CN103695419B - A kind of Viral nucleic acid extraction reagent - Google Patents
A kind of Viral nucleic acid extraction reagent Download PDFInfo
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- CN103695419B CN103695419B CN201310745655.7A CN201310745655A CN103695419B CN 103695419 B CN103695419 B CN 103695419B CN 201310745655 A CN201310745655 A CN 201310745655A CN 103695419 B CN103695419 B CN 103695419B
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- nucleic acid
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- biotin labeled
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 47
- 230000003612 virological effect Effects 0.000 title claims abstract description 31
- 238000000605 extraction Methods 0.000 title claims abstract description 30
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 122
- 239000000523 sample Substances 0.000 claims abstract description 109
- 239000011616 biotin Substances 0.000 claims abstract description 61
- 229960002685 biotin Drugs 0.000 claims abstract description 61
- 235000020958 biotin Nutrition 0.000 claims abstract description 61
- 241000700721 Hepatitis B virus Species 0.000 claims abstract description 18
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- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
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- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
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- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Present invention firstly provides a kind of biotin labeled specific probe, described biotin labeled specific probe is biotin labeled hepatitis C virus probe, and its sequence is: biotin (T)nTGGTACTGCCTGATAGGGTGCTTGCG, wherein n is the number selected from 3～30, and preferably n is the number selected from 5～20.The present invention correspondingly provides a kind of Viral nucleic acid extraction reagent, comprises above-mentioned biotin labeled specific probe in described reagent.The present invention also provides for a kind of biotin labeled hepatitis B virus probe and one biotin labeled HIV (human immunodeficiency virus) probe.Use a kind of Viral nucleic acid extraction reagent comprising above-mentioned 3 kinds of biotin labeled specific probes, be useful for this 3 kinds of viral blood screenings.
Description
Technical field
The present invention relates to a kind of Viral nucleic acid extraction reagent and corresponding Viral nucleic acid extraction reagent box, and this virus core
The biotin labeled specific probe in reagent is extracted in acid.
Background technology
The extraction and purification technology of nucleic acid is a basic fundamental of Biochemistry and Molecular Biology.Along with molecular biosciences
Learning a skill and be widely used in biology, medical science and association area thereof, the extraction and purification technology of nucleic acid is also further developed.
Nucleic acid in cell always together with various protein bound.The extraction of nucleic acid is primarily referred to as nucleic acid and albumen
The biomacromolecule materials such as matter, polysaccharide, fat separate.The extracting method of most of nucleic acid, the most all includes that cell cracks, and removes
The protein being combined with nucleic acid and the biomacromolecule such as polysaccharide, lipid, remove other unwanted nucleic acid molecules, precipitate nucleic acids,
Remove the impurity such as salt, organic solvent, the step such as purification of nucleic acid.
First, cell cracking can pass through the method realizations such as mechanism, chemical action, enzyme effect.Concrete, mechanism
The physical disruption method such as including hypotonic lysis, ultrasonic degradation, microwave cracking, freezing-thawing and cracking and grain breakage, these method machines
Tool power makes cell breakage, but mechanical force also can cause nucleic acid chain break, thus is not suitable for dividing of high molecular long nucleic acid
From.Chemical action is under certain pH environment and Denaturing, cell rupture, and protein denaturation precipitation, nucleic acid is released to
Aqueous phase, Denaturing can by heating, add surfactant (SDS, Triton X-100, Tween20, NP-40, CTAB,
Sar-cosyl, Chelex-100 etc.) or strong ionic agent (guanidinium isothiocyanate, guanidine hydrochloride, creatine guanidine etc.) and obtain.And enzyme effect
Mainly by adding lysozyme or protease (E.C. 3.4.21.64, plant rennet or pronase) so that cell rupture, nucleic acid
Release.
Secondly, ferment treatment refers to during nucleic acid extraction, can make unwanted mass degradation by adding suitable enzyme,
It is beneficial to the isolation and purification of nucleic acid, can degrade egg as added protease (E.C. 3.4.21.64 or pronase) in lysate
White matter, inactivation nuclease (DNase and RNase), DNase and RNase is also used for removing unwanted nucleic acid.
