CN112980830A - Kit for magnetic bead method nucleic acid extraction, magnetic bead and preparation method thereof - Google Patents

Kit for magnetic bead method nucleic acid extraction, magnetic bead and preparation method thereof Download PDF

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CN112980830A
CN112980830A CN201911311308.7A CN201911311308A CN112980830A CN 112980830 A CN112980830 A CN 112980830A CN 201911311308 A CN201911311308 A CN 201911311308A CN 112980830 A CN112980830 A CN 112980830A
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mmol
nucleic acid
magnetic bead
mol
cell lysate
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CN112980830B (en
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黄超杰
鲁津津
黄硕
路通
耿春雨
陈芳
蒋慧
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MGI Tech Co Ltd
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MGI Tech Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The application discloses a kit for magnetic bead method nucleic acid extraction, magnetic beads and a preparation method thereof. The kit for extracting nucleic acid by the magnetic bead method comprises cell lysate, magnetic beads and washing liquid, wherein the cell lysate contains guanidine isothiocyanate, Triton X-100, 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid and N-lauroyl sarcosine sodium. According to the kit for extracting nucleic acid by the magnetic bead method, the components of the lysis buffer solution are optimized, so that cells can be quickly and fully lysed, protein can be denatured, the protein and the nucleic acid can be effectively separated, and the nucleic acid can be released; improves the working efficiency of cell lysate and shortens the time of nucleic acid extraction.

Description

Kit for magnetic bead method nucleic acid extraction, magnetic bead and preparation method thereof
Technical Field
The application relates to the field of nucleic acid extraction, in particular to a kit for extracting nucleic acid by a magnetic bead method, a magnetic bead and a preparation method of the magnetic bead.
Background
Nucleic acids are substances that carry genetic information within cells, and play an extremely important role in the inheritance, mutation, and protein biosynthesis of organisms. With the rapid development of molecular biological detection technology, molecular diagnostic technology based on nucleic acid information becomes a novel clinical means and idea for effective disease diagnosis, especially in the fields of infectious diseases, tumors and genetic diseases. The newly developed molecular diagnosis technology for infectious diseases effectively makes up the defects of the traditional method, and provides infection information of bacteria, fungi or viruses, a drug-resistant gene result of pathogens and even homology analysis of the pathogens for clinicians.
The development of molecular diagnostic technology based on nucleic acid information has higher requirements on nucleic acid extraction and purification, and the rapid and efficient performance of clinical pathogen molecular detection is an important factor in the clinical application of molecular detection besides the need of obtaining high-quality nucleic acid molecules. For example, in clinical treatment, for some acute infection patients, rapid and accurate determination of pathogen is the most critical link for treatment. In addition, for some acute virus-induced epidemics, field detection is sometimes required; for some special departments and posts, rapid and accurate diagnosis is needed, for example, customs law enforcement officers need to rapidly diagnose whether the hot passengers are caused by specific viruses or germs, the process is often required to be completed within 0.5-2 hours, otherwise great inconvenience may be brought to the passengers, and even potential safety hazards may be caused to other passengers.
At present, the method for extracting microbial nucleic acid aiming at clinical samples such as serum, saliva, throat swabs and the like on the market mainly adopts a magnetic bead method, an adsorption column method and a Chelex100 boiling method for quickly extracting nucleic acid. The magnetic bead method mainly comprises the steps of firstly, performing nucleic acid separation by adopting a mode of incubation of biological active enzymes such as lysozyme and proteinase K and combined with wall breaking denaturation of a lysate, and then performing operations such as nucleic acid purification by adopting magnetic beads; the method can be combined with automatic extraction equipment to realize automatic operation, reduce pollution and operation errors caused by operators, and obtain the nucleic acid with high yield and good purity; however, the extraction steps are complicated, the operation period is long, the whole extraction needs 1.5 to 3 hours, even longer extraction needs overnight extraction, and the requirement of rapid detection is difficult to meet.
In addition, the existing nucleic acid extraction kit by the magnetic bead method has the following defects: firstly, in a cracking link, biological active enzymes such as lysozyme, proteinase K and the like are generally adopted and then are combined with strong protein denaturants such as guanidine hydrochloride or guanidine isothiocyanate and the like, incubation is carried out at 56-65 ℃, proteins and other components in sample tissues are damaged, nucleic acid is released, and then the nucleic acid is precipitated by adding isopropanol or absolute ethyl alcohol and the like in a certain proportion; the general operation time of the process is 25 minutes to 1 hour, even overnight incubation, and the requirement of rapid detection or field detection is difficult to meet. Secondly, after the magnetic beads are adsorbed, two washing solutions are usually adopted for two-step washing, firstly, a high-salt reagent washing solution containing alcohols such as absolute ethyl alcohol or isopropanol and the like in a certain proportion is adopted for washing to remove non-specific foreign proteins adsorbed on the magnetic beads, and then, a second-step washing is carried out by a washing solution containing a high-proportion absolute ethyl alcohol low-salt reagent to remove salt ions of the magnetic beads; in addition, after washing is finished, since washing is carried out by using PCR inhibitors such as ethanol and the like, subsequent PCR amplification is influenced, the operation of uncovering and placing for 5-10 minutes before elution is often needed, so that ethanol is volatilized and removed, the operation not only prolongs the extraction operation time, but also easily causes cross contamination among samples when the samples are directly uncovered and placed and exposed in the air. Thirdly, the existing magnetic bead method for extracting nucleic acid usually needs to elute the nucleic acid adsorbed by the magnetic bead, then absorbs the supernatant, removes the magnetic bead, and then adopts the nucleic acid dissolved in the supernatant to perform PCR amplification, which inevitably causes nucleic acid loss, especially, part of nucleic acid is abandoned in the transfer process of the nucleic acid solution absorbing the supernatant and eluting, which is not beneficial to the nucleic acid extraction and detection of trace samples, and indirectly reduces the detection sensitivity.
Disclosure of Invention
The application aims to provide an improved kit for extracting nucleic acid by a magnetic bead method, a magnetic bead and a preparation method thereof.
The following technical scheme is adopted in the application:
one aspect of the application discloses a kit for magnetic bead method nucleic acid extraction, which comprises cell lysate, magnetic beads and washing liquid, wherein the cell lysate contains guanidine isothiocyanate, Triton X-100, 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid and N-lauroyl sarcosine sodium.
In the kit for extracting nucleic acid by the magnetic bead method, Triton X-100, 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid and N-lauroyl sarcosine sodium are added into a cell lysate, and guanidine isothiocyanate is matched, so that cells can be quickly and sufficiently destroyed, protein is denatured, effective separation of protein and nucleic acid is promoted, and nucleic acid is released; thereby improving the working efficiency of the cell lysate and shortening the time of releasing nucleic acid. The cell lysis solution is a solution combining cell lysis and magnetic beads, that is, when the cell lysis solution is used, the cell lysis solution, the magnetic beads and cells to be treated are mixed together for reaction, and when the cells are broken to release nucleic acids, the nucleic acids and the magnetic beads are combined; therefore, the cell lysate can shorten the time for releasing nucleic acid, save the time for combining nucleic acid and magnetic beads, and further improve the efficiency of the nucleic acid extraction kit by the magnetic bead method.
It can be understood that the key point of the application is to add guanidinium isothiocyanate, Triton X-100, 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid and N-lauroyl sarcosine sodium into a cell lysate, and as for conventional components in the cell lysate, such as lysis buffer and the like, reference can be made to the existing magnetic bead method nucleic acid extraction kit; however, in order to ensure the efficiency and quality of the cell lysate, other components such as lysis buffer are limited, and the details are shown in the following technical scheme. In addition, other additives or functional reagents may be added to the cell lysate of the present application to provide the cell lysate with corresponding functions, as long as the properties of guanidinium isothiocyanate, Triton X-100, and the like are not affected, and for example, an antifoaming agent, a metal ion chelating agent, and the like may be added.
Preferably, in the cell lysate, the concentration of guanidinium isothiocyanate is 2-4 mol/L, the content of Triton X-100 is 1-5 vol%, the content of 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid is 5-50 g/L, and the content of N-lauroylsarcosine sodium is 1-10 g/L.
It should be noted that, although the key point of the present application is to add guanidinium isothiocyanate, Triton X-100, 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid and sodium N-lauroyl sarcosinate into the cell lysate, the efficiency of the cell lysate can be improved to different degrees as long as the four components are added into the cell lysate; however, further studies in this application have found that better cell lysis and bead binding effects can be achieved with specific concentrations of Triton X-100, 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid and N-lauroyl sarcosine sodium in combination with high concentrations of guanidinium isothiocyanate.
Preferably, the cell lysate further comprises polyethylene glycol.
Preferably, the content of polyethylene glycol in the cell lysate is 50-200 g/L.
Preferably, the polyethylene glycol is PEG4000, PEG6000 or PEG 8000.
In the present application, the addition of polyethylene glycol to the cell lysate further concentrates the precipitated nucleic acids, promotes the binding of nucleic acids to magnetic beads, further improves the efficiency of binding nucleic acids to magnetic beads, and shortens the time for binding magnetic beads. It can be understood that the effect of concentrating the precipitated nucleic acid can be achieved by adding polyethylene glycol; however, in order to ensure the functions and effects of other components of the cell lysate, the content of polyethylene glycol is preferably 50-200 g/L; in the range, the concentration of nucleic acid can be well guaranteed, the combination of the nucleic acid and magnetic beads is promoted, and the phenomenon that cell lysate is too viscous due to too high dosage of polyethylene glycol is avoided.
Preferably, the cell lysate further comprises a lysis buffer, a metal ion chelating agent, a negative charge neutralizing agent, and an antifoaming agent.
Wherein, the lysis buffer solution is used for providing a reaction environment for cell lysis and combination of the magnetic beads and the nucleic acids; metal ion chelating agents are used to bind divalent metal ions, such as Mg ions, Ca ions, Mn ions, Fe ions, and the like; the negative charge neutralizer is used for combining with phosphate radicals in nucleic acid and neutralizing the negative charge of the phosphate radicals of the nucleic acid, so that the acting force of the nucleic acid and water molecules is weakened, the solubility is reduced, and the nucleic acid is precipitated; the antifoaming agent is used for eliminating foam generated by cell lysate.
The metal ion chelating agent, the negative charge neutralizing agent and the defoaming agent are further added into the cell lysate, and the key point is that the components are matched with each other, so that the efficiency of the cell lysate is further improved. Wherein, the specific lysis buffer can refer to a conventional magnetic bead method; the role of the metal ion chelating agent is to bind divalent metal ions, and metal ion chelating agents having such an effect and causing no adverse effect on nucleic acids may be used herein; the negative charge neutralizer is used for neutralizing the negative charge of the nucleic acid phosphate to precipitate the nucleic acid, and reagents capable of neutralizing the negative charge of the nucleic acid phosphate can be used in the application; according to the method, the nucleic acid is precipitated by using the negative charge neutralizer without using isopropanol or absolute ethyl alcohol, so that the influence of the isopropanol or absolute ethyl alcohol on the subsequent process or detection is avoided; the function of the defoamer is to eliminate foam, and conventional defoamers can be used in this application. The key point of the application is the organic combination of each component, and the specific selection of each functional component can be selected conventionally according to the definition of the functional component; however, in order to achieve better effects, the components are defined in detail in the application, and the technical scheme is described in detail below.
Preferably, the lysis buffer is Tris-HCL, and the concentration of the Tris-HCL in the cell lysate is 10 mmol/L-100 mmol/L.
It should be noted that Tris-HCL is only a buffer solution which is used conventionally in cell lysates, and does not exclude the possibility of using other buffer solutions. However, in the present application, it is preferable to use Tris-HCl of 10mmol/L to 200mmol/L as a lysis buffer in view of cell lysis and magnetic bead binding.
