Matched kit for purifying viral nucleic acid in sewage by magnetic beads and nucleic acid extraction method

Abstract

The invention provides a matched kit for purifying viral nucleic acid in sewage by using magnetic beads. The matched kit comprises the following components: nucleic acid extraction magnetic beads, nucleic acid purification magnetic beads, a magnetic bead activating solution, a nucleic acid releasing agent, a cleaning solution and an eluent, the invention also relates to a nucleic acid extraction method, by using the kit provided by the invention, complicated pretreatment operations such as centrifugation or pre-precipitation on a sewage sample are not needed, and the concentration and enrichment of viruses in sewage and the subsequent virus nucleic acid extraction operation can be simply and rapidly completed.

Description

Technical Field

[0001] The invention relates to the technical field of nucleic acid extraction, and in particular to a matching kit for purifying viral nucleic acid in sewage using magnetic beads and a nucleic acid extraction method. Background Art

[0002] In recent years, sewage monitoring has been widely used in the field of disease monitoring and prevention as an emerging method. By analyzing and detecting pathogens in sewage, the spread of diseases can be detected and tracked early. Sewage monitoring can not only detect areas where viruses exist early, but also provide important data for public health decision-making. Common viruses that can currently be detected through sewage include: new coronavirus, polio virus, norovirus, rotavirus, hepatitis A virus, adenovirus, new enterovirus, coxsackievirus and echovirus, etc.

[0003] Although the virus has been greatly diluted in the water environment, it still has strong infectivity and resistance, and can survive for a long time in the water environment, which can easily cause human infection and virus transmission, posing certain risks to human health. It is very necessary to establish an accurate, sensitive and rapid detection method for viral nucleic acid in the water environment.

[0004] A variety of methods for detecting viruses in water based on different detection principles have been reported, but these methods have low sensitivity and low virus concentrations in water. If water samples are tested directly, false negatives are very likely to occur. The pretreatment step for virus particles in sewage has become an indispensable operation. The pretreatment operation can effectively concentrate virus particles in water, remove interfering substances, and improve the accuracy of detection.

[0005] Currently, the commonly used methods for concentrating viruses in sewage are: polyethylene glycol precipitation method, aluminum salt coagulation precipitation method, and centrifugal ultrafiltration method. Polyethylene glycol precipitation method: Add polyethylene glycol to sewage. Under certain salt concentration conditions, polyethylene glycol can cause virus particles to form polymers. Under a certain centrifugal force, the aqueous solution and the virus particle polymers are separated, and the precipitate formed by the virus particle polymers is collected for subsequent nucleic acid extraction. Aluminum salt coagulation precipitation method: Add aluminum salt coagulant to sewage. The aluminum hydroxide colloid produced by the hydrolysis of aluminum salt can adsorb and wrap virus particles. The virus-containing colloid is collected by centrifugation, and then the wrapped virus particles are released by chemical dissolution for subsequent nucleic acid extraction. Centrifugal ultrafiltration method: Use an ultrafiltration cup embedded with an ultrafiltration membrane to retain virus particles with a molecular weight greater than the ultrafiltration membrane cutoff molecular weight in the ultrafiltration cup through centrifugation, and collect the virus concentrate in the ultrafiltration cup for subsequent nucleic acid extraction.

[0006] The above common virus concentration methods require the addition of various precipitants to assist in the collection and concentration of virus particles, and the process involves centrifugal concentration operations, which require corresponding centrifugal equipment. This step will limit the convenience and automation of the enrichment of viral nucleic acids in sewage.

[0007] CN117143962A discloses a convenient magnetic bead method for the configuration and application of a water virus nucleic acid extraction kit, which mainly packs the water sample into more than 50mL / bottle, centrifuges at 4°C, 2,500xg for 30 minutes, and transfers the supernatant after centrifugation from each bottle to a clean reagent bottle as a sample to be tested for subsequent viral nucleic acid extraction. The above method requires a long-term centrifugal concentration operation on the sewage sample, and cannot quickly and efficiently complete the pre-treatment concentration and enrichment operation of the virus particles in the sewage. In addition, a centrifugal device is required in the process, and there are many complex operations and cumbersome pre-treatments, and it is impossible to achieve a fast, convenient and automated operation for the purification of viral nucleic acids in sewage.

