A magnetic bead method kit for efficiently extracting nucleic acid from sewage samples

Abstract

The present application relates to the technical field of nucleic acid extraction, and in particular to a magnetic bead method kit for efficiently extracting nucleic acid of sewage samples, The magnetic bead method kit for efficiently extracting nucleic acid of sewage samples provided by the present application comprises a reagent combination of a lysis solution, a binding solution, a washing solution, an elution solution, a magnetic bead suspension and protease K, and a method for extracting nucleic acid of sewage samples using the kit is also provided. The magnetic bead method kit provided by the present application can be used for nucleic acid extraction of concentrated sewage virus samples in cooperation with a traditional enrichment method, and can also be used for direct nucleic acid extraction of microorganisms in sewage samples without an enrichment step, so that the sample only needs 1-5 mL, thereby avoiding the problem that enrichment to heavy metals and humic acid inhibits extraction and subsequent detection. Meanwhile, the magnetic bead method kit for efficiently extracting nucleic acid of sewage samples provided by the present application is matched with an automatic nucleic acid extraction instrument, so that convenient, efficient and high-speed nucleic acid extraction is realized, and the kit is suitable for large quantities of sewage sample processing and nucleic acid extraction.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid extraction technology, specifically to a magnetic bead-based reagent kit for efficiently extracting nucleic acids from wastewater samples. Background Technology

[0002] With population growth, rapid economic development, and improved living standards, my country's total wastewater discharge is constantly increasing. Urban wastewater treatment plants are crucial infrastructure for ensuring water environment safety and achieving sustainable urban development. my country's urban wastewater treatment plants generally employ the activated sludge process, in which microorganisms are the primary agents for removing pollutants and form the basis for stable system operation. Therefore, understanding the community structure and function of microorganisms in wastewater treatment is essential for controlling and improving the stability of wastewater treatment. Detecting the presence of microbial nucleic acids in wastewater can serve as an important basis for determining the types and concentrations of microorganisms in the wastewater.

[0003] Wastewater contains a wide variety of viruses. Different viruses, and even the same type, vary in their pathogenicity and survival ability, posing different risks to humans. Many institutions worldwide are researching virus surveillance methods. Among these, using wastewater to monitor the spread of a virus at the regional or city level has proven to be a feasible approach. Wastewater surveillance, as a leading indicator of community infection changes, can effectively assist relevant departments in controlling the spread of viruses before an outbreak occurs. Currently, there is no unified standard for assessing viral pollution in water bodies. A thorough understanding of their distribution and characteristics in water bodies has significant practical value for preventing viral pollution, ensuring water quality safety, and reducing risks to human health. Research shows that detecting viral nucleic acids in wastewater can serve as an important basis for determining the type and concentration of viruses in wastewater.

[0004] The main steps in detecting viruses in wastewater involve extracting viral nucleic acids from the wastewater and then detecting them using methods such as qPCR or sequencing. Currently, the most commonly used methods for extracting viral nucleic acids from wastewater are enrichment followed by extraction. Enrichment methods include immunoassay, flocculation precipitation, and centrifugation ultrafiltration; extraction methods mainly include silica-based membrane column extraction kits and magnetic bead kits. General virus enrichment methods have the following drawbacks: 1) For example, flocculation precipitation may enrich many interfering substances such as heavy metals and humic acids, or introduce new interfering substances such as EDTA, while concentrating the virus, which can interfere with subsequent nucleic acid extraction and detection; 2) Some virus concentration methods are costly and time-consuming, with the total time for enrichment, concentration, and nucleic acid extraction exceeding three to four hours. These methods are more suitable for small-scale laboratory research projects and not for large-scale wastewater sample processing and nucleic acid extraction. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a magnetic bead method kit for efficient extraction of nucleic acid from sewage samples. The magnetic bead method kit for efficient extraction of nucleic acid from sewage samples provided by the present invention can quickly and efficiently extract microbial nucleic acid from sewage and is suitable for large-scale sewage sample processing and nucleic acid extraction.

[0006] This invention provides a reagent combination for nucleic acid extraction from wastewater samples, including a lysis buffer, a binding buffer, a washing buffer A, and a washing buffer B;

[0007] The lysis buffer includes water, guanidine thiocyanate, sodium chloride, Triton X-100, sodium lauroyl sarcosinate, tris(hydroxymethyl)aminomethane, disodium ethylenediaminetetraacetate, CTAB, PVP 360, guanidine hydrochloride, Tween-20, and sodium citrate.

[0008] The binding solution comprises water, guanidine hydrochloride, sodium chloride, polyvinyl alcohol, acetic acid, sodium acetate, and isopropanol, wherein the polyvinyl alcohol is selected from at least one of polyvinyl alcohol PEG 8000, polyvinyl alcohol PEG 6000, and polyvinyl alcohol PEG 2000;

[0009] The washing solution A includes water, guanidine thiocyanate, guanidine hydrochloride, polyvinyl alcohol PEG 8000, sodium chloride, Tween-20, tris(hydroxymethyl)aminomethane, disodium ethylenediaminetetraacetate, and isopropanol.

[0010] The washing solution B comprises water, guanidine thiocyanate, guanidine hydrochloride, Tween-20, Triton X-100, and isopropanol.

[0011] Compared to other extraction reagents, the wastewater sample nucleic acid extraction reagent combination provided by this invention can be used in conjunction with traditional enrichment methods to extract nucleic acids from concentrated wastewater samples, or the enrichment step can be skipped to directly extract nucleic acids from wastewater samples. This can better ensure the extraction effect of nucleic acids in high-concentration or low-concentration samples, thereby obtaining more accurate technical results. In practical use, it covers a wider range of samples and is more conducive to promotion and application.

[0012] Preferably, the concentrations of each component in the lysis buffer are as follows: the lysis buffer consists of water, 1-4M guanidine thiocyanate, 0.2-0.5M sodium chloride, 0.1 vol%-12 vol% Triton X-100, 0.1 wt%-5 wt% sodium lauroyl sarcosinate, 1-50 mM tris(hydroxymethyl)aminomethane, 1-50 mM disodium ethylenediaminetetraacetate, 0.5 vol%-2 vol% CTAB, 1 vol%-5 vol% PVP 360, 0.1-5M guanidine hydrochloride, 0.01 vol%-1 vol% Tween-20, and 0.1 wt%-5 wt% sodium citrate.