Preferably, the metal ion chelating agent is Ethylene Diamine Tetraacetic Acid (EDTA), the concentration of the EDTA in the cell lysis solution is 5 mmol/L-50 mmol/L, and the pH value is 8.0.
It should be noted that a certain amount of EDTA can bind with divalent metal ions such as Mg, Ca, Mn, Fe, etc., inhibit the activity of nuclease, and ensure the integrity of nucleic acid; it will be appreciated that other types of metal ion chelating agents besides EDTA are not excluded, as long as they do not adversely affect the nucleic acid or other components of the cell lysate.
Preferably, the negative charge neutralizer is NaCl, and the concentration of NaCl in the cell lysate is 0.28mol/L to 0.5 mol/L.
In the NaCl with a specific proportion, sodium ions can neutralize negative charges of nucleic acid phosphate radicals, so that the acting force between nucleic acid and water molecules is weakened, the solubility is reduced, and the precipitation of the nucleic acid is facilitated; it will be appreciated that other types of negative charge neutralizers besides NaCl are not excluded, as long as they do not adversely affect the nucleic acid or other components of the cell lysate.
Preferably, the antifoaming agent is Antifoam204, and the concentration of Antifoam204 in the cell lysate is 0.01 vol% to 0.1 vol%. Among them, Antifoam204, i.e., antifoaming agent 204, can be commercially available.
It should be noted that the function of the defoaming agent is to eliminate the foam generated by the reagent, which is beneficial to the production and extraction operations. It will be appreciated that other types of anti-foaming agents besides Antifoam204 are not excluded, as long as they do not adversely affect the nucleic acid or other components of the cell lysate.
Preferably, the cell lysate consists of 10 mmol/L-100 mmol/L Tris-HCl, 5 mmol/L-50 mmol/L EDTA with pH8.0, 2 mol/L-4 mol/L guanidine isothiocyanate, 1 vol% -5 vol% Triton X-100, 5-50 g/L3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid, 1-10 g/L N-lauroyl sarcosine sodium, 0.28 mol/L-0.5 mol/L NaCl, 0.01 vol% -0.1% Antifoam204 and 50-200 g/L polyethylene glycol.
Preferably, the cell lysate consists of 20 mmol/L-50 mmol/L Tris-HCl, 10 mmol/L-20 mmol/L EDTA with pH8.0, 3 mol/L-4 mol/L guanidine isothiocyanate, 2 vol% -5 vol% Triton X-100, 10-50 g/L3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid, 2-10 g/L N-lauroyl sarcosine sodium, 0.28 mol/L-0.3 mol/L LNaCl, 0.05 vol% -0.1% Antifoam204 and 50-100 g/L polyethylene glycol.
Preferably, the cell lysate consists of 25mmol/L of LTris-HCL, 10mmol/L of ethylenediaminetetraacetic acid (pH8.0), 4mol/L of guanidinium isothiocyanate, 2 vol% of TritonX-100, 50g/L of 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid, 5g/L of N-lauroylsarcosine sodium, 0.28mol/L of NaCl, 0.1 vol% of Antifoam204, and 100g/L of polyethylene glycol.
It should be noted that, in the cell lysate of the present application, the components are matched with each other at specific concentration and dosage, the whole lysis process can be rapidly completed within 5-10 minutes, the lysis intensity and speed can be further improved under the condition of heating incubation at 70 ℃, and compared with the lysis step of the existing magnetic bead method, the nucleic acid extraction kit of the magnetic bead method of the present application shortens the time for releasing nucleic acid and combining magnetic beads, and shortens the detection time of the kit. It is understood that the specific concentrations and amounts described above are merely preferred embodiments of one implementation of the present application and that other than the specific concentrations and amounts described above may be used with lesser requirements. The cell lysate of this application cooperates this application modified magnetic bead can be better capture and combine nucleic acid, optimizes the extraction effect.
Preferably, the washing solution does not contain ethanol or isopropanol, and the washing solution contains NaCl and polyethylene glycol.
Compared with the conventional magnetic bead method washing solution, the washing solution of the present invention does not use ethanol or isopropanol, but uses sodium chloride to reduce the solubility of nucleic acid, so that the nucleic acid is in a precipitation state and is convenient to combine with magnetic beads; and the concentration effect of polyethylene glycol on nucleic acid is utilized to further promote the combination of the nucleic acid and the magnetic beads. By adopting the washing liquid, on one hand, two-step washing of the existing magnetic bead method is combined into one-step washing, namely, the washing liquid can be used for effectively removing impurities such as protein and the like remained on the magnetic beads only by once washing, so that the operation steps and time of washing are saved; on the other hand, because no ethanol is used, the cover opening and the placement are not needed after washing, so that the time and the operation steps are further saved, and the cross contamination of the sample caused by the cover opening and the placement is also avoided; on the other hand, the washing liquid of the application is partially remained without influencing the subsequent PCR reaction, so that the washing liquid is combined with the improved magnetic beads, the nucleic acid is not required to be eluted after the washing, the magnetic beads adsorbing the nucleic acid are directly added into a PCR reaction system for amplification reaction, and the time and the operation steps are further saved.
It can be understood that the key point of the washing solution of the present application is to replace ethanol or isopropanol with NaCl and polyethylene glycol, and as for other components in the washing solution, such as washing buffer or other functional components, reference can be made to the existing magnetic bead method washing solution; however, in the present application, in order to ensure the function of the washing solution, the washing buffer is preferably defined, and the details are described in the following technical solutions.
Preferably, the concentration of NaCl in the washing liquid is 0.14-2 mol/L, and the content of polyethylene glycol is 50-200 g/L.
NaCl can reduce the solubility of nucleic acid and precipitate nucleic acid, and the effect can be achieved to different degrees only by adding NaCl; however, in order to more effectively reduce the nucleic acid solubility, the concentration of NaCl is preferably 0.14mol/L to 2 mol/L. The effect of reducing the nucleic acid solubility is poor due to too low NaCl concentration; the concentration is too high, on one hand, the nucleic acid solubility is difficult to further reduce after being reduced to a certain degree, and on the other hand, the washing of the magnetic bead impurities by the washing liquid is not facilitated.
Preferably, the polyethylene glycol is PEG4000, PEG6000 or PEG 8000.
In the present application, the addition of polyethylene glycol to the washing solution serves to further concentrate the precipitated nucleic acids, promote the binding of the nucleic acids to the magnetic beads, further improve the efficiency of binding of the nucleic acids to the magnetic beads, and shorten the binding time of the magnetic beads. It is understood that the effect of concentrating nucleic acid can be achieved by adding polyethylene glycol; however, in order to ensure the function and effect of the washing solution, the content of the polyethylene glycol is preferably 5 to 20 percent; in the range, the concentration of nucleic acid can be well guaranteed, the combination of the nucleic acid and magnetic beads is promoted, and the phenomenon that cell lysate is too viscous due to too high dosage of polyethylene glycol is avoided.
Preferably, the wash solution further comprises a wash buffer. Preferably, the washing buffer is Tris-HCL, and the concentration of the Tris-HCL in the washing solution is 10 mmol/L-100 mmol/L.
It should be noted that Tris-HCL is a buffer solution used in a relatively conventional manner, and it is not excluded that other buffer solutions may be used. However, in the present application, it is preferable to use 10mmol/L to 100mmol/L Tris-HCl as a buffer solution for the washing solution, in view of the washing effect of the washing solution on the magnetic beads and the inability of the washing solution to affect the subsequent PCR reaction.
Preferably, the washing solution consists of 10 mmol/L-100 mmol/L Tris-HCL, 0.14 mol/L-2 mol/L NaCl and 50-200 g/L polyethylene glycol.
Preferably, the washing solution consists of 20 mmol/L-50 mmol/L Tris-HCL, 0.3 mol/L-0.6 mol/L NaCl and 80-120 g/L polyethylene glycol.
Preferably, the washing solution consists of 20mmol/L Tris-HCL, 0.5mol/L NaCl and 100g/L polyethylene glycol.
It should be noted that, the washing liquid of the present application, each component cooperates under specific concentration and dosage, not only can realize the one-step washing of the present application, but also improve the efficiency of the washing liquid, save the time of washing and nucleic acid extraction. It is understood that the specific concentrations and amounts described above are merely preferred embodiments of one implementation of the present application and that other than the specific concentrations and amounts described above may be used with lesser requirements.
Preferably, in the kit, the magnetic bead core is a nano microsphere with superparamagnetism, the outer part of the magnetic bead core is coated with resin and a silicon hydroxyl modified material, the particle size is 200-350 nm, the particles are uniform, and the magnetic response time is 1-2 minutes. The magnetic bead has the characteristics of high temperature resistance, no release of metal ions at high temperature and the like.
In an implementation manner of the present application, the magnetic beads of the present application are prepared by the following method:
a) preparing the nano microspheres: FeCl is added3-6H2Adding O, anhydrous sodium acetate and PEG2000 into the ethylene glycol solution, uniformly mixing, and stirring for 30 minutes to 1 hour at room temperature under the nitrogen atmosphere; then reacting for 4-8 hours at 150-200 ℃ in nitrogen atmosphere; after the reaction is finished, removing the supernatant of the reaction product under the action of a magnetic field to obtain nano microspheres, and cleaning the nano microspheres by using deionized water for later use;
b) modifying the surface groups of the nano microspheres: and (b) adding the nano microspheres prepared in the step a) into a mixed solution of absolute ethyl alcohol and ammonia water, stirring under the condition of introducing nitrogen, slowly dropwise adding a mixed solution of tetraethoxysilane and polysiloxane, reacting at room temperature for 3-6 hours, removing the supernatant of a reaction product under the action of a magnetic field, and cleaning by using deionized water to prepare the magnetic beads.
The magnetic bead is coated with a layer of polysiloxane outside the superparamagnetic microsphere and is subjected to silicon hydroxyl modification, the process enables the magnetic bead to have the characteristic of resisting high temperature of 95 ℃, and metal ions of an inner core of the magnetic bead cannot be released under the high-temperature condition, so that the magnetic bead can be directly added into a PCR system, and the activity of DNA polymerase cannot be interfered.
Preferably, in step a), FeCl3-6H2The weight ratio of the O, the anhydrous sodium acetate and the PEG2000 is 2-3: 6-8: 1-2.
Preferably, in step a), every 20-30 g of FeCl3-6H2And adding 800 mL-1L of ethylene glycol solution correspondingly to the O.
Preferably, in step b), every 20-30 g of FeCl3-6H2Adding 400-1L of anhydrous ethanol and 20-60 mL of 14% ammonia water mixed solution into the product corresponding to the O in the step a), and slowly dropwise adding 15-30 mL of tetraethoxysilane and polysiloxane mixed solution.
Preferably, in the step b), the rotation speed of stirring is 400-1000 rpm/min.
Preferably, in the step b), the speed of slowly dripping the mixed solution of the tetraethoxysilane and the polysiloxane is 0.05-0.3 mL/min.
Preferably, step b) further comprises resuspending the magnetic beads with deionized water or a buffer to obtain a magnetic bead suspension.
The nucleic acid extraction kit adopting the magnetic bead method has the advantages that cell lysis solution, magnetic beads and washing solution are respectively improved; the improvement of the cell lysate shortens the cell lysis time and promotes the combination of nucleic acid and magnetic beads; the improvement of the magnetic beads can ensure that the magnetic beads adsorbing the nucleic acid are directly added into a PCR reaction system without eluting the nucleic acid, thereby saving the procedure and avoiding the loss of the nucleic acid; the improvement of the washing liquid changes the existing two-step washing into one-step washing, saves the washing process, does not need to be opened for placing after washing due to no addition of alcohol reagents, not only further saves the washing reagents, but also avoids sample cross contamination caused by opening the cover for placing. It can be understood that the improvement of the cell lysis solution, the magnetic beads and the washing solution in the kit of the present application has advantages separately compared with the existing magnetic bead method; the improved cell lysate, magnetic beads and washing solution can be combined in the kit of the application, and can also be used in combination with the existing magnetic bead method reagent alone.
Therefore, the other side of the application discloses a cell lysis solution for magnetic bead method nucleic acid extraction, a washing solution for magnetic bead method nucleic acid extraction, and a magnetic bead for magnetic bead method nucleic acid extraction, respectively. Wherein, the cell lysis solution, the washing solution and the magnetic beads are the cell lysis solution, the washing solution and the magnetic beads in the kit; therefore, the cell lysis solution, the washing solution and the magnetic beads for the magnetic bead method nucleic acid extraction of the present application are specifically limited, and reference is made to the kit for the magnetic bead method nucleic acid extraction of the present application.
On the basis of the magnetic bead preparation method of the kit, the application also discloses a preparation method of the magnetic bead for magnetic bead method nucleic acid extraction. The preparation method is the preparation method of the magnetic beads in the kit.
The application also discloses a use method of the nucleic acid extraction kit by the magnetic bead method, which specifically comprises the following steps:
step 1: taking a sample into a centrifuge tube, adding magnetic beads and cell lysate, uniformly mixing by oscillation, incubating at room temperature for 5min, and uniformly mixing by oscillation for 1-2 times in the middle;
step 2: placing the centrifuge tube on a magnetic frame, standing for 1min, completely adsorbing the magnetic beads, and then absorbing the supernatant liquid;
and step 3: taking down the centrifugal tube from the magnetic frame, adding a washing solution, and uniformly mixing by oscillation;
and 4, step 4: placing the centrifuge tube on a magnetic frame, standing for 1min, completely adsorbing the magnetic beads, and then absorbing the supernatant liquid;
and 5: and taking down the centrifugal tube from the magnetic frame, adding 25-50 mu L of PCR amplification reaction liquid, oscillating and whirling to ensure that magnetic beads are completely distributed in the PCR amplification reaction liquid, and thus completing the nucleic acid extraction.
Wherein, the step 5 is directly connected with the subsequent PCR amplification detection, so that the magnetic beads for adsorbing the nucleic acid are directly resuspended by adopting the PCR amplification reaction solution; it will be appreciated that if the PCR amplification assay is not performed immediately, conventional water or nucleic acid buffer may be used to resuspend the nucleic acid-adsorbed magnetic beads.
Subsequently, the mixture can be directly transferred to a PCR tube for routine PCR amplification or detection based on PCR amplification.
In the application method, the PCR amplification reaction solution typically comprises PCR Buffer, primers, probes, Taq enzyme or/and MMLV enzyme, and water. It is understood that the specific dosage of each reagent in the above steps can be adjusted according to the object of nucleic acid extraction; for example, the amount of sample may be adjusted to the particular sample obtained; the amount of magnetic beads and the amount of cell lysate can also be adaptively adjusted according to the amount of a specific sample to be processed, for example, if the amount of the sample is large, the amount of the magnetic beads and the amount of the cell lysate are correspondingly increased, and if the amount of the sample is small, the amount of the sample is correspondingly decreased. The incubation time and the number of times of mixing by intermediate shaking in step 1 can also be adjusted according to the specific sample amount. In the step 2 and the step 4, the standing time on the magnetic frame is based on the actual and effective complete precipitation of the magnetic beads, generally speaking, the standing time for 1min can basically meet the requirement, the standing time can be further optimized and shortened or prolonged, and the standing time for 1min on the magnetic frame is only used for reference.
The beneficial effect of this application lies in:
according to the kit for extracting nucleic acid by the magnetic bead method, the components of the lysis buffer solution are optimized, so that cells can be quickly and fully lysed, protein can be denatured, the protein and the nucleic acid can be effectively separated, and the nucleic acid can be released; improves the working efficiency of cell lysate and shortens the time of nucleic acid extraction.
Drawings
FIG. 1 is a scanning electron micrograph of a magnetic bead prepared in an example of the present application;
FIG. 2 shows the real-time fluorescence PCR detection result of Candida albicans in the present application example using the cell lysate in the first and second protocols to extract the nucleic acid of the serum sample B1 simulating bacterial infection;
FIG. 3 shows the real-time fluorescence PCR detection result of transmissible gastroenteritis virus (TGEV) obtained by extracting nucleic acid from serum sample simulating virus infection with cell lysates of different formulations in the present embodiment;
FIG. 4 shows the real-time fluorescence PCR detection result of fowlpox virus by using cell lysates of different formulations to extract nucleic acid of serum sample simulating virus infection in the present embodiment;
FIG. 5 shows the results of real-time fluorescence PCR detection of E.coli from a serum sample simulating bacterial infection by using different nucleic acid extraction methods in the examples of the present application;
FIG. 6 shows the real-time fluorescence PCR detection results of enterococcus faecalis from serum samples of simulated bacterial infection extracted by different nucleic acid extraction methods in the examples of the present application;
FIG. 7 shows the real-time fluorescence PCR detection results of Candida albicans extracted from serum sample nucleic acid simulating bacterial infection by different nucleic acid extraction methods in the present example;
FIG. 8 shows the real-time fluorescence PCR detection result of transmissible gastroenteritis virus from swine by using different nucleic acid extraction methods to extract nucleic acid simulating virus infection of serum samples in the present embodiment;
FIG. 9 shows the real-time fluorescence PCR detection results of fowlpox virus in the present application, wherein different nucleic acid extraction methods are used to extract nucleic acid from serum sample simulating virus infection.
Detailed Description
The existing magnetic bead method generally has the problem of long cracking time; after adsorbing nucleic acid, the magnetic beads are usually cleaned by ethanol, and at least two steps of cleaning are needed; in order to remove ethanol, the sample must be placed after being cleaned, so that the cross contamination risk of the sample is increased; after the magnetic beads are cleaned, the nucleic acid is eluted and then added into the PCR reaction solution for detection, and the nucleic acid elution process easily causes part of the nucleic acid to be abandoned along with the magnetic beads, so that the extracted nucleic acid cannot be completely put into the next detection link, and the detection sensitivity is reduced.
In view of the above problems, the present application first optimizes a cell lysate, which is also referred to as a lysis buffer, since it is a buffer in which magnetic beads are bound to nucleic acids in addition to cell lysis. The cell lysate of the present application, in addition to the basic lysis buffer, contains guanidinium isothiocyanate, Triton X-100, 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid and N-lauroylsarcosine sodium. The mutual matching of the four components is utilized to realize the rapid and sufficient cell destruction, protein denaturation, effective separation of protein and nucleic acid and release of nucleic acid. On the basis, in a preferred scheme of the application, a metal ion chelating agent is further added for binding metal ions, so that the nuclease activity is inhibited, and the integrity of nucleic acid is ensured; in order to promote the precipitation of nucleic acid, a negative charge neutralizer is added into a cell lysate, and is used for neutralizing the negative charge of phosphate radical of nucleic acid and reducing the solubility of nucleic acid, so that the nucleic acid is precipitated, which is completely different from the conventional thinking of precipitating nucleic acid by isopropanol or ethanol; in order to gather the precipitated nucleic acids together and facilitate the binding of the nucleic acids to the magnetic beads, polyethylene glycol, such as PEG4000, PEG6000, PEG 8000; finally, in order to avoid that the cell lysate generates foam to influence the effect of the cell lysate, the cell lysate of the present application is further added with an antifoaming agent.
The cell lysate is optimized from the aspects of cell disruption, protein denaturation, nucleic acid precipitation, magnetic bead combination, defoaming and the like, so that the cell lysate can rapidly and fully lyse cells and release nucleic acid, the working efficiency of the cell lysate is improved, and the lysis time is saved.
Further optimization of the present application improves the washing solution, and the existing washing solution is usually washed with ethanol solution, because ethanol has the function of precipitating nucleic acid and maintaining the stability of the binding of nucleic acid and magnetic beads. This application adopts polyethylene glycol and NaCl, utilizes certain concentration NaCl to neutralize the negative charge of nucleic acid phosphate radical, reduces the solubility of nucleic acid, makes nucleic acid keep deposiing, utilizes the polyethylene glycol, for example the function of PEG4000, PEG6000, the concentrated nucleic acid of PEG8000 to play the effect that stable nucleic acid and magnetic bead combine, consequently, the washing liquid of this application need not adopt the ethanol.
The washing liquid can effectively remove impurities such as residual protein on magnetic beads by one-step washing through the optimization of each component, reduces the washing times and operation steps, and shortens the washing time. In addition, no alcohol reagent is added into the washing liquid, and the washing liquid does not need to be placed after being opened, so that the working procedures and time are saved, and the cross contamination of samples caused by the working procedures and the time is avoided; the magnetic beads are combined, PCR reaction liquid can be directly added into the washing liquid to carry out PCR amplification after the washing liquid is washed, the subsequent PCR amplification cannot be influenced by the residual washing liquid, a nucleic acid elution step after washing is omitted, the detection time is saved, and the nucleic acid loss caused by the nucleic acid elution step is avoided.
The application further improves the magnetic bead, and current magnetic bead is adsorbing nucleic acid to after the washing, generally need uncapping to place, then elute nucleic acid and can carry out next PCR and detect. The application creatively provides that if the washed magnetic beads absorbed with the nucleic acid are directly added into a PCR reaction system, the step of elution can be omitted, and the extracted nucleic acid can be completely used for PCR detection, so that the detection sensitivity is improved; however, the present application has found that there are at least two problems with this: firstly, after the magnetic beads adsorbing nucleic acid are cleaned, although the magnetic beads are precipitated under the action of magnetic force, the cleaning solution of the supernatant is removed, a trace amount of cleaning solution still remains, the existing cleaning solution of the conventional magnetic bead method can influence the PCR reaction, and the problem can be solved by adopting the improved cleaning solution; however, the second and greater problem is that the existing conventional silicon hydroxyl magnetic beads or carboxyl hydroxyl magnetic beads are PCR inhibitors or release new inhibitors in the PCR link, and cannot be directly used in the PCR amplification system.
Therefore, the magnetic bead is improved, the inner core of the improved magnetic bead is a nano microsphere with superparamagnetism, the outer part of the improved magnetic bead is coated with resin and a silicon hydroxyl modified material through special treatment, the particle size is 200-350 nm, the particles are uniform, the magnetic response time is controlled within 1-2 minutes, and the improved magnetic bead has the characteristics of high temperature resistance, no release of metal ions at high temperature and the like; moreover, the magnetic beads can be directly added into a PCR reaction system after adsorbing nucleic acid, PCR amplification is not affected, the elution operation steps are saved, nucleic acid loss caused by elution is avoided, the nucleic acid adsorbed by the magnetic beads can be completely added into the PCR reaction, the detection sensitivity is increased, the detection accuracy is ensured, and the omission factor is reduced. The magnetic bead of this application combines the lysis buffer of this application, and capture that can be more abundant combines nucleic acid, improves nucleic acid extraction effect and quality.
In the kit of the application, lysis buffer, magnetic bead and washing liquid are all improved respectively, consequently, can solitary use to produce corresponding improvement effect. However, in the preferred embodiment of the present application, the improved lysis buffer, magnetic beads and washing solution are assembled into a preferred kit; according to the kit, through the improvement of a lysis buffer solution, magnetic beads and a washing solution, the extraction and purification of pathogen DNA can be completed within 10-15 minutes in the whole nucleic acid extraction process, and the high-sensitivity molecular detection of the pathogen can be completed within 1 hour by combining a downstream nucleic acid detection method based on a PCR or isothermal amplification technology, so that the detection efficiency is improved, and the kit can be used for the instant detection or the field detection of the pathogen.
