CN102816754A - Extraction method for hepatitis B virus nucleic acid - Google Patents
Extraction method for hepatitis B virus nucleic acid Download PDFInfo
- Publication number
- CN102816754A CN102816754A CN2012102457313A CN201210245731A CN102816754A CN 102816754 A CN102816754 A CN 102816754A CN 2012102457313 A CN2012102457313 A CN 2012102457313A CN 201210245731 A CN201210245731 A CN 201210245731A CN 102816754 A CN102816754 A CN 102816754A
- Authority
- CN
- China
- Prior art keywords
- hepatitis
- magnetic bead
- virus
- nucleic acid
- damping fluid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000700721 Hepatitis B virus Species 0.000 title claims abstract description 50
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 28
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 28
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 28
- 238000000605 extraction Methods 0.000 title claims abstract description 19
- 239000011324 bead Substances 0.000 claims abstract description 63
- 230000005291 magnetic effect Effects 0.000 claims abstract description 63
- 238000000034 method Methods 0.000 claims abstract description 24
- 210000002966 serum Anatomy 0.000 claims abstract description 19
- 239000002245 particle Substances 0.000 claims abstract description 17
- 239000000243 solution Substances 0.000 claims abstract description 11
- 241000700605 Viruses Species 0.000 claims abstract description 8
- 239000012535 impurity Substances 0.000 claims abstract description 8
- 238000013016 damping Methods 0.000 claims description 30
- 239000012530 fluid Substances 0.000 claims description 30
- 230000008878 coupling Effects 0.000 claims description 29
- 238000010168 coupling process Methods 0.000 claims description 29
- 238000005859 coupling reaction Methods 0.000 claims description 29
- 239000006228 supernatant Substances 0.000 claims description 19
- 238000005406 washing Methods 0.000 claims description 16
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical group CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 14
- 230000004913 activation Effects 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 11
- 239000002953 phosphate buffered saline Substances 0.000 claims description 8
- 239000006166 lysate Substances 0.000 claims description 7
- 239000000872 buffer Substances 0.000 claims description 4
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 4
- 230000000694 effects Effects 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 3
- 230000002101 lytic effect Effects 0.000 claims description 3
- 239000013543 active substance Substances 0.000 claims description 2
- CCIVGXIOQKPBKL-UHFFFAOYSA-N ethanesulfonic acid Chemical compound CCS(O)(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-N 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 abstract description 16
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 3
- 230000009089 cytolysis Effects 0.000 abstract description 3
- 239000007853 buffer solution Substances 0.000 abstract description 2
- 239000006148 magnetic separator Substances 0.000 description 10
- 238000012360 testing method Methods 0.000 description 8
- 239000000284 extract Substances 0.000 description 7
- 230000005298 paramagnetic effect Effects 0.000 description 7
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 6
- 239000000427 antigen Substances 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 230000003612 virological effect Effects 0.000 description 5
- 238000005336 cracking Methods 0.000 description 4
- 238000013019 agitation Methods 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 208000002672 hepatitis B Diseases 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 230000008021 deposition Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 206010057248 Cell death Diseases 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 241000700739 Hepadnaviridae Species 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 241000282405 Pongo abelii Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000000274 adsorptive effect Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 244000144992 flock Species 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 description 1
- 238000001216 nucleic acid method Methods 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an extraction method for hepatitis B virus nucleic acid. According to the method, activated magnetic beads are coupled with specific antibodies to hepatitis B viruses so as to allow the coupled magnetic beads to specifically adsorb hepatitis B virus particles in serum containing the hepatitis B virus particles, then a simple buffer solution is used to remove needless impurities, and finally a virus lysis solution is added for lysis of virus cells so as to obtain the hepatitis B virus nucleic acid. Because of the introduction of the magnetic beads, automation becomes simpler, and high specific reaction of antigen-antibodies enables shorter extraction time and strong specificity to be obtained. Reagents used in the method are simple and easily available.
Description
Technical field
The present invention relates to technical field of molecular biology, particularly relate to a kind of method of from serum, extracting hepatitis B virus nucleic acid.