[0008] CN117384897A discloses a magnetic bead method for the enrichment and extraction of viral nucleic acid from water bodies. The virus-containing water sample is transferred to a filter containing water-based microporous filter paper for filtration, and the obtained filter membrane is transferred to a centrifuge tube, a lysate is added, and the sample is placed in a 60°C metal bath for 20 minutes. The obtained sample is subjected to nucleic acid extraction. Although the above method improves the efficiency of virus concentration in sewage compared to the polyethylene glycol precipitation method and the aluminum salt coagulation precipitation method, the sewage sample still needs to be filtered and concentrated by a filter, and there are also difficulties in the pre-treatment operation, which is time-consuming and difficult to realize automated operation, and it is impossible to realize the rapid and convenient automated operation of purifying viral nucleic acid in sewage.

[0009] Therefore, it is necessary to develop a new type of magnetic beads and a matching kit for purifying viral nucleic acids in sewage. Summary of the invention

[0010] In order to overcome the shortcomings of the prior art, the first object of the present invention is to provide a kit for purifying viral nucleic acids in sewage using magnetic beads, comprising the following components:

[0011] Nucleic acid extraction magnetic beads, nucleic acid purification magnetic beads, magnetic bead activation solution, nucleic acid release agent, cleaning solution and elution solution;

[0012] Wherein, the nucleic acid extraction magnetic beads are prepared by the following method:

[0013] Add olefin-based magnetic beads, monomers, crosslinking agents and initiators to the reaction system, transfer to a flask after ultrasonic dispersion, and start heating after mechanical stirring;

[0014] The reaction system is rapidly heated up, refluxed at a constant temperature, and then washed with ethanol to obtain wrinkled porous polymer magnetic beads;

[0015] Dispersing the prepared wrinkled porous polymer magnetic beads in a dispersion liquid to obtain nucleic acid extraction magnetic beads;

[0016] The nucleic acid purification magnetic beads are at least one of silicon hydroxy magnetic beads, carboxyl magnetic beads, and amino magnetic beads in the range of 50nm-10um.

[0017] Preferably, in the method for preparing magnetic beads for nucleic acid extraction, the magnetic bead activation solution is a NaCl solution with a concentration of 0.1-1 M.

[0018] Preferably, the nucleic acid releaser includes at least one of guanidine isothiocyanate, guanidine hydrochloride, sodium acetate, acetic acid, sodium hydroxide, sodium chloride, and bromocresol purple indicator.

[0019] Preferably, the cleaning solution at least includes an acid guanidine compound and an alcohol compound.

[0020] Preferably, the guanidine acid compound is guanidine hydrochloride and / or guanidine isothiocyanate.

[0021] Preferably, the alcohol compound is anhydrous ethanol and / or isopropanol.

[0022] Preferably, the alcohol compound is used in an amount of 20%-100%.

[0023] Preferably, the eluent is tris(hydroxymethyl)aminomethane and / or ethylenediaminetetraacetic acid.

[0024] The second object of the present invention is to provide a method for extracting nucleic acid based on the above-mentioned magnetic beads in a kit for purifying viral nucleic acid in sewage, comprising the following steps:

[0025] Take environmental water samples, add activated magnetic beads at one time or in batches, vortex mix, and complete nucleic acid capture;

[0026] After the nucleic acid is captured, magnetic separation is performed to collect the captured magnetic beads;

[0027] Add nucleic acid release agent to the capture magnetic beads and vortex to mix;

[0028] Magnetic separation, transfer the supernatant to a clean centrifuge tube;

[0029] Add cleaning solution and nucleic acid purification magnetic beads to the supernatant respectively, vortex mix, perform magnetic separation, and discard the supernatant;

[0030] After washing at least twice, keep the magnetic state, let it stand and evaporate;

[0031] Add appropriate amount of eluent, vortex mix, and elute thoroughly;

[0032] After magnetic separation, transfer the eluate to a clean centrifuge tube for subsequent detection.

[0033] Preferably, the method for preparing activated magnetic beads comprises:

[0034] Take an appropriate amount of nucleic acid extraction magnetic beads into a centrifuge tube, separate them by magnetic absorption, and discard the dispersion;

[0035] Add magnetic bead activation solution, vortex mix at high speed, separate by magnetic absorption, and discard the supernatant;

[0036] Disperse with magnetic bead activation solution and set aside.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention provides a matching kit for purifying viral nucleic acids in sewage using magnetic beads, comprising the following components: nucleic acid extraction magnetic beads, nucleic acid purification magnetic beads, magnetic bead activation solution, nucleic acid releaser, cleaning solution and elution solution; the present invention also relates to a nucleic acid extraction method. When the kit of the present invention is used, there is no need to perform cumbersome pre-treatment operations such as centrifugation or pre-precipitation on the sewage sample, and the concentration and enrichment of viruses in the sewage and the subsequent extraction of viral nucleic acids can be completed simply and quickly.

