction in sludge. Before extracting the virus from the sludge, ethylenediaminetetraacetate and lysozyme are added to the original sludge sample to pretreat the original sludge. By pretreating the sludge with ethylenediaminetetraacetate, the outer wall layer containing lipopolysaccharide can be removed from the G-part of the virus base pair, which helps to dissolve the lipid compounds in the sludge, thereby weakening the binding force between the sludge and the virus, and further promoting the shedding of the virus in the sludge from the surface of the sludge solid particles; lysozyme can directly act on the negatively charged virus protein, form complex salts with viral genes such as DNA and RNA, inhibit the denaturation of virus cells, and thus improve the virus enrichment efficiency in the sludge; and the residual lysozyme can stabilize the virus cells in the sludge, overcoming the defect that the virus in the sludge is easily denatured after extraction, resulting in inaccurate analysis of the virus type in the sludge, and can improve the amplification efficiency of the virus gene in the sludge in the qPCR amplification system, so as to improve the detection limit of the virus gene in the sludge. The EDTA-coupled lysozyme pretreatment sludge of the present application can promote the efficiency of virus extraction and analytical detection in sludge, thereby reflecting the actual occurrence characteristics and distribution of viruses in municipal sludge, and can provide technical guidance for the sanitary and safe disposal of sludge in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0025] Figure 1 The comparison of virus extraction efficiency measured by different pretreatment methods in the examples of the present application and the comparative examples is shown;

[0026] Figure 2 The effects of different pretreatment methods on the subsequent extraction efficiency of human viruses from sludge were demonstrated;

[0027] Figure 3 The effects of different pretreatment methods on the subsequent extraction efficiency of enveloped viruses from sludge were demonstrated;

[0028] Figure 4 The effects of different pretreatment methods on the subsequent extraction efficiency of non-enveloped viruses from sludge were demonstrated. DETAILED DESCRIPTION

[0029] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0030] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0031] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0032] The weight of the relevant components mentioned in the specification of the examples of this application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the specification of the examples of this application, it is within the scope disclosed in the specification of the examples of this application. Specifically, the mass described in the specification of the examples of this application can be a mass unit known in the biochemical field such as μg, mg, g, kg, etc.

[0033] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0034] In the present application, nine viruses with relatively high contents in sludge, including enterovirus 71, rotavirus, SARS-CoV-2 variant, hepatitis A virus, astrovirus, human parechovirus, poliovirus, and viroid, are used to represent typical pathogenic microorganisms, and the effects of different sludge pretreatment methods on virus extraction efficiency are evaluated to establish a complete method for extracting and analyzing pathogenic microorganisms in sludge.

[0035] Example 1

[0036] Example 1 of the present application provides a method for improving the efficiency of virus extraction in sludge, comprising the following steps:

[0037] (1) Sludge pretreatment (EDTA-Na2+ enzyme): First, take 50 ml of original sludge, add 0.2 g / g-TSS EDTA-Na2 (i.e., add 0.2 g EDTA-Na2 per gram of dry sludge), stir at -4°C for 1 hour, then add 15%-TSS lysozyme (i.e., add 15 wt% lysozyme per gram of dry sludge), stir at -4°C for 30 minutes, and store at -4°C for 2 hours, waiting for the subsequent sludge virus DNA extraction step.

[0038] The sludge virus DNA extraction can be carried out using the current existing technology. The sludge virus extraction steps of the embodiment of the present application are as follows:

[0039] (2) Sludge virus extraction: 50 ml of the pretreated sludge sample was placed in a clean centrifuge tube, and after ultrasonication for 15 minutes, the sludge was centrifuged at 8000 rpm for 30 minutes. The supernatant was discarded and the original volume was supplemented with 3% beef extract buffer (pH = 9). After thorough vortexing for five minutes to mix well, the sludge was placed in a shaker at 200 rpm and a temperature of 4±0.5°C for 2 hours (shaking box reaction, manually shaking up and down every 15 minutes to make the system uniform); then After that, ultrasonicate for 15 minutes (80% power), mix thoroughly with a vortex machine, and then centrifuge at 8000 rpm for half an hour. Finally, pass the supernatant through 0.45 and 0.22 micron water filter membranes in sequence; add 1 mg / L ferric chloride to the liquid phase after passing through the membrane and let it settle at 4±0.5°C for 12 hours to flocculate the virus, then centrifuge at 12000 rpm for 30 minutes at 4±0.5°C, and resuspend the obtained flocs with 10 ml of oxalic acid standard buffer (pH 6.8). Use an RNA extraction kit to extract RNA from the resuspended sample, and then use an RNA reverse transcription kit for reverse transcription, and then establish a PCR reaction system to determine the total number of viral genes.

