CN103695419A - Viral nucleic acid extraction reagent - Google Patents
Viral nucleic acid extraction reagent Download PDFInfo
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- CN103695419A CN103695419A CN201310745655.7A CN201310745655A CN103695419A CN 103695419 A CN103695419 A CN 103695419A CN 201310745655 A CN201310745655 A CN 201310745655A CN 103695419 A CN103695419 A CN 103695419A
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- nucleic acid
- biotin labeled
- specific probe
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- probe
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Images
Abstract
The invention firstly provides a biotin-labeled specific probe which is a biotin-labeled hepatitis c virus probe and has a sequence of biotin-(T)n-TGGTACTGCCTGATAGGGTGCTTGCG, wherein n is the number selected from 3 to 30, preferably 5 to 20. Correspondingly, the invention further provides a viral nucleic acid extraction reagent which comprises the biotin-labeled specific probe. The invention further provides the biotin-labeled hepatitis c virus probe and a biotin-labeled human immunodeficiency virus probe. The viral nucleic acid extraction reagent comprising the three biotin-labeled specific probes can be used for blood screening for three viruses.
Description
Technical field
The present invention relates to a kind of viral nucleic acid and extract reagent and corresponding viral nucleic acid extraction test kit, and this viral nucleic acid extracts the biotin labeled specific probe in reagent.
Background technology
The extraction and purification technology of nucleic acid is a basic fundamental of Biochemistry and Molecular Biology.Along with Protocols in Molecular Biology is widely used in biology, medical science and association area thereof, the extraction and purification technology of nucleic acid is also further developed.
Nucleic acid always combines with range protein in cell.The extraction of nucleic acid mainly refers to biomacromolecule separating substances such as nucleic acid and protein, polysaccharide, fat.The extracting method of most of nucleic acid, generally all comprises lysis, the biomacromolecules such as the protein that removal is combined with nucleic acid and polysaccharide, lipid, remove other unwanted nucleic acid molecule, precipitate nucleic acids, removes the impurity such as salt, organic solvent, the steps such as purification of nucleic acid.
First, lysis can realize by methods such as mechanical effect, chemical action, enzyme effects.Concrete, mechanical effect comprises the physics cleavage methods such as hypotonic cracking, ultrasonic degradation, microwave cracking, freezing-thawing and cracking and grain breakage, these methods make cytoclasis by mechanical force, but mechanical force also can cause nucleic acid chain break, thereby are not suitable for the separation of high molecular long-chain nucleic acid.Chemical action is under certain pH environment and Denaturing, cell rupture, protein denaturation precipitation, nucleic acid is released to water, and Denaturing can be by heating, add tensio-active agent (SDS, Triton X-100, Tween20, NP-40, CTAB, sar-cosyl, Chelex-100 etc.) or strong ionic agent (guanidinium isothiocyanate, Guanidinium hydrochloride, creatine guanidine etc.) to obtain.And enzyme effect is mainly by adding N,O-Diacetylmuramidase or proteolytic enzyme (Proteinase K, plant protease or pronase) so that cell rupture, nucleic acid discharges.
Secondly, enzyme is processed and is referred in nucleic acid extraction process, can be by adding suitable enzyme to make unwanted mass degradation, be beneficial to the isolation and purification of nucleic acid, as added the proteolytic enzyme (Proteinase K or pronase) can degrade proteins in lysate, deactivation nuclease (DNase and RNase), DNase and RNase are also for removing unwanted nucleic acid.