The kits of the present application can be used for nucleic acid extraction of the following sample types: plasma, serum, saliva, cerebrospinal fluid, alveolar lavage fluid, ascites, urine, cervical swabs, buccal swabs, pharyngeal swabs, culture cell supernatants, blood, tissues, etc., and the types of pathogens that can be extracted include gram-negative bacteria, gram-positive bacteria, candida, mycoplasma, chlamydia, DNA viruses, RNA viruses, etc. After washing the magnetic beads having the nucleic acids adsorbed thereon, the reaction system such as the subsequent PCR or Real Time PCR is directly added to the magnetic bead precipitate having the nucleic acids adsorbed thereon without elution, and the reaction is carried out after mixing the reaction system and the magnetic bead precipitate.
The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.
The reagents, consumables and apparatus used in the following examples include:
the main reagents are as follows: premix Ex TaqTM(Probe qPCR) RR390A and One Step PrimeScriptTMRT-PCR Kit (Perfect Real Time) RR064A was purchased from TaKaRa, porcine transmissible gastroenteritis, porcine epidemic diarrhea, porcine rotavirus triple live vaccine was purchased from Havidae, fowlpox virus vaccine was purchased from Youbei, and normal goat serum AR0009 was purchased from Boshide.
The main consumables are as follows: 200 μ L of eight-tube PCR-0208-C, 0.2mL clear PCR eight-tube flat-cover PCR-02FCP-C, and 1.5mL of DNase-free centrifuge tube MCT-150-C were purchased from Axygen.
The main equipment is as follows: vortex oscillator adopts Vortex QL-901, fluorescent quantitative PCR instrument adopts YARui biological MA6000P, and constant temperature oscillation metal bath is laboratory conventional equipment.
The samples used in the following examples were prepared as follows:
in order to better illustrate the performance advantages of the kit in the embodiment in the aspect of extracting the virus sample, a swine transmissible gastroenteritis virus vaccine is taken as an RNA virus representative, a fowlpox virus vaccine is taken as a DNA virus representative, serum samples simulating virus infection are respectively prepared, and then the kit, other comparison kits or comparison reagents in the embodiment are respectively used for extracting RNA and DNA. Wherein the preparation steps of the serum sample simulating virus infection are as follows:
operation 1: sucking 2.5mL goat serum, adding into a pig transmissible gastroenteritis, pig epidemic diarrhea and pig rotavirus triple live vaccine bottle, and blowing, beating and mixing uniformly for later use;
operation 2: sucking 2.5mL goat serum, adding into a chicken pox virus vaccine bottle, and blowing, beating and mixing uniformly for later use;
operation 3: adding the mixed solution obtained in the operation 1 and the operation 2 together and uniformly mixing to obtain 5mL of serum sample stock solution simulating virus infection, wherein the stock solution is marked as V1;
and operation 4: adding 9mL goat serum into 1mL V1 serum solution obtained in the step 3, uniformly mixing by vortex, marking as V2, and sequentially diluting according to 10-fold gradient, marking as V3, V4, V5 and V6.
To better illustrate the performance advantages of the kit of this example in the extraction of viral samples, serum samples were prepared to mimic bacterial infection using E.coli standard strains as representative of gram-negative strains, E.faecalis as representative of gram-positive strains, and Candida albicans as representative of Candida, and RNA and DNA were extracted using the kit of this example, other comparative kits, or comparative reagents, respectively. Wherein the preparation steps of the serum sample simulating bacterial infection are as follows:
operation 1: preparing 3 centrifuge tubes with 1.5mL, adding 1.5mL of Escherichia coli liquid with OD value of 1 into a first centrifuge tube, adding 1.5mL of enterococcus faecalis liquid with OD value of 1 into a second centrifuge tube, adding 1.5mL of Candida albicans liquid with OD value of 1 into a third centrifuge tube, centrifuging at 8000rpm for 5min through the 3 centrifuge tubes, removing supernatant, respectively adding 1mL of goat serum to blow and stir bottom strain precipitates uniformly, sucking out the liquid after blowing and stirring uniformly, adding 2mL of goat serum into a 15mL centrifuge tube in the same tube, and uniformly mixing to obtain 5mL of mixed bacterial liquid marked as B1.
Operation 2: and adding 1mL of B1 serum solution obtained in the operation 1 into 9mL of normal goat serum, uniformly mixing by vortex, marking as B2, and sequentially performing 10-fold gradient dilution operation to obtain B3, B4, B5 and B6 respectively.
The primers and probes used in the following examples:
the adopted nucleic acid detection technology is TaqMan probe method fluorescent quantitative PCR technology, and a primer and a probe are respectively designed aiming at a detection object, and the sequences of the primer and the probe are shown in Table 1.
TABLE 1 primers and probes for the examples
Figure BDA0002324598710000131
In Table 1, all probes were labeled at their 5 'end with FAM fluorophore and at their 3' end with TAMRA quencher.
Example 1 preliminary optimization of cell lysate formulation
Two cell lysate formulations were designed for preliminary screening of the assay protocol.
The formula of the first scheme is as follows: 25mM Tris-HCl, 10mM EDTA (pH8.0), 1% SDS, 1% 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid, 0.5% sodium N-lauroyl sarcosinate, 1M NaCl, 0.1% Antifoam204, 10% PEG 6000.
The formula of the second scheme is as follows: 25mM Tris-HCl, 10mM EDTA (pH8.0), 4M guanidinium isothiocyanate, 5 vol% Triton X-100, 10 g/L3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid, 5g/L sodium N-lauroylsarcosine, 0.28M NaCl, 0.1 vol% Antifoam204, 100g/LPEG 6000.
The washing solution used in this example was formulated with 20mM Tris-HCl, 0.5M NaCl, 100g/LPEG 6000.
The magnetic beads of this example were prepared as follows:
a) preparing nano microspheres: 30g of FeCl was weighed3-6H2Adding 80g of anhydrous sodium acetate and 20g of PEG2000 into a three-neck flask, adding 1L of glycol solution, mechanically stirring and uniformly mixing, introducing nitrogen, and stirring at room temperature to continuously react for 1 hour; transferring the solution into a reaction kettle, introducing nitrogen to remove air above the reaction kettle, putting the reaction kettle into an oven, and reacting for 6 hours at 200 ℃; after the reaction is finished, after the reaction kettle is cooled to room temperature, transferring the product to a beaker, adding a magnetic field to the beaker, removing the supernatant under the action of the magnetic field, and cleaning the magnetic beads by using deionized water4 times, obtaining the nano-microsphere of the embodiment;
b) surface group modification of nano magnetic beads: and (b) transferring the nano microspheres prepared in the step a) into a three-mouth beaker, adding 1L of absolute ethyl alcohol and 60mL of 14% ammonia water solution, mechanically stirring under the condition of introducing nitrogen, setting the rotating speed at 800rpm/min, slowly dripping 30mL of mixed solution of tetraethoxysilane and polysiloxane by using a peristaltic pump, and adjusting the dripping speed to 0.15 mL/min. The reaction was carried out at room temperature for 5 hours. And transferring the product to a new beaker, separating the magnetic beads under the action of a magnetic field, removing the supernatant solution, and washing the magnetic beads for 4 times by using deionized water to obtain the magnetic beads of the embodiment.
Washing magnetic beads with deionized water, removing supernatant, and then resuspending with a magnetic bead buffer solution to obtain a magnetic bead suspension; in this example, the suspension of magnetic beads was diluted to a concentration of 5mg/mL with a bead buffer. Wherein the magnetic bead buffer solution consists of 5mM Tris-HCl and 10g/LPEG 6000.
The magnetic beads prepared in this example were observed by a scanning electron microscope, and the results are shown in fig. 1, and fig. 1 shows that the magnetic beads with uniform particle size were obtained.
In the embodiment, a serum sample B1 simulating bacterial infection is used as a nucleic acid extraction sample, and the content of Candida albicans nucleic acid in the extracted nucleic acid is used as a judgment standard of nucleic acid extraction quality.
The nucleic acid extraction steps are as follows:
step 1: a200-microliter serum sample B1 is taken into a 1.5-mL centrifuge tube, 10-microliter magnetic beads prepared in the embodiment and 200-microliter cell lysate are added, the mixture is fully shaken and uniformly mixed, incubated at 70 ℃ for 5min, and shaken and uniformly mixed for 2 times in the middle. Wherein, the cell lysate is a formula of a scheme I or a formula of a scheme II.
Step 2: placing the centrifuge tube on a magnetic frame, standing for 1min, completely adsorbing the magnetic beads, and then absorbing the supernatant liquid.
And step 3: the centrifuge tube was removed from the magnetic holder, 200. mu.L of the washing solution of this example was added, and the mixture was shaken for 10 seconds to mix.
And 4, step 4: placing the centrifuge tube on a magnetic frame, standing for 1min, completely adsorbing the magnetic beads, and then absorbing the supernatant liquid.
And 5: the centrifuge tube was removed from the magnetic stand, 25. mu.L of LPCR amplification solution was added, and the magnetic beads were distributed into the PCR amplification solution by vortexing.
Step 6: transferring the PCR amplification solution into a PCR tube for the next PCR amplification.
Wherein, the 25 muL PCR amplification solution comprises: 2 XPCR Mix, 2.5 μ Lprimermix (including 1 μ L10nM CalF, 1 μ L10nM CalR and 0.5 μ L10nM CalP), 5 μ L H2O, 5 mu LDNA template.
The PCR amplification program adopted by the Candida albicans detection is set as follows: pre-denaturation at 95 ℃ for 3min, then 40 cycles: FAM fluorescence signals were collected at 95 ℃ for 10s and 55 ℃ for 30s during the cycle at 55 ℃.
In this example, three repeated PCR amplification assays of Candida albicans were performed on the nucleic acid extracted using the recipe of scheme one and the nucleic acid extracted using the recipe of scheme two, respectively, and the results are shown in Table 2 and FIG. 2.
TABLE 2 PCR amplification detection of Ct values for Candida albicans
Figure BDA0002324598710000151
The results in table 2 and fig. 2 show that the cell lysate of the formula of the first scheme has poor nucleic acid extraction performance on candida represented by candida albicans, and cannot meet the detection requirement, and the cell lysate of the formula of the second scheme can extract candida albicans DNA, so that the cell lysate of the formula of the second scheme and the washing liquid correspondingly used by the cell lysate are adopted as verification formulas in the present example.
Example 2 optimization of cell lysate formulation
In order to further optimize the formulation of the cell lysate, this example performs an optimization test on the concentrations of the main components such as guanidine isothiocyanate, triton x100, PEG6000, and 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid in the formulation of the second embodiment of example 1. The optimized scheme is shown in table 3, and formula 9 in table 3 is not suitable for cell lysate because of crystallization phenomenon after being placed at room temperature after preparation.
TABLE 3 optimization of cell lysate formulations
Numbering Component 1 Component 2 Component 3 Component 4 Component 5 Component 6 Component 7 Component 8 Component 9
Formulation 1 2M 1vol％ 0 5 5 0.28M 0.1vol％ 10mM 10mM
Formulation
2 2M 2vol％ 100 10 5 0.28M 0.1vol％ 10mM 10mM
Formulation
3 2M 5vol％ 200 50 5 0.28M 0.1vol％ 10mM 10mM
Formulation
4 3M 5vol％ 0 10 5 0.28M 0.1vol％ 25mM 10mM
Formulation 5 3M 1vol％ 100 50 5 0.28M 0.1vol％ 25mM 10mM
Formulation
6 3M 2vol％ 200 5 5 0.28M 0.1vol％ 25mM 10mM
Formulation 7 4M 2vol％ 0 50 5 0.28M 0.1vol％ 25mM 10mM
Formulation
8 4M 5vol％ 100 5 5 0.28M 0.1vol％ 25mM 10mM
Formulation
9 4M 1vol％ 200 10 5 0.28M 0.1vol％ 25mM 10mM
In Table 3, component 1 is guanidinium isothiocyanate, component 2 is Triton X-100, component 3 is PEG6000, component 4 is 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid, component 5 is sodium N-lauroylsarcosine, component 6 is NaCl, component 7 is Antifoam204, component 8 is Tris, and component 9 is EDTA. In Table 3, the unit of component 3, component 4 and component 5 is "g/L".