[0040] Among them, the RNA extraction kit is the Solarbio RNA virus genome extraction kit, and the steps for extracting the total viral genetic material RNA are as follows:

[0041] Before extraction, 60 ml of absolute ethanol was added to the rinse solution. All centrifugation steps were performed at 2-8°C.

[0042] 1) Take 1.5 ml of the virus supernatant from step (2), centrifuge at 12000 rpm for 5 min, aspirate as much of the supernatant as possible, and discard the precipitate (if there is no precipitate, this step can be omitted);

[0043] 2) Add 60ul of 10mg / ml proteinase K to the viral supernatant, mix thoroughly, and digest at 65℃ for 10min. During this period, invert the centrifuge tube several times to mix well.

[0044] 3) Pretreatment of adsorption column: Take out the adsorption column from the package, put it into the collection tube, add 700ul of column washing solution, leave it at room temperature for 2 minutes, centrifuge it at 2-8℃ and 12000rpm for 2min, discard the waste liquid, and put the adsorption column into the collection tube for later use;

[0045] 4) Add 500ul of binding solution to the virus supernatant and mix thoroughly. Then add 400ul of anhydrous ethanol to the tube and mix thoroughly. Flocculent precipitation may appear at this time, which does not affect the extraction of RNA. The solution and flocculent precipitation can be added to the adsorption column and allowed to stand for 2 minutes (the maximum volume of the adsorption column is 750ul, which can be added several times. After one adsorption and centrifugation, add the remaining mixed liquid to the column and let it stand for centrifugation);

[0046] 5) Centrifuge at 12000 rpm for 2 min, discard the waste liquid, and place the adsorption column into a collection tube;

[0047] 6) Add 700ul of rinse solution to the adsorption column (check whether anhydrous ethanol has been added before use), centrifuge at 12000rpm for 1min, discard the waste liquid, and place the adsorption column in a collection tube;

[0048] 7) Add 500ul of rinse solution to the adsorption column, centrifuge at 12000rpm for 1min, discard the waste liquid, and place the adsorption column in a collection tube;

[0049] 8) Centrifuge at 12000 rpm for 2 minutes, and place the adsorption column at room temperature or in a 50°C incubator for several minutes to remove the residual rinse solution in the adsorption column. Otherwise, the ethanol in the rinse solution will affect subsequent experiments such as enzyme digestion and PCR.

[0050] 9) Place the adsorption column in a clean centrifuge tube, add 50ul-100ul of RNase free dH2O preheated in a 65°C water bath to the center of the adsorption column, let it stand at room temperature for 5 minutes, and centrifuge at 12000rpm for 2 minutes to obtain high-quality viral genomic RNA.

[0051] The extracted RNA needs to be reverse transcribed into cDNA in time for easy storage. The steps for reverse transcription of RNA are as follows:

[0052] 1) Add the following reaction components to an RNase-free centrifuge tube on ice:

[0053] 1-5 μg total RNA

[0054] 2 pmole of the gene-specific primers in Table 1 below

[0055] Add RNase-free ddH2O to 14.5uL;

[0056] 2) After keeping at 70℃ for 5 minutes, cool on ice for 2 minutes, collect the reaction solution by centrifugation and add the following components:

[0057] 4uL 5×M-MLV Buffer

[0058] 1 μL dNTPs

[0059] 0.5 μL RNasin

[0060] 1 μL M-MLV;

[0061] 3) Incubate at 42°C for 60 min. If using random primers, first incubate the centrifuge tube at 25°C for 10 min, then incubate at 42°C for 60 min.