In addition, the separation of nucleic acid and purifying: the high electric charge phosphoric acid skeleton of nucleic acid makes it compare protein, polysaccharide, fat waits other biological macromolecular substance to have more wetting ability, according to the difference of their physico-chemical properties, with selective precipitation, chromatography, the methods such as density gradient centrifugation can be by separate nucleic acid, purifying, the ordinary method of existing separation and purification nucleic acid has: (1) phenol extraction/precipitator method: the water by centrifugation after lysis containing nucleic acid, add isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1 volumes) mixed solution, two-phase through vortex oscillation mix (being applicable to separated small molecules amount nucleic acid) or simply put upside down mix (being applicable to separation of high molecular weight nucleic acid) after centrifugation, again by nucleinate organic solvent deposit, by precipitation condensed nucleic acid, changing nucleic acid dissolves the kind of damping fluid and removes some impurity molecule, (2) density gradient centrifugation: double-stranded DNA, single stranded DNA, RNA and protein have different density, thereby can through density gradient centrifugation, form the pure sample area band of different densities, this method is applicable to the preparation of a large amount of sample of nucleic acid, (3) chromatography, chromatography is to utilize the difference of some physico-chemical property of different substances and the method for separating and analyzing set up, comprise the methods such as adsorption chromatography, affinity chromatography, ion exchange chromatography, under certain ionic environment, nucleic acid can be optionally adsorbed onto tripoli, silica gel or glass surface and with other biological molecular separation, other selective adsorption method, usings modified or coated magnetic bead as solid phase carrier, and magnetic bead can be by magnetic field separation and without centrifugal, and the nucleic acid that is bonded to solid phase carrier can be with low salt buffer or water elution.
Paramagnetic particle method is the method for extracting nucleic acid just growing up in recent years, its reagents series is not containing large organic solvents of toxicity such as chloroform, phenol, the nucleic acid quality of extracting purifying is good, output is high, extraction step is simpler, do not need centrifugal, easily be automated, these advantages and feature make its method of extracting nucleic acid have the development trend that substitutes all other methods.
Magnetic bead is by magnetic grain and the various particle with certain magnetic and special surface structure forming containing the Material cladding of activity functional groups.In biomagnetism separation, magnetic bead surfaces active group is a very important factor.Use at present more magnetic bead surfaces aglucon to have monoclonal antibody (Monoclonal antibody), Streptavidin (Streptavidin), oligonucleotide (Oligonucleotides), and on magnetic microsphere surface, introduce amino (NH2) with silylating reagent, hydroxyl (OH), sulfydryl (SH), carboxyl (COOH), sulfonic group (SO
3-) etc., on this basis, also can introduce polysaccharide compound (as Mierocrystalline cellulose, agarose), vitamin H, antibiotin etc.
The domestic existing method based on magnetic bead extraction nucleic acid is applied in clinical detection and nucleic acid blood screening, these paramagnetic particle method reagent all comprise following shortcoming: 1) nucleic acid extraction process steps is many, sample process length consuming time, RNA long-term exposure easily, by RNA enzyme enzymolysis, has reduced the sensitivity of reagent.2) conventional paramagnetic particle method extracts needs heating, and solution causes Aerosol Pollution, cause sample negative and positive to be checked regardless of.3) conventional paramagnetic particle method does not have mark specific oligonucleotide chain, can not specific enrichment viral nucleic acid, and magnetic bead is easily coated with by human genome nucleic acid, has reduced the ability of magnetic bead viral adsorption nucleic acid, has reduced the sensitivity of paramagnetic particle method.
Summary of the invention
First the present invention provides a kind of biotin labeled specific probe, and described biotin labeled specific probe is biotin labeled hepatitis C virus probe, and its sequence is: vitamin H-(T)
n-TGGTACTGCCTGATAGGGTGCTTGCG, wherein n is selected from 3～30 number, and preferably n is selected from 5～20 number.
Those skilled in the art are known, and specific probe sequence of the present invention can be all that its 5 ' end is connected with vitamin H by a plurality of T, can be also that its 3 ' end is connected with vitamin H by a plurality of T; Therefore, in sequence of the present invention, all represent being connected and a plurality of T and being connected that probe 3 ' is held of a plurality of T and probe 5 ' end being connected between a plurality of T and described specific probe sequence.
The present invention also provides a kind of viral nucleic acid to extract reagent, contains biotin labeled specific probe as above in described reagent.The specific oligonucleotide sequences that is marked with vitamin H relies on its sequence conservation specificity to hybridize with the viral nucleic acid in sample to be checked, its biotin moiety again can be with mark the magnetic bead of Streptomycin sulphate be combined, thereby reach the effect of separated object nucleic acid specifically.Compare other non-specific methods, use viral nucleic acid provided by the invention to extract reagent and carry out nucleic acid extraction, the nucleic acid product purity of gained is higher and nucleic acid extraction efficiency is higher.
In a kind of concrete embodiment, in described reagent, also contain another kind of biotin labeled specific probe, the biotin labeled specific probe of described another kind is biotin labeled hepatitis B virus probe, its sequence is: vitamin H-(T)
m-TTGGGTGGCTTTGGGGCATGG, wherein m is selected from 3～30 number.
According to the present invention, in the concrete embodiment of another kind, in described reagent, also contain another biotin labeled specific probe, described another biotin labeled specific probe is biotin labeled human immunodeficiency virus probe, and its sequence is: vitamin H-(T)
q-CCAGGCCAGATGAGAGAACCAAGGG, wherein q is selected from 3～30 number.In a kind of concrete reagent of the present invention, described pack is containing biotin labeled hepatitis B virus probe, biotin labeled hepatitis C virus probe and biotin labeled human immunodeficiency virus probe.Its object is to solve the defect of existing hepatitis B virus DNA, HCV RNA, human immunodeficiency virus nucleic acid RNA extraction, the viral nucleic acid that provide that a kind of method is easy, detection sensitivity is high, can be used for automatization extracts reagent, apply this reagent and can accomplish specific extraction to above-mentioned three kinds of viral nucleic acids, nucleic acid one pipe that is applicable to blood screening project extracts.
In the present invention, preferably, described reagent comprises the magnetic bead containing specific probe, and the described magnetic bead containing specific probe is for being combined and obtaining with described biotin labeled specific probe through the magnetic bead of Streptavidin modification.Those skilled in the art are known, the Streptavidin of vitamin H in described biotin labeled specific probe in the present invention in the described magnetic bead of modifying through Streptavidin is combined correspondingly, but because being combined with a plurality of Streptavidin molecules on same magnetic bead, therefore, on same magnetic bead, can connect identical biotin labeled specific probe, also can connect different biotin labeled specific probes, for example on same magnetic bead, be connected with biotin labeled hepatitis C virus probe and biotin labeled hepatitis B virus probe.In a kind of concrete embodiment, in described reagent, also comprise cell pyrolysis liquid.Those skilled in the art hold intelligible, and the cell pyrolysis liquid in the present invention can be mixed to form a kind of solution with the described magnetic bead containing specific probe, and described viral nucleic acid extracts reagent; Also can be that described cell pyrolysis liquid and the described magnetic bead solution that contains specific probe are packed respectively sale and uses.
The present invention also provides a kind of viral nucleic acid to extract test kit, comprises that viral nucleic acid as above extracts reagent, also comprises elutriant and/or washings.Described washings is for rinsing separation by described containing the impurity on the magnetic bead of specific probe, and described elutriant is for eluting target nucleotide for PCR and react from the described magnetic bead containing specific probe.In a specific embodiment of the present invention, the specific probe after wash-out, vitamin H and Streptavidin are all connected on magnetic bead, and the target nucleotide stemming from sample to be tested enters in elutriant, are used further to downstream experiment.The experiment of described downstream refers to that detection, clone, sequential analysis, pcr amplification, molecular hybridization and cDNA are synthetic etc.
The present invention also provides a kind of reagent as above or the application of test kit as above in blood screening.
The present invention also provides a kind of biotin labeled specific probe, and described biotin labeled specific probe is biotin labeled hepatitis B virus probe, and its sequence is: vitamin H-(T)
m-TTGGGTGGCTTTGGGGCATGG, wherein m is selected from 3～30 number.
The present invention also provides another kind of biotin labeled specific probe, and described biotin labeled specific probe is the human immunodeficiency virus probe of thing element mark, and its sequence is: vitamin H-(T)
q-CCAGGCCAGATGAGAGAACCAAGGG, wherein q is selected from 3～30 number.
Same, above-mentioned two kinds of biotin labeled specific probes can corresponding two-strain nucleic acid extracting reagent.Wherein viral nucleic acid extraction reagent comprises the magnetic bead containing specific probe, and the described magnetic bead containing specific probe is combined with biotin labeled hepatitis B virus probe and is obtained for the magnetic bead of modifying through Streptavidin; Another kind of viral nucleic acid extracts in reagent and also comprises that the magnetic bead containing specific probe, the described magnetic bead containing specific probe are that the magnetic bead of modifying through Streptavidin is combined and is obtained with biotin labeled human immunodeficiency virus probe.
Use method of the present invention to extract the nucleic acid obtaining and not only can be used for corresponding three kinds of viral nucleic acid blood source examinations, also can be applicable in these three kinds of viral detection in Gene Mutation, genotype tests and gene sequencing.
In the present invention, containing the magnetic bead of specific probe and the specificity virus nucleic acid hybridization in sample to be tested, the DNA of human body self, protein, rRNA, tRNA and other impurity are not adsorbed, be adsorbed on the nucleic acid of magnetic bead surfaces can be directly as the template of pcr amplification, or add target nucleotide molecule that elutriant makes to be adsorbed on magnetic bead surfaces and be released to and in elutriant, be used further to downstream experiment.
Accompanying drawing explanation
Fig. 1 extracts the augmentation detection result of the various hypotypes of HCV that obtain for the reagent that provides in embodiment 3;
The amplification that HIV-1 C-type virus C nucleic acid (50IU/ml) duplicate detection that Fig. 2 obtains for the reagent that provides in embodiment 4 extracts is 30 times.
Embodiment
Following examples are for the present invention is done to more detailed description, but the present invention is not limited to following embodiment.
In the present embodiment, provide a kind of viral nucleic acid to extract test kit, in described test kit, comprise viral nucleic acid and extract reagent, in described reagent, comprise three kinds with biotin labeled specific probe, i.e. biotin labeled hepatitis B virus probe, biotin labeled hepatitis C virus probe and biotin labeled human immunodeficiency virus probe.Concrete, in described test kit, comprise following several component:
1. contain the magnetic bead of specific probe: the described magnetic bead containing specific probe comprises following three kinds:
Magnetic bead containing HBV capture probe: magnetic bead-Streptavidin-vitamin H-TTTTTTTTTTTTTT-TTGGGTGGCTTTGGGGCATGG;
Magnetic bead containing HCV capture probe: magnetic bead-Streptavidin-vitamin H-TTTTTTTTTTTTTT-TGGTACTGCCTGATAGGGTGCTTGCG;
Magnetic bead containing HIV-I capture probe: magnetic bead-Streptavidin-vitamin H-TTTTTTTTTTTTTT-CCAGGCCAGATGAGAGAACCAAGGG.
Streptavidin MagneSphere described in the present invention can be purchased, and also can synthesize voluntarily.As described in Streptavidin MagneSphere be with γ Fe
2o
3and Fe
3o
4synthetic homogeneous, superparamagnetic, the single diversity poly microballoon of magneticsubstance is magnetic source, and each microsphere is coated with one deck poly material, carries the magnetic macromolecular microsphere of carboxylate salt by letex polymerization or the preparation of diffuse-aggregate method; Poly material (being for example polystyrene) core particle surface cover one deck magnet of modifying through carboxylate salt, Streptomycin sulphate covalently bind on this surface again; Described Streptavidin MagneSphere for example dilutes and is kept in PBS damping fluid.
Biotin labeled specific probe of the present invention is customization acquisition, and described biotin labeled specific probe is for example kept in TE solution.
