CN106607013B - A kind of nanometer of multidimensional chromato-graphy nucleic acid extraction medium and preparation method thereof - Google Patents
A kind of nanometer of multidimensional chromato-graphy nucleic acid extraction medium and preparation method thereof Download PDFInfo
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- CN106607013B CN106607013B CN201611230329.2A CN201611230329A CN106607013B CN 106607013 B CN106607013 B CN 106607013B CN 201611230329 A CN201611230329 A CN 201611230329A CN 106607013 B CN106607013 B CN 106607013B
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Abstract
The invention discloses a kind of nanometer of multidimensional chromato-graphy nucleic acid extraction media; extract in its substrate and the infiltration substrate with polymeric carbohydrate; the extract includes the guanidine hydrochloride of first layer, SDS, EDTA mixture; positioned at second layer protective agent; positioned at the guanidine hydrochloride of third layer, SDS, EDTA mixture and protective agent positioned at the 4th layer, guanidine hydrochloride, SDS, EDTA mixture of the first layer are different from the guanidine hydrochloride of third layer, SDS, EDTA mixture content.Substrate can be very good to save the integrality of nucleic acid, prevent the degradation of nucleic acid.
Description
Technical field
The present invention relates to a kind of nucleic acid extraction material more particularly to a kind of nanometer of multidimensional chromato-graphy nucleic acid extraction medium and its systems
Preparation Method.
Background technique
Currently, the principle of nucleic acid extraction is generally the integrality that guarantees nucleic acid primary structure, the dirt of other molecules is excluded
Dye.The problem of nucleic acid extraction should be noted that: simplifying step, shortens extraction time, reduces degradation of the chemical factor to nucleic acid, reduces object
Degradation of the reason factor to nucleic acid: mechanical shear stress and high temperature prevent the biodegrade of nucleic acid, and nucleic acid extraction is roughly divided into 3 big portions
Point: broken, extraction, purifying.
It is broken, extract before, existing technology is with the different various liquid samples of container collection/acquisition, then by liquid
Sample transport to locality (laboratory) is handled, in this way, the condition requirement of liquid sample transport is harsher, and liquid
The body sample exposure aerial time is longer, may result in liquid sample degradation.In addition, liquid sample is in transportational process
In, it is readily volatilized, it is also easy to pollute, denaturation, corruption or still have potential infection risk, be easy to cause virus, spread of germs, infect
To other people.
The extraction of nucleic acid, existing technology are to instill liquid to be detected sample substep on each liquid Extraction medium, are divided more
Step completes broken, extraction, purifying on liquid Extraction medium, and obtained nucleic acid is also liquid.Step is complex, in addition, liquid
State nucleic acid preservation, the requirement of the condition of transport are harsher, and can degrade during liquid nucleic acid preservation, lead to the later period
Testing result is unreliable.
Therefore, it is necessary to the new nucleic acid extraction medium of one kind is provided to solve the above problems.
Summary of the invention
For the above-mentioned problems in the prior art, the present invention provides a kind of receiving for more conveniently, safely extraction nucleic acid
Rice multidimensional chromato-graphy nucleic acid extraction medium and preparation method thereof.
To achieve the above object, the present invention adopts the following technical scheme: a kind of nanometer of multidimensional chromato-graphy nucleic acid extraction medium,
Substrate with polymeric carbohydrate and penetrate into extract in the substrate, the extract include first layer guanidine hydrochloride,
SDS, EDTA mixture are located at second layer protective agent, positioned at the guanidine hydrochloride of third layer, SDS, EDTA mixture and positioned at the 4th layer
Protective agent, the guanidine hydrochloride of the first layer, the guanidine hydrochloride of SDS, EDTA mixture and third layer, SDS, EDTA mixture content
It is different.
Specifically, the protective agent is hydroxyethyl starch.
To achieve the above object, the present invention also adopts the following technical scheme that a kind of nanometer of multidimensional chromato-graphy nucleic acid extraction medium
Preparation method, it is characterised in that: its step are as follows, first step: provide absorption substrate；Second step: in the base
Be added dropwise on material the first buffer until the substrate water suction saturation, wherein the first buffer in parts by weight: hydrochloric acid
Guanidine accounts for the 0.7-10.0% of buffer, and SDS accounts for the 0.7-10.2% of buffer, and EDTA accounts for the 0.06-6.0% of buffer；Third
Step: the substrate that the first buffer is added dropwise is dry；Four steps: be added dropwise to the substrate has 50-100mg/ml
The protective agent of hydroxyethyl starch, the protective agent are covered on the first buffer, are completed until protective agent is absorbed by the substrate
Sedimentation；5th step: dry；6th step: second of buffer being added dropwise on the substrate and is saturated up to substrate absorbs water, and second
Kind of buffer is covered on above-mentioned protective agent, wherein second of buffer in parts by weight: guanidine hydrochloride accounts for buffer
0.5-8.8%, SDS account for the 1-5.5% of buffer, and EDTA accounts for the 0.05-0.06% of buffer；7th step: second will be added dropwise
The substrate of kind buffer is dry；8th step: the substrate is carried out the protection with 50-100mg/ml hydroxyethyl starch is added dropwise
Agent, the protective agent are covered on second of buffer.9th step: dry.
Specifically, weighed with assay balance, and confirm whether substrate water suction is saturated, of poor quality is twice less than when adjacent
0.001g, surface substrate water suction have been saturated.
Specifically, confirmation Extraction medium has dried the method completed are as follows: the substrate is placed measures quality when adjacent twice
Difference is less than 0.001g, and Extraction medium completes drying.
Specifically, in the first step, substrate is dried, drying mode are as follows: it is dry in -5 DEG C -5 DEG C of vacuum,
It is placed under room temperature relative humidity≤30% again and reaches balance.
Compared with prior art, the beneficial effects of the present invention are: increase the buffer of two layers of different specific weight on substrate
After two layers of protective agent, when being acquired to liquid sample, substrate can absorb a certain amount of liquid sample, is solidified, just
In transport, save (room temperature)；And the treatment fluid in matrix can be by virus, clasmatosis in the curing process, and completes nucleic acid
Extraction, purifying, so that cured sample be made to lose infectiousness.Processed substrate can be very good to save the complete of nucleic acid
Property, prevent the degradation of nucleic acid.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiments of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's all other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Nanometer multidimensional chromato-graphy nucleic acid extraction medium of the present invention is the single layer or composite multi-layer of a kind of polymeric carbohydrate
Substrate, the buffer in infiltration substrate and the protective agent (extract for extracting nucleic acid) of plane, substrate can be for such as filter paper
Material.The substrate can be cotton fiber filter paper, and the hydroxyl on filter paper fibre has hydrophily, can adsorb denatured protein.
Cotton fiber filter paper will not be reacted, to keep treatment fluid as inert solid support with the chemical component in buffer
Activity has certain adsorptivity, adsorbable buffer.Wherein the aperture of the substrate is less than 10um, guarantees substrate after treatment
Its aperture can reach nanoscale requirement.It, will be in extract layering and substrate by multiple infiltration mode.The extract packet
Guanidine hydrochloride, SDS, EDTA mixture of first layer are included, second layer protective agent, guanidine hydrochloride, SDS, EDTA positioned at third layer are located at
Mixture and positioned at the 4th layer of protective agent, the guanidine hydrochloride of the first layer, SDS, EDTA mixture and third layer guanidine hydrochloride,
SDS, EDTA mixture content are different.
Specifically, providing the buffer of two kinds of ingredient specific gravity, buffer is to contain guanidine hydrochloride, SDS (dodecyl sulphate
Sodium), the aqueous solution of EDTA (ethylenediamine tetra-acetic acid), the first buffer in parts by weight: guanidine hydrochloride accounts for buffer
0.7-10.0%, SDS account for the 0.7-10.2% of buffer, and EDTA accounts for the 0.06-6.0% of buffer.Second of buffer according to
Parts by weight meter: guanidine hydrochloride accounts for the 0.5-8.8% of buffer, and SDS accounts for the 1-5.5% of buffer, and EDTA accounts for the 0.05- of buffer
0.06%.Wherein the main function of guanidine hydrochloride is to make protein denaturation and inhibitory enzyme activity, and the main function of SDS is that can crack carefully
Born of the same parents make albuminous degeneration, discharge nucleic acid, the main function of EDTA are as follows: enzyme inhibitor.
Additionally, it is provided a kind of protective agent is a kind of solution of hydroxyethyl starch containing 50-100mg/ml.
The multidimensional chromato-graphy nucleic acid extraction medium the preparation method is as follows:
Embodiment one:
First step: the substrate of one square centimeter of absorption is provided, substrate is dried, drying mode are as follows: -5 DEG C -
It is dry in 5 DEG C of vacuum, then be placed under room temperature relative humidity≤30% and reach balance；
Second step: under the conditions of room temperature relative humidity >=80%, the first buffer is added dropwise on substrate until substrate
Water suction saturation.Wherein, 0.7%, the EDTA that 0.7%, the SDS that guanidine hydrochloride accounts for buffer accounts for buffer accounts for the 0.06% of buffer.
Specially each 100ul (drop) buffer, is weighed, and confirm whether substrate water suction is saturated with assay balance, when
Adjacent of poor quality twice less than 0.001g, surface substrate water suction has been saturated.It is as follows:
Described above, after the 4th time plus buffer, substrate has absorbed water saturation the substrate.
Third step: under room temperature relative humidity≤20%, the substrate drying 30min of the first buffer will be added dropwise.
Drying time determines experiment: by the drying disposed within of above-mentioned substrate, recording certain time rear substrate in drying process
The variation of weight, see the table below:
| Substrate sheets state | Quality (g) |
| It is completely wet | 0.4075 |
| (drying is visually observed: substrate surface no liquid residual) after 20min | 0.2172 |
| After 30min | 0.1221 |
| After 40min | 0.1223 |
Experiment condition: room temperature；Relative humidity≤20%
Experiment conclusion: substrate places 30 minutes and 40 minutes basic indifferences of weight of placement, illustrates that substrate is placed 30 minutes
Drying can be completed.
Four steps: substrate is carried out the protective agent with 50mg/ml hydroxyethyl starch is added dropwise, which is covered in the
On a kind of buffer, to reach substrate liquid measure every square centimeter in 30-50ul, protective agent is absorbed by substrate completely completes sedimentation；
Specifically, protective agent, 25ul droplet size, at 1 square centimeter is continuously added dropwise to substrate by atomizing humidifier
After the protective agent for spraying 50ul on substrate, it is sprayed at 0.5M or so above substrate, then rotation sprinkling settles 10min-
15min or so, in 5-10min, the visible liquid of naked eyes is absorbed by substrate completely completes sedimentation.
5th step: in environmental drying 30min of the relative humidity less than 20%.
6th step: under the conditions of room temperature relative humidity >=80%, second of buffer is added dropwise on substrate until substrate
Water suction saturation, second of buffer are covered on above-mentioned protective agent.Wherein, 0.5%, the SDS that guanidine hydrochloride accounts for buffer accounts for buffering
1%, EDTA of liquid accounts for the 0.05% of buffer.
7th step: under room temperature relative humidity≤20%, the substrate drying 30min of second of buffer will be added dropwise.
8th step: be added dropwise to substrate has the protective agent of 50mg/ml hydroxyethyl starch to reach every square centimeter
In 30-50ul, protective agent is covered on second of buffer liquid measure, and protective agent is absorbed by substrate completely completes sedimentation.
9th step: dry.
In this way, being acquired after increasing the buffer and two layers of protective agent of two layers of different specific weight on substrate when to sample
When, substrate can absorb a certain amount of liquid sample, is solidified, and is readily transported, saves (room temperature)；And matrix in the curing process
On treatment fluid can by virus, clasmatosis, so that cured sample be made to lose infectiousness.Processed substrate can be fine
Preservation nucleic acid integrality, prevent the degradation of nucleic acid.
The step of according to embodiment one, for the first buffer, protective agent, second of buffer of different proportion ingredient
It is tested, confirms implementation result.
Embodiment two:
First step: the substrate of one square centimeter of absorption is provided, substrate is dried, drying mode are as follows: 5 DEG C
It is dry in vacuum, then be placed under room temperature relative humidity≤30% and reach balance；
Second step: under the conditions of room temperature relative humidity >=80%, the first buffer is added dropwise on substrate until substrate
Water suction saturation.Wherein, 10.2%, the EDTA that 10.0%, the SDS that guanidine hydrochloride accounts for buffer accounts for buffer accounts for the 6.0% of buffer.
Third step: under room temperature relative humidity≤20%, the substrate drying 30min of the first buffer will be added dropwise.
Four steps: substrate is carried out the protective agent with 100mg/ml hydroxyethyl starch is added dropwise, is often put down with reaching substrate
For square centimetre of liquid measure in 30-50ul, protective agent is absorbed by substrate completely completes sedimentation；
5th step: in the environmental drying 30min of relative humidity≤20%.
6th step: under the conditions of room temperature relative humidity >=80%, second of buffer is added dropwise on substrate until substrate
Water suction saturation.Wherein, 5.5%, the EDTA that 8.8%, the SDS that guanidine hydrochloride accounts for buffer accounts for buffer accounts for the 0.06% of buffer.