[0062] 4) Heat at 95°C for 5 min to terminate the reaction and place on ice for subsequent experiments or store at -20°C.

[0063] The extracted DNA / cDNA was mixed with viral primers to construct a qPCR reaction system. The viral primer sequences are shown in Table 1. The cycle threshold (Ct value) obtained by the machine test was compared with the standard curve to determine the final concentration of the virus (gene copy number), and each sample was repeated three times. The test and construction of the standard curve were completed by MegaGene Testing Company.

[0064] Table 1 Virus primer sequences

[0065]

[0066] The specific method for establishing the standard curve is as follows: the PCR product obtained after the sample is amplified with the primer as the template is tested by agarose gel electrophoresis, the target DNA fragment is recovered by gel excision, connected to Pgem-T Easy Vector (3015bp), and then transferred into DH5a competent Escherichia coli, which is spread on a culture dish containing ampicillin and cultured overnight. The monoclonal strain is picked, and after qPCR verification, the culture is expanded, and the plasmid DNA is extracted for standby use. According to the concentration ratio, 109 copies / uL, 108 copies / uL, 107 copies / uL, 106 copies / uL, and 105 copies / uL of plasmid DNA are diluted respectively for the construction of the standard curve. The concentrations of the 9 viruses and the corresponding copy numbers are shown in Table 2.

[0067] Table 2 Concentration and copy number of plasmid DNA used in the experiment

[0068]

[0069] The construction of the qPCR reaction system was based on the kit method (new 2×SYBR Green PCR Mastermix, Solebio), and the primer concentration and nucleic acid volume were shown in Table 3.

[0070] Table 3 qPCR reaction system

[0071]

[0072] The reaction procedure of the qPCR test is shown in Table 4 , and the selected temperature range is suitable for most viral primers to produce corresponding fluorescence reactions.

[0073] Table 4 qPCR reaction procedure

[0074]

[0075] Example 2

[0076] The difference from Example 1 is that 0.1 g / g-TSS EDTA-Na2 and 30%-TSS lysozyme are added in step (1), and other conditions are the same.

[0077] Example 3

[0078] The difference from Example 1 is that 0.3 g / g-TSS EDTA-Na2 and 10%-TSS lysozyme are added in step (1), and other conditions are the same.

[0079] Comparative Example

[0080] The difference between this embodiment and Example 1 is that the sludge pretreatment step in step (1) is different, and the other steps are the same. Specifically, the following different pretreatment methods are performed respectively:

[0081] (1) Enzyme: Take 50 ml of original sludge, add 15%-TSS lysozyme, stir at -4°C for 30 minutes, and store at -4°C for 2 hours, waiting for the subsequent sludge viral DNA extraction step.

[0082] (2) Alkali: Take 50 ml of original sludge, add 0.2 g / g-TSS NaOH, stir at -4 °C for 1 hour, and wait for the subsequent sludge viral DNA extraction step.

[0083] (3) EDTA-Na2: Take 50 ml of original sludge, add 0.2 g / g-TSS EDTA-Na2, stir at -4°C for 1 hour, and wait for the subsequent sludge viral DNA extraction step.

[0084] (4) Enzyme + alkali: Take 50 ml of the original sludge, add 15%-TSS lysozyme, and stir at -4°C for 30 minutes, add 0.2 g / g-TSS NaOH, stir at -4°C for 1 hour, and wait for the subsequent sludge virus DNA extraction step.

[0085] (5) Alkali + enzyme: Take 50 ml of original sludge, add 0.2 g / g-TSS NaOH, stir at -4 °C for 1 hour, then add 15%-TSS lysozyme, stir at -4 °C for 30 minutes, and wait for the subsequent sludge virus DNA extraction step.

[0086] (6) Enzyme + EDTA-Na2: Take 50 ml of original sludge, add 15%-TSS lysozyme, and stir at -4°C for 30 minutes. Add 0.2 g / g-TSS EDTA-Na2, stir at -4°C for 1 hour, and wait for the subsequent sludge virus DNA extraction step.