[0038] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail below. The specific implementation of the present invention is given in detail by the following embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0040] Figure 1 This is an optical microscope image of wrinkled porous polymer magnetic beads;

[0041] Figure 2 This is a scanning electron micrograph of wrinkled porous polymer magnetic beads;

[0042] Figure 3 This is a scanning electron micrograph of the surface of wrinkled porous polymer magnetic beads;

[0043] Figure 4 Directly extract nucleic acid from 10-100mL of wastewater containing anaerobic bacteria and E. coli (add 800bp\350bp PCR product);

[0044] Figure 5 Directly extract nucleic acid from 10-500mL wastewater containing anaerobic bacteria and E. coli (add 350bp\280bp PCR product);

[0045] Figure 6Gel run after PCR amplification of 280bp of eluate extracted from samples 1-10;

[0046] Figure 7 Gel run after 350bp PCR amplification of the eluate extracted from samples 1-10. DETAILED DESCRIPTION

[0047] The present invention is further described below in conjunction with specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0048] In view of the shortcomings of existing commercially available kits, the tedious and time-consuming pre-treatment and concentration operations of sewage samples such as centrifugation and pre-precipitation are eliminated, and the rapid enrichment and concentration of viral nucleic acids in sewage samples and the subsequent nucleic acid extraction operations are achieved; the present invention provides a matching kit for purifying viral nucleic acids in sewage using magnetic beads, comprising the following components:

[0049] Nucleic acid extraction magnetic beads, nucleic acid purification magnetic beads, magnetic bead activation solution, nucleic acid release agent, cleaning solution and elution solution;

[0050] Wherein, the nucleic acid extraction magnetic beads are prepared by the following method:

[0051] Add olefin-based magnetic beads, monomers, crosslinking agents and initiators to the reaction system, transfer to a flask after ultrasonic dispersion, and start heating after mechanical stirring;

[0052] The reaction system is heated up rapidly, refluxed at a constant temperature, and then washed with ethanol to obtain wrinkled porous polymer magnetic beads; they have ion exchange function, prefer to adsorb nucleic acids and are easy to desorb; they solve the time-consuming and labor-intensive operation of conventional methods relying on centrifugation and water enrichment pretreatment, and improve the efficiency and accuracy of nucleic acid detection in sewage. Generally, the volume percentage of olefin double-bond magnetic beads in the reaction system is 0.1-0.5%, including but not limited to: any one of silicon-based olefin magnetic beads, polymer olefin magnetic microspheres, and wrinkled macroporous polymer olefin magnetic microspheres. The volume percentage of the monomer in the reaction system is 0.5-5%; the monomer includes but is not limited to: any one or more of 4-aminostyrene, 4-dimethylaminostyrene, N-vinylpyrrolidone, N,N-dimethylallylamine, benzylvinyltrimethylammonium chloride, allyltrimethylammonium chloride, (3-acrylamidopropyl)trimethylammonium chloride, dimethylaminopropylacrylamide, 1-vinylimidazole, 1-allylimidazole, 1-allyl-3-methylimidazole chloride, N,N,N-trimethyl-(1-(4-vinylphenyl))methylammonium chloride, polyethyleneimine, and N,N-dimethylvinylbenzylamine. The volume percentage of the crosslinking agent in the reaction system is 0.5-5%, including but not limited to any one or more of divinylbenzene, N,N-methylenebisacrylamide, and dimethyldiallylammonium chloride. The volume percentage of the initiator in the reaction system is 0.1-1%, including but not limited to any one of azobisisobutyronitrile, dimethyl azobisisobutyrate, and potassium persulfate. The reaction system of the magnetic beads is mainly a reflux reaction, and the system includes but is not limited to any one or more of anhydrous ethanol, acetonitrile, isopropanol, and dimethyl sulfoxide. The wrinkled porous polymer magnetic beads thus formed have a surface controlled by a reaction process to form a multi-fold porous structure on the surface of the magnetic beads. Compared with traditional smooth spherical magnetic beads, they have a larger effective specific surface area. The wrinkled structure on the surface of the magnetic beads can effectively reduce the steric hindrance on the surface of the magnetic beads when enriching viral particles, and have a strong adsorption effect on viral nucleic acids. By changing the adsorption environment of the magnetic beads reagent, viral nucleic acid adsorption and release can be achieved, with high efficiency, high reusability, and good application prospects.

[0053] The prepared wrinkled porous polymer magnetic beads are dispersed in a dispersion liquid to obtain nucleic acid extraction magnetic beads, such as a 0.1-0.5M NaCl solution; it should be understood that the dispersion liquid can also be a common dispersion liquid in the art, such as pure water. The final concentration of the magnetic beads after dispersion is 20mg / mL-60mg / mL.