Background technology
Hepatitis B virus (Hepatitis B virus) is called for short hepatitis B virus (HBV).Be a kind of dna virus, belong to Hepadnaviridae (hepadnavividae).Root so far as is known, HBV just only has susceptibility to people and orangutan, initiation hepatitis B disease.Up to the present, according to some relatively more authoritative statisticses, should there be hepatitis B virus (HBV) the infected 1.2 hundred million in China, accounts for 9.09% of China's total population basically, wherein has 1/4 to be the chronic viral hepatitis B patient, approximately is 30,000,000.
The test kit that detects to hepatitis B nucleic acid in the prior art is very many, and the process for extracting of its amplifying nucleic acid is also very many, for example: boiling method, post extraction method, paramagnetic particle method etc.Equal existing problems in above-mentioned these process for extracting, wherein boiling method is easier but impurity that introduce is too much, and can not realize robotization; Post extraction method extraction step is more, though purity is very high, robotization realizes having any problem; Paramagnetic particle method realizes that robotization is very simple, but agents useful for same is expensive.
Summary of the invention
In view of this, the objective of the invention is to propose a kind of method of extracting hepatitis B virus nucleic acid, so that it is process for extracting both can be realized robotization, simple and feasible again.
Based on above-mentioned purpose, the method for extraction hepatitis B virus nucleic acid provided by the invention may further comprise the steps:
(1) activation magnetic bead: adopt acvator activation magnetic bead, and with the magnetic bead after the damping fluid washing activation;
(2) antibody coupling: with the specific antibody coupling of activatory magnetic bead and hepatitis B virus, and with the magnetic bead after the damping fluid washing coupling; Add cancellation liquid,, and wash with damping fluid with the magnetic bead mixing after the coupling; After the washing, magnetic bead is resuspended in the damping fluid the last time, places 2～8 ℃ to preserve down;
(3) specificity combines: the magnetic bead after the coupling is mixed with serum and shakes mixing, make said magnetic bead and hepatitis B virus particle in the serum carry out specificity and combine; After effect for some time, said magnetic bead is washed the enrichment and the removal of impurity with phosphate buffered saline buffer; Add lysate lytic virus cell, make hepatitis B virus nucleic acid be discharged in the solution; Said solution is boiled, get supernatant.
Alternatively, said acvator is 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide.
Alternatively, said lysate is the solution that contains tensio-active agent.
Used damping fluid is 2-(N-morpholine) ethyl sulfonic acid damping fluid when washing in said step (1) and (2) alternatively.
Alternatively, said method is applied to the direct detection of clinical sample.
Saidly can find out from top; The method of extraction hepatitis B virus nucleic acid provided by the invention is through the specific antibody coupling with activatory magnetic bead and hepatitis B virus; Make magnetic bead after coupling viral adsorption particle specifically in having the serum of hepatitis B virus particles; Can redundant impurities be removed with simple damping fluid, add employing virus cracking liquid afterwards with the virocyte cracking, thereby obtain viral nucleic acid.Because the introducing of magnetic bead can make robotization become simply, the reaction of the high specific of antigen-antibody makes extraction time become shorter, high specificity; Used reagent is fairly simple to be easy to get.
Embodiment
For making the object of the invention, technical scheme and advantage clearer, below in conjunction with specific embodiment, to further explain of the present invention.
The principle of work of marked by magnetic bead antibody is: the carboxyl of carboxyl magnetic bead surfaces through the acvator activation after can with antibody coupling.
The principle of work of antigen antibody reaction is: antigen can specificity combine to be based on antigenic determinant (epi-position) and intermolecular complementary structure property of antibody hypervariable region and affinity with antibody.This specific character is to be determined by antigen, antibody molecule sterie configuration.Except that both molecular configuration height complementations, epitope must contact with the antibody hypervariable region closely, and enough bonding forces are just arranged.Antigen antibody reaction can be divided into two stages: the fs is antigen and antibody generation specificity bonded stage, and this elementary reaction is fast, only needs several seconds to several minutes, but visible reaction do not occur; Subordinate phase is visible step of reaction; This stage immune complex is under proper temperature, pH, ionogen and complement influence; Macroscopic reactions such as deposition, aggegation, cytolysis, complement combination mediation occur, this elementary reaction is slow, often needs several minutes to several hours.In serological reaction, more than two stages often can not be strict separately, often receive the influence of reaction conditions (like temperature, pH, ionogen, antigen-antibody ratio etc.).