[0013] In some embodiments, the concentrations of the components in the lysis buffer are as follows: the lysis buffer consists of water, 0.5–4 M guanidine thiocyanate, 0.1–2 M sodium chloride, 0.1 vol%–12 vol% Triton X-100, 0.1 wt%–1 wt% sodium lauroyl sarcosinate, 10–50 mM tris(hydroxymethyl)aminomethane, 5–25 mM disodium ethylenediaminetetraacetate, 0.5 vol%–2 vol% CTAB, 5 vol% PVP 360, 1 M guanidine hydrochloride, 0.5 vol%–1 vol% Tween-20, and 0.2 wt%–1 wt% sodium citrate. Within this concentration range, the components of the lysis buffer are tightly coupled, enabling more efficient lysis of wastewater samples and releasing the nucleic acids within the lysis buffer system, thereby achieving a higher nucleic acid extraction efficiency.

[0014] In some specific embodiments, the lysis buffer consists of water, 3M guanidine thiocyanate, 0.5M sodium chloride, 1 vol% Triton X-100, 1 wt% sodium lauroyl sarcosinate, 20 mM tris(hydroxymethyl)aminomethane, 20 mM disodium ethylenediaminetetraacetate, 2 vol% CTAB, 5 vol% PVP 360, 1M guanidine hydrochloride, 1 vol% Tween-20, and 1 wt% sodium citrate. Experiments have shown that within this concentration range, the lysis buffer is most effective for extracting nucleic acids from wastewater samples with high or low concentrations.

[0015] Preferably, the concentration of each component in the binding solution is as follows: the binding solution is composed of water, 0.5-5M guanidine hydrochloride, 0.1-2M sodium chloride, 1wt%-10wt% polyvinyl alcohol PEG 8000, 1wt%-10wt% polyvinyl alcohol PEG 6000, 1wt%-10wt% polyvinyl alcohol PEG 2000, 1vol%-10vol% acetic acid, 0.1-2M sodium acetate, and 10vol%-60vol% isopropanol.

[0016] In some embodiments, the concentrations of the components in the binding solution are as follows: the binding solution consists of water, 2-4M guanidine hydrochloride, 0.5M sodium chloride, 5wt%-10wt% polyvinyl alcohol PEG 8000, 5wt%-10wt% polyvinyl alcohol PEG 6000, 5wt%-10wt% polyvinyl alcohol PEG 2000, 1vol%-5vol% acetic acid, 0.5M sodium acetate, and 30vol%-35vol% isopropanol. Within this concentration range, the components of the binding solution are closely combined, which can better cooperate with the magnetic beads to adsorb the lysed nucleic acids in the sample during magnetic bead nucleic acid extraction, thereby obtaining a higher nucleic acid extraction efficiency.

[0017] In some specific embodiments, the binding solution consists of water, 4M guanidine hydrochloride, 0.5M sodium chloride, 5wt% polyvinyl alcohol PEG 8000, 5wt% polyvinyl alcohol PEG 6000, 5wt% polyvinyl alcohol PEG 2000, 5vol% acetic acid, 0.5M sodium acetate, and 35vol% isopropanol. Experiments have shown that within this concentration range, the binding solution is most effective for extracting nucleic acids from wastewater samples with high or low concentrations.

[0018] Preferably, the washing solution A contains the following components: water, 0.5–4 M guanidine thiocyanate, 0.1–2 M guanidine hydrochloride, 1 wt%–10 wt% polyvinyl alcohol PEG 8000, 0.1–10 M sodium chloride, 0.01 vol%–5 vol% Tween-20, 1–50 mM tris(hydroxymethyl)aminomethane, 1–50 mM disodium ethylenediaminetetraacetate, and 10 vol%–60 vol% isopropanol.

[0019] In some embodiments, the concentrations of each component in the washing solution A are as follows: Washing solution A consists of water, 1-2M guanidine thiocyanate, 1M guanidine hydrochloride, 5wt%-10wt% polyvinyl alcohol PEG 8000, 0.5-1M sodium chloride, 0.5vol%-5vol% Tween-20, 20-50mM tris(hydroxymethyl)aminomethane, 20-50mM disodium ethylenediaminetetraacetate, and 30vol%-50vol% isopropanol. Within this concentration range, the components of washing solution A are tightly combined, enabling more efficient removal of protein impurities and some other impurities adsorbed when magnetic beads bind to nucleic acids.

[0020] In some specific embodiments, the washing solution A consists of water, 2M guanidine thiocyanate, 1M guanidine hydrochloride, 10wt% polyvinyl alcohol PEG 8000, 0.5M sodium chloride, 0.5vol% Tween-20, 20mM tris(hydroxymethyl)aminomethane, 20mM disodium ethylenediaminetetraacetate, and 50vol% isopropanol. Experiments show that within this concentration range, washing solution A is most effective for extracting nucleic acids from wastewater samples with high or low concentrations.

[0021] Preferably, the washing solution B contains the following components: water, 0.1-1M guanidine thiocyanate, 0.1-2M guanidine hydrochloride, 0.01 vol%-0.5 vol% Tween-20, 0.1 vol%-5 vol% Triton X-100, and 10 vol%-60 vol% isopropanol.

[0022] In some embodiments, the concentrations of each component in the washing solution B are as follows: Washing solution B consists of water, 1M guanidine thiocyanate, 0.5–2M guanidine hydrochloride, 0.05 vol%–0.5 vol% Tween-20, 0.15 vol%–5 vol% Triton X-100, and 30 vol%–50 vol% isopropanol. Within this concentration range, the components of washing solution B are well-matched and can work with elution solution A to perform a secondary removal of protein impurities and some other impurities adsorbed when magnetic beads bind to nucleic acids.