[0054] The nucleic acid purification magnetic beads are at least one of silicon hydroxy magnetic beads, carboxyl magnetic beads, and amino magnetic beads in the range of 50nm-10um, and the nucleic acid purification magnetic beads are monodisperse or polydisperse magnetic beads.

[0055] In some embodiments, in the method for preparing magnetic beads for nucleic acid extraction, the magnetic bead activation solution is a 0.1-1M NaCl solution. It should be understood that the magnetic bead activation solution can also be a common magnetic bead activation solution in the art.

[0056] In some embodiments, the nucleic acid release agent includes at least one of guanidine isothiocyanate, guanidine hydrochloride, sodium acetate, acetic acid, sodium hydroxide, sodium chloride, and bromocresol purple indicator; the final concentration of guanidine isothiocyanate or guanidine hydrochloride in the nucleic acid release agent is 1M-5M, and the final concentration of sodium acetate and acetic acid in the nucleic acid release agent is 50mM-500mM. It should be understood that the magnetic bead activation solution can also be a common nucleic acid release agent in the art.

[0057] In some embodiments, the cleaning solution at least comprises an acid guanidine compound and an alcohol compound, wherein the acid guanidine compound is guanidine hydrochloride and / or guanidine isothiocyanate with a concentration of 0.2M-1M, and the alcohol compound is anhydrous ethanol and / or isopropanol.

[0058] In some embodiments, the alcohol compound is used in an amount of 20%-100%; 20%-100% refers to the proportion of alcohol in the cleaning solution. If the alcohol compound content is low, the cleaning solution is not effective, which is not conducive to the cleaning of nucleic acids. Finally, the nucleic acid purity is poor. And when the alcohol content is low, the nucleic acid is easily eluted from the cleaning solution, resulting in a large amount of nucleic acid loss.

[0059] In some embodiments, the eluent is one or more of Tris (tris(hydroxymethylaminomethane)) and EDTA (ethylenediaminetetraacetic acid), and the final concentration is 0.01%-0.15%. It should be understood that the eluent may also be a common eluent in the art.

[0060] The present invention also relates to a nucleic acid extraction method of a matching kit for purifying viral nucleic acid in sewage based on magnetic beads, comprising the following steps:

[0061] Take environmental water samples, add activated magnetic beads at one time or in batches, vortex mix, and complete nucleic acid capture;

[0062] After the nucleic acid is captured, magnetic separation is performed to collect the captured magnetic beads;

[0063] Add nucleic acid release agent to the capture magnetic beads and vortex to mix;

[0064] Magnetic separation, transfer the supernatant to a clean centrifuge tube;

[0065] Add cleaning solution and nucleic acid purification magnetic beads to the supernatant respectively, vortex mix, perform magnetic separation, and discard the supernatant;

[0066] After washing at least twice, keep the magnetic state, let it stand and evaporate;

[0067] Add appropriate amount of eluent, vortex mix, and elute thoroughly;

[0068] Magnetic separation, the eluate is transferred to a clean centrifuge tube for subsequent detection. Specifically, the steps of extracting and purifying nucleic acids in environmental water (and nucleic acids of Gram-negative bacteria in environmental water) include:

[0069] Step 1: Take an environmental water sample, add activated magnetic beads at one time or in batches, vortex mix, and complete nucleic acid capture: The batch addition in this step can be a one-time addition or a batch addition. For example, in some embodiments, take an environmental water sample of a suitable volume, add 1 / 2 volume of activated magnetic beads, vortex mix for 15-20 minutes, and then add another 1 / 2 volume of activated magnetic beads to complete nucleic acid capture; in some embodiments, for a small volume of 0.5-2mL water sample, all activated magnetic beads can be added at one time. See Table 1 for recommended capture time.

[0070] It should be understood that a higher vortex mixing speed and a slightly longer vortex mixing time are beneficial to the rapid and efficient capture of nucleic acids.

[0071] Step 2: After nucleic acid capture is completed, magnetic separation is performed and the captured magnetic beads are collected;

[0072] Step 3: Add nucleic acid release agent to the captured magnetic beads and vortex mix; for example, add 600 μL nucleic acid release agent to the magnetic beads and vortex mix for 15 minutes; it should be understood that a longer vortex time is beneficial to reduce the interference of Gram-negative bacteria in environmental water.