Therefore, the present invention is based on above-mentioned two kinds of experimental principles, propose a kind of method of extracting hepatitis B virus nucleic acid, said method comprising the steps of:
(1) activation magnetic bead: adopt acvator activation magnetic bead, and with the magnetic bead after the damping fluid washing activation;
(2) antibody coupling: with the specific antibody coupling of activatory magnetic bead and hepatitis B virus, and with the magnetic bead after the damping fluid washing coupling; Add cancellation liquid,, and wash with damping fluid with the magnetic bead mixing after the coupling; After the washing, magnetic bead is resuspended in the damping fluid the last time, places 2～8 ℃ to preserve down;
(3) specificity combines: the magnetic bead after the coupling is mixed with serum and shakes mixing, make said magnetic bead and hepatitis B virus particle in the serum carry out specificity and combine; After effect for some time, said magnetic bead is washed the enrichment and the removal of impurity with phosphate buffered saline buffer; Add lysate lytic virus cell, make hepatitis B virus nucleic acid be discharged in the solution; Said solution is boiled, get supernatant.
As one embodiment of the present of invention, used experiment material and the device as follows:
BioMagPlus carboxyl magnetic bead, 2.5mL, 1.5 μ m, 20mg/mL;
1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDAC), 0.10g;
15mL tip centrifuge tube;
The magnetic separator frame;
0.05M L-one water morpholino b acid buffered soln (MES damping fluid, pH 5.2), 2 * 175mL;
Cancellation liquid (1M Glycine, pH 8.0), 25mL;
Phosphate buffered saline buffer (PBS damping fluid), 125mL.
Wherein, PBS damping fluid: 8g NaCl, 0.2g KCl, 1.44g Na
2HPO
4And 0.24gKH
2PO
4, be dissolved in the 800ml zero(ppm) water, with the pH value to 7.4 of HCl regulator solution, adding distil water is settled to 1L again; Be mixed with the PBS damping fluid of 0.1%Tween 20 at last.
Lysate: 0.1mol/L Tris-HCl, (pH 8.0), 0.1%TritonX-100,0.05%Tween20,1mg/ml Proteinase K, 50mmol/L KCl.
The concrete operations step of said extraction hepatitis B virus nucleic acid method is following:
One, the coupling of magnetic bead and antibody
(1), activation
1, pipette in BioMagPlus carboxyl magnetic bead to the 15mL tip centrifuge tube of 0.5mL (10mg), and be placed on the magnetic separator frame up to supernatant become fully thorough clearly after, carefully move with suction pipe and to abandon supernatant.
2, add the washing of the abundant mixing of 5mL MES damping fluid, with centrifuge tube be placed on the magnetic separator frame up to supernatant become clear after, carefully move with suction pipe and to abandon supernatant.
3, repeating step is 2 three times; After the last washing, magnetic bead is resuspended in the 5mL MES damping fluid.
4, EDAC was placed room temperature 30 minutes from the refrigeration taking-up, accurately take by weighing required EDAC (for example, about 1.6mg EDAC/mg magnetic bead) adding and be equipped with in the centrifuge tube of magnetic bead.
5, thermal agitation shakes up.
6, under the room temperature, centrifuge tube is placed on the rotation mixing appearance priming reaction 30 minutes, in the reaction process, note not allowing the magnetic bead deposition to flock together.
7, with centrifuge tube be placed on the magnetic separator frame up to supernatant become clear after, carefully move with suction pipe and to abandon supernatant.
8, repeating step is 2 four times, obtains the activation magnetic bead.
(2), magnetic bead after the activation and antibody coupling
1, calculating needs the specific anti scale of construction of link coupled hepatitis B virus, and every mg activatory carboxyl magnetic bead can coupling 20～500ug antibody.