It addition, the separation of nucleic acid and purification: the high electric charge phosphate backbones of nucleic acid make its than protein, polysaccharide, fat etc. its
Allogene macromolecular substances is more hydrophilic, according to the difference of they physicochemical properties, by selective precipitation, chromatography, density gradient
The method such as centrifugal can be by separate nucleic acid, purification, and the conventional method of existing isolated and purified nucleic acid has: (1) phenol extraction/sedimentation method: will
Centrifugation after cell lysis separates containing the aqueous phase of nucleic acid, adds isopyknic phenol: chloroform: isoamyl alcohol (25: 24: 1 volume) mixed liquor,
Biphase through vortex oscillation mixing (be applicable to separate small-molecular-weight nucleic acid) or simple reverse mixing (is applicable to separate high molecular core
Acid) centrifugation afterwards, then by nucleinate organic solvent deposit, by precipitation concentration nucleic acid, change nucleic acid and dissolve buffer
Kind and remove some impurity molecule；(2) density－gradient centrifuga－tion method: double-stranded DNA, single stranded DNA, RNA and protein have not
Same density, thus the pure sample product zone of different densities can be formed through density gradient centrifugation, this method is applicable to a large amount of sample of nucleic acid
Preparation；(3) chromatography, chromatography is to utilize the difference of some physicochemical property of different material and the method for separating and analyzing set up,
The method such as including adsorption chromatography, affinity chromatograph, ion-exchange chromatography, under certain ionic environment, nucleic acid can be by optionally
It is adsorbed onto tripoli, silica gel or glass surface and separates with other biological molecule；Other selective absorption method, with modified
Or coated magnetic bead is as solid phase carrier, magnetic bead can be by magnetic field separation without centrifugal, and the nucleic acid being bound to solid phase carrier can
With low salt buffer or water elution.
Paramagnetic particle method is the method for extracting nucleic acid the most just grown up, and its reagents series does not contains the toxicity such as chloroform, phenol
Big organic solvent, the Nucleic acid quality of extraction purification is good, yield is high, and extraction step is simpler, it is not necessary to centrifugal, it is easy to accomplish from
Dynamicization, these advantages and feature make its method extracting nucleic acid have the development trend substituting other methods all.
Magnetic bead is to be had certain magnetic and special by magnetic grain and the various Material claddings containing activity functional groups
The particle of surface texture.In biomagnetism separates, magnetic bead surfaces active group is a very important factor.Currently used
More magnetic bead surfaces aglucon have monoclonal antibody (Monoclonal antibody), Streptavidin (Streptavidin),
Oligonucleotide (Oligonucleotides), and with silylating reagent at magnetic microsphere surface introducing amino (-NH2), hydroxyl
Base (-OH), sulfydryl (-SH), carboxyl (-COOH), sulfonic group (SO3-) etc., on this basis, polysaccharide compound also can be introduced
(such as cellulose, agarose), biotin, antibiotin etc..
Domestic existing method based on magnetic bead extraction nucleic acid is applied in Clinical detection and nucleic acid blood screening, these magnetic beads
Method reagent all comprises following shortcoming: 1) nucleic acid extraction process steps is many, and sample process is the longest, and RNA exposes for a long time easily by RNA
Enzyme enzymolysis, reduces the sensitivity of reagent.2) conventional paramagnetic particle method extracts and needs heating, and solution causes Aerosol Pollution, causes and treats
These negative and positive of sample regardless of.3) conventional paramagnetic particle method does not has labelling specific oligonucleotide chain, it is impossible to specific enriching virus nucleic acid,
Magnetic bead is easily coated by human genome nucleic acid, reduces the ability of magnetic bead viral adsorption nucleic acid, reduces the sensitivity of paramagnetic particle method.
Summary of the invention
Present invention firstly provides a kind of biotin labeled specific probe, described biotin labeled specific probe is
Biotin labeled hepatitis C virus probe, its sequence is: biotin-(T)n-TGGTACTGCCTGATAGGGTGCTTGCG,
Wherein n is the number selected from 3～30, and preferably n is the number selected from 5～20.