[0073] Step 4: Magnetic separation, transfer the supernatant to a clean (or new) centrifuge tube;

[0074] Step 5: Add cleaning solution and nucleic acid purification magnetic beads to the supernatant, vortex mix, perform magnetic separation, and discard the supernatant; for example, add 300 μL of isopropanol and 40 μL of nucleic acid purification magnetic beads to the collected supernatant, vortex mix for 6 minutes, perform magnetic separation, and discard the supernatant;

[0075] Step 6: After washing at least twice, keep the magnetic state, let it stand, and evaporate to dryness; if it can be washed twice, for the first wash: add 500μL of washing solution to the purified magnetic beads, vortex mix for 1min, magnetically separate, and discard the supernatant; for the second wash: add 500μL of freshly prepared 80% ethanol, vortex mix for 30-60s, magnetically separate, and discard the supernatant.

[0076] It should be understood that after the above operation is completed, the washing can be repeated once with 80% ethanol.

[0077] After cleaning at least twice, the alcohol removal operation can be performed: keep the magnetic state, let it stand, and evaporate the ethanol.

[0078] It should be understood that over-drying will affect the subsequent elution efficiency.

[0079] Step 7: Add an appropriate amount of elution buffer, vortex mix, and elute thoroughly; when eluting, add an appropriate amount of elution buffer or self-prepared elution buffer (such as 20-50 μL), vortex mix for 4-6 minutes, and elute thoroughly.

[0080] It will be appreciated that a temperature of 50-60°C is beneficial for adequate elution.

[0081] Step 8: Magnetic separation, transfer the eluate to a clean centrifuge tube for subsequent testing. The clean centrifuge tube can be a new centrifuge tube or a PCR tube.

[0082] It should be understood that extraction and purification can be performed manually. If automatic instrument purification is required, the automatic instrument purification process can be edited by the user based on a full understanding of the process and in combination with different instrument operations.

[0083] In some embodiments, the method for preparing activated magnetic beads comprises:

[0084] Take an appropriate amount of nucleic acid extraction magnetic beads into a centrifuge tube, separate them by magnetic absorption, and discard the dispersion;

[0085] Add magnetic bead activation solution, vortex mix at high speed, separate by magnetic absorption, and discard the supernatant;

[0086] Disperse with magnetic bead activation solution and set aside. Specifically, the activation steps of nucleic acid extraction magnetic beads (capture magnetic beads preparation) are:

[0087] Use a pipette to take an appropriate amount of nucleic acid extraction magnetic beads into a centrifuge tube, separate them by magnetic absorption, and discard the dispersion;

[0088] The reference dosage of magnetic beads for nucleic acid extraction is shown in Table 1.

[0089] Add 200 μL of magnetic bead activation solution, vortex mix at high speed for 1 min, separate by magnetic absorption, and discard the supernatant;

[0090] Repeat the above steps to activate once more;

[0091] Disperse with 100-200 μL magnetic bead activation solution and set aside.

[0092] Table 1 Recommended dosage of magnetic beads for nucleic acid extraction

[0093]

[0094] It should be understood that if capture time adjustment is required, it can be optimized at will.

[0095] It should be understood that the amount of magnetic beads and capture time corresponding to other water sample volumes can be appropriately adjusted and optimized by referring to this table.

[0096] Embodiment 1

[0097] Synthesis process of 5um wrinkled porous polymer magnetic beads

[0098] 1. Add 6g of 5um polymer matrix olefin magnetic microspheres to 500mL of anhydrous ethanol and 1000mL of acetonitrile, stir and ultrasonically disperse for 10min, add 5mL of monomer 4-dimethylaminostyrene, 5g of benzylvinyltrimethylammonium chloride, 10mL of 1-allylimidazole, 5mL of crosslinking agent divinylbenzene, 20g of N-methylenebisacrylamide, and 6g of initiator dimethyl azobisisobutyrate, then continue ultrasonication until uniform dispersion, transfer to a flask and mechanically stir for 10min before starting to heat up.

[0099] 2. Rapidly heat up to the internal temperature of 75±5℃ and reflux at constant temperature for 2h.

[0100] 3. After the reaction is completed, magnetic separation is performed, and the beads are washed with ethanol for 4-5 times and dried to obtain wrinkled porous polymer magnetic beads.

[0101] Embodiment 2

[0102] Modification of 30-40um wrinkled porous polymer-based olefin magnetic microspheres

[0103] 1. Add 8g 30-40um wrinkled porous polymer matrix olefin magnetic microspheres to 500mL isopropanol and 1000ml dimethyl sulfoxide, stir and ultrasonically disperse for 10min, add monomer 4-aminostyrene 10g, allyltrimethylammonium chloride 10g, dimethylaminopropyl acrylamide 25g, 1-allyl-3-methylimidazole chloride 30g, cross-linking agent divinylbenzene 50mL, dimethyldiallylammonium chloride 25mL, initiator potassium persulfate 15g, continue ultrasonication until uniform dispersion, transfer to a flask and mechanically stir for 10min before starting to heat up.

[0104] 2. Rapidly heat up to the internal temperature of 75±5℃ and reflux at constant temperature for 2h.

[0105] 3. After the reaction is completed, magnetic separation is performed, and the beads are washed with ethanol for 4-5 times and dried to obtain wrinkled porous polymer magnetic beads.

[0106] Embodiment 3

[0107] The components of the magnetic bead application kit in this embodiment are shown in Table 2 below:

[0108] Table 2 Magnetic beads application kit group 1

[0109]

[0110]

[0111] Embodiment 4

[0112] The components of the magnetic bead application kit in this embodiment are shown in Table 3 below:

[0113] Table 3 Components of the two-component kit for magnetic bead application

[0114]

[0115] Embodiment 5

[0116] 1. Preparation of sewage samples: Take 10ml, 20ml, 50ml and 100ml of anaerobic wastewater (taken from the company's sewage treatment pool), add 10ug 800bp and 10ug 350bp nucleic acid fragments to different volumes of sewage samples and mix them evenly as sewage samples to be extracted.

[0117] 2. Activation of magnetic beads for nucleic acid extraction:

[0118] 2.1 Use a pipette to take an appropriate amount of nucleic acid extraction magnetic beads (concentration 50 mg / mL, 35 um wrinkled porous polymer magnetic beads) into a centrifuge tube, separate by magnetic absorption, and discard the dispersion;

[0119] The reference dosage of magnetic beads for nucleic acid extraction is shown in Table 4.

[0120] 2.2 Add 200 μL of magnetic bead activation solution, i.e. 0.2 M / L NaCl solution, vortex mix at high speed for 1 min, separate by magnetic absorption, and discard the supernatant;

[0121] 2.3 Repeat 2.2 activation once;

[0122] 2.4 Disperse with 200 μL magnetic bead activation solution, i.e. 0.2 M / L NaCl solution, and set aside.

[0123] Table 4 Amount of magnetic beads for nucleic acid extraction

[0124]

[0125] 3. Extraction and purification of nucleic acids in environmental water

[0126] 3.1 Add activated magnetic beads in batches: Take the prepared sewage sample, add 1 / 2 volume of activated magnetic beads (see Table 4), vortex mix for 15-20 minutes, and then add another 1 / 2 volume of activated magnetic beads (see Table 4) to complete nucleic acid capture;

[0127] 3.2 After nucleic acid capture is completed, magnetic separation is performed and the captured magnetic beads are collected;

[0128] 3.3 Add 600 μL of nucleic acid release agent (corresponding to the nucleic acid release agent in Example 4) to the magnetic beads and vortex mix for 15 minutes;

[0129] 3.4 Magnetic separation, transfer the supernatant to a new centrifuge tube;

[0130] 3.5 Add 300 μL of isopropanol and 40 μL of nucleic acid purification magnetic beads (MSi010-DNA-0507 silicon hydroxy magnetic beads, manufacturer: Suzhou Yingruicheng Biochemical Technology Co., Ltd., model: MSi010-DNA-0507 silicon hydroxy magnetic beads) to the supernatant collected in 3.4, vortex mix for 6 minutes, magnetically separate, and discard the supernatant;

[0131] 3.6 Washing 1: Add 500 μL of washing solution (80% ethanol) to the nucleic acid purification magnetic beads, vortex mix for 1 min, magnetically separate, and discard the supernatant;

[0132] 3.7 Wash 2: Add 500 μL of freshly prepared 80% ethanol, vortex mix for 30-60 seconds, magnetically separate, discard the supernatant; and repeat the wash with 80% ethanol;

[0133] 3.8 Alcohol removal: Keep the magnetic state, let it stand, and evaporate the ethanol;

[0134] 3.9 Elution: Add 20-50 μL of elution buffer or your own elution buffer (Tris-HCl, concentration is 5 mM), vortex mix for 4-6 minutes, and elute thoroughly;

[0135] 3.10 Magnetic separation, transfer the eluate to a new centrifuge tube or PCR tube for subsequent detection.

[0136] 3.11 Take the above eluate and run the gel for detection. The bands from left to right are: DNA market, 850bp DNA fragment, nucleic acid fragment extracted from 10ml sewage sample, nucleic acid fragment extracted from 20ml sewage sample, nucleic acid fragment extracted from 50ml sewage sample, and nucleic acid fragment extracted from 100ml sewage sample.

[0137] like Figure 4 As shown, the product of the present invention can purify and enrich nucleic acids in sewage without the need for cumbersome pretreatments such as centrifugal concentration and pre-precipitation; it can quickly and effectively complete the enrichment and purification of nucleic acids of different sizes and fragments in sewage samples of various sizes ranging from 10ml to 100ml.