The washing solution of this example was the same as that of example 1, and the same magnetic beads as those of example 1 were used for nucleic acid extraction. In this example, the serum sample B1 which simulates bacterial infection as in example 1 was used as a nucleic acid extraction sample, and the content of Candida albicans nucleic acid in the extracted nucleic acid was used as a criterion for determining the amount of extracted nucleic acid. Nucleic acid extraction and Candida albicans detection were the same as in example 1. The extraction procedure and detection method were the same as in example 1, in this example, three replicates of PCR amplification of Candida albicans were performed, and the results are shown in Table 4.
TABLE 4 nucleic acid samples extracted according to different formulations and PCR amplification detection Ct values of Candida albicans
Figure BDA0002324598710000161
According to the detection results in Table 4, Ct values of four components of guanidinium isothiocyanate, Triton X100, PEG6000 and 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid at different concentrations are further counted, and the results are shown in Table 5.
TABLE 5 nucleic acid samples extracted from different concentrations of each component Candida albicans PCR amplification detection Ct values
Component 1 Ct Component 2 Ct Component 3 Ct Component 4 Ct
2M 24.04 1vol％ 23.4 0 38.1 5g/L 23.3
3M 23.47 2vol％ 22.3 10g/100mL 15.7 10g/L 23.5
4M 21.32 5vol％ 23.2 20g/100mL 15.1 50g/L 22.0
In Table 5, component 1 is guanidinium isothiocyanate, component 2 is Triton X-100, component 3 is PEG6000, and component 4 is 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid; the concentration corresponding to each component is the concentration of the component in the cell lysate, and the corresponding Ct value is the average of three repeated Ct values obtained by detecting all formulations with the same concentration of the component in nine formulations, for example, guanidine isothiocyanate in component 1, the Ct value corresponding to 2M is 24.04, and the Ct value is the average of three repeated Ct values detected by detecting all formulations with guanidine isothiocyanate concentration of 2M in nine formulations, specifically, the average of three repeated Ct values in formulation 1, the average of three repeated Ct values in formulation 2, and the average of three repeated Ct values in formulation 3, (40.00+16.60+15.52) ÷ 3 ÷ 24.04. The rest components are similar.
The results in tables 4 and 5 show that PEG6000 has a key effect on nucleic acid binding, but when the concentration of PEG6000 is too high, the cell lysate is more viscous, and the variation coefficient of 200g/L PEG6000 is larger and the data is unstable in parallel sample test data, so that 100g/L PEG6000 can meet the requirement.
To further optimize the formulation of the cell lysate, this example further optimizes the assay for the concentrations of guanidinium isothiocyanate, Antifoam204, Tris-HCl, EDTA (pH8.0), etc., in the formulation of protocol two of example 1. The optimization scheme is shown in table 6.
Table 6 further optimization of cell lysate formulations
Numbering Component 1 Component 2 Component 3 Component 4 Component 5 Component 6 Component 7 Component 8 Component 9
Formulation 10 4M 2vol％ 100 50 5 0.28M 0.1vol％ 25mM 10mM
Formulation
11 4M 2vol％ 100 50 5 0M 0.1vol％ 25mM 10mM
Formulation
12 4M 2vol％ 100 50 5 0.5M 0.1vol％ 25mM 10mM
Formulation 13 4M 2vol％ 100 50 5 0.28M 0vol％ 25mM 10mM
Formulation
14 4M 2vol％ 100 50 5 0.28M 1vol％ 25mM 10mM
Formulation
15 4M 2vol％ 100 50 5 0.28M 0.1vol％ 50mM 10mM
Formulation
16 4M 2vol％ 100 50 5 0.28M 0.1vol％ 100mM 10mM
Formulation 17 4M 2vol％ 100 50 5 0.28M 0.1vol％ 25mM 20mM
Formulation
18 4M 2vol％ 100 50 5 0.28M 0.1vol％ 25mM 25mM
In Table 6, component 1 is guanidinium isothiocyanate, component 2 is Triton X-100, component 3 is PEG6000, component 4 is 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid, component 5 is N-lauroylsarcosine sodium, component 6 is NaCl, component 7 is Antifoam204, component 8 is Tris, and component 9 is EDTA. Wherein the unit of component 3, component 4 and component 5 is "g/L".
Three replicates of PCR amplification of Candida albicans were performed in this example and the results are shown in Table 7.
TABLE 7 nucleic acid samples extracted according to different formulations Candida albicans PCR amplification detection Ct values
Figure BDA0002324598710000171
Figure BDA0002324598710000181
The results in tables 6 and 7 show that the optimum concentration of NaCl is 0.28M, the main function of the Antifoam204 is to eliminate the foam generated by the lysate, the effect is satisfactory at a concentration of 0.1%, the effect on the extraction of nucleic acids is not affected when the concentration reaches 1%, and the foam removal effect is consistent with 0.1 vol%, so that the recommended concentration of the Antifoam204 is 0.1 vol%. The effect of the concentration of Tris-HCl and EDTA on the extraction performance is not significant, and the concentration of 25mM and 10mM are recommended by comprehensive consideration. From the above data, it can be seen that the cell lysate is preferably formulated with 25mM Tris-HCl, 10mM EDTA (pH8.0), 4M guanidinium isothiocyanate, 2 vol% Triton X-100, 50 g/L3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid, 5g/L sodium N-lauroylsarcosine, 0.28M NaCl, 0.1 vol% Antifoam204, and 100g/L PEG6000, and the subsequent examples are all performed using this formulation.
Example 3 cleaning solution formulation optimization
To further optimize the wash liquor formulation, this example was optimized for wash liquor major component concentrations, as shown in table 8. The cell lysate of this example was the optimized formulation of example 2, and the same magnetic beads as those of example 1 were used for nucleic acid extraction. In this example, the serum sample B1 which simulates bacterial infection as in example 1 was used as a nucleic acid extraction sample, and the content of Candida albicans nucleic acid in the extracted nucleic acid was used as a criterion for determining the amount of extracted nucleic acid. Nucleic acid extraction and Candida albicans detection were the same as in example 1. Similarly, the nucleic acid samples extracted, the extraction procedure and the detection method for each formulation were the same as in example 1, in this case, three replicates of PCR amplification of Candida albicans were performed, and the results are shown in Table 9.
Table 8 further optimization of the detergent formulations
Figure BDA0002324598710000182
Figure BDA0002324598710000191
TABLE 9 PCR amplification detection Ct values of nucleic acid samples extracted from washing solutions of different formulations of Candida albicans
Figure BDA0002324598710000192
According to the detection results in table 9, Ct values of three components, PEG6000, NaCl and Tris, at different concentrations were further counted, and the results are shown in table 10.
TABLE 10 PCR amplification detection Ct values of nucleic acid samples extracted from washing solutions of different formulations of Candida albicans
PEG6000 Ct CV％ NaCl Ct CV％ Tris Ct CV％
50g/L 19.164 2.3 0.14M 16.24 2.85 10mM 16.10 3.65
100g/L 15.052 1.8 0.5M 15.80 3.61 20mM 16.09 4.88
150g/L 15.067 5.1 1M 16.10 2.92 50mM 16.08 2.70
200g/L 15.101 5.3 2M 16.24 5.11 100mM 16.12 3.26
The results in tables 8, 9 and 10 show that the concentration of PEG6000 is between 100g/L and 200g/L, the extraction performance is the best, and the difference of the extraction performance between different concentrations is not significant, but the concentration of PEG reaches more than 150g/L, the washing liquid becomes sticky, the difference of the coefficient of variation between different parallel samples is large, and therefore the optimal concentration is 100 g/L; the optimum concentration of NaCl is 0.5M, the influence of the concentration of Tris-HCl on the extraction performance is not obvious, and the recommended concentration is 20mM by comprehensive consideration. From the above data, it can be seen that the washing solution is preferably formulated with 20mmol/L Tris-HCl, 0.5mol/L NaCl, 100g/L PEG, and the subsequent examples are all performed using the formulation.
EXAMPLE 4 comparative experiments on viral DNA and RNA extraction from serum by different nucleic acid extraction methods
This example comparatively analyzes the nucleic acid extraction performance of the kit of this example, and the nucleic acid extraction performance of the conventional magnetic bead method and Chelex100 boiling method for extracting DNA virus and RNA virus in serum.
The conventional magnetic bead method comprises components such as a proteinase K solution, a lysis solution, a protein washing solution, a salt ion washing solution, an eluent and the like. The detailed formula of each component in the conventional magnetic bead method is as follows:
the lysate consisted of 50mM Tris-HCl, 10mM EDTA (pH8.0), 5M guanidinium isothiocyanate, 10% Triton X-100.
The protein wash consisted of 20mM Tris-HCl, 10mM EDTA (pH8.0), 3M guanidinium isothiocyanate, 56% ethanol.
The salt ion washing solution consists of 10mM Tris-HCl and 80% ethanol.
The eluent was RNasefree deionized purified water.
The formula of the cracking solution of the Chelex100 boiling method is as follows: 10mM Tris-HCL, 2mM EDTA, 1% NP-40, 5% Chelex 100.
The nucleic acid extraction steps of the conventional magnetic bead method are as follows:
step 1: putting 200 mu L of sample into a 1.5mL centrifuge tube, adding 20 mu L of proteinase K solution and 200 mu L of lysate, fully shaking and uniformly mixing, incubating for 15min at 56 ℃, and shaking and uniformly mixing for 2 times in the middle.
Step 2: adding 250 μ L isopropanol solution, shaking thoroughly, mixing, adding 20 μ L magnetic bead solution, shaking thoroughly, mixing, standing at room temperature for 5min, and shaking for 2 times.
And step 3: placing the centrifuge tube on a magnetic frame, standing for 1min, completely adsorbing the magnetic beads, and then absorbing the supernatant liquid.
And 4, step 4: the centrifuge tube was removed from the magnetic stand, 500. mu.L of protein wash was added, shaken for 10s and mixed.
And 5: placing the centrifuge tube on a magnetic frame, standing for 1min, completely adsorbing the magnetic beads, and then absorbing the supernatant liquid.
Step 6: taking the centrifuge tube off the magnetic frame, adding 500 μ L of desalting ion washing solution, shaking for 10s, and mixing.
And 7: placing the centrifuge tube on a magnetic frame, standing for 1min, completely adsorbing the magnetic beads, and then absorbing the supernatant liquid.
And 8: and 6-7 repeating the steps.
And step 9: and (4) sucking and discarding the liquid residue at the bottom of the tube by using a pipettor, and opening the cover at room temperature and placing for 5 min.
Step 10: add 100. mu.L of eluent into the tube and incubate at 56 ℃ for 5 min.
Step 11: and (3) placing on a magnetic frame, standing for 2min, and sucking the supernatant into a new centrifugal tube for later use, thereby completing the nucleic acid extraction.
The Chelex100 boiling method comprises the following steps of nucleic acid extraction:
step 1: 100 mu L of sample is taken and put into a 1.5mL centrifuge tube, 100 mu L of Chelex100 lysate is added, the mixture is fully shaken and mixed evenly, the incubation is carried out for 5min at the temperature of 90 ℃, and the mixture is shaken and mixed evenly for 2 times in the middle.
Step 2: a1.5 mL centrifuge tube was placed on a centrifuge at 8000rpm and centrifuged for 5 min.
And step 3: transferring 100 mu L of supernatant to a new centrifuge tube for standby, and completing the nucleic acid extraction.
The cell lysate in the kit of this example is the optimized formulation of example 2, the washing solution is the optimized formulation of example 3, and the rest refer to example 1.