[0023] In some specific embodiments, the washing solution B consists of water, 1M guanidine thiocyanate, 1M guanidine hydrochloride, 0.5 vol% Tween-20, 1 vol% Triton X-100, and 50 vol% isopropanol. Experiments have shown that within this concentration range, the washing solution B is most effective for extracting nucleic acids from wastewater samples with high or low concentrations.

[0024] Preferably, the wastewater sample nucleic acid extraction reagent combination provided by the present invention further includes washing solution C and elution solution, wherein the concentrations of each component in washing solution C and elution solution are as follows:

[0025] The washing solution C is composed of water, 1-50 mM tris(hydroxymethyl)aminomethane and 10 vol%-80 vol% anhydrous ethanol;

[0026] The eluent consists of water and 1–50 mM trihydroxymethylaminomethane.

[0027] In some embodiments,

[0028] The washing solution C is composed of water, 5-20 mM tris(hydroxymethyl)aminomethane, and 50 vol%-80 vol% anhydrous ethanol.

[0029] In some specific embodiments,

[0030] The washing solution C is composed of water, 10 mM tris(hydroxymethyl)aminomethane, and 80 vol% anhydrous ethanol;

[0031] The eluent consists of water and 10 mM tris(hydroxymethyl)aminomethane.

[0032] Experiments show that at this concentration, the washing solution C and elution solution are most effective at extracting nucleic acids from wastewater samples with high or low concentrations.

[0033] Preferably, the wastewater sample nucleic acid extraction reagent combination provided by the present invention further includes magnetic bead suspension and proteinase K;

[0034] The magnetic beads in the magnetic bead suspension are superparamagnetic silica magnetic beads, and the concentration of the superparamagnetic silica magnetic beads in the magnetic bead suspension is 20–100 mg / mL.

[0035] The wastewater sample nucleic acid extraction reagent combination described in this invention is applicable to the extraction of various bacteria and viruses, including Escherichia coli, Bacillus subtilis, novel coronavirus, African swine fever virus, hepatitis B virus, etc., and this invention does not limit them.

[0036] The present invention also provides a method for extracting nucleic acid from sewage samples, wherein the nucleic acid of the sample to be tested is extracted using the aforementioned combination of sewage sample nucleic acid extraction reagents.

[0037] Preferably, the extraction method includes the following steps:

[0038] The sample was lysed with a lysis buffer containing proteinase K, then a binding buffer and isopropanol were added, and the sample was adsorbed onto carboxyl magnetic beads. After separating the magnetic beads, the sample was washed sequentially with washing buffer A, washing buffer B, and washing buffer C, and then the nucleic acid was obtained by elution.

[0039] Preferably, the washing solutions A, B, and C are used to wash the clothes 1 to 2 times independently.

[0040] In some specific embodiments, after washing twice with washing solution C, the nucleic acid eluted by the elution solution contains fewer impurities and has higher purity.

[0041] This invention provides a wastewater sample nucleic acid extraction reagent kit comprising a lysis buffer, binding buffer, washing buffer, elution buffer, magnetic bead suspension, and proteinase K. This kit is suitable for assembling a high-efficiency magnetic bead method reagent kit for extracting nucleic acids from wastewater samples. A method for extracting nucleic acids from wastewater samples using this kit is also provided. Compared with existing technologies, the high-efficiency magnetic bead method reagent kit for extracting nucleic acids from bacteria, viruses, and other microorganisms in wastewater provided by this invention can be used in conjunction with traditional enrichment methods to extract viral nucleic acids from concentrated viral samples in wastewater. Alternatively, it can skip the enrichment step and directly extract nucleic acids from microorganisms in wastewater samples, avoiding the problems of heavy metal accumulation and humic acid inhibition during extraction and subsequent detection that can occur with enrichment methods. Furthermore, this kit requires only 1-5 mL of wastewater sample to achieve the detection sensitivity of 50 mL of concentrated extraction using traditional enrichment methods. In addition, the high-efficiency magnetic bead method reagent kit for extracting nucleic acids from wastewater samples, when used with an automated nucleic acid extractor, can process 24 wastewater samples for viral nucleic acid extraction within 38 minutes, significantly reducing the cost and time of nucleic acid extraction from wastewater samples. This provides a convenient, fast, and efficient nucleic acid extraction process suitable for large-scale wastewater sample processing and nucleic acid extraction. Attached Figure Description

[0042] Figure 1 This is a schematic diagram of the process for enriching wastewater samples using the aluminum salt precipitation coagulation method in Example 1.

[0043] Figure 2This is a schematic diagram of the process for treating colloidal samples enriched with viruses in wastewater, as shown in Example 1.

[0044] Figure 3 The figure shows the nucleic acid extraction results of different lysis buffer formulations in Example 2. Figure 3 Image 'a' shows the results of the enrichment method. Figure 3 b is the result of the direct method extraction;

[0045] Figure 4 The image shows the nucleic acid extraction results for different binding solution formulations in Example 3. Figure 4 Image 'a' shows the results of the enrichment method. Figure 4 b is the result of the direct method extraction;

[0046] Figure 5 This is a graph showing the nucleic acid extraction results of different detergent A formulations in Example 4. Figure 5 Image 'a' shows the results of the enrichment method. Figure 5 b is the result of the direct method extraction;

[0047] Figure 6 This is a graph showing the nucleic acid extraction results of different detergent B formulations in Example 5. Figure 6 Image 'a' shows the results of the enrichment method. Figure 6 b is the result of the direct method extraction;

[0048] Figure 7 The image shows the nucleic acid extraction results in Example 6.

[0049] Figure 8 The image shows the electrophoresis results of nucleic acid extraction in Example 6. Detailed Implementation

[0050] This invention provides a magnetic bead-based reagent kit for efficiently extracting nucleic acids from wastewater samples. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art will clearly be able to modify or appropriately alter and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0051] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0052] This invention provides a highly efficient magnetic bead-based reagent kit and extraction method for extracting nucleic acids from sewage samples. This method can be used in conjunction with traditional enrichment methods to extract concentrated virus solutions from sewage samples, or it can skip the enrichment step and directly extract nucleic acids from sewage samples.

[0053] The magnetic bead method kit for efficient extraction of nucleic acids from sewage samples includes lysis buffer, binding buffer, washing buffer A, washing buffer B, washing buffer C, elution buffer, magnetic bead suspension, and proteinase K.

[0054] The main steps of the method for extracting nucleic acid from sewage samples include: lysing the sample with a lysis buffer containing proteinase K, adding a binding buffer and isopropanol, and adsorbing it with carboxyl magnetic beads; after separating the magnetic beads, washing the sample sequentially with washing buffer A, washing buffer B, and washing buffer C, and then eluting to obtain the nucleic acid from the sewage sample.

[0055] The present invention will be further illustrated below with reference to the embodiments.

[0056] Example 1: Detailed steps for nucleic acid extraction from wastewater samples

[0057] A. Manual extraction steps (1 mL sample system)

[0058] Preparation:

[0059] Isopropanol (analytical grade)

[0060] 15mL centrifuge tubes, 1.5mL centrifuge tubes (DNase-free, RNase-free)

[0061] Single-channel pipettes: 20μL, 200μL, 1000μL, 5000μL

[0062] Vortex oscillator

[0063] Vertical mixer

[0064] Water bath or metal bath (Beaver 2016C)

[0065] Magnetic separator: Beaver magnetic separator, part number: 60201, is available.

[0066] Note: The kit must be equilibrated to room temperature before each use.

[0067] 1. Prepare the lysis buffer

[0068] The lysis buffer was placed in a water bath at 65°C for 5–10 minutes until the reagent precipitate was completely dissolved.

[0069] 2. Pyrolysis

[0070] Take a 15mL centrifuge tube and add 1mL of sewage sample (or 200μL of virus concentrate and 800μL of purified water), then add 2mL of lysis buffer, and then add 100μL of proteinase K working solution (in specific use, proteinase K is prepared by mixing glycerol and water to make a working solution). Adjust the speed to a suitable speed and vortex for 1min to mix it thoroughly. Then place the centrifuge tube in 65℃ for 10min, and vortex for 30s every 5min.

[0071] 3. Combining

[0072] Add 2 mL of binding solution, 2 mL of isopropanol (self-prepared), and 20 μL of magnetic bead suspension to the above 15 mL centrifuge tube. Mix vertically at room temperature for 10 min. Place the centrifuge tube on a magnetic separator until the solution is clear. Remove the supernatant with a pipette and remove the centrifuge tube.

[0073] 4. Washing

[0074] (1) Add 0.8 mL of washing solution A, use a pipette to draw the liquid in the tube and blow it to the bottom of the tube to mix it. Transfer the magnetic bead suspension in the tube to a 1.5 mL centrifuge tube, vortex for 1 min, place the centrifuge tube on a magnetic separator until the solution is clear, remove the supernatant with a pipette and remove the centrifuge tube.

[0075] (2) Add 0.8 mL of washing solution B (check if isopropanol has been added), vortex for 1 min, place the centrifuge tube on a magnetic separator until the solution is clear, remove the supernatant with a pipette and remove the centrifuge tube.

[0076] (3) Add 0.8 mL of washing solution C (check if anhydrous ethanol has been added), vortex for 1 min, place the centrifuge tube on a magnetic separator until the solution is clear, remove the supernatant with a pipette and remove the centrifuge tube.

[0077] (4) Add 0.8 mL of washing solution C, vortex for 1 min, place the centrifuge tube on a magnetic separator until the solution is clear, remove the supernatant with a pipette and keep the centrifuge tube on the magnetic rack.

[0078] Note: Step (4) should remove as many residual droplets as possible.

[0079] 5. Drying

[0080] Keep the centrifuge tubes open and place them on a magnetic rack. Let them sit at room temperature for 3 to 10 minutes until there is no obvious liquid sheen on the surface.

[0081] 6. Washing

[0082] Add 50-100 μL of elution buffer, vortex for 1 min to mix the magnetic beads thoroughly, heat at 65 °C for 3 min and then vortex for 1 min. Place the centrifuge tube on a magnetic separator until the solution is clear, and transfer the supernatant to a new 1.5 mL centrifuge tube. This is the purified nucleic acid.

[0083] B. Automated extraction step (1 mL sample system)

[0084] Preparation:

[0085] One magnetic rod sleeve, and seven 24-hole deep hole plates (Cat.:43019, 43020).

[0086] Nucleic acid extraction equipment: Beaver Rosetta 24 automated nucleic acid extractor

[0087] 15mL centrifuge tubes, 1.5mL centrifuge tubes (DNase-free, RNase-free)

[0088] Single-channel pipettes: 20μL, 200μL, 1000μL, 5000μL

[0089] Vortex oscillator

[0090] Water bath 65℃

[0091] Isopropanol (analytical grade)

[0092] Note: The kit must be equilibrated to room temperature before each use.

[0093] 1. Remove the reagent kit from the refrigerator 30 minutes in advance and allow it to equilibrate to room temperature. Place the lysis buffer vial in a water bath at 65°C 10 minutes in advance to dissolve and then mix well.

[0094] 2. Inspect consumables and mark plate numbers: Check all deep hole plates for damage or cracks on the bottom; check the magnetic rod sleeves for unevenness or misalignment. After inspection, place the magnetic rod sleeves into the deep hole plates and gently and evenly press them down above each hole to reset them. Mark the sides of the 24-hole deep hole plates with numbers 2, 3, 4, 5, 6, and 8, corresponding to the instrument plate numbers.

[0095] 3. Confirm that the instrument is in normal condition and the program is working properly (users can adjust the extraction program parameters, such as magnetic attraction time, drying time, elution volume, etc., according to actual usage).

[0096] 4. Add reagents and samples

[0097] Follow these steps:

[0098] a. Plate No. 3: Add 3 mL of washing solution A to each well.

[0099] b. Plate No. 4: Add 3 mL of washing solution B to each well.