2, antibody is added in the 5mL MES damping fluid be mixed with the antibody liquid storage.
3, draw 50 μ L antibody liquid storages in 950 μ L MES damping fluids, be made into the antibody liquid of 1:20.
4, antibody liquid is joined in the centrifuge tube that the activation magnetic bead is housed, the thermal agitation mixing under the room temperature, places centrifuge tube on the rotation mixing appearance and to carry out linked reaction, about 16～24 hours of the time of said linked reaction.
5, with centrifuge tube be placed on the magnetic separator frame up to supernatant become clear after, carefully move with suction pipe and to abandon supernatant.
6, magnetic bead is in resuspended 5mL MES damping fluid, and vibration shakes up, with centrifuge tube be placed on the magnetic separator frame up to supernatant become clear after, carefully move with suction pipe and to abandon supernatant.
7, repeating step 6 once.
8, add 5mL cancellation liquid, vibration shakes up, and under the room temperature centrifuge tube is placed rotation mixing appearance last 30 minute.
9, with centrifuge tube be placed on the magnetic separator frame up to supernatant become clear after, carefully move with suction pipe and to abandon supernatant.
(3), the magnetic bead after washing and the storage coupling
1, in the solution of step (two) gained, add the 5ml damping fluid, and thermal agitation shakes up and washs, with centrifuge tube be placed on the magnetic separator frame up to supernatant become clear after, carefully move with suction pipe and to abandon supernatant.
2, repeating step is 1 three times.
3, after the last washing, magnetic bead is resuspended in the 2ml lavation buffer solution, this moment, the concentration of magnetic bead was about 5mg/mL.
4, place 2～8 ℃ to preserve down.
Wherein, should be noted that:
1, can not use during coupling and contain the amino (for example, tris (Tris)) or the damping fluid of carboxyl (for example acetate, Citrate trianion etc.).
2, the non-coupling adsorptive that produces in the time of can removing coupling through the washing after the coupling.
3, the time of prolongation vibration helps resuspended magnetic bead.
Two, the magnetic bead after the coupling combines and prepares nucleic acid with hepatitis B virus particle specificity in the serum.
1, magnetic bead is hanged, mix, draw 50 μ L magnetic beads and mix with 100 μ L test serums.
2, concussion mixing 30s places 5min, 2 times repeatedly.
3, be placed on the magnetic separator frame magnetic bead absorption 1min.
4, carefully supernatant is siphoned away, as far as possible wash clean.
5, with the PBS damping fluid of 200 μ L that magnetic bead is resuspended, it is even to be mixed.
6, repeating step 3, step 4.
7, repeating step 5, step 6 are twice.
8, add 50 μ L lysates, with the magnetic bead mixing.
9,100 ℃ are boiled 5min.
10, as on the magnetic separator frame, supernatant promptly can be used as template amplification; As need not, preserve down for-20 ℃.
Three, experimental design:
20 parts of serum samples that contain the hepatitis B virus of different concns of picking extract, and with commercial kit the serum before and after extracting are detected the viral nucleic acid number change before and after relatively extracting afterwards.
It is clinical that 20 parts of serum comes from, extract with the test kit of Shanghai section China, amplification quantitatively, test result is seen table 1.
The clinical serum HBV DNA of table 1 section China's kit measurement copy number
| Sequence number | HBV DNA quantitative values | Sequence number | HBV DNA quantitative values |
| 1 | 3.77E+03 | 11 | 6.04E+07 |
| 2 | 3.61E+03 | 12 | 5.28E+07 |
| 3 | 6.25E+07 | 13 | 6.81E+03 |
[0077]?
| 4 | 6.48E+07 | 14 | 6.55E+03 |
| 5 | 1.70E+03 | 15 | 3.69E+06 |
| 6 | 2.65E+03 | 16 | 5.54E+06 |
| 7 | 1.20E+08 | 17 | 2.49E+05 |
| 8 | 1.68E+08 | 18 | 4.40E+04 |
| 9 | 1.70E+05 | 19 | 6.35E+04 |
| 10 | 2.11E+05 | 20 | 5.63E+05 |
Through after the step 2, the serum of crossing through nucleic acid extraction to be measured, and carried out coefficient correction according to the variation of sample volume, test result is seen table 2.