[0087] (7) Alkali + enzyme + EDTA-Na2: Take 50 ml of original sludge, add 0.2 g / g-TSS NaOH, stir at -4°C for 1 hour, then add 15%-TSS lysozyme, stir at -4°C for 30 minutes, and finally add 0.2 g / g-TSSEDTA-Na2, stir at -4°C for 1 hour, and wait for the subsequent sludge virus DNA extraction step.

[0088] (8) Raw mud: without any pretreatment process.

[0089] The virus extraction efficiencies measured by different pretreatment methods in Example 1 of the present application and various comparative examples are shown in Tables 5 and Figure 1 shown.

[0090] Table 5 Extraction efficiency of different pretreatment methods

[0091]

[0092] From Table 5 and Figure 1It can be seen that after the embodiment of the present application, that is, EDTA-Na2+ enzyme pretreatment, the extraction efficiencies of viruses such as enterovirus 71, rotavirus, SARS-CoV-2 variant, hepatitis A virus, astrovirus, human parechovirus, poliovirus, viroid, and rhinovirus in the sludge are 27.10%, 88.53%, 404.56%, 192.11%, 1034.3%, 132.23%, 226.15%, 290.39%, and 347.42%, respectively. Among them, the extraction efficiencies of SARS-CoV-2 variant, hepatitis A virus, astrovirus, human parechovirus, poliovirus, viroid, and rhinovirus are more than 100%, which is significantly better than the virus extraction efficiencies obtained by other treatment methods, indicating that after EDTA-Na2+ enzyme pretreatment, the extraction efficiency of most viruses in the sludge is significantly improved.

[0093] from Figure 1 The effects of different pretreatment methods on the gene extraction efficiency of human viruses (enterovirus 71, human parechovirus, poliovirus, viroids), enveloped viruses (SARS-CoV-2 variants, rhinoviruses) and non-enveloped viruses (rotavirus, hepatitis A virus, astrovirus) in subsequent sludge can also be further evaluated.

[0094] Figure 2 The effects of different pretreatment methods on the extraction efficiency of human viruses (enterovirus 71, human parechovirus, poliovirus, viroid) in subsequent sludge were demonstrated. Figure 2 It can be seen that the extraction efficiency of three human viruses, human parechovirus, poliovirus and viroid, in the subsequent sludge by EDTA-Na2+ enzyme pretreatment process exceeded that of the original sludge, which were 132.23%, 226.15% and 290.39% respectively.

[0095] Figure 3 The effects of different pretreatment methods on the extraction efficiency of enveloped viruses (SARS-CoV-2 variants, rhinovirus) in subsequent sludge were demonstrated. Figure 3 It can be seen that the EDTA-Na2+ enzyme pretreatment process has the highest improvement in the extraction efficiency of SARS-CoV-2 variants and rhinovirus, two enveloped viruses in the subsequent sludge, which are 404.56% and 347.42% respectively.

[0096] Figure 4 The effects of different pretreatment methods on the extraction efficiency of non-enveloped viruses (rotavirus, hepatitis A virus, astrovirus) in subsequent sludge are demonstrated. Figure 4It can be seen that the extraction efficiency of hepatitis A virus and astrovirus in the subsequent sludge during the EDTA-Na2+ enzyme pretreatment process exceeded that of the original sludge, which were 192.11% and 1034.3% respectively. This shows that the EDTA-Na2+ enzyme pretreatment process is conducive to the extraction and enrichment of the total genes of three major types of viruses, including human viruses, enveloped viruses and non-enveloped viruses in the subsequent sludge.

[0097] Figure 1 In the figure, the nine vertical columns corresponding to each treatment method represent enterovirus 71, rotavirus, SARS-CoV-2 variant, hepatitis A virus, astrovirus, human parechovirus, poliovirus, viroid, and rhinovirus from left to right.

[0098] Figure 2 In the figure, the four vertical columns corresponding to each treatment method represent enterovirus 71, human parechovirus, poliovirus, and viroid from left to right.

[0099] Figure 3 In the figure, the two vertical columns corresponding to each treatment method represent SARS-CoV-2 variants and rhinovirus from left to right.

[0100] Figure 4 In the figure, the three vertical columns corresponding to each treatment method represent rotavirus, hepatitis A virus, and astrovirus from left to right.

[0101] The embodiment of the present application utilizes EDTA coupled with lysozyme to pretreat the sludge, dissociate the adsorption of the virus on the surface of the sludge solid particles and make it fall off, and then the beef extract buffer (BE) elution method is combined with proteinase K to extract the total viral gene. On this basis, the total viral gene RNA is reverse transcribed, and then the virus type and content are determined by the QPCR amplification system. Among them, the binding effect between the virus and the sludge solid particles can be weakened by the EDTA and lysozyme pretreatment method, and the amplification factors that affect the sludge virus gene can be shielded, which can effectively improve the subsequent QPCR amplification efficiency, thereby improving the concentration and detection efficiency of the virus in the sludge. The embodiment of the present application optimizes the sludge pretreatment step to improve the extraction efficiency of the virus in the sludge, provides a theoretical basis for the occurrence characteristics and inactivation mechanism of the virus in the subsequent sludge stabilization treatment process, and facilitates the optimization research of the subsequent sludge stabilization treatment technology, thereby reducing the risk of virus transmission caused by municipal sludge.

[0102] The raw materials required for the method established in the examples of the present application are cheap and readily available, the reaction conditions are mild, and the operation is convenient.

[0103] The above embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present application. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the attached claims.

Claims

1. A method for improving the efficiency of virus extraction from sludge, characterized in that: The following steps are involved: Before extracting the virus from the sludge, ethylenediaminetetraacetate and lysozyme are added to the original sludge sample to pre-treat the original sludge; Extracting viruses from the pretreated sludge; Among them, the extraction efficiency of SARS-CoV-2 variants, hepatitis A virus, astrovirus, and rhinovirus was improved by adding EDTA first and then lysozyme; Alternatively, lysozyme is added first and then EDTA is added, and the extraction efficiency of rotavirus and viroid is improved.

2. A method for improving the efficiency of virus extraction from sludge as claimed in claim 1, characterized in that: The pre-processing comprises the following steps: Adding the ethylenediaminetetraacetate to the original sludge sample and stirring evenly to perform a first reaction; After the first reaction is completed, lysozyme is added to carry out the second reaction.

3. A method for improving the efficiency of virus extraction from sludge as claimed in claim 2, characterized in that: Based on the dry weight of the sludge, 0.1-0.3 g of the ethylenediaminetetraacetic acid salt is added to each gram of dry sludge.

4. A method for improving the efficiency of virus extraction from sludge as claimed in claim 2, characterized in that: Based on the dry weight of the sludge, add 10-30% lysozyme to each gram of dry sludge.

5. A method for improving the efficiency of virus extraction from sludge as claimed in claim 2, characterized in that: The temperature of the first reaction is -4±0.5°C and the time is 1~1.5 h.

6. A method for improving the efficiency of virus extraction from sludge as claimed in claim 2, characterized in that: The temperature of the second reaction is -4±0.5°C and the time is 2~2.5 h.

7. A method for improving the efficiency of virus extraction from sludge as claimed in claim 1, characterized in that: The ethylenediaminetetraacetate is any one of EDTA-Na2, EDTA-Na3, Na4EDTA·4H2O, and EDTA-K2.

8. A method for improving the efficiency of virus extraction from sludge according to any one of claims 1 to 7, characterized in that: The virus extraction comprises the following steps: Adding beef extract buffer to the pretreated sludge, mixing and performing a third reaction, then ultrasonicating and centrifuging, and collecting the supernatant; The supernatant is filtered, and an iron flocculant is added to the filtrate to precipitate the sludge virus; The settled sludge virus is resuspended with oxalic acid buffer, and RNA is extracted and reverse transcribed after resuspension; the type and content of the sludge virus are determined by QPCR amplification system.

9. A method for improving the efficiency of virus extraction from sludge as claimed in claim 8, characterized in that: The temperature of the third reaction is 4±0.5°C and the time is 2-2.5 h; and / or the filtration is performed using 0.45 um and 0.22 um water filter membranes in sequence.

10. A method for improving the efficiency of virus extraction from sludge according to claim 8, characterized in that: The iron flocculant is any one of ferric chloride, potassium ferric oxalate and ferric sulfate.

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