[0100] c. Plate No. 5: Add 3 mL of washing solution C to each well. Then gently place the magnetic rod sleeve into Plate No. 5, confirming that the placement is accurate.

[0101] d. Plate No. 6: Add 1 mL of washing solution C to each well.

[0102] e. Plate #8: Add 50-100 μL of elution buffer to each well.

[0103] Note: After inserting the pipette into the bottom of the well, add the eluent to the central recessed area; do not add it to the sides. This product can automatically eluent volumes as low as 50 μL, but it's important to note that elution efficiency is related to the volume of eluent. A larger volume of eluent results in higher elution efficiency and a greater total amount of nucleic acids eluted.

[0104] f. Plate #2: Add 2 mL of lysis buffer, 100 μL of proteinase K working solution (for actual use, proteinase K is prepared using glycerol and water), 2 mL of binding buffer, 2 mL of isopropanol (prepared by the user), and 20 μL of magnetic bead suspension to each well in sequence. Then, add 1 mL of wastewater sample (or 200 μL of virus concentrate and 800 μL of purified water) to the corresponding position in each well.

[0105] g. Place the deep hole plates No. 2, 3, 4, 5, 6, and 8 into the corresponding numbered positions inside the instrument, and confirm that the deep hole plates are fully inserted into the slots.

[0106] 5. Immediately run the preset program in the instrument.

[0107] 6. After the program finishes running, immediately remove plate #8. Transfer the supernatant from each well to the corresponding 1.5mL centrifuge tube; this is the purified nucleic acid.

[0108] Step 3) The sample can be a sewage sample or a concentrated virus solution obtained after enriching a sewage sample. The extraction volume is 0.2 mL to 5 mL.

[0109] In step 2), the superparamagnetic silica beads are carboxyl beads with a concentration of 20-100 mg / mL.

[0110] Example 2: Adjustment and Testing of Lysis Buffer Formulation in a Magnetic Bead Method Kit for High-Efficiency Extraction of Nucleic Acid from Wastewater Samples

[0111] The table below shows the lysis buffer formulations for experiments 1-7. Seven different formulations were prepared according to the table, and used for direct extraction or extraction after enrichment of viral nucleic acid from wastewater samples. The viral nucleic acid concentration was then measured after extraction. The experimental procedures were the same except for the lysis buffer. For the direct extraction method, 1 mL of a wastewater sample with 50 copies / mL of virus was directly extracted. For the enrichment method, 50 mL of a 50 copies / mL wastewater sample was enriched into 1 mL of virus concentrate, and then 200 μL of the supernatant was extracted.

[0112]

[0113] Experimental results are as follows Figure 3As shown, among the seven formulations, formulations 1-7 can all be used for direct extraction of viruses from wastewater samples, but formulations 2 and 3 show the best results (a lower Ct value indicates a higher nucleic acid concentration and better extraction effect), while the others show only average results. Formulations 1-3 can all be used for enrichment extraction of concentrated viruses from wastewater samples, but formulation 3 shows the best results, while the others show relatively poor results. Therefore, formulation 3 is the optimal lysis buffer formulation that can be used for both methods simultaneously.

[0114] Example 3: Adjustment and Testing of the Binding Solution Formulation in a Magnetic Bead Method Kit for High-Efficiency Extraction of Nucleic Acids from Wastewater Samples

[0115] The table below shows the binding solution formulations for experiments 1-3. Three different binding solutions were prepared according to the table, and used for direct extraction or extraction after enrichment of viral nucleic acid from wastewater samples. The viral nucleic acid concentration was then measured after extraction. The experimental procedures were the same except for the binding solution. For the direct extraction method, 1 mL of a wastewater sample with 50 copies / mL of virus was directly extracted. For the enrichment method, 50 mL of a 50 copies / mL wastewater sample was enriched into 1 mL of virus concentrate, and then 200 μL of the supernatant was extracted.

[0116]

[0117] Experimental results are as follows Figure 4 As shown, among the three formulations, formulations 1 and 2 can both be used for direct extraction of viruses from wastewater samples, but formulation 2 is the most effective. Both formulations 1 and 2 can be used for enrichment extraction of concentrated viruses from wastewater samples, but formulation 2 is the most effective. Therefore, formulation 2 is the optimal conjugate formulation that can be used for both methods simultaneously.

[0118] Example 4: Adjustment and Testing of Washing Solution A Formula in a Magnetic Bead Method Kit for High-Efficiency Extraction of Nucleic Acid from Wastewater Samples

[0119] The table below shows the formulations of washing solution A. Four different formulations of washing solution A were prepared according to the table (Experiments 1-4). These were then used for direct extraction or extraction after enrichment of viral nucleic acid from wastewater samples. The viral nucleic acid concentration was then measured after extraction. The experimental procedures were the same except for washing solution A. For the direct extraction method, 1 mL of a wastewater sample with 50 copies / mL of virus was directly extracted. For the enrichment method, 50 mL of a 50 copy / mL wastewater sample was enriched into 1 mL of virus concentrate, and then 200 μL of the supernatant was extracted.

[0120]

[0121]

[0122] Experimental results are as follows Figure 5As shown, among the four formulations, formulations 1 and 2 can both be used for direct extraction of viruses from wastewater samples, with formulation 2 showing the best results, while formulations 3 and 4 are less effective. Formulations 1 and 2 can also be used for enrichment extraction of concentrated viruses from wastewater samples, with formulation 2 showing the best results, while formulations 3 and 4 are less effective. Therefore, formulation 2 is the optimal conjugate formulation that can be used for both methods simultaneously.

[0123] Example 5: Adjustment and Testing of Washing Buffer B Formula in the Magnetic Bead Method Kit for High-Efficiency Extraction of Nucleic Acid from Wastewater Samples

[0124] The table below shows the experimental formulation of washing solution B. Three different formulations of washing solution A were prepared according to the table (Experiments 1-3). These were then used for direct extraction or extraction after enrichment of viral nucleic acid from wastewater samples. The viral nucleic acid concentration was then measured after extraction. The experimental procedures were the same except for washing solution B. For the direct extraction method, 1 mL of a wastewater sample with 50 copies / mL of virus was directly extracted. For the enrichment method, 50 mL of a 50 copy / mL wastewater sample was enriched into 1 mL of virus concentrate, and then 200 μL of the supernatant was extracted.

[0125]

[0126] Experimental results are as follows Figure 6 As shown, among the three formulations, formulations 1-3 can all be used for direct extraction of viruses from wastewater samples, with formulations 2 and 3 showing the best results. Formulations 1-3 can also be used for enrichment extraction of concentrated viruses from wastewater samples, with formulation 3 showing the best results, while formulations 1 and 2 are less effective. Therefore, formulation 3 is the optimal conjugate formulation that can be used for both methods simultaneously.

[0127] Based on Examples 2-5, the specific components of the magnetic bead method kit for efficient extraction of nucleic acid from sewage samples in this invention are as follows:

[0128] The lysis buffer consisted of water, 3M guanidine thiocyanate, 0.5M sodium chloride, 1 vol% Triton X-100, 1 wt% sodium lauroyl sarcosinate, 20 mM tris(hydroxymethyl)aminomethane, 20 mM disodium ethylenediaminetetraacetate, 2 vol% CTAB, 5 vol% PVP 360, 1M guanidine hydrochloride, 1 vol% Tween-20 and 1 wt% sodium citrate;

[0129] The binding solution consists of water, 4M guanidine hydrochloride, 0.5M sodium chloride, 5wt% polyvinyl alcohol PEG 8000, 5wt% polyvinyl alcohol PEG 6000, 5wt% polyvinyl alcohol PEG 2000, 5vol% acetic acid, 0.5M sodium acetate and 35vol% isopropanol;

[0130] Washing solution A consists of water, 2M guanidine thiocyanate, 1M guanidine hydrochloride, 10wt% polyvinyl alcohol PEG 8000, 0.5M sodium chloride, 0.5vol% Tween-20, 20mM tris(hydroxymethyl)aminomethane, 20mM disodium ethylenediaminetetraacetate and 50vol% isopropanol.

[0131] Washing solution B consists of water, 1M guanidine thiocyanate, 1M guanidine hydrochloride, 0.5 vol% Tween-20, 1 vol% Triton X-100 and 50 vol% isopropanol;

[0132] The washing solution C is composed of water, 10 mM tris(hydroxymethyl)aminomethane, and 80 vol% anhydrous ethanol;

[0133] The eluent consists of water and 10 mM tris(hydroxymethyl)aminomethane.

[0134] Example 6: Parameter Adjustment and Operation of Automated Nucleic Acid Extractor

[0135] The final operating procedure suitable for wastewater samples was obtained by adjusting different instrument operating parameters, as shown in the table below.

[0136]

[0137] The automated nucleic acid extraction parameters shown in the table above were used, and the effects of automated and manual extraction were compared. The results are shown in the table below. The results indicate that the efficiency of automated and manual extraction is similar, meeting the ideal performance requirements of the magnetic bead method kit for efficient extraction of nucleic acids from wastewater samples.

[0138]

[0139] Example 1: Nucleic acid extraction test was performed on the concentrated virus solution enriched by aluminum salt precipitation coagulation method.

[0140] Aluminum salt coagulation and precipitation method

[0141] Principle: When aluminum salt coagulant is added to wastewater, the aluminum hydroxide colloid produced by the hydrolysis of aluminum salt can adsorb and encapsulate virus particles. The virus-containing colloid is collected by centrifugation, and then the encapsulated virus particles are released by chemical dissolution for subsequent nucleic acid extraction and real-time fluorescent RT-PCR detection.

[0142] The enrichment operation diagram is attached. Figure 1 and Figure 2 As shown.

[0143] Experimental objective:

[0144] After enrichment of wastewater using aluminum salt precipitation coagulation, the virus concentrate is automatically extracted and tested.

[0145] Experimental steps:

[0146] 1. To test the effectiveness of one-step and two-step methods on a 1mL wastewater sample system, 5000 copies of SARS-CoV-2 pseudovirus were added to 50mL of wastewater sample and enriched to 1mL. The supernatant was collected, mixed well, and then extracted using both the two-step and one-step methods.

[0147] 2. The instrument used was the Beaver Rosetta 24 nucleic acid extractor, and the consumables were a dedicated 24-channel deep well plate and a dedicated magnetic rod sleeve.

[0148] 3. The kit uses the magnetic bead method kit for high-efficiency extraction of nucleic acid from sewage samples provided by this invention.

[0149] 4. The experimental groups used both two-step and one-step extraction methods.

[0150] Two-step method:

[0151] (1) 200 μL sample + 800 μL pure water + 2 mL lysis buffer + 100 μL proteinase K were heated at 65 °C for 10 min to lyse.

[0152] (2) Then add 2 mL of binding solution + 2 mL of isopropanol + 100 μL of magnetic bead suspension and mix vertically at room temperature for 10 min.

[0153] (3) Wash 4 times according to the kit instructions, then elute with 50 μL. qPCR sample loading: 5 μL.

[0154] One-step method:

[0155] (1) 200 μL sample + 800 μL pure water + 2 mL lysis buffer + 2 mL binding buffer + 2 mL isopropanol + 100 μL magnetic bead suspension + 100 μL PK, heat at 65℃ for 15 min.

[0156] (2) Wash 4 times according to the kit instructions, then elute with 50 μL. qPCR sample loading: 5 μL.

[0157] 4. The extracted eluent was tested using the purchased Novizan qPCR detection kit.

[0158] The experimental results are shown in the table below:

[0159]

[0160] Results analysis: Compared with the two-step method, the one-step manual extraction method yielded a Ct value 0.6 higher. Both methods had Ct values ​​<35, indicating positive detection, demonstrating that the automated extraction sensitivity of this kit can reach 100 copies / mL.

[0161] Example 2: Direct extraction and testing of low concentrations of SARS-CoV-2 nucleic acid from wastewater samples.