Skilled person will appreciate that, specific probe sequence of the present invention may each be its 5 ' end by multiple T
It is connected with biotin, it is also possible to be that its 3 ' end is connected with biotin by multiple T；Therefore, in sequence of the present invention multiple T with described
Connection between specific probe sequence all represents connection that multiple T and probe 5 ' hold and the connection that multiple T holds with probe 3 '.
The present invention also provides for a kind of Viral nucleic acid extraction reagent, containing the most biotin labeled in described reagent
Specific probe.Be marked with the specific oligonucleotide sequences of biotin by its sequence conservation can with specificity with treat sample
Viral nucleic acid hybridization in Ben, its biotin moiety can be combined with the magnetic bead that marked streptomycin again, thus reach specifically
Separate the effect of purpose nucleic acid.Comparing other non-specific method, the Viral nucleic acid extraction reagent using the present invention to provide is carried out
Nucleic acid extraction, the nucleic acid product purity of gained is higher and nucleic acid extraction is in hgher efficiency.
In a kind of specific embodiment, possibly together with another kind of biotin labeled specific probe in described reagent,
The biotin labeled specific probe of described another kind is biotin labeled hepatitis B virus probe, and its sequence is: biological
Element-(T)m-TTGGGTGGCTTTGGGGCATGG, wherein m is the number selected from 3～30.
According to the present invention, in another kind of specific embodiment, possibly together with another biotin labeling in described reagent
Specific probe, another biotin labeled specific probe described be biotin labeled HIV (human immunodeficiency virus) visit
Pin, its sequence is: biotin-(T)q-CCAGGCCAGATGAGAGAACCAAGGG, wherein q is the number selected from 3～30.At this
In bright a kind of concrete reagent, described reagent comprises biotin labeled hepatitis B virus probe, biotin labeled third type
Hepatitis virus probe and biotin labeled HIV (human immunodeficiency virus) probe.Its object is to solve existing hepatitis B virus
The defect that DNA, HCV RNA, HIV (human immunodeficiency virus) nucleic acid RNA extract, it is provided that a kind of method is easy, detection spirit
Sensitivity is high, can be used for the Viral nucleic acid extraction reagent of automatization, applies this reagent above-mentioned three kinds of viral nucleic acids can be accomplished spy
The opposite sex is extracted, it is adaptable to nucleic acid one pipe of blood screening project extracts.
In the present invention, it is preferred to, described reagent includes the magnetic bead containing specific probe, described containing specific probe
Magnetic bead is that the magnetic bead modified through Streptavidin is combined with described biotin labeled specific probe and to obtain.People in the art
Member is knowable, and the biotin in described biotin labeled specific probe in the present invention is modified through Streptavidin with described
Magnetic bead in Streptavidin combine correspondingly, but divide because multiple Streptavidin can be combined with on same magnetic bead
Son, therefore, same magnetic bead can connect identical biotin labeled specific probe, it is also possible to connect different biologies
The specific probe of element labelling, the most same magnetic bead connects and has biotin labeled hepatitis C virus probe and biotin
The hepatitis B virus probe of labelling.In a kind of specific embodiment, described reagent also includes cell pyrolysis liquid.Ability
Field technique personnel are easy to understand, and the cell pyrolysis liquid in the present invention can be mixed to form with the described magnetic bead containing specific probe
A kind of solution, the most described Viral nucleic acid extraction reagent；Can also be described cell pyrolysis liquid and the described magnetic containing specific probe
Pearl solution is packed sale respectively and uses.
The present invention also provides for a kind of Viral nucleic acid extraction reagent box, including Viral nucleic acid extraction reagent as above, also
Including eluent and/or cleaning mixture.Described cleaning mixture separates for being rinsed by the impurity on the described magnetic bead containing specific probe,
And described eluent reacts for PCR for being eluted from the described magnetic bead containing specific probe by target nucleotide.?
In a specific embodiment of the present invention, specific probe, biotin and Streptavidin after eluting are both connected to magnetic bead
On, and stem from the target nucleotide in sample to be tested and enter in eluent, it is used further to downstream experiment.The experiment of described downstream refers to inspection
Survey, clone, sequence analysis, PCR amplification, molecule hybridization and cDNA synthesis etc..