In contrast, in the present example, serum samples V1, V2, V3, V4, V5 and V6 simulating virus infection were used as nucleic acid extraction samples, and magnetic bead method, conventional magnetic bead method and Chelex100 boiling method of the kit of the present example were used to extract nucleic acids of serum samples V1, V2, V3, V4, V5 and V6, respectively, and then the content of the transmissible gastroenteritis virus nucleic acid and the content of the fowlpox virus nucleic acid in the extracted nucleic acids were measured to determine the quality of nucleic acid extraction.
The PCR amplification reaction system of the porcine transmissible gastroenteritis virus is 25 mu L and comprises the following components: 12.5 μ L of 2 XPCR MIX, 2.5 μ L of Primer MIX (including 1 μ L of 10nM upstream Primer, 1 μ L of 10nM downstream Primer, 0.5 μ L of 10nM probe), 5 μ L H2O, 5 mu LDNA template.
The PCR amplification program adopted by the detection of the transmissible gastroenteritis virus of swine is as follows: keeping the temperature at 50 ℃ for 20min, pre-denaturing at 95 ℃ for 3min, and then entering 40 cycles: FAM fluorescence signals were collected at 95 ℃ for 10s and 55 ℃ for 30s during the cycle at 55 ℃.
The chicken pox virus PCR amplification reaction system is 25 mu L, which comprises: 12.5 μ L of 2 XPCR MIX, 2.5 μ L of PrimerMIX (including 1 μ L of 10nM upstream primer, 1 μ L of 10nM downstream primer, 0.5 μ L of 10nM probe), 5 μ L H2O, 5 mu LDNA template.
The PCR amplification program adopted by the detection of the chicken pox virus is set as follows: pre-denaturation at 95 ℃ for 3min, then 40 cycles: FAM fluorescence signals were collected at 95 ℃ for 10s and 55 ℃ for 30s during the cycle at 55 ℃.
The results of the test of the porcine transmissible gastroenteritis virus and the results of the test of the fowlpox virus were shown in Table 11 and FIG. 3, respectively, when three replicates of each of the extracted nucleic acid samples were conducted, and in Table 12 and FIG. 4, respectively.
TABLE 11 detection results of transmissible gastroenteritis virus of swine of nucleic acid samples obtained by different nucleic acid extraction methods
Figure BDA0002324598710000221
TABLE 12 detection results of fowlpox Virus in nucleic acid samples obtained by different nucleic acid extraction methods
Figure BDA0002324598710000222
The results of table 11, table 12, fig. 3 and fig. 4 show that the Chelex100 boiling method is suitable for extracting DNA virus samples and is not suitable for extracting nucleic acids of RNA viruses, and it can be seen that the kit of this example is consistent with the conventional magnetic bead method in terms of the performance of extracting nucleic acids of DNA viruses and RNA viruses in high concentration samples, and is significantly superior to the conventional magnetic bead method and the Chelex100 method in terms of the performance of extracting nucleic acids of DNA viruses and RNA viruses in low concentration samples.
Example 5 comparison of bacterial DNA extraction in serum with different nucleic acid extraction methods
This example comparatively analyzes the nucleic acid extraction performance of the kit of this example, and the nucleic acid extraction performance of the conventional magnetic bead method and Chelex100 boiling method for extracting bacterial DNA from serum.
The conventional magnetic bead method for extracting bacterial nucleic acid adopted in the embodiment is composed of lysozyme solution, proteinase K solution, lysis solution, protein washing solution, salt ion washing solution and eluent. The lysate, the protein washing solution, the salt ion washing solution and the eluent were the same as in example 4.
The formulation of the Chelex100 boiling lysate used in this example was the same as in example 4.
The conventional magnetic bead method for extracting bacterial nucleic acid comprises the following steps:
step 1: centrifuging 200 μ L sample in 1.5mL centrifuge tube at 10000rpm for 5min, removing supernatant, adding 100 μ L lysozyme solution, shaking thoroughly, mixing, incubating at 37 deg.C for 15min, and shaking for 2 times.
Step 2: adding 20 μ L proteinase K solution and 300 μ L lysate, shaking thoroughly, mixing, incubating at 65 deg.C for 15min, and shaking for 2 times.
And step 3: adding 350 μ L isopropanol solution, shaking thoroughly, mixing, adding 20 μ L magnetic bead solution, shaking thoroughly, mixing, standing at room temperature for 5min, and shaking for 2 times.
And 4, step 4: placing the centrifuge tube on a magnetic frame, standing for 1min, completely adsorbing the magnetic beads, and then absorbing the supernatant liquid.
And 5: the centrifuge tube was removed from the magnetic stand, 500. mu.L of protein wash was added, shaken for 10s and mixed.
Step 6: placing the centrifuge tube on a magnetic frame, standing for 1min, completely adsorbing the magnetic beads, and then absorbing the supernatant liquid.
And 7: taking the centrifuge tube off the magnetic frame, adding 500 μ L of desalting ion washing solution, shaking for 10s, and mixing.
And 8: placing the centrifuge tube on a magnetic frame, standing for 1min, completely adsorbing the magnetic beads, and then absorbing the supernatant liquid.
And step 9: and 7, repeating the steps 7-8.
Step 10: and (4) sucking and discarding the liquid residue at the bottom of the tube by using a liquid transfer device, and uncovering and drying for 5min at room temperature.
Step 11: add 100. mu.L of eluent into the tube and incubate at 56 ℃ for 5 min.
Step 12: and (3) placing on a magnetic frame, standing for 2min, and sucking the supernatant into a new centrifugal tube for later use, thereby completing the nucleic acid extraction.
The procedure for extracting nucleic acid by Chelex100 boiling method was the same as in example 4.
The cell lysate in the kit of this example is the optimized formulation of example 2, the washing solution is the optimized formulation of example 3, and the rest refer to example 1.
In the present embodiment, the serum samples B1, B2, B3, B4, B5 and B6 simulating bacterial infection are used as nucleic acid extraction samples, the magnetic bead method, the conventional magnetic bead method and the Chelex100 boiling method of the kit of the present embodiment are used to extract nucleic acids of the serum samples B1, B2, B3, B4, B5 and B6, and then the contents of escherichia coli nucleic acid, enterococcus faecalis nucleic acid and candida albicans nucleic acid in the extracted nucleic acids are detected to serve as the determination criteria of nucleic acid extraction quality.
The PCR amplification reaction systems of the escherichia coli nucleic acid, the enterococcus faecalis nucleic acid and the candida albicans nucleic acid are the same, except that corresponding primers and probes are added into each detection system, and the PCR amplification reaction system is 25 mu L and comprises the following components: 12.5 μ L of 2 XPCRMIX, 2.5 μ L of LPrimer MIX (including 1 μ L of 10nM upstream primer, 1 μ L of 10nM downstream primer, 0.5 μ L of 10nM probe), 5 μ L H2O, 5 mu LDNA template.
The PCR amplification programs of the escherichia coli nucleic acid, the enterococcus faecalis nucleic acid and the candida albicans nucleic acid are the same, and are set as follows: pre-denaturation at 95 ℃ for 3min, then 40 cycles: FAM fluorescence signals were collected at 95 ℃ for 10s and 55 ℃ for 30s during the cycle at 55 ℃.
The results of detection of E.coli nucleic acids are shown in Table 13 and FIG. 5, the results of detection of E.faecalis nucleic acids are shown in Table 14 and FIG. 6, and the results of detection of C.albicans nucleic acids are shown in Table 15 and FIG. 7.
TABLE 13 results of E.coli detection of nucleic acid samples obtained by different nucleic acid extraction methods
Sample(s) Ct value by conventional paramagnetic particle method Ct value of Chelex100 boiling method Ct value of the kit of this example by paramagnetic particle method
B1 15.10 16.88 11.92
B2 19.13 20.87 15.09
B3 21.72 23.91 20.21
B4 24.84 25.62 23.57
B5 27.72 30.10 26.50
B6 35.90 37.02 30.54
TABLE 14 detection results of enterococcus faecalis for nucleic acid samples obtained by different nucleic acid extraction methods
Sample(s) Ct value by conventional paramagnetic particle method Ct value of Chelex100 boiling method Ct value of the kit of this example by paramagnetic particle method
B1 15.06 19.81 15.68
B2 19.46 20.89 18.11
B3 22.81 24.28 22.99
B4 25.88 29.76 27.35
B5 29.67 33.24 29.82
B6 33.62 NA 32.50
TABLE 15 detection results of Candida albicans in nucleic acid samples obtained by different nucleic acid extraction methods
Sample(s) Ct value by conventional paramagnetic particle method Ct value of Chelex100 boiling method Ct value of the kit of this example by paramagnetic particle method
B1 25.55 28.23 22.80
B2 28.93 30.84 25.37
B3 31.20 35.38 28.33
B4 33.53 36.84 31.11
B5 34.89 36.32 33.49
B6 40.00 NA 37.68
The results in tables 13, 14, 15, 5, 6 and 7 show that the Chelex100 boiling method is inferior in performance of extracting samples of gram-positive bacteria, candida and the like to the conventional paramagnetic particle method and the paramagnetic particle method of the kit of the present example, and is inferior in sensitivity by one order of magnitude, and is not suitable for extracting nucleic acids of gram-positive bacteria, fungi and the like. The performance of the magnetic bead method of the kit for extracting nucleic acid of high-concentration gram-negative bacteria, gram-positive bacteria, candida and the like is consistent with that of the conventional magnetic bead method, and the performance of the magnetic bead method for extracting bacterial nucleic acid in a low-concentration sample is obviously superior to that of the conventional magnetic bead method and the Chelex100 boiling method.
EXAMPLE 6 comparative experiments on viral DNA and RNA extraction from serum by different nucleic acid extraction methods
Patent application CN105420230A A lysate (hereinafter referred to as contrast test) for extracting nucleic acid by a magnetic bead method, which mainly provides a lysate improved magnetic bead method kit and a corresponding nucleic acid extraction magnetic bead method, and the patent application is similar to the application in part, namely, the lysis process is accelerated by optimizing cell lysate; the difference is that the specific components of the lysate of patent application CN105420230A are different from the specific components of the cell lysate of the present application; therefore, the optimization effect and function of the two are also different.
This example comparatively analyzes the nucleic acid extraction performance of the magnetic bead method and the comparative test magnetic bead method of the kit of this example for extracting DNA viruses and RNA viruses from serum.
Wherein, the lysate and the washing solution of the comparative experiment are the final optimized lysate and washing solution formula in patent application CN105420230A, namely the lysate and washing solution formula of 'example 3', specifically, the lysate is composed of 0.3N sodium hydroxide, 0.45M potassium chloride, 0.03% N-lauroyl sarcosine sodium, 5mM EDTA, 0.45M Tris-HCL, 1% triton X-100 and 3mM DTT. The wash was 0.3M potassium chloride and the pH was adjusted to 4 with acetic acid. Wherein, bromophenol blue is not added in the lysis solution, and lime is not added in the washing solution, because the bromophenol blue and lime are only used for visual observation through color change, and have no promotion effect on the lysis and the washing; the real-time fluorescent PCR detection is needed in the follow-up of the test, so that the influence of the dye on the follow-up PCR detection is avoided, and therefore, bromophenol blue and lime are not added.
The nucleic acid extraction procedure of the comparative experiment is referred to patent application CN 105420230A.
The cell lysate in the kit of this example is the optimized formulation of example 2, the washing solution is the optimized formulation of example 3, and the rest refer to example 1.
In this example, serum samples V1, V3, V5 and V6 simulating virus infection were used as nucleic acid extraction samples, and the magnetic bead method of the kit of this example and the magnetic bead method of the CN105420230A comparative test were used to extract nucleic acids of serum samples V1, V3, V5 and V6, respectively, and then the content of transmissible gastroenteritis virus nucleic acid and fowlpox virus nucleic acid in the extracted nucleic acids were measured to determine the quality of nucleic acid extraction.
The reaction system and reaction conditions for detecting transmissible gastroenteritis virus and fowl pox virus are the same as in example 4.
The results of the test of the porcine transmissible gastroenteritis virus and the results of the test of the fowlpox virus are shown in Table 16 and FIG. 8, respectively, when three replicates of each of the extracted nucleic acid samples were performed, and in Table 17 and FIG. 9, respectively.
TABLE 16 detection results of transmissible gastroenteritis virus of swine of nucleic acid samples obtained by different nucleic acid extraction methods
Figure BDA0002324598710000251
Figure BDA0002324598710000261
TABLE 17 detection results of fowlpox Virus on nucleic acid samples obtained by different nucleic acid extraction methods
Figure BDA0002324598710000262
The results in table 16, table 17, fig. 8 and fig. 9 show that the magnetic bead method of patent application CN105420230A has a poor effect of extracting viral DNA from virus-infected serum samples, while the magnetic bead method of the kit of this example has a significantly better effect of extracting fowlpox virus nucleic acid than the comparative test, as shown in table 17. The magnetic bead method of the kit of this example also has a higher effect on the extraction of viral RNA, as shown in Table 16.
In general, the nucleic acid extraction step of the kit magnetic bead method comprises cell lysis solution lysis, magnetic frame loading and washing solution washing, and no elution step is needed after washing; the conventional magnetic bead method mainly comprises an enzyme digestion step, a lysis solution cracking step, a step of combining the cracked nucleic acid with magnetic beads, a magnetic frame mounting step, a first washing step, a second washing step, a cover opening step, a placing step and an elution step; the Chelex100 boiling method mainly comprises lysis of lysate, combination of nucleic acid and magnetic beads and centrifugal separation; the patent application CN105420230A magnetic bead method mainly comprises lysis solution cracking, magnetic frame mounting and washing solution washing; the statistics of the time used in each step of the above methods and the total time are shown in table 18.
TABLE 18 time taken for each step of the different nucleic acid extraction methods and Total time consumed (min)
Step (ii) of Magnetic bead method of the present example Conventional magnetic bead method Chelex100 boiling method Magnetic bead method of comparative test
Enzymatic digestion step 0 15 0 0
Lysis solution 5 15 5 5
Magnetic bead binding 0 5 5 0
Upper magnetic rack 2 2 0 2
First washing 3 3 0 3
Second washing 0 5 0 0
Cover opening placement 0 10 0 0
Elution is carried out 0 5 0 0
Total time of day 10 60 10 10
In table 18, the "bead binding" time of the Chelex100 boiling method is actually the "centrifugation" time after its lysis, the Chelex100 boiling method has no bead binding, the Chelex100 boiling method has only lysis and centrifugation steps in practice, and therefore, the time of the other steps is 0; the time for mounting the magnetic frame is the sum of the total two times of time for mounting the magnetic frame; the lysis time of "lysis solution" in the "magnetic bead method of this example" is 5min, which is actually the time for binding magnetic beads, that is, the lysis of cells releases nucleic acid and the binding of magnetic beads is performed simultaneously, so the "binding of magnetic beads" time is 0; "comparative test magnetic bead method" is the magnetic bead method protocol of patent application CN105420230A "example 3".
The results in Table 18 show that the kit, Chelex100 boiling method and comparative test of this example achieve rapid nucleic acid extraction over time; however, according to the above tests, the kit of this example has a better extraction effect on viral DNA, viral RNA and bacterial nucleic acid, and the magnetic bead method of this example has a better nucleic acid extraction effect than the conventional magnetic bead method, Chelex100 boiling method and comparative test, regardless of high or low concentration of virus or bacteria. From the results in Table 18, the magnetic bead kit method of this example is very similar to the magnetic bead method of the comparative test in comparison of the respective steps, but as can be seen from the results in tables 16 and 17 of example 6, the nucleic acid extraction effect of the magnetic bead kit method of this example is significantly superior to that of the comparative test.
Therefore, the kit magnetic bead method has the characteristics of simple operation, short extraction time and the like, a single sample can finish the high-quality extraction of nucleic acid within about 10 minutes, and the kit magnetic bead method is suitable for the nucleic acid extraction of various pathogenic bacteria such as virus DNA, virus RNA, bacterial nucleic acid and the like and can meet the requirements of field detection and point-of-care testing (POCT).
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.
SEQUENCE LISTING
<110> Shenzhen Hua Dazhi science and technology Limited
<120> kit for magnetic bead method nucleic acid extraction, magnetic bead and preparation method thereof
<130> 19I29285
<160> 15
<170> PatentIn version 3.3
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Claims (10)

1. The utility model provides a kit for magnetic bead method nucleic acid extraction, includes cell lysate, magnetic bead and washing liquid, its characterized in that: the cell lysate contains guanidinium isothiocyanate, Triton X-100, 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid and N-lauroyl sarcosine sodium.
2. The kit of claim 1, wherein: in the cell lysate, the concentration of guanidinium isothiocyanate is 2-4 mol/L, the content of Triton X-100 is 1-5 vol%, the content of 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid is 5-50 g/L, and the content of N-sodium lauroylsarcosine is 1-10 g/L;
preferably, the cell lysate further contains polyethylene glycol;
preferably, the content of polyethylene glycol in the cell lysate is 50-200 g/L;
preferably, the polyethylene glycol is PEG4000, PEG6000 or PEG 8000;
preferably, the cell lysis solution further comprises a lysis buffer solution, a metal ion chelating agent, a negative charge neutralizing agent and an antifoaming agent;
preferably, the lysis buffer is Tris-HCL, and the concentration of the Tris-HCL in the cell lysate is 10 mmol/L-100 mmol/L;
preferably, the metal ion chelating agent is ethylenediamine tetraacetic acid, the concentration of the ethylenediamine tetraacetic acid in the cell lysis solution is 5 mmol/L-50 mmol/L, and the pH value is 8.0;
preferably, the negative charge neutralizer is NaCl, and the concentration of the NaCl in the cell lysate is 0.28-0.5 mol/L;
preferably, the antifoaming agent is Antifoam204, and the concentration of Antifoam204 in the cell lysate is 0.01 vol% to 0.1 vol%.
3. The kit of claim 2, wherein: the cell lysate is composed of 10 mmol/L-100 mmol/L Tris-HCL, 5 mmol/L-50 mmol/L EDTA with pH8.0, 2 mol/L-4 mol/L guanidine isothiocyanate, 1 vol% -5 vol% Triton X-100, 5-50 g/L3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid, 1-10 g/L N-lauroyl sarcosine sodium, 0.28 mol/L-0.5 mol/L NaCl, 0.01 vol% -0.1% Antifoam204 and 50-200 g/L polyethylene glycol;
preferably, the cell lysate consists of 20 mmol/L-50 mmol/L Tris-HCL, 10 mmol/L-20 mmol/L EDTA with pH8.0, 3 mol/L-4 mol/L guanidine isothiocyanate, 2 vol% -5 vol% Triton X-100, 10-50 g/L3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid, 2-10 g/L N-lauroyl sarcosine sodium, 0.28 mol/L-0.3 mol/L NaCl, 0.05% -0.1 vol% Antifoam204 and 50-100 g/L polyethylene glycol;
preferably, the cell lysate consists of 25mmol/L of LTris-HCL, 10mmol/L of ethylenediaminetetraacetic acid (EDTA) at pH8.0, 4mol/L of guanidinium isothiocyanate, 2 vol% of Triton X-100, 50g/L of 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid, 5g/L of N-lauroylsarcosine sodium, 0.28mol/L of LNaCl, 0.1 vol% of Antifoam204, and 100g/L of polyethylene glycol.
4. The kit according to any one of claims 1 to 3, characterized in that: the washing solution does not contain ethanol or isopropanol, and contains NaCl and polyethylene glycol;
preferably, in the washing solution, the concentration of NaCl is 0.14-2 mol/L, and the content of polyethylene glycol is 50-200 g/L;
preferably, the polyethylene glycol is PEG4000, PEG6000 or PEG 8000;
preferably, the washing solution further comprises a washing buffer;
preferably, the washing buffer solution is Tris-HCL, and the concentration of the Tris-HCL in the washing solution is 10 mmol/L-100 mmol/L;
preferably, the washing solution consists of 10 mmol/L-100 mmol/L Tris-HCL, 0.14 mol/L-2 mol/L NaCl and 50-200 g/L polyethylene glycol;
preferably, the washing solution consists of 20 mmol/L-50 mmol/L Tris-HCL, 0.3 mol/L-0.6 mol/L NaCl and 80-120 g/L polyethylene glycol;
preferably, the washing solution consists of 20mmol/L Tris-HCL, 0.5mol/L NaCl and 100g/L polyethylene glycol.
5. The kit according to any one of claims 1 to 3, characterized in that: the magnetic bead comprises a magnetic bead core and a magnetic bead core, wherein the magnetic bead core is a nano microsphere with superparamagnetism, the magnetic bead core is coated with resin and a silicon hydroxyl modified material, the particle size of the magnetic bead is 200-350 nm, and the magnetic response time is 1-2 minutes.
6. A cell lysis solution for magnetic bead method nucleic acid extraction is characterized in that: the cell lysate contains guanidinium isothiocyanate, Triton X-100, 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid and N-lauroyl sarcosine sodium;
preferably, in the cell lysate, the concentration of guanidinium isothiocyanate is 2-4 mol/L, the content of Triton X-100 is 1-5 vol%, the content of 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid is 5-50 g/L, and the content of N-lauroylsarcosine sodium is 1-10 g/L;
preferably, the cell lysate further contains polyethylene glycol;
preferably, the content of polyethylene glycol in the cell lysate is 50-200 g/L;
preferably, the polyethylene glycol is PEG4000, PEG6000 or PEG 8000;
preferably, the cell lysis solution further comprises a lysis buffer solution, a metal ion chelating agent, a negative charge neutralizing agent and an antifoaming agent;
preferably, the lysis buffer is Tris-HCL, and the concentration of the Tris-HCL in the cell lysate is 10 mmol/L-100 mmol/L;
preferably, the metal ion chelating agent is ethylenediamine tetraacetic acid, the concentration of the ethylenediamine tetraacetic acid in the cell lysis solution is 5 mmol/L-50 mmol/L, and the pH value is 8.0;
preferably, the negative charge neutralizer is NaCl, and the concentration of the NaCl in the cell lysate is 0.14 mol/L-0.5 mol/L;
preferably, the antifoaming agent is Antifoam204, and the concentration of Antifoam204 in the cell lysate is 0.01 vol% to 0.2 vol%.
7. Cell lysate according to claim 6, characterized in that: the cell lysate is composed of 10 mmol/L-100 mmol/L Tris-HCL, 5 mmol/L-50 mmol/L EDTA with pH8.0, 2 mol/L-4 mol/L guanidine isothiocyanate, 1 vol% -5 vol% Triton X-100, 5-50 g/L3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid, 1-10 g/L N-lauroyl sarcosine sodium, 0.28 mol/L-0.5 mol/L NaCl, 0.01 vol% -0.1 vol% Antifoam204 and 50-200 g/L polyethylene glycol;
preferably, the cell lysate consists of 20 mmol/L-50 mmol/L Tris-HCl, 10 mmol/L-20 mmol/L EDTA with pH8.0, 3 mol/L-4 mol/L guanidine isothiocyanate, 2 vol% -5 vol% Triton X-100, 10-50 g/L3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid, 2-10 g/L N-lauroyl sarcosine sodium, 0.28 mol/L-0.3 mol/L LNaCl, 0.05% -0.1 vol% Antifoam204 and 50-100 g/L polyethylene glycol;
preferably, the cell lysate consists of 25mmol/L of LTris-HCL, 10mmol/L of ethylenediaminetetraacetic acid (pH8.0), 4mol/L of guanidinium isothiocyanate, 2 vol% of Triton X-100, 50g/L of 3- [3- (cholamidopropyl) dimethylamino ] -1-propanesulfonic acid, 5g/L of sodium N-lauroylsarcosine, 0.28mol/L of NaCl, 0.1 vol% of Antifoam204, and 100g/L of polyethylene glycol.
8. A washing solution for magnetic bead method nucleic acid extraction is characterized in that: the washing solution does not contain ethanol or isopropanol, and contains NaCl and polyethylene glycol;
preferably, in the washing solution, the concentration of NaCl is 0.14-2 mol/L, and the content of polyethylene glycol is 50-200 g/L;
preferably, the polyethylene glycol is PEG4000, PEG6000 or PEG 8000;
preferably, the washing solution further comprises a washing buffer;
preferably, the washing buffer solution is Tris-HCL, and the concentration of the Tris-HCL in the washing solution is 10 mmol/L-100 mmol/L;
preferably, the washing solution consists of 10 mmol/L-100 mmol/L Tris-HCL, 0.14 mol/L-2 mol/L NaCl and 50-200 g/L polyethylene glycol;
preferably, the washing solution consists of 20 mmol/L-50 mmol/L Tris-HCL, 0.3 mol/L-0.6 mol/L NaCl and 80-120 g/L polyethylene glycol;
preferably, the washing solution consists of 20mmol/L Tris-HCL, 0.5mol/L NaCl and 100g/L polyethylene glycol.
9. A magnetic bead for magnetic bead method nucleic acid extraction which characterized in that: the magnetic bead comprises a magnetic bead core and a magnetic bead core, wherein the magnetic bead core is a nano microsphere with superparamagnetism, the magnetic bead core is coated with resin and a silicon hydroxyl modified material, the particle size of the magnetic bead is 200-350 nm, and the magnetic response time is 1-2 minutes.
10. The method for preparing a magnetic bead according to claim 9, wherein: comprises the following steps of (a) carrying out,
a) preparing the nano microspheres: FeCl is added3-6H2Adding O, anhydrous sodium acetate and PEG2000 into the ethylene glycol solution, uniformly mixing, and stirring for 30 minutes to 1 hour at room temperature under the nitrogen atmosphere; then in nitrogen atmosphereReacting for 4-8 hours at 150-200 ℃; after the reaction is finished, removing the supernatant of the reaction product under the action of a magnetic field to obtain nano microspheres, and cleaning the nano microspheres by using deionized water for later use;
b) modifying the surface groups of the nano microspheres: adding the nano-microspheres prepared in the step a) into a mixed solution of absolute ethyl alcohol and ammonia water, stirring under the condition of introducing nitrogen, slowly dropwise adding a mixed solution of tetraethoxysilane and polysiloxane, reacting at room temperature for 3-6 hours, removing the supernatant of a reaction product under the action of a magnetic field, and cleaning by using deionized water to obtain the magnetic beads;
preferably, in said step a), FeCl3-6H2The weight ratio of the O, the anhydrous sodium acetate and the PEG2000 is 2-3: 6-8: 1-2;
preferably, in the step a), every 20-30 g of FeCl3-6H2Correspondingly adding 800 mL-1L of glycol solution into the O;
preferably, in the step b), every 20-30 g of FeCl3-6H2Adding 400-1L of a mixed solution of anhydrous ethanol and 20-60 mL of 14% ammonia water into the product obtained in the step a) corresponding to the O, and slowly and correspondingly dropwise adding 15-30 mL of a mixed solution of tetraethoxysilane and polysiloxane;
preferably, in the step b), the rotating speed of the stirring is 400-1000 rpm/min;
preferably, in the step b), the speed of slowly dripping the mixed solution of the tetraethoxysilane and the polysiloxane is 0.05-0.3 mL/min;
preferably, the step b) further comprises resuspending the magnetic beads with deionized water or a buffer to obtain a magnetic bead suspension.
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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113462683A (en) * 2021-07-22 2021-10-01 上海思路迪生物医学科技有限公司 Alcohol-free cleaning solution suitable for extracting nucleic acid from multiple samples and nucleic acid extraction kit
CN113584128A (en) * 2021-07-29 2021-11-02 翌圣生物科技(上海)股份有限公司 Solid phase particulate material for processing biological samples and biological sample processing method
CN113699145A (en) * 2021-09-06 2021-11-26 上海伯杰医疗科技有限公司 Lysis binding solution based on paramagnetic particle method pathogen nucleic acid extraction, product and application thereof
CN113913494A (en) * 2021-09-14 2022-01-11 苏州锐讯生物科技有限公司 Kit for quickly and conveniently extracting virus DNA in body fluid and extraction method
CN114058615A (en) * 2021-11-25 2022-02-18 重庆威斯腾生物医药科技有限责任公司 General reagent combination for extracting nucleic acid RNA from saliva based on full-automatic nano magnetic bead method
CN114196669A (en) * 2021-12-24 2022-03-18 南京诺唯赞生物科技股份有限公司 Rinsing liquid for nucleic acid extraction
CN114277026A (en) * 2021-12-21 2022-04-05 深圳市易瑞生物技术股份有限公司 Magnetic bead for nucleic acid extraction, preparation method thereof and kit for magnetic bead method nucleic acid extraction
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CN114934041A (en) * 2022-04-27 2022-08-23 北京金豪制药股份有限公司 Reagent and method for extracting nucleic acid
CN116555248A (en) * 2023-07-10 2023-08-08 北京凡知医学科技有限公司 Kit and method for extracting high molecular weight DNA in bacteria
CN116891849A (en) * 2023-09-11 2023-10-17 成都斯马特科技有限公司 Kit for rapidly extracting nucleic acid from nasopharyngeal swab and extraction method
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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100029925A1 (en) * 2008-05-23 2010-02-04 Life Technologies Corporation, A Delaware Corporation Methods and kits for extraction of dna
CN102181434A (en) * 2009-08-05 2011-09-14 公安部物证鉴定中心 Method for extracting purified DNA from human exfoliative cells
US20130017552A1 (en) * 2010-04-08 2013-01-17 Greiner Bio - One Gmbh Method and kit for separating viral and prokaryotic nucleic acids from eukaryotic nucleic acids
US20150252354A1 (en) * 2012-09-28 2015-09-10 Cepheid Methods for dna and rna extraction from fixed paraffin-embedded tissue samples
US20150275269A1 (en) * 2012-10-26 2015-10-01 The Emerther Company Method for purifying nucleic acid and kit
CN105368820A (en) * 2015-12-22 2016-03-02 南京先进激光技术研究院 Whole blood DNA (deoxyribonucleic acid) extraction kit based on paramagnetic particle method and application of extraction kit
CN105420230A (en) * 2016-01-14 2016-03-23 北京纳捷诊断试剂有限公司 Lysis solution for extracting nucleic acid through magnetic bead method
CN109022417A (en) * 2018-08-13 2018-12-18 益善生物技术股份有限公司 A kind of paramagnetic particle method nucleic acid extraction conversion reagent box and its application method
CN109750030A (en) * 2019-01-21 2019-05-14 上海科华生物工程股份有限公司 A kind of quick, nucleic acid extraction kit for not heating based on paramagnetic particle method

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100029925A1 (en) * 2008-05-23 2010-02-04 Life Technologies Corporation, A Delaware Corporation Methods and kits for extraction of dna
CN102181434A (en) * 2009-08-05 2011-09-14 公安部物证鉴定中心 Method for extracting purified DNA from human exfoliative cells
US20130017552A1 (en) * 2010-04-08 2013-01-17 Greiner Bio - One Gmbh Method and kit for separating viral and prokaryotic nucleic acids from eukaryotic nucleic acids
US20150252354A1 (en) * 2012-09-28 2015-09-10 Cepheid Methods for dna and rna extraction from fixed paraffin-embedded tissue samples
US20150275269A1 (en) * 2012-10-26 2015-10-01 The Emerther Company Method for purifying nucleic acid and kit
CN105368820A (en) * 2015-12-22 2016-03-02 南京先进激光技术研究院 Whole blood DNA (deoxyribonucleic acid) extraction kit based on paramagnetic particle method and application of extraction kit
CN105420230A (en) * 2016-01-14 2016-03-23 北京纳捷诊断试剂有限公司 Lysis solution for extracting nucleic acid through magnetic bead method
CN109022417A (en) * 2018-08-13 2018-12-18 益善生物技术股份有限公司 A kind of paramagnetic particle method nucleic acid extraction conversion reagent box and its application method
CN109750030A (en) * 2019-01-21 2019-05-14 上海科华生物工程股份有限公司 A kind of quick, nucleic acid extraction kit for not heating based on paramagnetic particle method

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113462683A (en) * 2021-07-22 2021-10-01 上海思路迪生物医学科技有限公司 Alcohol-free cleaning solution suitable for extracting nucleic acid from multiple samples and nucleic acid extraction kit
CN113584128A (en) * 2021-07-29 2021-11-02 翌圣生物科技(上海)股份有限公司 Solid phase particulate material for processing biological samples and biological sample processing method
CN113699145A (en) * 2021-09-06 2021-11-26 上海伯杰医疗科技有限公司 Lysis binding solution based on paramagnetic particle method pathogen nucleic acid extraction, product and application thereof
CN113913494A (en) * 2021-09-14 2022-01-11 苏州锐讯生物科技有限公司 Kit for quickly and conveniently extracting virus DNA in body fluid and extraction method
CN114058615A (en) * 2021-11-25 2022-02-18 重庆威斯腾生物医药科技有限责任公司 General reagent combination for extracting nucleic acid RNA from saliva based on full-automatic nano magnetic bead method
CN114277026A (en) * 2021-12-21 2022-04-05 深圳市易瑞生物技术股份有限公司 Magnetic bead for nucleic acid extraction, preparation method thereof and kit for magnetic bead method nucleic acid extraction
CN114196669B (en) * 2021-12-24 2023-02-28 南京诺唯赞生物科技股份有限公司 Rinsing liquid for nucleic acid extraction
CN114196669A (en) * 2021-12-24 2022-03-18 南京诺唯赞生物科技股份有限公司 Rinsing liquid for nucleic acid extraction
CN114317524A (en) * 2021-12-28 2022-04-12 国家粮食和物资储备局科学研究院 Reagent, kit and extraction method for extracting DNA of grain kernel attaching fungi
CN114657231A (en) * 2022-04-27 2022-06-24 珠海宝锐生物科技有限公司 Rapid DNA extraction kit with magnetic beads for fluorescent quantitative PCR detection and extraction method thereof
CN114934041A (en) * 2022-04-27 2022-08-23 北京金豪制药股份有限公司 Reagent and method for extracting nucleic acid
CN114934041B (en) * 2022-04-27 2023-12-15 北京金豪制药股份有限公司 Reagent and method for extracting nucleic acid
CN116555248A (en) * 2023-07-10 2023-08-08 北京凡知医学科技有限公司 Kit and method for extracting high molecular weight DNA in bacteria
CN116555248B (en) * 2023-07-10 2023-10-20 北京凡知医学科技有限公司 Kit and method for extracting high molecular weight DNA in bacteria
CN116891849A (en) * 2023-09-11 2023-10-17 成都斯马特科技有限公司 Kit for rapidly extracting nucleic acid from nasopharyngeal swab and extraction method
CN116949036A (en) * 2023-09-11 2023-10-27 成都斯马特科技有限公司 Kit for rapidly extracting nucleic acid from ascites and extraction method
CN116891849B (en) * 2023-09-11 2023-11-17 成都斯马特科技有限公司 Kit for rapidly extracting nucleic acid from nasopharyngeal swab and extraction method
CN116949036B (en) * 2023-09-11 2023-12-15 成都斯马特科技有限公司 Kit for rapidly extracting nucleic acid from ascites and extraction method

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