The described Streptavidin MagneSphere being purchased is combined with the described biotin labeled specific probe of customization, prepares the described magnetic bead containing specific probe.In a concrete embodiment, every mg Streptavidin MagneSphere is combined with the biotin labeled specific probe of 50pmol, and each reaction (for the sample to be tested of every person-portion) needs Streptavidin MagneSphere 20 μ g and each 1pmol of biotin labeled specific probe.
Magnetic bead containing specific probe provided by the invention can be placed in 4 ℃ or-20 ℃ of preservations, and convenient transportation, and with strong points, adsorption efficiency is high, can from testing sample, extract and purifying HBV/HCV/HIV-I viral nucleic acid single-mindedly.
2. cell pyrolysis liquid: described cell pyrolysis liquid is the solution containing tensio-active agent, preferably containing the solution of 0.01%～10% SDS, LLS, Chelex-100, Triton X-100, Tween20 or NP-40.
3. Triton X-100 and NaCl, the KCl of 100mmol/L～2mol/L or the solution that LiCl forms washings: described washings is for example by 0.1% to 2%(volume ratio).
4. elutriant: the solution that described elutriant is 8.0 for the pH value that consists of 10mM Tris-HCl and 1mM EDTA.Embodiment 2
The present embodiment provides test kit described in embodiment 1 to carry out fluorescence quantitative PCR detection and result thereof for extracting method and the gained viral nucleic acid of the HBV/HCV/HIV-I virus (being HBV DNA in the present embodiment) of sample to be tested.
First, sample to be tested of the present invention is: the concentration of Products in China calibrating institute (Zhong Jian institute) hepatitis B virus (HBV) nucleic acid quantification standardization is respectively 5.0 * 10
6iU/ml, 5.0 * 10
4iU/ml and 5.0 * 10
2the HBV DNA positive serum of 3 kinds of concentration of IU/ml.
The step of nucleic acid extraction and detection is as follows:
1) lysis: sample to be tested is added in centrifuge tube, add cell pyrolysis liquid lysing cell to discharge nucleic acid;
2) nucleic acid absorption: add the magnetic bead containing specific probe in solution, HBV/HCV/HIV-I nucleic acid (only containing HBV-DNA in the sample to be tested of the present embodiment) is adsorbed by specific probe and is connected on magnetic bead, and protein, other non-specific sequence and impurity are not adsorbed; Centrifuge tube is placed on magnetic separator, and after separation, magnetic bead is adsorbed in centrifugal tube wall completely; Wherein, the capture probe with vitamin H can be combined in magnetic bead surfaces well together with the specific nucleic acid molecule being extracted;
3) remove impurity: add washings, the magnetic bead that effects on surface absorption has nucleic acid and is attached to centrifugal tube wall washs, and the impurity on magnetic bead is rinsed separated;
4) nucleic acid reclaims: be adsorbed on the nucleic acid of magnetic bead surfaces can be directly as the template of pcr amplification; Or add elutriant to be heated to the Tm value temperature of capture probe, the nucleic acid molecule desorb that is adsorbed on magnetic bead surfaces is entered in elutriant, be used further to downstream experiment.
5) fluorescence quantitative PCR detection: what above-mentioned wash-out was obtained is transferred in PCR reaction tubes containing viral nucleic acid solution, uses quantitative real time PCR Instrument to carry out detection by quantitative.
To totally 3 concentration (wherein 5.0 * 10 in above-mentioned sample to be tested
6iU/ml, 5.0 * 10
4iU/ml and 5.0 * 10
2the sample to be tested of IU/ml is labeled as respectively sample 1, sample 2 and sample 3) each concentration repeat to test 8 times, result is as shown in table 1.
Table 1
From the variation coefficient in table 1 (CV), the equal <0.6% of the variation coefficient of Ct value, the equal <13% of the concentration variation coefficient, illustrate and use the reproducible of method separation and purification viral nucleic acid of the present invention, even different people operation, also can obtain consistent result.Thereby guaranteed the stable of subsequent experimental, difference and the mistake of avoiding manual operation to cause.