7th step: under room temperature relative humidity≤20%, the substrate drying 30min of second of buffer will be added dropwise.
8th step: be added dropwise to substrate has the protective agent of 100mg/ml hydroxyethyl starch to reach every square centimeter
For liquid measure in 30-50ul, protective agent is absorbed by substrate completely completes sedimentation.
9th step: dry.
Embodiment three:
First step: the substrate of one square centimeter of absorption is provided, substrate is dried, drying mode are as follows: 0 DEG C
It is dry in vacuum, then be placed under room temperature relative humidity≤30% and reach balance；
Second step: under the conditions of room temperature relative humidity >=80%, the first buffer is added dropwise on substrate until substrate
Water suction saturation.Wherein, 5.0%, the EDTA that 5.0%, the SDS that guanidine hydrochloride accounts for buffer accounts for buffer accounts for the 3.0% of buffer.
Third step: under room temperature relative humidity≤20%, the substrate drying 30min of the first buffer will be added dropwise.
Four steps: substrate is carried out the protective agent with 100mg/ml hydroxyethyl starch is added dropwise, is often put down with reaching substrate
For square centimetre of liquid measure in 30-50ul, protective agent is absorbed by substrate completely completes sedimentation；
5th step: in the environmental drying 30min of relative humidity≤20%.
6th step: under the conditions of room temperature relative humidity >=80%, second of buffer is added dropwise on substrate until substrate
Water suction saturation.Wherein, 3.2%, the EDTA that 4.0%, the SDS that guanidine hydrochloride accounts for buffer accounts for buffer accounts for the 0.055% of buffer.
7th step: under room temperature relative humidity≤20%, the substrate drying 30min of second of buffer will be added dropwise.
8th step: be added dropwise to substrate has the protective agent of 75mg/ml hydroxyethyl starch to reach every square centimeter
For liquid measure in 30-50ul, protective agent is absorbed by substrate completely completes sedimentation.
9th step: dry.
Example IV:
First step: the substrate of one square centimeter of absorption is provided, substrate is dried, drying mode are as follows: 0 DEG C
It is dry in vacuum, then be placed under room temperature relative humidity≤30% and reach balance；
Second step: under the conditions of room temperature relative humidity >=80%, the first buffer is added dropwise on substrate until substrate
Water suction saturation.Wherein, 1.0%, the EDTA that 1.0%, the SDS that guanidine hydrochloride accounts for buffer accounts for buffer accounts for the 0.1% of buffer.
Third step: under room temperature relative humidity≤20%, the substrate drying 30min of the first buffer will be added dropwise.
Four steps: substrate is carried out the protective agent with 100mg/ml hydroxyethyl starch is added dropwise, is often put down with reaching substrate
For square centimetre of liquid measure in 30-50ul, protective agent is absorbed by substrate completely completes sedimentation；
5th step: in the environmental drying 30min of relative humidity≤20%.
6th step: under the conditions of room temperature relative humidity >=80%, second of buffer is added dropwise on substrate until substrate
Water suction saturation.Wherein, 5.0%, the EDTA that 7.8%, the SDS that guanidine hydrochloride accounts for buffer accounts for buffer accounts for the 0.055% of buffer.
7th step: under room temperature relative humidity≤20%, the substrate drying 30min of second of buffer will be added dropwise.
8th step: be added dropwise to substrate has the protective agent of 100mg/ml hydroxyethyl starch to reach every square centimeter
For liquid measure in 30-50ul, protective agent is absorbed by substrate completely completes sedimentation.
9th step: dry.
Embodiment five:
First step: the substrate of one square centimeter of absorption is provided, substrate is dried, drying mode are as follows: 5 DEG C
It is dry in vacuum, then be placed under room temperature relative humidity≤30% and reach balance；
Second step: under the conditions of room temperature relative humidity >=80%, the first buffer is added dropwise on substrate until substrate
Water suction saturation.Wherein, 10.0%, the EDTA that 9.8%, the SDS that guanidine hydrochloride accounts for buffer accounts for buffer accounts for buffer
0.055%.
Third step: under room temperature relative humidity≤20%, the substrate drying 30min of the first buffer will be added dropwise.
Four steps: substrate is carried out the protective agent with 75mg/ml hydroxyethyl starch is added dropwise, to reach every square of substrate
In 30-50ul, protective agent is absorbed by substrate completely completes sedimentation for centimetre liquid measure；
5th step: in the environmental drying 30min of relative humidity≤20%.
6th step: under the conditions of room temperature relative humidity >=80%, second of buffer is added dropwise on substrate until substrate
Water suction saturation.Wherein, 1.2%, the EDTA that 0.6%, the SDS that guanidine hydrochloride accounts for buffer accounts for buffer accounts for the 0.055% of buffer.
7th step: under room temperature relative humidity≤20%, the substrate drying 30min of second of buffer will be added dropwise.
8th step: be added dropwise to substrate has the protective agent of 75mg/ml hydroxyethyl starch to reach every square centimeter
For liquid measure in 30-50ul, protective agent is absorbed by substrate completely completes sedimentation.
9th step: dry.
By above-mentioned multiple EXPERIMENTAL EXAMPLEs, formation can nanometer multidimensional chromato-graphy nucleic acid extraction medium, which can
To solidify liquid sample, and the treatment fluid in matrix can solve virus, clasmatosis well in the curing process
Certainly the prior art liquid sample transport save it is not convenient present on problem, colleague simplifies the extraction step of nucleic acid.
The above is only the embodiment of the present invention, are not intended to limit the scope of the invention, all to be said using the present invention
Bright book content, which is known, is directly or indirectly used in other relevant technical fields, is included in scope of patent protection of the invention.
Claims (5)
1. a kind of nanometer of multidimensional chromato-graphy nucleic acid extraction medium, substrate and the infiltration substrate with polymeric carbohydrate
Middle extract, it is characterised in that: the substrate is cotton fiber filter paper, and the hydroxyl on filter paper fibre has hydrophily, the extraction
Object includes the guanidine hydrochloride of first layer, SDS, EDTA mixture, is located at second layer protective agent, positioned at the guanidine hydrochloride of third layer, SDS,
EDTA mixture and positioned at the 4th layer of protective agent, the salt of the guanidine hydrochloride of the first layer, SDS, EDTA mixture and third layer
Sour guanidine, SDS, EDTA mixture content are different；The protective agent is hydroxyethyl starch.
2. the preparation method of a kind of nanometer of multidimensional chromato-graphy nucleic acid extraction medium, it is characterised in that: its step are as follows,
First step: providing the substrate of absorption, and the substrate is cotton fiber filter paper, and the hydroxyl on filter paper fibre has hydrophilic
Property；
Second step: being added dropwise the first buffer on the substrate until substrate water suction saturation, wherein the first buffering
Liquid is in parts by weight: guanidine hydrochloride accounts for the 0.7-10.0% of buffer, and SDS accounts for the 0.7-10.2% of buffer, and EDTA accounts for slow
The 0.06-6.0% of fliud flushing；
Third step: the substrate that the first buffer is added dropwise is dry；
Four steps: the substrate is carried out the protective agent with 50-100mg/mL hydroxyethyl starch is added dropwise, which covers
It is placed on the first buffer, until protective agent is absorbed by the substrate completes sedimentation；
5th step: dry；
6th step: being added dropwise second of buffer on the substrate until substrate water suction saturation, second of buffer are covered in
On above-mentioned protective agent, wherein second of buffer in parts by weight: guanidine hydrochloride accounts for the 0.5-8.8% of buffer, and SDS is accounted for
The 1-5.5% of buffer, EDTA account for the 0.05-0.06% of buffer；
7th step: the substrate that second of buffer is added dropwise is dry；
8th step: the substrate is carried out the protective agent with 50-100mg/mL hydroxyethyl starch, the protective agent is added dropwise
It is covered on second of buffer；
9th step: dry.
3. the preparation method of as claimed in claim 2 nanometer of multidimensional chromato-graphy nucleic acid extraction medium, it is characterised in that: with analysis day
It is flat to weigh, and confirm whether substrate water suction is saturated, when it is adjacent it is of poor quality twice be less than 0.001g, show that substrate water suction has been saturated.
4. the preparation method of as claimed in claim 2 nanometer of multidimensional chromato-graphy nucleic acid extraction medium, it is characterised in that: confirmation is extracted
Medium has dried the method completed are as follows: the substrate place when it is adjacent measure twice it is of poor quality be less than 0.001g, Extraction medium is complete
At drying.
5. the preparation method of as claimed in claim 2 nanometer of multidimensional chromato-graphy nucleic acid extraction medium, it is characterised in that: in the first step
In rapid, substrate is dried, drying mode are as follows: it is dry in -5 DEG C -5 DEG C of vacuum, then be placed in room temperature relative humidity≤
Reach balance under 30%.
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