CN113913494A - Kit for quickly and conveniently extracting virus DNA in body fluid and extraction method - Google Patents
Kit for quickly and conveniently extracting virus DNA in body fluid and extraction method Download PDFInfo
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- 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
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- 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
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- 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
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/706—Specific hybridization probes for hepatitis
Abstract
The invention discloses a kit for quickly and conveniently extracting virus DNA from body fluid and an extraction method thereof, wherein the kit comprises four components, wherein the first component comprises 2-6 mol/L guanidine isothiocyanate, 0.5-12% TritonX-100, 10-300 mmol/L tris (hydroxymethyl) aminomethane and 5-15% PEG 4000; the second component comprises 1-50 mmol/L sodium dihydrogen citrate, 0.005-0.05 mmol/L tris (hydroxymethyl) aminomethane, 5-30% PEG4000 and 0.005-0.05% magnetic bead content; the third component contains 10-200 mmol/L sodium dihydrogen citrate and 1-10% TritonX-100; the fourth component comprises mineral oil. The method for extracting the DNA by utilizing the kit can enter a PCR link through cracking/combining and cleaning, requires less manual operation and waiting time compared with the conventional magnetic bead extraction method, is efficient and safe in reagent used for extraction, avoids harm to a human body to the maximum extent, and further improves the sensitivity because the DNA in a sample can be completely used for PCR amplification.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a kit and an extraction method for quickly and conveniently extracting virus DNA from body fluid.
Background
The molecular biology technology of nucleic acid can be used for detecting infectious diseases, genetic diseases, transgene, phylogeny, species origin and other common means. Numerous advances have been made in various nucleic acid molecular biology techniques including fluorescent quantitative PCR, digital PCR, Next Generation Sequencing (NGS), etc. However, the favorable detection results of these techniques are all based on the efficiency of nucleic acid purification and recovery. A good nucleic acid purification technique should be simple, safe and cheap, and the recovered nucleic acid should reach sufficient concentration and purity.
Generally, the nucleic acid isolation method includes phenol-chloroform extraction, freeze thawing, boiling, silica gel membrane adsorption, magnetic bead separation, etc., and the extraction step generally includes cell lysis (organic solvent or inorganic solvent), precipitation, centrifugation, rinsing, elution, nucleic acid dissolution, etc. At present, in the process of extracting the DNA of the body fluid virus, a silica gel membrane adsorption or magnetic bead separation method is generally adopted, the steps of heating digestion by proteinase K, mercaptoethanol treatment, rinsing by alcohol-containing washing liquor, heating elution and the like are often used, the operation is relatively complicated, and some existing kits contain toxic substances with pungent odor, such as mercaptoethanol and the like, which can damage the health of human bodies. In addition, due to the influence of the volume of the final eluate, it is often difficult to use all of the recovered DNA for detection, which in turn affects the detection sensitivity.
In the step of extracting the virus DNA from the body fluid, a detergent (such as SDS, Tween-20, TritonX-100, NP-40, etc.) is used for destroying the shell or membrane structure of cells or virus proteins to free nucleic acids into a solution, a guanidinium salt or perchlorate (such as guanidine hydrochloride, guanidine isothiocyanate, potassium perchlorate, etc.) is used for denaturing proteins, releasing the bound nucleic acids and promoting the binding of the nucleic acids with silicon matrix membranes or magnetic beads, and simultaneously has the function of cracking the shell or membrane structure of the virus, if an organic solvent (such as phenol/chloroform) is used, the cell structure can be destroyed, and the proteins are denatured to release the nucleic acids. DNA combined on the surface of a silicon substrate membrane or magnetic beads can be further removed by rinsing with alcoholic salt solution, cell debris, non-specifically adsorbed protein and over-concentrated salt ions can be further removed, and DNA with higher purity can be eluted by eluent such as pure water and the like, so that the DNA can be applied to downstream experiments (such as PCR, enzyme digestion, connection, sequencing and the like); nucleic acid released into the solution through phenol-formaldehyde can be precipitated by adding a high-concentration alcohol solution after adjusting the pH value, and then the high-purity DNA can be obtained after the nucleic acid is washed by the alcohol solution and dried.
The magnetic bead separation and purification technology is receiving more and more attention in the nucleic acid extraction technology, because the method not only can effectively purify nucleic acid, but also can achieve the purpose of automatically purifying sample nucleic acid in batches by applying the technology, and is particularly suitable for large-scale rapid detection of infectious diseases or purification and concentration of nucleic acid with large volume and low concentration. The main principle is that a silicon substrate shell is coated outside a superparamagnetic iron oxide core, or iron oxide is filled in a skeleton in other forms to construct a magnetic microsphere, functional groups such as hydroxyl, carboxyl, amino and the like are modified on the surface of the microsphere, the functional groups can specifically adsorb nucleic acid molecules in a solution under certain salt ion concentration and pH, and then the magnetic beads are separated under the action of a magnetic field, rinsed and eluted, so that the purpose of extracting the nucleic acid is achieved.
Magnetic bead purification nucleic acid is often along with operations such as incessant mixing, shaking, and the use instrument easily realizes, but if need manual operation, then is difficult to reach the requirement, and then leads to recovery efficiency to seriously descend, is the common fault of most magnetic bead recovery kit at present. In general, DNA recovered from magnetic beads needs to be eluted by an eluent at a certain temperature (e.g., 70 ℃), and in order to improve the elution efficiency or compensate for losses caused by evaporation, a larger volume of the eluent is often needed, which results in a relatively low final nucleic acid concentration, and may reduce the final detection sensitivity if the DNA content of the sample is low.
In general, magnetic beads are a strong inhibitor for PCR, and when the magnetic beads are directly added to a PCR solution in a DNA-bound state, the fluorescence intensity of a fluorophore in real-time fluorescence quantitative PCR is severely inhibited, resulting in false negative results.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides a kit and an extraction method for quickly and conveniently extracting virus DNA in body fluid.
The purpose of the invention is realized by the following technical scheme:
a kit for quickly and conveniently extracting virus DNA in body fluid comprises a first component, a second component, a third component and a fourth component, wherein the first component contains 2-6 mol/L guanidine isothiocyanate, 0.5-12% TritonX-100(V/V), 10-300 mmol/L tris (hydroxymethyl) aminomethane and 5-15% PEG4000 (w/V); the second component comprises 1-50 mmol/L sodium dihydrogen citrate, 5-50 mmol/L Trisbase, 5-30% PEG4000(w/V), and 0.005-0.05% (w/V) of magnetic bead; the third component comprises 10-200 mmol/L sodium dihydrogen citrate and 1-10% TritonX-100; the fourth component mainly contains mineral oil.
Preferably, the first component is 3.62mol/L guanidinium isothiocyanate, 7.7% Triton X-100(V/V), 24mmol/L tris (hydroxymethyl) aminomethane, 6.66% PEG4000 (w/V); the second component comprises 14.5mmol/L sodium dihydrogen citrate, 23mmol/L tris (hydroxymethyl) aminomethane, 20% PEG4000(w/V), 0.015% (w/V) magnetic bead and 1 μm diameter magnetic bead; the third component contains 63mmol/L sodium dihydrogen citrate and 5% TritonX-100.
Preferably, the magnetic beads are Fe coupled with oligo (dT)30 prepared based on carboxyl magnetic beads with the average diameter of 0.5-1 μm3O4Magnetic particles.
Preferably, the kit is used for quickly and conveniently extracting the virus DNA in the body fluid.
Preferably, the method for rapidly and conveniently extracting the virus DNA from the body fluid, which is serum, plasma or saliva, by using the kit comprises the following steps:
s1, adding a sample into a centrifuge tube, sequentially adding the first component and the second component, uniformly mixing by oscillation, collecting liquid to the bottom of the centrifuge tube by instantaneous centrifugation, and standing at room temperature;
s2, placing the centrifugal tube obtained in the step S1 on a magnetic frame and standing until the magnetic beads are fully enriched on the wall of the centrifugal tube;
s3, keeping the centrifugal tube under the magnetic force, sucking the supernatant and discarding;
s4, adding the third component and the fourth component, oscillating, mixing uniformly, and performing instantaneous centrifugation to collect liquid to the bottom of the tube;
s5, placing the centrifuge tube on a magnetic rack until the magnetic beads are fully enriched on the wall of the centrifuge tube;
s6, keeping the centrifugal tube on the magnetic frame, making the pipette tip go deep into the bottom of the tube under the condition of not contacting the magnetic beads on the tube wall, slowly sucking out the liquid and discarding;
s7, standing the centrifugal tube on a magnetic frame for 1-5 min, and removing residual liquid at the bottom of the centrifugal tube; adding a PCR solution prepared in advance into a centrifuge tube, sucking and mixing uniformly the magnetic beads by a pipette, transferring the magnetic bead-PCR mixed solution into the PCR tube, and carrying out real-time fluorescence quantitative PCR, wherein the PCR solution contains all components except template DNA: the Taqman probe method reaction mixed solution containing the hot start Taq DNA polymerase and a specific primer probe required by detecting HBV.
Wherein, the reaction mixed liquid of the Taqman probe method is a qualified product and meets the following conditions: (1) using serum containing hepatitis B virus with the volume of 200 mul and the concentration of 30IU/ml as a sample, and obtaining a positive amplification result by using a primer probe required by the hepatitis B virus detection of the kit after extracting DNA by using the kit; (2) with ultrapure water as the negative sample, a negative result was obtained.
The primer probes required by the hepatitis B virus detection are as follows:
designing a primer probe:
HBV forward primer: 5'-TGTATTCCCATCCCATCATCCTG-3'
HBV reverse primer: 5'-TGGCACTAGTAAACTGAGCCA-3'
HBV probe:
5’-FAM-ACCTATGGGAGTGGGCCTCAGTCCG-3’-BHQ1
the invention has the beneficial effects that: the kit is used for extracting virus DNA from body fluids such as serum, plasma, saliva and the like, and the method does not need heating, proteinase K treatment or elution treatment, and finally the DNA exists in a form of being combined on magnetic beads, so that the kit can be directly used for real-time fluorescent quantitative PCR, and the utilization rate and detection sensitivity of a sample are greatly improved.
Drawings
FIG. 1: example one of the kits of the present invention was used to extract DNA from hepatitis B serum and test the results.
FIG. 2: example A test result after DNA extraction was performed on hepatitis B serum using the other kit of the present invention.
FIG. 3: example a third branch of the kit of the invention is used for extracting DNA from hepatitis B serum and then detecting the result.
FIG. 4: and (3) carrying out qPCR program detection result atlas on the 1 st sample extracted by the kit.
FIG. 5: and (3) carrying out qPCR program detection result atlas on the 2 nd sample extracted by the kit.
FIG. 6: and (3) carrying out qPCR program detection result atlas on the 3 rd sample extracted by the kit.
FIG. 7: and (3) carrying out qPCR program detection result atlas on the 4 th sample extracted by the kit.
FIG. 8: and (3) carrying out qPCR program detection result atlas on the 5 th sample extracted by the kit.
FIG. 9: and (3) carrying out qPCR program detection result atlas on the 6 th sample extracted by the kit.
FIG. 10: and (3) carrying out qPCR program detection result atlas on the 7 th sample extracted by the kit.
FIG. 11: and (3) carrying out qPCR program detection result atlas on the 8 th sample extracted by the kit.
FIG. 12: and (3) carrying out qPCR program detection result atlas on the 9 th sample extracted by the kit.
FIG. 13: and (3) carrying out qPCR program detection result atlas on the 10 th sample extracted by the kit.
FIG. 14: and (3) carrying out qPCR program detection result atlas on the 11 th sample extracted by the kit.
FIG. 15: and (3) carrying out qPCR program detection result atlas on the 12 th sample extracted by the kit.
FIG. 16: and (3) carrying out qPCR program detection result atlas on the 13 th sample extracted by the kit.
FIG. 17: and (3) carrying out qPCR program detection result atlas on the 14 th sample extracted by the kit.
FIG. 18: and (3) carrying out qPCR program detection result atlas on the 15 th sample extracted by the kit.
FIG. 19: and (3) carrying out qPCR program detection result atlas on the 16 th sample extracted by the kit.
FIG. 20: and (3) carrying out qPCR program detection result atlas on the 17 th sample extracted by the kit.
FIG. 21: and (3) carrying out qPCR program detection result atlas on the 18 th sample extracted by the kit.
FIG. 22: and (3) carrying out qPCR program detection result atlas on the 19 th sample extracted by the kit.
FIG. 23: and (3) carrying out qPCR program detection result atlas on the 20 th sample extracted by the kit.
FIG. 24: and (3) carrying out qPCR program detection result atlas on the 21 st sample extracted by the kit.
FIG. 25: and (3) carrying out qPCR program detection result atlas on the 22 nd sample extracted by the kit.
FIG. 26: and (3) carrying out qPCR program detection result atlas on the 23 rd sample extracted by the kit.
Detailed Description
Objects, advantages and features of the present invention will be illustrated and explained by the following non-limiting description of preferred embodiments. The embodiments are merely exemplary for applying the technical solutions of the present invention, and any technical solution formed by replacing or converting the equivalent thereof falls within the scope of the present invention claimed.
The invention discloses a kit for quickly and conveniently extracting virus DNA from body fluid and an extraction method. The kit for rapidly and conveniently extracting the virus DNA in the body fluid comprises a first component, a second component, a third component and a fourth component, wherein the first component contains 2-6 mol/L guanidine isothiocyanate, 0.5-12% TritonX-100(V/V), 10-300 mmol/L tris (hydroxymethyl) aminomethane and 5-15% PEG4000 (w/V); the second component comprises 1-50 mmol/L sodium dihydrogen citrate, 5-50 mmol/L Trisbase, 5-30% PEG4000(w/V), and 0.005-0.05% (w/V) of magnetic bead; the third component comprises 10-200 mmol/L sodium dihydrogen citrate and 1-10% TritonX-100; the fourth component comprises mineral oil. The magnetic beads in the present invention are oligo-dT coupled magnetic beads prepared based on carboxyl magnetic beads.
The method for extracting DNA by using the kit can enter a PCR link after cracking/combining and cleaning, requires less manual operation and shorter waiting time compared with the conventional magnetic bead extraction method, and has the advantages of high efficiency and safety of reagents used for extraction, avoids the damage to a human body to the greatest extent, and further improves the sensitivity because the DNA in a sample can be completely used for PCR amplification. The method specifically comprises the following steps:
s1, adding 200 mu L of sample into a 1.5mL centrifuge tube, sequentially adding 300 mu L of first component and 100 mu L of second component, shaking and uniformly mixing for 15S, performing instantaneous centrifugation to collect liquid to the bottom of the centrifuge tube, and standing for 10min at room temperature;
s2, placing the centrifugal tube obtained in the step S1 on a magnetic frame and standing for 8min, or observing that magnetic beads are fully enriched on the wall of the centrifugal tube;
s3, keeping the centrifugal tube under the magnetic force, carefully sucking the supernatant and discarding;
s4, adding 600 mu L of third component and 200 mu L of fourth component, shaking and uniformly mixing for 5S, and performing instantaneous centrifugation to collect liquid to the bottom of the tube;
s5, placing the centrifuge tube on a magnetic frame for 3min, or observing that magnetic beads are fully enriched on the wall of the centrifuge tube;
s6, keeping the centrifugal tube on the magnetic frame, making the pipette tip go deep into the bottom of the tube under the condition of not contacting the magnetic beads on the tube wall, slowly sucking out the liquid and discarding;
s7, standing the centrifugal tube on a magnetic frame for 1min, and discarding residual liquid at the bottom of the centrifugal tube; adding the prepared PCR solution (containing all components except the template DNA) into a centrifuge tube, blowing and sucking the magnetic beads uniformly by using a pipette, transferring the magnetic bead-PCR mixed solution into the PCR tube, and carrying out real-time fluorescence quantitative PCR.