[0138] Embodiment 6

[0139] 1.1. Preparation of sewage samples: Take 10ml, 20ml, 50ml, 100ml and 500ml of anaerobic wastewater (taken from the company's sewage treatment pool) respectively, add 10ug 350bp and 10ug 280bp nucleic acid fragments to different volumes of sewage samples and mix them evenly as sewage samples to be extracted.

[0140] Table 5 Quantity of samples No. 1-5

[0141]

[0142] 1.2 Preparation of sewage samples: Take 10ml, 20ml, 50ml, 100ml and 500ml of anaerobic wastewater (taken from the company's sewage treatment pool) respectively, add 10ug 350bp and 10ug 280bp nucleic acid fragments and Escherichia coli liquid as interfering bacteria to sewage samples of different volumes and mix them evenly as sewage samples to be extracted.

[0143] Table 6 Quantity of samples No. 6-10

[0144]

[0145] 2. Activation of magnetic beads for nucleic acid extraction:

[0146] 2.1 Use a pipette to take an appropriate amount of nucleic acid extraction magnetic beads (concentration 50 mg / mL, 35 um wrinkled porous polymer magnetic beads) into a centrifuge tube, separate by magnetic absorption, and discard the dispersion; the reference amount of nucleic acid extraction magnetic beads is shown in Table 7.

[0147] 2.2 Add 200 μL of magnetic bead activation solution, i.e. 0.2 M / L NaCl solution, vortex mix at high speed for 1 min, separate by magnetic absorption, and discard the supernatant;

[0148] 2.3 Repeat 2.2 activation once;

[0149] 2.4 Disperse with 200 μL magnetic bead activation solution, i.e. 0.2 M / L NaCl solution, and set aside.

[0150] Table 7 Amount of magnetic beads for nucleic acid extraction

[0151]

[0152]

[0153] 3. Extraction and purification of nucleic acids in environmental water

[0154] 3.1 Add activated magnetic beads in batches: Take the prepared sewage sample, add 1 / 2 volume of activated magnetic beads (see Table 7), vortex mix for 15-20 minutes, and then add another 1 / 2 volume of activated magnetic beads (see Table 7) to complete nucleic acid capture;

[0155] 3.2 After nucleic acid capture is completed, magnetic separation is performed and the captured magnetic beads are collected;

[0156] 3.3 Add 600 μL of nucleic acid release agent (the nucleic acid release agent selected in Example 4) to the magnetic beads and vortex mix for 15 minutes;

[0157] 3.4 Magnetic separation, transfer the supernatant to a new centrifuge tube;

[0158] 3.5 Add 300 μL of isopropanol and 40 μL of nucleic acid purification magnetic beads (MSi010-DNA-0507 silicon hydroxy magnetic beads, manufacturer: Suzhou Yingruicheng Biochemical Technology Co., Ltd., model: MSi010-DNA-0507 silicon hydroxy magnetic beads) to the supernatant collected in 3.4, vortex mix for 6 minutes, magnetically separate, and discard the supernatant;

[0159] 3.5 Washing 1: Add 500 μL of washing solution to the nucleic acid purification magnetic beads, vortex mix for 1 min, perform magnetic separation, and discard the supernatant;

[0160] 3.6 Wash 2: Add 500 μL of freshly prepared 80% ethanol, vortex mix for 30-60 seconds, magnetically separate, discard the supernatant; and repeat the wash with 80% ethanol;

[0161] 3.7 Alcohol removal: Keep the magnetic state, let it stand, and evaporate the ethanol;

[0162] 3.8 Elution: Add 20-50 μL of elution buffer or self-prepared elution buffer (Tris-HCl, concentration is 5 mM), vortex mix for 4-6 minutes, and elute thoroughly;

[0163] 3.9 Magnetic separation, transfer the eluate to a new centrifuge tube or PCR tube for subsequent detection.

[0164] 3.10 Take the above eluate and run the gel for detection. The bands from left to right are: Sample No. 1: nucleic acid fragments extracted from 500 ml of waste water, Sample No. 2: nucleic acid fragments extracted from 100 ml of waste water, Sample No. 3: nucleic acid fragments extracted from 50 ml of waste water, Sample No. 4: nucleic acid fragments extracted from 20 ml of waste water, Sample No. 5: nucleic acid fragments extracted from 10 ml of waste water, DNA market, Sample No. 6: nucleic acid fragments extracted from 10 ml of waste water, Sample No. 7: nucleic acid fragments extracted from 20 ml of waste water, Sample No. 8: nucleic acid fragments extracted from 50 ml of waste water, Sample No. 9: nucleic acid fragments extracted from 100 ml of waste water, Sample No. 10: nucleic acid fragments extracted from 500 ml of waste water.