The present embodiment provides test kit described in embodiment 1 to carry out fluorescence quantitative PCR detection and result thereof for extracting method and the gained viral nucleic acid of the HBV/HCV/HIV-I virus (being HCV RNA in the present embodiment) of sample to be tested.
Sample to be tested of the present invention is: the srea careHCV somatotype reference material (totally 20 kinds) after diluting 10 times.In the present invention, use two kinds of contradistinction systems (the RealTime HCV m2000 of Abbott Laboratories detection system, Roche AmpliPrep/COBAS TaqMan Test detection system) and test kit of the present invention to detect the various hypotypes of HCV in sample to be tested serum.
Extraction of the present invention and detection method: add 200 μ l samples to be tested in 1.5ml centrifuge tube, add subsequently 600 μ lRNA extracting solution 1(cell pyrolysis liquid and mixed solution containing the magnetic bead of specific probe): described RNA extracting solution 1 is by 0.5%(W/V) LLS, 2% (V/V) Triton X-100,1mol/L GuScN, magnetic bead and the biotin labeled specific probe solution of 50 μ l (total amount 1pmol) that 200 μ g/ml Streptavidins are modified mix; Through simple, add 100 μ l RNA extracting solutions 2 after centrifugal, described RNA extracting solution 2 is by Tris(pH8.0 ± 0.2 of 200mmol/L) and 200mmol/LNaCl form; Concussion mixes 10s, simply centrifugal after room temperature placement 10min.Be placed on magnetic separator, after separated 5min, treat that magnetic bead is adsorbed in tube wall completely, from managing bottom slowly by supernatant liquor sucking-off; Add 600 μ l washingss, described washings is comprised of the Triton X-100 of 0.2% (V/V) and the KCl of 200mmol/L, and concussion mixes magnetic bead is suspended completely, again this pipe is placed in to magnetic bead separator after simple centrifugal, treat that magnetic bead is adsorbed in tube wall completely, from managing bottom by the complete sucking-off of liquid.With pipettor, get 50 μ lPCR reaction solutions and repeatedly several times the magnetic bead that is adsorbed in tube wall is washed down completely, be transferred in PCR reaction tubes, use quantitative real time PCR Instrument to carry out detection by quantitative.Wherein, PCR response procedures is in Table 2.
Table 2
Use above-mentioned two kinds of contradistinction systems detected result detecting and the detected result that the aforesaid method using in the present embodiment obtains all to list in table 3.
Table 3
Corresponding with last row of table 3, Fig. 1 extracts the augmentation detection result figure of the various hypotypes of HCV (20 kinds) that obtain for the reagent providing in the present embodiment, and the ordinate zou in Fig. 1 is fluorescent value.
Embodiment 4
The present embodiment provides test kit described in embodiment 1 to carry out fluorescence quantitative PCR detection and result thereof for extracting method and the gained viral nucleic acid of the HBV/HCV/HIV-I virus (being HIV-1RNA in the present embodiment) of sample to be tested.
Sample to be tested of the present invention is: the HIV-1 of National Institute for Food and Drugs Control type nucleic acid blood screening reagent National reference sensitivity reference material 3.79 * 10 of purchase
4iU/ml is diluted to 50IU/ml, is packed as 30 pipes and repeats experiment for 30 times.
Extraction of the present invention and detection method: add 200 μ l samples to be tested in 1.5ml centrifuge tube, add subsequently 600 μ lRNA extracting solution 1(cell pyrolysis liquid and mixed solution containing the magnetic bead of specific probe): described RNA extracting solution 1 is by 0.5%(W/V) LLS, 2% (V/V) Triton X-100,1mol/L GuScN, magnetic bead and the biotin labeled specific probe solution of 50 μ l (total amount 1pmol) that 200 μ g/ml Streptavidins are modified mix; Through simple, add 100 μ l RNA extracting solutions 2 after centrifugal, described RNA extracting solution 2 is by Tris(pH8.0 ± 0.2 of 200mmol/L) and 200mmol/LNaCl form; Concussion mixes 10s, simply centrifugal after room temperature placement 10min.Be placed on magnetic separator, after separated 5min, treat that magnetic bead is adsorbed in tube wall completely, from managing bottom slowly by supernatant liquor sucking-off; Add 600 μ l washingss, described washings is comprised of the Triton X-100 of 0.2% (V/V) and the KCl of 200mmol/L, and concussion mixes magnetic bead is suspended completely, again this pipe is placed in to magnetic bead separator after simple centrifugal, treat that magnetic bead is adsorbed in tube wall completely, from managing bottom by the complete sucking-off of liquid.With pipettor, get 50 μ lPCR reaction solutions and repeatedly several times the magnetic bead that is adsorbed in tube wall is washed down completely, be transferred in PCR reaction tubes, use quantitative real time PCR Instrument to carry out detection by quantitative.As shown in Figure 2, the ordinate zou in Fig. 2 is fluorescent value to its amplification, and as seen from Figure 2, amplification is all positive.
Claims (10)
1. a biotin labeled specific probe, described biotin labeled specific probe is biotin labeled hepatitis C virus probe, its sequence is: vitamin H-(T)
n-TGGTACTGCCTGATAGGGTGCTTGCG, wherein n is selected from 3～30 number, and preferably n is selected from 5～20 number.
2. viral nucleic acid extracts a reagent, contains biotin labeled specific probe as claimed in claim 1 in described reagent.
3. reagent according to claim 2, it is characterized in that, in described reagent, also contain another kind of biotin labeled specific probe, the biotin labeled specific probe of described another kind is biotin labeled hepatitis B virus probe, and its sequence is: vitamin H-(T)
m-TTGGGTGGCTTTGGGGCATGG, wherein m is selected from 3～30 number.
4. according to reagent described in claim 2 or 3, it is characterized in that, in described reagent, also contain another biotin labeled specific probe, described another biotin labeled specific probe is biotin labeled human immunodeficiency virus probe, and its sequence is: vitamin H-(T)
q-CCAGGCCAGATGAGAGAACCAAGGG, wherein q is selected from 3～30 number.
5. according to the reagent described in any one in claim 2～4, it is characterized in that, described reagent comprises the magnetic bead containing specific probe, and the described magnetic bead containing specific probe is for being combined and obtaining with described biotin labeled specific probe through the magnetic bead of Streptavidin modification.
6. reagent according to claim 5, is characterized in that, also comprises cell pyrolysis liquid in described reagent.
7. viral nucleic acid extracts a test kit, comprises that the viral nucleic acid as described in any one in claim 2～6 extracts reagent, also comprises elutriant and/or washings.
8. reagent or the application of test kit as claimed in claim 7 in blood screening as described in any one in claim 2～6.
9. a biotin labeled specific probe, described biotin labeled specific probe is biotin labeled hepatitis B virus probe, its sequence is: vitamin H-(T)
m-TTGGGTGGCTTTGGGGCATGG, wherein m is selected from 3～30 number.
10. a biotin labeled specific probe, described biotin labeled specific probe is the human immunodeficiency virus probe of thing element mark, its sequence is: vitamin H-(T)
q-CCAGGCCAGATGAGAGAACCAAGGG, wherein q is selected from 3～30 number.
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| WO2016154992A1 (en) * | 2015-04-02 | 2016-10-06 | 武汉格蓝丽富科技有限公司 | Controllable method for extracting nucleic acids |
| CN107022543A (en) * | 2017-05-25 | 2017-08-08 | 郑州市第六人民医院 | One kind extracts hbv nucleic acid with magnetic bead, extracts reagent, extracting method, quantitative detection kit |
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