The present invention also provides for a kind of reagent as above or test kit application in blood screening as above.
The present invention also provides for a kind of biotin labeled specific probe, and described biotin labeled specific probe is made a living
The hepatitis B virus probe of thing element labelling, its sequence is: biotin-(T)m-TTGGGTGGCTTTGGGGCATGG, wherein m is
Selected from the number of 3～30.
The present invention also provides for another kind of biotin labeled specific probe, and described biotin labeled specific probe is
The HIV (human immunodeficiency virus) probe of thing element labelling, its sequence is: biotin-(T)q-
CCAGGCCAGATGAGAGAACCAAGGG, wherein q is the number selected from 3～30.
Same, the biotin labeled specific probe of above two can corresponding two-strain nucleic acid extracting reagent.Its
Middle a kind of Viral nucleic acid extraction reagent includes that the magnetic bead containing specific probe, the described magnetic bead containing specific probe are through strepto-
The magnetic bead that Avidin is modified is combined with biotin labeled hepatitis B virus probe and obtains；Another kind of Viral nucleic acid extraction reagent
In also include the magnetic bead containing specific probe, the described magnetic bead containing specific probe be through Streptavidin modify magnetic bead and life
The HIV (human immunodeficiency virus) probe of thing element labelling combines and obtains.
The method using the present invention is extracted the nucleic acid obtained and be cannot be only used for corresponding three kinds of viral nucleic acid blood source examinations, also
Can be applicable in the detection in Gene Mutation of these three virus, gene type detection and gene sequencing.
In the present invention, the magnetic bead containing specific probe and the specificity virus nucleic acid hybridization in sample to be tested, and human body
The DNA of self, protein, rRNA, tRNA and other impurity are not adsorbed, and adsorbing the nucleic acid in magnetic bead surfaces can directly make
For the template of PCR amplification, or add eluent and make absorption be released in eluent again at the target nucleotide molecule of magnetic bead surfaces
Test for downstream.
Accompanying drawing explanation
Fig. 1 extracts the augmentation detection result of the various hypotype of HCV obtained for the reagent provided in embodiment 3；
Fig. 2 extracts, for the reagent provided in embodiment 4, HIV-1 type viral nucleic acid (50IU/ml) duplicate detection 30 obtained
Secondary amplification.
Detailed description of the invention
Following example for doing more detailed description to the present invention, but the present invention is not limited to following embodiment.
Embodiment 1
The present embodiment provides a kind of Viral nucleic acid extraction reagent box, described test kit comprises viral nucleic acid and extracts examination
Agent, described reagent comprises three kinds with biotin labeled specific probe, the most biotin labeled hepatitis B virus probe,
Biotin labeled hepatitis C virus probe and biotin labeled HIV (human immunodeficiency virus) probe.Concrete, described examination
Agent box comprises following several component:
1. the magnetic bead containing specific probe: the described magnetic bead containing specific probe comprises following three kinds:
Magnetic bead containing HBV capture probe: magnetic bead-Streptavidin-biotin-TTTTTTTTTTTTTT-
TTGGGTGGCTTTGGGGCATGG；
Magnetic bead containing HCV capture probe: magnetic bead-Streptavidin-biotin-TTTTTTTTTTTTTT-
TGGTACTGCCTGATAGGGTGCTTGCG；
Magnetic bead containing HIV-I capture probe: magnetic bead-Streptavidin-biotin-TTTTTTTTTTTTTT-
CCAGGCCAGATGAGAGAACCAAGGG。
Heretofore described Streptavidin MagneSphere can be purchased, it is also possible to synthesizes voluntarily.Such as described Streptavidin magnetic
Pearl is with γ Fe2O3And Fe3O4Homogeneous, superparamagnetic, single diversity poly microsphere of magnetic material synthesis are magnetic source, each
Microsphere is coated one layer of poly material, is polymerized by emulsion or diffuse-aggregate method prepares the magnetic high score carrying carboxylate
Sub-microsphere；Through poly material (for example, polystyrene) core particle surface one layer of Magnet of cover that carboxylate is modified, streptomycin
It covalently bind in again on this surface；Described Streptavidin MagneSphere such as dilutes and is saved in PBS.
Biotin labeled specific probe of the present invention obtains for customization, described biotin labeled specific probe
Such as it is saved in TE solution.
The described Streptavidin MagneSphere being purchased is combined with the described biotin labeled specific probe of customization, preparation
Obtain the described magnetic bead containing specific probe.In a specific embodiment, every mg Streptavidin MagneSphere and 50pmol
Biotin labeled specific probe combines, 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.
The magnetic bead containing specific probe that the present invention provides can be placed in 4 DEG C or-20 DEG C of preservations, and convenient transport, for
Property strong, adsorption efficiency is high, can exclusively extract and purification HBV/HCV/HIV-I viral nucleic acid from testing sample.
2. cell pyrolysis liquid: described cell pyrolysis liquid is the solution containing surfactant, preferably containing 0.01%～10%
The solution of SDS, LLS, Chelex-100, Triton X-100, Tween20 or NP-40.
3. cleaning mixture: described cleaning mixture is for example, by 0.1% to 2%(volume ratio) Triton X-100 and 100mmol/L
～the solution of NaCl, KCl or LiCl composition of 2mol/L.
4. eluent: described eluent is the solution that pH value is 8.0 being made up of 10mM Tris-HCl and 1mM EDTA.
Embodiment 2
The present embodiment provides HBV/HCV/HIV-I virus for extracting in sample to be tested of the test kit described in embodiment 1
Method and the gained viral nucleic acid of (for HBV DNA in the present embodiment) carry out fluorescence quantitative PCR detection and result thereof.
First, the sample to be tested of the present invention is: Products in China calibrating institute (Zhong Jian institute) hepatitis B virus (HBV) core
The concentration of acid cut amount standardization is respectively 5.0 × 106IU/ml、5.0×104IU/ml and 5.0 × 1023 kinds of IU/ml dense
The HBV DNA positive serum of degree.
The step of nucleic acid extraction and detection is as follows:
1) cell cracking: added by sample to be tested in centrifuge tube, adds cell pyrolysis liquid cell lysis to discharge nucleic acid；
2) nucleic acid absorption: add the magnetic bead containing specific probe, HBV/HCV/HIV-I nucleic acid (the present embodiment in solution
Sample to be tested in only containing HBV-DNA) adsorbed by specific probe and be connected on magnetic bead, and protein, other is non-specific
Sequence and impurity are not adsorbed；Being placed on magnetic separator by centrifuge tube, separated rear magnetic bead is adsorbed in centrifugal tube wall completely；
Wherein, the capture probe with biotin can be combined in magnetic bead surfaces well together with the specific nucleic acid molecules being extracted
On；
3) go the removal of impurity: add cleaning mixture, surface adsorption had nucleic acid and is attached to the magnetic bead of centrifugal tube wall and washs,
Impurity on magnetic bead is rinsed and separates；
4) nucleic acid reclaims: adsorbing the nucleic acid in magnetic bead surfaces can be directly as the template of PCR amplification；Or addition eluent
It is heated to the Tm value temperature of capture probe, makes absorption enter in eluent, under being used further in the nucleic acid molecules desorbing of magnetic bead surfaces
Trip experiment.
5) fluorescence quantitative PCR detection: the above-mentioned solution containing viral nucleic acid afforded is transferred in PCR reaction tube, fortune
Detection by quantitative is carried out with quantitative real time PCR Instrument.
To totally 3 concentration (wherein 5.0 × 10 in above-mentioned sample to be tested6IU/ml、5.0×104IU/ml and 5.0 × 102IU/
The sample to be tested of ml is respectively labeled as 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 coefficient of variation (CV) in table 1, the coefficient of variation of Ct value all < 0.6%, the concentration coefficient of variation all < 13%, say
The isolated and purified viral nucleic acid of method of the bright utilization present invention reproducible, even different people's operations, also can obtain consistent
Result.Thus ensure that stablizing of subsequent experimental, it is to avoid difference that manual operation causes and mistake.
Embodiment 3
The present embodiment provides HBV/HCV/HIV-I virus for extracting in sample to be tested of the test kit described in embodiment 1
Method and the gained viral nucleic acid of (for HCV RNA in the present embodiment) carry out fluorescence quantitative PCR detection and result thereof.
The sample to be tested of the present invention is: the srea careHCV typing reference material (totally 20 kinds) after diluting 10 times.The present invention
Two kinds of contradistinction systems of middle use (Abbott Laboratories' RealTime HCV m2000 detecting system, Roche AmpliPrep/COBAS TaqMan
Test detecting system) and the test kit of the present invention detect the various hypotypes of HCV in sample to be tested serum.
The extraction of the present invention and detection method: add 200 μ l samples to be tested in 1.5ml centrifuge tube, be subsequently added 600 μ
LRNA extracting solution 1(cell pyrolysis liquid and the mixed liquor of the magnetic bead containing specific probe): described RNA extracting solution 1 is by 0.5%
(W/V) magnetic bead that LLS, the GuScN of Triton X-100,1mol/L of 2% (V/V), 200 μ g/ml Streptavidins are modified and
50 μ l biotin labeled specific probe solution (total amount 1pmol) mixes；After simple being centrifuged, add 100 μ l RNA carry
Taking liquid 2, described RNA extracting solution 2 is by Tris(pH8.0 ± 0.2 of 200mmol/L) and 200mmol/LNaCl form；Concussion mixing
10s, room temperature is the most centrifugal after placing 10min.It is placed on magnetic separator, after separating 5min, treats that magnetic bead is adsorbed in pipe completely
Wall, slowly by supernatant sucking-off bottom pipe；Adding 600 μ l cleaning mixture, described cleaning mixture is by the Triton X-of 0.2% (V/V)
The KCl composition of 100 and 200mmol/L, concussion mixing makes magnetic bead suspend completely, again this pipe is placed in Beads enrichment after being simply centrifuged
Device, treats that magnetic bead is adsorbed in tube wall completely, by complete for liquid sucking-off bottom pipe.50 μ lPCR reactant liquors are taken the most several with pipettor
Secondary the magnetic bead being adsorbed in tube wall is washed down completely, be transferred in PCR reaction tube, use quantitative real time PCR Instrument to carry out detection by quantitative.
Wherein, PCR response procedures is shown in Table 2.
Table 2
The testing result using the detection of above two contradistinction system and the inspection using the said method in the present embodiment to obtain
Survey result to be all shown in Table 3.
Table 3
Corresponding with the last string of table 3, Fig. 1 extracts, for the reagent provided in the present embodiment, the various hypotype of HCV obtained
The augmentation detection result figure of (20 kinds), the vertical coordinate in Fig. 1 is fluorescent value.
Embodiment 4
The present embodiment provides HBV/HCV/HIV-I virus for extracting in sample to be tested of the test kit described in embodiment 1
Method and the gained viral nucleic acid of (for HIV-1RNA in the present embodiment) carry out fluorescence quantitative PCR detection and result thereof.
The sample to be tested of the present invention is: National Institute for Food and Drugs Control HIV-1 type nucleic acid blood screening reagent state of purchase
Family's reference material sensitivity reference material 3.79 × 104IU/ml is diluted to 50IU/ml, is packed as 30 pipes and is used for 30 repetitions in fact
Test.
The extraction of the present invention and detection method: add 200 μ l samples to be tested in 1.5ml centrifuge tube, be subsequently added 600 μ
LRNA extracting solution 1(cell pyrolysis liquid and the mixed liquor of the magnetic bead containing specific probe): described RNA extracting solution 1 is by 0.5%
(W/V) magnetic bead that LLS, the GuScN of Triton X-100,1mol/L of 2% (V/V), 200 μ g/ml Streptavidins are modified and
50 μ l biotin labeled specific probe solution (total amount 1pmol) mixes；After simple being centrifuged, add 100 μ l RNA carry
Taking liquid 2, described RNA extracting solution 2 is by Tris(pH8.0 ± 0.2 of 200mmol/L) and 200mmol/LNaCl form；Concussion mixing
10s, room temperature is the most centrifugal after placing 10min.It is placed on magnetic separator, after separating 5min, treats that magnetic bead is adsorbed in pipe completely
Wall, slowly by supernatant sucking-off bottom pipe；Adding 600 μ l cleaning mixture, described cleaning mixture is by the Triton X-of 0.2% (V/V)
The KCl composition of 100 and 200mmol/L, concussion mixing makes magnetic bead suspend completely, again this pipe is placed in Beads enrichment after being simply centrifuged
Device, treats that magnetic bead is adsorbed in tube wall completely, by complete for liquid sucking-off bottom pipe.50 μ lPCR reactant liquors are taken the most several with pipettor
Secondary the magnetic bead being adsorbed in tube wall is washed down completely, be transferred in PCR reaction tube, use quantitative real time PCR Instrument to carry out detection by quantitative.
Its amplification is as in figure 2 it is shown, the vertical coordinate in Fig. 2 is fluorescent value, as it is clear from fig. 2 that all positives of amplification.
Claims (6)
1. a Viral nucleic acid extraction reagent, it is biotin labeled for containing biotin labeled specific probe in described reagent
Hepatitis C virus probe, its sequence is: biotin-(T)n-TGGTACTGCCTGATAGGGTGCTTGCG, wherein n is selected from 3
～the number of 30；Possibly together with another kind of biotin labeled specific probe in described reagent, described another kind is biotin labeled
Specific probe is biotin labeled hepatitis B virus probe, and its sequence is: biotin-(T)m-
TTGGGTGGCTTTGGGGCATGG, wherein m is the number selected from 3～30.
Reagent the most according to claim 1, it is characterised in that biotin labeled special possibly together with another in described reagent
Property probe, another biotin labeled specific probe described is biotin labeled HIV (human immunodeficiency virus) probe, its
Sequence is: biotin-(T)q-CCAGGCCAGATGAGAGAACCAAGGG, wherein q is the number selected from 3～30.
Reagent the most according to claim 1 and 2, it is characterised in that described reagent includes the magnetic bead containing specific probe,
The described magnetic bead containing specific probe is that the magnetic bead modified through Streptavidin is tied with described biotin labeled specific probe
Conjunction obtains.
Reagent the most according to claim 3, it is characterised in that also include cell pyrolysis liquid in described reagent.
Reagent the most according to claim 1 and 2, it is characterised in that described n is the number selected from 5～20.
6. a Viral nucleic acid extraction reagent box, extracts examination including the viral nucleic acid as described in any one in Claims 1 to 5
Agent, also includes eluent and/or cleaning mixture.
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| CN104974999A (en) * | 2015-04-02 | 2015-10-14 | 武汉格蓝丽富科技有限公司 | Controllable nucleic acid extraction method |
| 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 |
| CN117844986A (en) * | 2024-02-04 | 2024-04-09 | 广州维伯鑫生物科技有限公司 | Nucleic acid composition, kit and method for detecting abalone herpesvirus |
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| CN1661098A (en) * | 2004-12-29 | 2005-08-31 | 深圳益生堂生物企业有限公司 | Chop for diagnosing absence type gene of alpha thalassemia |
| CN101597653A (en) * | 2009-04-22 | 2009-12-09 | 亚能生物技术（深圳）有限公司 | Fluorescence quantitative PCR detection kit for hepatitis B virus |
| CN102816871A (en) * | 2012-09-10 | 2012-12-12 | 广州达健生物科技有限公司 | Fluorescence quantitation PCR (Polymerase Chain Reaction) detection kit for hepatitis c viruses (HCV) |
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| CN1661098A (en) * | 2004-12-29 | 2005-08-31 | 深圳益生堂生物企业有限公司 | Chop for diagnosing absence type gene of alpha thalassemia |
| CN101597653A (en) * | 2009-04-22 | 2009-12-09 | 亚能生物技术（深圳）有限公司 | Fluorescence quantitative PCR detection kit for hepatitis B virus |
| CN102816871A (en) * | 2012-09-10 | 2012-12-12 | 广州达健生物科技有限公司 | Fluorescence quantitation PCR (Polymerase Chain Reaction) detection kit for hepatitis c viruses (HCV) |
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