Example 1:
the kit and hepatitis B serum which is preserved at 3 branches and 80 ℃ and has the volume of 200 mu L and the concentration of 30IU/mL are taken for nucleic acid extraction and subsequent PCR detection.
After the serum is unfrozen, the steps are carried out according to the standard steps of kit extraction, the finally obtained magnetic beads combined with the hepatitis B virus DNA are suspended by a PCR solution, and the magnetic beads are operated on a real-time fluorescence quantitative PCR instrument ABI7500 according to the following procedure 1 (the fluorescence channel is detected as FAM):
table 1: PCR running program
Designing a primer probe:
HBV forward primer: 5'-TGTATTCCCATCCCATCATCCTG-3'
HBV reverse primer: 5'-TGGCACTAGTAAACTGAGCCA-3'
HBV probe:
5’-FAM-ACCTATGGGAGTGGGCCTCAGTCCG-3’-BHQ1
as shown in fig. 1-3, 3 technical replicates were all well detected.
Example 2:
taking 5.12mL of African Swine Fever (ASFV) negative pig saliva and a nucleic acid standard (5 x 10) of African swine fever5Copy/ml) 4. mu.L, extracted and amplified for detection as follows.
S1, adding 5.12mL negative pig saliva into 4 μ L nucleic acid standard substance to obtain 390 copies/mL sample, which is named ASFVL 6;
s2, loading ASFVL6 into a 1.5mL centrifuge tube according to 200 mu L per branch, and loading 23 branches in a total way, namely repeating 23 technologies;
s3, extracting the nucleic acid DNA of the 23 samples by using the kit;
s4, preparing a real-time fluorescent quantitative PCR solution by using a four-color African swine fever virus detection kit developed by the sharpening biology, suspending 23 magnetic beads in centrifugal tubes respectively, suspending each magnetic bead by 150 mu L, splitting each magnetic bead into 3 technologies of 50 mu L, and repeatedly performing real-time fluorescent quantitative PCR detection to detect 23 multiplied by 3 which is 69 holes in total;
s5, detection using qPCR program (fluorescent channels FAM, VIC, ROX, Cy 5):
table 2: qPCR run program
Designing a primer probe:
p72 forward primer: 5'-TTAGCTTCCTTGCTTTTCTG-3', respectively;
p72 reverse primer: 5'-TCCCTCTGAATCGGTAGTAA-3', respectively;
p72 probe: 5 '-FAM-TCTGGGTTTTGCAGCTATCC-3' -BHQ 1.
B646L-1 forward primer: 5'-ACAAGATCAGCCGTAGTGAT-3', respectively;
B646L-1 reverse primer: 5'-ACCCAGTGGTCATATTAACG-3', respectively;
B646L-1 probe: 5 '-HEX-acgtaatccGTGTCCCAACT-3' -BHQ 2.
B646L-2 forward primer: 5'-AATGACTCCTGGGATAAACC-3', respectively;
B646L-2 reverse primer: 5'-TGGACTTTTTATACGCCAGT-3', respectively;
probe B646L-2: 5 '-ROX-tactggggcgtccaggtata-3' -BHQ 3.
B646L-3 forward primer: 5'-CATGAATGTTGCATAGGAGA-3'
B646L-3 reverse primer: 5'-AGGGGATAAAATGACTGGAT-3'
Probe B646L-3: 5 '-Cy 5-agttccctccaccgatacct-3' -BHQ 3.
S6, judging that the sample is positive if the amplification curve of more than or equal to 1 hole is repeated in 3 techniques of each sample, and otherwise, judging that the sample is negative;
s7, according to the data of the ROX fluorescence channel, the detection result is that the positive rate reaches 22/23, namely 95.6%, and the condition of determining the lowest limit of detection (LOD) is met. Specifically, with reference to fig. 4-26 and table 3, fig. 16 shows negatives, corresponding to sample name S8, with the remainder being positives.
Table 3: ct values for 23 samples.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein, and any reference signs in the claims are not intended to be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art. The invention has various embodiments, and all technical solutions formed by adopting equivalent transformation or equivalent transformation are within the protection scope of the invention.
Claims (6)
1. Kit for rapidly and conveniently extracting virus DNA in body fluid is characterized in that: the composition comprises a first component, a second component, a third component and a fourth component, wherein the first component contains 2-6 mol/L of guanidinium isothiocyanate, 0.5-12% of Triton X-100(V/V), 10-300 mmol/L of tris (hydroxymethyl) aminomethane and 5-15% of PEG4000 (w/V); the second component comprises 1-50 mmol/L sodium dihydrogen citrate, 5-50 mmol/L tris (hydroxymethyl) aminomethane, 5-30% PEG4000(w/V), and 0.005-0.05% (w/V) of magnetic bead content; the third component contains 10-200 mmol/L sodium dihydrogen citrate and 1-10% TritonX-100; the fourth component comprises mineral oil.
2. The kit for rapidly and conveniently extracting viral DNA from body fluid according to claim 1, wherein: the first component is 3.62mol/L of guanidinium isothiocyanate, 7.7 percent of Triton X-100(V/V), 24mmol/L of tris (hydroxymethyl) aminomethane, and 6.66 percent of PEG4000 (w/V); the second component comprises 14.5mmol/L sodium dihydrogen citrate, 23mmol/L tris (hydroxymethyl) aminomethane, 20% PEG4000(w/V), 0.015% magnetic bead (w/V), and the diameter of the magnetic bead is 1 μm; the third component contains 63mmol/L sodium dihydrogen citrate and 5% TritonX-100.
3. The kit for rapidly and conveniently extracting viral DNA from body fluid according to claim 1, wherein: the magnetic beads are Fe coupled with oligo (dT)30 and prepared on the basis of carboxyl magnetic beads, the average diameter of the magnetic beads is 0.5-1 mu m3O4Magnetic particles.
4. The use of the kit according to any one of claims 1 to 3 for rapid and convenient extraction of viral DNA from body fluids.
5. The method for rapidly and conveniently extracting the virus DNA from the body fluid by using the kit according to claim 4, which is characterized in that: the body fluid is serum, plasma or saliva, and comprises the following steps:
s1, adding a sample into a centrifuge tube, sequentially adding the first component and the second component, uniformly mixing by oscillation, collecting liquid to the bottom of the centrifuge tube by instantaneous centrifugation, and standing at room temperature;
s2, placing the centrifugal tube obtained in the step S1 on a magnetic frame and standing until the magnetic beads are fully enriched on the wall of the centrifugal tube;
s3, keeping the centrifugal tube on a magnetic frame, sucking the supernatant and discarding;
s4, adding the third component and the fourth component, oscillating, mixing uniformly, and performing instantaneous centrifugation to collect liquid to the bottom of the tube;
s5, placing the centrifuge tube on a magnetic rack until the magnetic beads are fully enriched on the wall of the centrifuge tube;
s6, keeping the centrifugal tube on the magnetic frame, making the pipette tip go deep into the bottom of the tube under the condition of not contacting the magnetic beads on the tube wall, slowly sucking out the liquid and discarding;
s7, standing the centrifugal tube on a magnetic frame for 1-5 min, and removing residual liquid at the bottom of the centrifugal tube; and adding the prepared PCR solution into a centrifuge tube, uniformly mixing magnetic beads by blowing and sucking with a pipettor, and transferring the magnetic bead-PCR mixed solution into the PCR tube to perform real-time fluorescence quantitative PCR.
6. The method for rapidly and conveniently extracting the virus DNA from the body fluid by using the kit according to claim 5, is characterized in that: the PCR solution in S7 contains all components except the template DNA, and comprises: the reaction mixed liquor qualified by the Taqman probe method containing the hot start Taq DNA polymerase and a specific primer probe required by detecting HBV.
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