The clinical serum of table 2 section China's kit measurement (after paramagnetic particle method extracts) HBV DNA copy number.
| Sequence number | HBV DNA quantitative values | Sequence number | HBV DNA quantitative values |
| 1 | 1.24E+03 | 11 | 1.03E+06 |
| 2 | 1.12E+03 | 12 | 4.33E+05 |
| 3 | 4.45E+05 | 13 | 1.21E+02 |
| 4 | 7.54E+05 | 14 | 1.39E+02 |
| 5 | 8.7E+01 | 15 | 4.88E+04 |
| 6 | 1.35E+03 | 16 | 3.24E+04 |
| 7 | 2.43E+06 | 17 | 1.23E+03 |
| 8 | 2.73E+06 | 18 | 2.03E+03 |
| 9 | 2.03E+03 | 19 | 3.23E+02 |
| 10 | 3.25E+03 | 20 | 3.22E+03 |
To directly measure with section China test kit with the nucleic acid that paramagnetic particle method extracts, test result is seen table 3.
The clinical serum HBV DNA of table 3 paramagnetic particle method extraction and determination copy number
| Sequence number | HBV DNA quantitative values | Sequence number | HBV DNA quantitative values |
| 1 | 4.86E+03 | 11 | 1.34E+08 |
| 2 | 4.03E+03 | 12 | 7.49E+07 |
| 3 | 5.89E+07 | 13 | 2.05E+04 |
| 4 | 8.33E+07 | 14 | 1.86E+04 |
| 5 | 1.87E+03 | 15 | 4.54E+06 |
| 6 | 5.72E+03 | 16 | 5.67E+06 |
[0084]?
| 7 | 1.09E+08 | 17 | 5.26E+05 |
| 8 | 2.43E+08 | 18 | 5.34E+04 |
| 9 | 8.40E+05 | 19 | 7.65E+04 |
| 10 | 5.34E+05 | 20 | 6.66E+05 |
From above-mentioned three forms: we can draw to draw a conclusion:
Can find out that from the data of table 1 and table 2 recovery that paramagnetic particle method extracts wherein has three parts of organic efficiencies to hang down about 60% slightly, but all can detect smoothly more than 90%.
Can find out that from the data of table 1 and table 3 paramagnetic particle method extracts nucleic acid quality (comprising the removal of quantity and impurity), effect is better than business-like test kit.
Need to prove that the method for extraction hepatitis B virus nucleic acid provided by the invention can be applied to the direct detection of clinical sample.
The method of extraction hepatitis B virus nucleic acid provided by the invention is through the specific antibody coupling with activatory magnetic bead and hepatitis B virus; Make magnetic bead after coupling viral adsorption particle specifically in having the serum of hepatitis B virus particles; Can redundant impurities be removed with simple damping fluid; Add employing virus cracking liquid afterwards with the virocyte cracking, thereby obtain viral nucleic acid.
Because the introducing of magnetic bead can make robotization become simply, the reaction of the high specific of antigen-antibody makes extraction time become shorter, high specificity; Used reagent is fairly simple to be easy to get.
The those of ordinary skill in affiliated field is to be understood that: the above is merely specific embodiment of the present invention; Be not limited to the present invention; All within spirit of the present invention and principle, any modification of being made, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (5)
1. a method of extracting hepatitis B virus nucleic acid is characterized in that, said method comprising the steps of:
(1) activation magnetic bead: adopt acvator activation magnetic bead, and with the magnetic bead after the damping fluid washing activation;
(2) antibody coupling: with the specific antibody coupling of activatory magnetic bead and hepatitis B virus, and with the magnetic bead after the damping fluid washing coupling; Add cancellation liquid,, and wash with damping fluid with the magnetic bead mixing after the coupling; After the washing, magnetic bead is resuspended in the damping fluid the last time, places 2 ~ 8 ℃ to preserve down;
(3) specificity combines: the magnetic bead after the coupling is mixed with serum and shakes mixing, make said magnetic bead and hepatitis B virus particle in the serum carry out specificity and combine; After effect for some time, said magnetic bead is washed the enrichment and the removal of impurity with phosphate buffered saline buffer; Add lysate lytic virus cell, make hepatitis B virus nucleic acid be discharged in the solution; Said solution is boiled, get supernatant.