[0162] Experimental objective: To conduct a direct extraction experiment of 1 mL of sewage sample using the kit, and to test the extraction efficiency of SARS-CoV-2 nucleic acid at concentrations of 50 copies / mL and 20 copies / mL.

[0163] Experimental steps:

[0164] 5.1. To test the effectiveness of direct extraction of viral nucleic acid from 1 mL of sewage sample, 50 copies and 20 copies of SARS-CoV-2 pseudovirus were added to 1 mL of sewage sample and mixed well. The samples were then extracted using the manual extraction method, the automated two-step method, and the one-step method provided in this invention.

[0165] 2. The instrument used was the Beaver Rosetta 24 nucleic acid extractor, and the consumables were a dedicated 24-channel deep well plate and a dedicated magnetic rod sleeve.

[0166] 3. The experimental groups included manual extraction, automated two-step extraction, and one-step extraction.

[0167] 4. The extracted eluent was analyzed using the purchased Novizan qPCR detection kit.

[0168] The experimental results are shown in the table below.

[0169]

[0170] Results analysis: Using this kit to directly extract low concentrations of SARS-CoV-2 from sewage samples, both manual and automated extraction methods can extract and detect positive results, with a sensitivity of 20-50 copies / mL.

[0171] Example 3: Low concentrations of SARS-CoV-2 nucleic acid were directly extracted from sewage samples and tested using the DaAn SARS-CoV-2 test kit.

[0172] Experimental Objective: To conduct a direct extraction experiment using the kit on a 1 mL wastewater sample, and to test the extraction efficiency of SARS-CoV-2 nucleic acid at concentrations of 50 copies / mL and 20 copies / mL. The nucleic acid extraction kit used in this experiment employed the high-efficiency magnetic bead method for extracting nucleic acid from wastewater samples provided by this invention, and qPCR detection was performed using the commercially available DaAn COVID-19 test kit.

[0173] Experimental steps:

[0174] 1. To test the effectiveness of direct extraction of viral nucleic acid from 1 mL of sewage sample, 50 copies and 20 copies of SARS-CoV-2 pseudovirus were added to 1 mL of sewage sample, mixed well, and then extracted using the manual extraction method and the automated one-step extraction method respectively.

[0175] 2. The instrument used was the Beaver Rosetta 24 nucleic acid extractor, and the consumables were a dedicated 24-channel deep well plate and a dedicated magnetic rod sleeve.

[0176] 3. The experimental group used both manual and automated one-step extraction methods.

[0177] 4. The extracted eluent was tested using the DaAn COVID-19 test kit. The DaAn COVID-19 test kit's positive interpretation criterion is a Ct value ≤ 30.

[0178] The experimental results are shown in the table below.

[0179]

[0180] Results Analysis: Using this kit to directly extract low-concentration SARS-CoV-2 from wastewater samples, both manual and automated extraction methods were successful, and the samples tested positive in the DaAn COVID-19 test kit with a sensitivity of 20-50 copies / mL. Based on the Ct value data and the positive criterion of Ct ≤ 30, it can be inferred that the sensitivity could even reach 10 copies / mL. The magnetic bead method kit for efficient nucleic acid extraction from wastewater samples provided by this invention has been validated by various commercially available nucleic acid test kits, demonstrating excellent extraction efficiency. It can extract high concentrations of viral nucleic acid from virus concentrates, and even in wastewater samples with concentrations as low as 20-50 copies / mL, the nucleic acid positive detection sensitivity is high.

[0181] Example 4: Test of the effectiveness of African swine fever virus nucleic acid extraction from wastewater

[0182] Experimental objective: To test the efficiency of the magnetic bead method kit for extracting nucleic acid from sewage samples for African swine fever virus extraction. The nucleic acid extraction kit used in this experiment was the magnetic bead method kit for extracting nucleic acid from sewage samples provided in this invention, and qPCR detection was performed using the Wens African swine fever virus detection kit.

[0183] Experimental Procedure: After wastewater collection, pre-centrifuge at 4℃, 2500g, for 30 min to collect the supernatant. Then, add Bondsheng African swine fever pseudovirus at concentrations of 100 copies / mL and 10 copies / mL as samples. Extract 1 mL of sample using an automated extraction system, followed by elution with 50 μL. Then, perform qPCR detection using the Wens African swine fever virus detection kit, with a sample loading volume of 15 μL MIX + 10 μL. (The instructions state that a Ct value ≤ 42 indicates a positive result.)

[0184] The experimental results are shown in the table below.

[0185]

[0186] Results Analysis: 1. At concentrations of 100 copies / mL and 10 copies / mL, the eluent obtained from automated extraction was detectable using the Wens African swine fever virus detection kit, indicating ideal extraction efficiency. Example 5: Hepatitis B virus nucleic acid extraction efficiency test in wastewater.

[0187] Experimental objective: To test the efficiency of the magnetic bead method kit for extracting nucleic acid from sewage samples for hepatitis B virus nucleic acid extraction. The nucleic acid extraction kit used in this experiment was the magnetic bead method kit for extracting nucleic acid from sewage samples provided in this invention, and qPCR detection was performed using the Fosun hepatitis B virus detection kit.

[0188] Experimental Procedure: Wastewater was collected from river water and pre-centrifuged at 4℃, 2500g, for 30 min to collect the supernatant. Then, 20 μL of high and low concentrations of positive control samples were added to 1 mL of the supernatant as samples. 1 mL of sample was extracted using an automated extraction system, followed by 50 μL elution. Then, qPCR detection was performed using the Fosun Hepatitis B Virus Detection Kit, with a sample loading volume of 15 μL MIX + 10 μL (Note: The Fosun program does not collect fluorescence in the first 5 cycles, so the actual Ct value is +5).

[0189] The experimental results are shown in the table below.

[0190]

[0191] Results analysis: 1. Strongly positive and borderline positive samples were detected by Fosun Hepatitis B Virus Nucleic Acid Detection Kit using the eluent obtained from automated extraction. The calculated Ct value for borderline positive samples met the quality control requirements of the instructions (the instructions state that 20 μL of quality control sample should be directly spotted to measure qpCR, and the Ct value should be <35).

[0192] Example 6: Test of Escherichia coli Nucleic Acid Extraction Efficacy in Wastewater

[0193] Experimental objective: To test the efficiency of the magnetic bead method kit for extracting nucleic acid from sewage samples using high efficiency, the kit was used to extract nucleic acid from E. coli in sewage samples. The kit was detected using a UV spectrophotometer.

[0194] Different concentrations of E. coli were added to wastewater samples, and extraction was performed using a wastewater extraction kit. 1 mL of sample was manually extracted, followed by 50 μL elution.

[0195] The experimental results are shown in the table below.

[0196] 0.668 36.71 1.84 1.84 1.67 71.89 3.59 1.88 3.34 138.29 6.91 1.88 6.68 230.65 11.53 1.89

[0197] Results analysis: From the ultraviolet spectrophotometer ( Figure 7 ) and agarose gel electrophoresis ( Figure 8The results showed that the amount of nucleic acid extracted from bacteria in wastewater by this kit was positively correlated with the bacterial concentration; the extraction amount increased proportionally with the increase of bacterial concentration, and the nucleic acid extraction efficiency did not decrease. Furthermore, agarose gel electrophoresis showed that bacterial DNA and RNA could be extracted simultaneously, and no nucleic acid degradation due to gel diffusion occurred, indicating ideal extraction results.

[0198] The above examples demonstrate that, compared to traditional virus extraction methods, the magnetic bead-based reagent kit for efficiently extracting nucleic acids from bacteria, viruses, and other microorganisms in wastewater provided by this invention can be used in conjunction with traditional enrichment methods to extract viral nucleic acids from concentrated wastewater virus samples. Alternatively, it can bypass the enrichment step and directly extract nucleic acids from microorganisms in wastewater samples, avoiding the problems of heavy metal accumulation and humic acid inhibition during extraction and subsequent detection that can occur with enrichment methods. Furthermore, this kit requires only 1 mL of wastewater sample to achieve the detection sensitivity of traditional enrichment methods that require 50 mL of concentrated extraction.

[0199] Comparative Example 1: Other Brands of Wastewater Reagent Kits as a Control

[0200] Experimental Objective: To compare the T brand wastewater nucleic acid extraction kit with competing products.

[0201] Experimental Procedure: Wastewater was collected and pre-centrifuged at 4℃, 2500g, for 30 min to obtain the supernatant. Then, Bondsheng COVID-19 pseudovirus at concentrations of 50 copies / mL and 20 copies / mL were added as samples. The wastewater nucleic acid extraction kit from competing brand T mainly includes lysis buffer, binding buffer, washing buffer, elution buffer, proteinase K, and magnetic beads. 1 mL of sample was manually extracted according to the respective instructions, followed by 50 μL elution. Then, qPCR detection was performed using the DaAn COVID-19 detection kit, with a sample loading volume of 20 μL MIX + 5 μL (Note: the DaAn program does not collect fluorescence in the first 10 cycles, so the actual Ct value is +10).

[0202] The experimental results are shown in the table below.

[0203]

[0204]

[0205] Results analysis: In the two low-concentration wastewater samples extracted by direct extraction method, the Ct value of the brand T wastewater kit was delayed by about 2-3 compared with the beaver kit, and the extraction effect was not as good as that of the beaver kit.

[0206] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A reagent kit for nucleic acid extraction from wastewater samples, characterized in that, This includes lysis buffer, binding buffer, washing buffer A, washing buffer B, washing buffer C, and elution buffer; The lysis buffer consists of water, 3M guanidine thiocyanate, 0.5M sodium chloride, 1 vol% Triton X-100, 1 wt% sodium lauroyl sarcosinate, 20 mM tris(hydroxymethyl)aminomethane, 20 mM disodium ethylenediaminetetraacetate, 2 vol% CTAB, 5 vol% PVP 360, 1M guanidine hydrochloride, 1 vol% Tween-20, and 1 wt% sodium citrate. The binding solution is composed of water, 4M guanidine hydrochloride, 0.5M sodium chloride, 5wt% polyvinyl alcohol PEG 8000, 5wt% polyvinyl alcohol PEG 6000, 5wt% polyvinyl alcohol PEG 2000, 5vol% acetic acid, 0.5M sodium acetate and 35vol% isopropanol; The washing solution A is composed of water, 2M guanidine thiocyanate, 1M guanidine hydrochloride, 10wt% polyvinyl alcohol PEG 8000, 0.5M sodium chloride, 0.5vol% Tween-20, 20mM tris(hydroxymethyl)aminomethane, 20mM disodium ethylenediaminetetraacetate and 50vol% isopropanol; The washing solution B is composed of water, 1M guanidine thiocyanate, 1M guanidine hydrochloride, 0.5 vol% Tween-20, 1 vol% Triton X-100 and 50 vol% isopropanol; The washing solution C is composed of water, 10 mM tris(hydroxymethyl)aminomethane and 80 vol% anhydrous ethanol; The eluent consists of water and 10 mM tris(hydroxymethyl)aminomethane.

2. The wastewater sample nucleic acid extraction reagent combination as described in claim 1, characterized in that, It also includes magnetic bead suspensions and proteinase K; The magnetic beads in the magnetic bead suspension are superparamagnetic silica magnetic beads, and the concentration of the superparamagnetic silica magnetic beads in the magnetic bead suspension is 20~100mg / mL.

3. A method for extracting nucleic acid from wastewater samples, characterized in that, Nucleic acid of the sample to be tested is extracted using the combination of nucleic acid extraction reagents for sewage samples as described in claim 1 or 2.

4. The extraction method as described in claim 3, characterized in that, Includes the following steps: The sample was lysed with a lysis buffer containing proteinase K, then a binding buffer and isopropanol were added, and the sample was adsorbed with carboxyl magnetic beads. After separating the magnetic beads, the samples were washed sequentially with washing solution A, washing solution B, and washing solution C, and then eluted to obtain nucleic acid from the wastewater sample.

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