[0165] like Figure 5-7 As shown, the product of the present invention can accurately extract and purify target fragments from sewage containing a variety of bacteria, and the sewage sample does not need to be centrifuged for tedious pretreatment operations, and can be directly extracted from the sewage sample. Furthermore, the product of the present invention can perform nucleic acid extraction processing on samples of various sizes of 10-500ml.

[0166] It should be understood that nucleic acid extraction and purification can be done manually or automatically. Based on a full understanding of the process, the automatic purification process can be edited by the user in combination with different instrument operations.

[0167] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the embodiments shown here.

Claims

1. A kit for purifying viral nucleic acid in sewage using magnetic beads, characterized in that: Includes the following components: Nucleic acid extraction magnetic beads, nucleic acid purification magnetic beads, magnetic bead activation solution, nucleic acid release agent, cleaning solution and elution solution; Wherein, the nucleic acid extraction magnetic beads are prepared by the following method: Add olefin-based magnetic beads, monomers, crosslinking agents and initiators to the reaction system, transfer to a flask after ultrasonic dispersion, and start heating after mechanical stirring; The reaction system is rapidly heated up, refluxed at a constant temperature, and then washed with ethanol to obtain wrinkled porous polymer magnetic beads; Dispersing the prepared wrinkled porous polymer magnetic beads in a dispersion liquid to obtain nucleic acid extraction magnetic beads; The nucleic acid purification magnetic beads are at least one of silicon hydroxy magnetic beads, carboxyl magnetic beads, and amino magnetic beads in the range of 50nm-10um.

2. The kit for purifying viral nucleic acid in sewage using magnetic beads according to claim 1, characterized in that: In the method for preparing magnetic beads for nucleic acid extraction, the magnetic bead activation solution is a NaCl solution with a concentration of 0.1-1M.

3. The kit for purifying viral nucleic acid in sewage using magnetic beads according to claim 1, characterized in that: The nucleic acid release agent includes at least one of guanidine isothiocyanate, guanidine hydrochloride, sodium acetate, acetic acid, sodium hydroxide, sodium chloride, and bromocresol purple indicator.

4. The kit for purifying viral nucleic acid in sewage using magnetic beads according to claim 1, characterized in that: The cleaning solution at least includes an acid guanidine compound and an alcohol compound.

5. The kit for purifying viral nucleic acid in sewage using magnetic beads as claimed in claim 4, characterized in that: The guanidine acid compound is guanidine hydrochloride and / or guanidine isothiocyanate.

6. The kit for purifying viral nucleic acid in sewage using magnetic beads as claimed in claim 4, characterized in that: The alcohol compound is anhydrous ethanol and / or isopropanol.

7. The kit for purifying viral nucleic acid in sewage using magnetic beads as claimed in claim 4 or 6, characterized in that: The alcohol compound is used in an amount of 20%-100%.

8. The kit for purifying viral nucleic acid in sewage using magnetic beads as claimed in claim 1, characterized in that: The eluent is tris(hydroxymethyl)aminomethane and / or ethylenediaminetetraacetic acid.

9. A method for extracting nucleic acid based on a kit for purifying viral nucleic acid in sewage using magnetic beads according to any one of claims 1 to 8, characterized in that: The following steps are involved: Take environmental water samples, add activated magnetic beads at one time or in batches, vortex mix, and complete nucleic acid capture; After the nucleic acid is captured, magnetic separation is performed to collect the captured magnetic beads; Add nucleic acid release agent to the capture magnetic beads and vortex to mix; Magnetic separation, transfer the supernatant to a clean centrifuge tube; Add cleaning solution and nucleic acid purification magnetic beads to the supernatant respectively, vortex mix, perform magnetic separation, and discard the supernatant; After washing at least twice, keep the magnetic state, let it stand and evaporate; Add appropriate amount of eluent, vortex mix, and elute thoroughly; After magnetic separation, transfer the eluate to a clean centrifuge tube for subsequent detection.

10. The method for extracting nucleic acid of the supporting kit for purifying viral nucleic acid in sewage using magnetic beads as claimed in claim 9, characterized in that: The method for preparing activated magnetic beads comprises: Take an appropriate amount of nucleic acid extraction magnetic beads into a centrifuge tube, separate them by magnetic absorption, and discard the dispersion; Add magnetic bead activation solution, vortex mix at high speed, separate by magnetic absorption, and discard the supernatant; Disperse with magnetic bead activation solution and set aside.

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