2. the method for extraction hepatitis B virus nucleic acid according to claim 1 is characterized in that, said acvator is 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide.
3. the method for extraction hepatitis B virus nucleic acid according to claim 1 is characterized in that, said lysate is the solution that contains tensio-active agent.
4. the method for extraction hepatitis B virus nucleic acid according to claim 1 is characterized in that, used damping fluid is 2-(N-morpholine) ethyl sulfonic acid damping fluid when washing in said step (1) and (2).
5. according to the method for any described extraction hepatitis B virus nucleic acid of claim 1 ~ 4, it is characterized in that said method is applied to the direct detection of clinical sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210245731.3A CN102816754B (en) | 2012-07-16 | 2012-07-16 | Extraction method for hepatitis B virus nucleic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210245731.3A CN102816754B (en) | 2012-07-16 | 2012-07-16 | Extraction method for hepatitis B virus nucleic acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102816754A true CN102816754A (en) | 2012-12-12 |
| CN102816754B CN102816754B (en) | 2014-05-14 |
Family
ID=47301204
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210245731.3A Active CN102816754B (en) | 2012-07-16 | 2012-07-16 | Extraction method for hepatitis B virus nucleic acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102816754B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103149350A (en) * | 2013-01-23 | 2013-06-12 | 苏州浩欧博生物医药有限公司 | Method for coupling of nanometer magnetic particles and immune body |
| EP3480601A4 (en) * | 2016-06-30 | 2020-03-18 | Shenzhen Yhlo Biotech Co., Ltd | INHIBIN B CHEMOLUMINESCENT IMMUNOASSAY KIT AND PREPARATION METHOD THEREOF |
| CN111187769A (en) * | 2020-04-01 | 2020-05-22 | 常州天地人和生物科技有限公司 | Method for one-step rapid and efficient extraction of viral nucleic acid |
| CN113322302A (en) * | 2021-06-02 | 2021-08-31 | 重庆医科大学 | Immunocapture molecular detection method for HBV complete virus particles |
| CN115786326A (en) * | 2022-11-04 | 2023-03-14 | 重庆医科大学 | Kit for extracting viral nucleic acid and method for extracting complete viral particle nucleic acid using the kit |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851617A (en) * | 2010-04-28 | 2010-10-06 | 长春市博坤生物科技有限公司 | Magnetic bead virus nucleic acid extraction method that can be used for high-throughput fully automated extraction |
| CN102230938A (en) * | 2011-06-22 | 2011-11-02 | 中国科学院武汉病毒研究所 | Kit and method for detecting influenza A virus based on immune magnetic bead enrichment |
| CN102229925A (en) * | 2011-05-13 | 2011-11-02 | 薛昱 | Enhanced magnetic-bead-based nucleic acid extraction method |
-
2012
- 2012-07-16 CN CN201210245731.3A patent/CN102816754B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851617A (en) * | 2010-04-28 | 2010-10-06 | 长春市博坤生物科技有限公司 | Magnetic bead virus nucleic acid extraction method that can be used for high-throughput fully automated extraction |
| CN102229925A (en) * | 2011-05-13 | 2011-11-02 | 薛昱 | Enhanced magnetic-bead-based nucleic acid extraction method |
| CN102230938A (en) * | 2011-06-22 | 2011-11-02 | 中国科学院武汉病毒研究所 | Kit and method for detecting influenza A virus based on immune magnetic bead enrichment |
Non-Patent Citations (1)
| Title |
|---|
| 陈晓东 等: "核酸提取方法在聚合酶链反应测定乙肝病毒核酸中的评价", 《中华检验医学杂志》, vol. 25, no. 4, 31 December 2002 (2002-12-31), pages 221 - 225 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103149350A (en) * | 2013-01-23 | 2013-06-12 | 苏州浩欧博生物医药有限公司 | Method for coupling of nanometer magnetic particles and immune body |
| EP3480601A4 (en) * | 2016-06-30 | 2020-03-18 | Shenzhen Yhlo Biotech Co., Ltd | INHIBIN B CHEMOLUMINESCENT IMMUNOASSAY KIT AND PREPARATION METHOD THEREOF |
