Method for eluting and enriching virions in high-heterogeneity household garbage sample

Abstract

The invention discloses a method for eluting and enriching virions in a high-heterogeneity household garbage sample, and relates to the technical field of biology. The elution and enrichment method comprises the following steps: sampling household garbage in a target area to obtain a representative garbage sample; performing homogenization pretreatment on the garbage sample to obtain a homogenized garbage sample; extracting viruses in the homogenized garbage sample into a liquid phase by adopting a neutral inorganic solution, and purifying to obtain a virus eluent; and enriching the viruses in the virus eluent by adopting a ferric salt flocculation method. The method not only fills the blank of the method for eluting and enriching the viruses in the high-heterogeneity household garbage, but also provides reliable technical support for researching the occurrence characteristics, the transmission mechanism and the potential ecological health risk of the viruses in the household garbage, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for eluting and enriching virus particles of highly heterogeneous domestic garbage samples. Background Art

[0002] The temporary storage, transportation, and treatment of HMSW are closely related to the health and safety of residents and employees. During these operations, microorganisms carried or bred in HMSW will inevitably pose potential risks to the surrounding people and the environment. Especially in the context of frequent outbreaks of viral infectious diseases, viral contamination in HMSW and the secondary transmission risks it causes have attracted widespread attention.

[0003] Existing studies have shown that HMSW is an important virus carrier. Some research institutions have found a variety of pathogenic viruses in domestic waste landfills and their leachate, including enterovirus, norovirus, hepatitis B virus, and herpes virus. In addition, high-risk viruses with the potential for transmission and spread have also been detected in domestic waste landfills and their leachate. These studies have confirmed that the viruses in HMSW pose a serious threat to the health of surrounding residents and operators.

[0004] Although the methods of viral nucleic acid extraction and macrovirome detection are relatively mature, there is still a lack of efficient and feasible methods for extracting viruses from domestic waste. This gap in the field limits the in-depth study of the characteristics of virus occurrence, transmission mechanism and potential ecological health risks in HMSW, and also hinders the formulation of relevant public health prevention and control measures.

[0005] At present, there are a few related patents that provide methods for extracting viruses in media such as wastewater and sludge, mainly including steps such as concentration and extraction of virus particles and nucleic acids. For example, Chinese invention patent CN117701369A discloses a method for extracting viruses in wastewater, which is based on the principle of ultrafiltration to construct a multi-channel device to achieve the concentration and separation of viruses in sewage. However, this method is mainly aimed at liquid matrices and cannot effectively contact and separate viral ions in solid media. Another Chinese invention patent (CN117778381A) discloses a method for extracting and enriching viruses in sludge based on EDTA-coupled lysozyme pretreatment, but the method of rupturing cells may cause high sudden toxicity virus preference to be too high. In addition, most of the existing methods are aimed at homogeneous media, and when dealing with high organic matter content and highly heterogeneous complex solid matrices, they often face problems such as complex operation, low virus recovery rate, and large detection interference. Therefore, there is still a lack of an effective method for extracting viruses in highly heterogeneous domestic waste. Summary of the invention

[0006] The purpose of the present invention is to provide a method for eluting and enriching virus particles from highly heterogeneous domestic waste samples, so as to solve the problems existing in the above-mentioned prior art, overcome the extremely unstable virus recovery rate and complex operation problems faced by the prior art when treating highly heterogeneous complex matrices in solid waste, and significantly improve the representativeness of the recovery rate of virus particles in highly heterogeneous samples. This method not only fills the gap in virus enrichment and extraction methods in highly heterogeneous domestic waste, but also provides reliable technical support for studying the characteristics of virus occurrence, transmission mechanism and potential ecological health risks in domestic waste, and has broad application prospects.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a method for eluting and enriching virus particles in highly heterogeneous domestic waste, comprising the following steps:

[0009] Sampling domestic waste in the target area to obtain representative waste samples;

[0010] Performing homogenization pretreatment on the garbage sample to obtain a homogenized garbage sample;

[0011] The virus in the homogenized garbage sample is extracted into a liquid phase using an inorganic neutral solution, and then purified to obtain a virus eluate;

[0012] The virus in the virus eluate is enriched by using an iron salt flocculation method.

[0013] Furthermore, the biomass components in the garbage sample are pre-treated by homogenization using a low-temperature freezing and crushing method.

[0014] Furthermore, the cryogen used in the low-temperature freezing and crushing method is dry ice or liquid nitrogen; and / or

[0015] When the refrigerant is dry ice, the mass ratio of the biomass component to the dry ice is (30-15): 1

[0016] When the cryogen is liquid nitrogen, the mass ratio of the biological component to the liquid nitrogen is (75-150):1.

[0017] Furthermore, the homogenization pretreatment time is ≥30s.

[0018] Furthermore, the non-biomass components in the garbage sample are crushed under sterile conditions to control the particle size to ≤ 2 cm.

[0019] Furthermore, the eluent used in the solution elution method includes the following components: NaCl 5-10 g / L, KCl 0.1-0.5 g / L, Na 2 HPO4 1-1.5g / L and KH 2 PO 4 0.1-0.5g / L.

[0020] Furthermore, the mass ratio of the homogenized garbage sample and the eluent is (3-5):10.

[0021] Furthermore, the iron salt flocculation method is to use 0.2 mol / L iron salt solution to flocculate the virus eluate.

[0022] Iron salts used include FeCl 3 , Fe 2 (SO 4 ) 3 wait.

[0023] Furthermore, the volume ratio of the iron salt solution to the virus eluate is 1:(50-100).

[0024] Furthermore, the flocculation treatment time is ≥10 min.

[0025] Furthermore, the operating temperature of the virus extraction into liquid phase and the iron salt flocculation method is ≤4°C.

[0026] The method for eluting and enriching virus particles from domestic waste samples provided by the present invention has the following advantages:

[0027] (1) Wide applicability and superior performance: The homogenization pretreatment overcomes the main problems of high heterogeneity and complex components of solid waste, significantly improving the representativeness of the extracted virus samples and making it suitable for various types of domestic waste and their different components; the multi-step treatment and optimized enrichment method effectively improves the virus enrichment rate, and the enrichment rate is significantly higher than that of the PEG precipitation method and the ultrafiltration method.

[0028] (2) Easy to operate: The process design is clear, no special equipment is required for virus extraction, and inorganic solvents are used to avoid the interference of organic matter dissolution from high-organic waste on subsequent operations and detection, making it easy to operate in a laboratory environment.

[0029] (3) Short processing time: The optimized steps and conditions significantly shorten the overall processing time and improve work efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 The present invention is a flow chart of the method for eluting and enriching virus particles from domestic waste samples;

[0032] Figure 2 It is a statistical diagram of the elution effect using IO-7 solution (the inorganic neutral eluent of the present invention) as the eluent;

[0033] Figure 3 It is a statistical diagram of the elution effect using O-7 solution (an organic neutral eluent used for technical effect comparison) as the eluent;

[0034] Figure 4 This is a comparison chart of the enrichment effect of viral nucleic acid in domestic waste by iron salt flocculation method and other enrichment methods (PEG precipitation method, ultrafiltration method). DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is specifically included. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] The present invention provides a method for eluting and enriching virus particles in highly heterogeneous domestic waste (see flow chart Figure 1 ), including the following steps:

[0041] Sampling domestic waste in the target area to obtain representative samples;

[0042] The samples were pre-treated by homogenization, where: the biomass component was pre-treated by homogenization using a low-temperature freezing crushing method; the non-biomass component was mechanically crushed under sterile conditions to control the particle size to ≤ 2 cm;

[0043] The virus in the homogenized garbage sample is extracted into the liquid phase using an inorganic neutral solution, and the virus eluate is obtained by repeated leaching, centrifugation and removal of free DNA;

[0044] The virus in the virus eluate was enriched by iron salt flocculation method.

[0045] In the embodiment, the preparation method of the inorganic neutral eluent (IO-7) is as follows: 8 g NaCl, 0.2 g KCl, 1.44 g Na 2 HPO 4 and 0.24 g KH 2 PO 4 Dissolve in 1 L of deionized water and adjust the pH to 7.4 with 1 mol / L HCl.

[0046] In the example, the preparation method of the organic neutral eluent (O-7) is as follows: 7.5 g beef extract and 1.88 g glycine are dissolved in 500 mL deionized water, and the pH value is adjusted to 7.4 with 1 mol / L NaOH.

[0047] The solution prepared above was transferred to a glass container and sterilized at 120° C. for 20 minutes. The sterilized solution was stored at 4° C. until use.

[0048] Example 1

[0049] (1) Homogenization pretreatment

[0050] Take 300g of kitchen waste and 20g of dry ice mixture (the mass ratio of kitchen waste and dry ice can be set in the range of 20-15:1) and place it in a sterile stainless steel crusher (model: SS-1022, 34000r / min) for 60s (30-60s can achieve the same effect), and crush the garbage sample at low temperature to obtain a kitchen waste sample with good homogeneity.

[0051] The g23 gene of T4 phage in kitchen waste was selected as the marker gene because the gene is highly conserved and the species to which it belongs, T4 phage, has been proven to be widely distributed in various habitats, so the conclusion formed is highly reliable. The comparative analysis of the content of viral marker gene (g23) in domestic waste before and after homogenization pretreatment can clarify the necessity and superiority of homogenization treatment of domestic waste. The results of homogenization are shown in Table 1.

[0052] Table 1 Contents of viral marker genes in kitchen waste samples before and after homogenization pretreatment

[0053]

[0054] Note: * is an organic eluent, used as a control treatment for the method of the present invention.

[0055] The results in Table 1 show that the g23 gene content in the pretreated samples is lower than that in the untreated samples, but the uniformity of the values ​​(SD value) is significantly improved, indicating that the homogenization pretreatment effectively reduces the interference caused by high heterogeneity in the samples and improves the accuracy of subsequent virus data processing and information extraction.

[0056] (2) Elution treatment

[0057] The elution treatment effect was tested by adding model virus to detect the internal standard recovery rate. 10 8 PFU / mL, 10 6 PFU / mL and 10 4 PFU / mL of Phi6 model virus. Then, incubate at 4°C for 1 h, and transfer the virus-carrying sample to a 50 mL sterile centrifuge tube using sterile tweezers.

[0058] Add 25mL IO-7 eluent to the above centrifuge tube, stir and mix thoroughly, let stand overnight at 4°C, and then treat with an ultrasonic instrument at 0.03W / mL power for 5min; centrifuge the mixed solution at 4°C and 1000g for 2min to precipitate domestic waste, and collect the supernatant; continue to add eluent to the precipitate for resuspending, and the eluent added each time just covers the precipitate, and repeat the low-speed centrifugation step; combine the supernatants obtained in the above steps, centrifuge at 4°C and 5000g for 8min, and collect the supernatant; filter the supernatant through 0.45μm and 0.22μm water filter membranes in turn, and collect the filtrate; add DNase I (to a final concentration of 20U / mL) to the filtrate, and culture at 37°C for 1h to further remove free extracellular DNA; filter the filtrate after enzyme treatment through a 0.22μm water filter membrane again to further remove miscellaneous bacteria to obtain a virus eluate.

[0059] The elution rate was determined by virus titer experiment. Figure 2-Figure 3 shown.

[0060] (3) Enrichment treatment

[0061] FeCl 3 The viruses in the virus elution were enriched by flocculation, PEG precipitation and ultrafiltration respectively. The g23 gene of T4 phage in kitchen waste was selected as the marker gene, and the elution and enrichment effects of each elution and enrichment method on viral nucleic acid in domestic waste were evaluated for the same reason as the homogenization part.

[0062] FeCl 3 The specific steps of the flocculation method are as follows: 3 The solution was added dropwise to the virus solution at a volume ratio of 1:100 (V 铁盐溶液 :V 病毒洗脱溶液 =1:100), the mixture was shaken at 4°C at 5000 rpm for 10 min; the supernatant was filtered through a 0.45 μm water filter, and the filter was collected for sequencing analysis.

[0063] The specific steps of the PEG precipitation method are as follows: adjust the pH value of the virus solution to 7.0, add PEG-6000 with a final concentration of 10% and NaCl with a final concentration of 0.5 mol / L to the virus solution, place it at 4°C overnight, centrifuge at 4°C, 8000r / min for 30min, retain the virus precipitate and resuspend it for sequencing analysis.

[0064] The specific steps of the ultrafiltration method are as follows: centrifuge the 30 kDa ultrafiltration tube added with the virus solution at 4°C and 4000 g, repeating the process several times until the volume of the retentate is about 1 mL, which is used for g23 gene content analysis.

[0065] FeCl 3 The enrichment effect of flocculation method and other enrichment methods (PEG precipitation method, ultrafiltration method) on viral nucleic acid in domestic waste is as follows Figure 4 FeCl 3 The enrichment multiples of flocculation, PEG precipitation and ultrafiltration were 238.7, 209.6 and 39.4 times, respectively. They were easy to operate and took less time.

[0066] Example 2

[0067] The method constructed by the present invention was further verified using classified and unclassified domestic waste.

[0068] According to "CJT313-2009 Sampling and Analysis Methods for Domestic Waste", domestic waste in the target area is sampled. During sampling, 2kg of kitchen waste, 2kg of other waste or 2kg of mixed waste are collected from each garbage bin. The collected samples are fully mixed and divided into four parts strictly according to the quartering method. The two parts of the waste in the opposite position are retained, and low-temperature transportation and storage are ensured before the subsequent steps.

[0069] Configure different concentrations (10 4 PFU / mL, 10 6 PFU / mL and 10 8 PFU / mL) of DNA model virus (T7 phage) and RNA model virus (Phi6) culture fluid were inoculated into different components of the above-mentioned domestic waste by hanging drop method and cultured at 4°C for 1 hour. The experimental groups with different components and different exogenous virus inoculation concentrations were recorded as kitchen waste (F1, F2, F3), other waste (R1, R2, R3), and mixed waste (M1, M2, M3). Three parallel samples (n=3) were set for each treatment.

[0070] 1. The biomass components (such as kitchen waste) are pre-treated by homogenization (the method is the same as in Example 1), and the non-biomass components (such as plastic bags, napkins, masks) are mechanically crushed under sterile conditions, and the particle size is controlled to be ≤ 2 cm.

[0071] 2. The method of Example 1 was used to perform elution treatment on each group to obtain virus eluate.

[0072] 3. Using the FeCl2 of Example 1 3 The virus eluates obtained from each group were enriched by the flocculation method, and the filter membranes were collected and stored in an ultra-low temperature refrigerator at -80°C for subsequent viral nucleic acid extraction and sequencing analysis.

[0073] The content of g23 gene in different components of domestic waste samples is shown in Table 2. The results show that the method of the present invention can effectively extract and enrich virus particles in highly heterogeneous domestic waste samples. Specifically, the content of g23 gene in mixed waste samples (M1-M3) is relatively high, and the content of g23 gene in other waste (R1-R3) and kitchen waste samples (F1-F3) also reaches the detection limit, proving that this method is suitable for the extraction of virus particles in highly heterogeneous domestic waste samples of different types and concentration ranges.

[0074] Table 2 Virus enrichment effect of different components of domestic waste samples

[0075]

[0076] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for eluting and enriching viral particles in highly heterogeneous domestic waste, characterized in that: The following steps are involved: Sampling domestic waste in the target area to obtain representative waste samples; Performing homogenization pretreatment on the garbage sample to obtain a homogenized garbage sample; The virus in the homogenized garbage sample is extracted into a liquid phase using a neutral inorganic solution, and then purified to obtain a virus eluate; The virus in the virus eluate is enriched by using an iron salt flocculation method.

2. The elution and enrichment method according to claim 1, characterized in that: The biomass components in the garbage sample are homogenized and pretreated by using a low-temperature freezing and crushing method.

3. The elution and enrichment method according to claim 2, characterized in that: The cryogen used in the low temperature freezing and crushing method is dry ice or liquid nitrogen; and / or When the refrigerant is dry ice, the mass ratio of the biomass component to the dry ice is (30-15): 1 When the cryogen is liquid nitrogen, the mass ratio of the biological component to the liquid nitrogen is (75-150):

1.

4. The elution and enrichment method according to claim 3, characterized in that: The homogenization pretreatment time is ≥30s.

5. The elution and enrichment method according to claim 1, characterized in that: The non-biomass components in the garbage sample are mechanically crushed under sterile conditions to control the particle size to ≤ 2 cm.

6. The elution and enrichment method according to claim 1, characterized in that: The eluent used in the solution elution method comprises the following components: NaCl 5-10 g / L, KCl 0.1-0.5 g / L, Na2HPO4 1-1.5 g / L, KH2PO4 0.1-0.5 g / L; and / or The mass ratio of the eluent to the homogenized garbage sample is 10:(3-5).

7. The elution and enrichment method according to claim 1, characterized in that: The iron salt flocculation method is to use 0.2 mol / L iron salt solution to flocculate the virus eluate.

8. The elution and enrichment method according to claim 7, characterized in that: The volume ratio of the iron salt solution to the virus elution solution is 1:(50-100).

9. The elution and enrichment method according to claim 7, characterized in that: The flocculation treatment time is ≥10 min.

10. The elution and enrichment method according to claim 1, characterized in that: The operating temperature of the virus extraction into liquid phase and the iron salt flocculation method is ≤4°C.

Images