| CN111187769A (en) * | 2020-04-01 | 2020-05-22 | 常州天地人和生物科技有限公司 | Method for one-step rapid and efficient extraction of viral nucleic acid |
| CN111187769B (en) * | 2020-04-01 | 2024-03-08 | 常州天地人和生物科技有限公司 | Method for rapidly and efficiently extracting viral nucleic acid in one step |
| CN113322302A (en) * | 2021-06-02 | 2021-08-31 | 重庆医科大学 | Immunocapture molecular detection method for HBV complete virus particles |
| CN113322302B (en) * | 2021-06-02 | 2023-08-11 | 重庆医科大学 | An immunocapture molecular detection method for intact HBV virus particles |
| CN115786326A (en) * | 2022-11-04 | 2023-03-14 | 重庆医科大学 | Kit for extracting viral nucleic acid and method for extracting complete viral particle nucleic acid using the kit |
| CN115786326B (en) * | 2022-11-04 | 2023-10-03 | 重庆医科大学 | A viral nucleic acid extraction kit and a method for extracting intact viral particle nucleic acid using the kit |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102816754B (en) | 2014-05-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102816754B (en) | Extraction method for hepatitis B virus nucleic acid | |
| US7329491B2 (en) | Methods for isolating nucleic acids | |
| CN114200127B (en) | Hepatitis B virus enrichment fluorescent PCR detection method | |
| CN103215253A (en) | Reagent kit for extracting virus DNA (Deoxyribonucleic Acid) or RNA (Ribonucleic Acid) by using paramagnetic particle method and use method of reagent kit | |
| CN101684138A (en) | Kit using nanometer magnetic beads for purifying nucleic acid | |
| EP1774334B1 (en) | Use of magnetic material to direct isolation of compounds and fractionation of multipart samples | |
| CN102533724A (en) | Cell lysis reagent for extracting and purifying nucleic acids in biological samples | |
| CN112226432B (en) | A kind of magnetic bead method rapid nucleic acid extraction kit and its application | |
| CN104673783A (en) | Kit for extracting DNA/RNA of virus through magnetic bead method and using method | |
| CN105018461B (en) | A kind of rapid screening method of aptamer | |
| CN113322302B (en) | An immunocapture molecular detection method for intact HBV virus particles | |
| CN117778309B (en) | Separation and purification method of animal stem cell exosome and application thereof | |
| CN105120986A (en) | One-step procedure for the purification of nucleic acids | |
| CN106754890B (en) | A kind of extraction kit and extraction method of viral RNA | |
| CN111187769A (en) | Method for one-step rapid and efficient extraction of viral nucleic acid | |
| CN106365207A (en) | Ultra-sensitive magnetic nanometer detection technology for hepatitis C virus (HCV) | |
| JPWO2006123781A1 (en) | Microbial and nucleic acid recovery method using fine particles and kit used therefor | |
| CN117871221A (en) | A method for enriching biological particles in liquid samples by using adsorption particles and ultrafiltration | |
| CN116539875A (en) | Reagent for detecting varicella-zoster virus infection and preparation method thereof | |
| CN108192892A (en) | A kind of kit and extracting method of hydroxyl nanometer magnetic bead method extraction RNA | |
| WO2022263500A1 (en) | Method for isolating non-vesicular mirna | |
| CN114717228A (en) | Method for rapidly extracting genome of sputum sample by using paramagnetic particle method | |
| CN115786326B (en) | A viral nucleic acid extraction kit and a method for extracting intact viral particle nucleic acid using the kit | |
| CN115094161A (en) | Enrichment real-time fluorescence RT-PCR detection method for hepatitis C virus | |
| CN121203016A (en) | A specific antibody and its application in the detection and enrichment of Cryptosporidium oocysts. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |