CN109306350A - A kind of nucleic acid releasing agent and nucleic acid on-site method for releasing - Google Patents
A kind of nucleic acid releasing agent and nucleic acid on-site method for releasing Download PDFInfo
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Abstract
The present invention provides a kind of nucleic acid releasing agent, belong to nucleic acid extraction technical field, the nucleic acid releasing agent is liquid preparation, and in the nucleic acid releasing agent, the volumn concentration of nonionic surface active agent is 0.2~2%；The quality volumn concentration of trehalose is 0.01~2%；The mass-volume concentration of Proteinase K is 0.1~5mg/ml；The concentration of potassium chloride is 50~200mmol/L；The concentration of guanidinium isothiocyanate is 20~100mmol/L；The concentration of bovine serum albumin(BSA) is 0.5~10mmol/L；The concentration of ethylenediamine tetra-acetic acid is 1~10mmol/L；The concentration of Tris-HCl is 0.2~5mmol/L, and the nucleic acid releasing agent further includes the water of surplus.The reagent can remove the recycling step of nucleic acid from, avoid nucleic acid from losing or pollute, can be realized the release and detection of site nucleic acid using the reagent.
Description
Technical field
The invention belongs to nucleic acid extraction technical field more particularly to a kind of nucleic acid releasing agent and nucleic acid on-site release sides
Method.
Background technique
Currently, detection of nucleic acids due to its high sensitivity, high specificity, it is easy to operate, required time is short the features such as, extensively
Applied to the nucleic acid amplification technologies such as the multiple fields such as clinical medicine, inspection and quarantine, especially PCR, isothermal duplication.However, nucleic acid
Amplification technique is more sensitive for the interfering substance in clinic or the sample directly obtained, will cause the false negative of testing result,
Therefore it needs to carry out sample nucleic extraction and purification before nucleic acid amplification.But all the time, the extraction and purifying of sample amplifying nucleic acid are whole
Process most time-consuming in a experiment, most cumbersome seriously affects the speed and field application of pattern detection.So far, nucleic acid
Extracting method mainly has boiling method, centrifugal column method and paramagnetic particle method.It is poly- to need repeatedly to change the steps such as pipe centrifugation when boiling method extracts, sample
The present treatment time is long, and there are still a small amount of suppression of amplification substance, has depression effect to following amplification；Centrifugal column method extraction effect
Preferably, but step gathers deficiency that is various, and should being easily lost or pollute there are nucleic acid in the process；Paramagnetic particle method extracts step and gathers simply, returns
High income is easy to automate extraction, but product price is sufficiently expensive, and current application is not extensive.
In conclusion current method for extracting nucleic acid, there are sample requirement amount is big, the time is long, the problem of extraction effect difference.
Summary of the invention
In view of this, the purpose of the present invention is to provide the reagent that one kind can carry out nucleic acid quick release, the reagent
The recycling step that nucleic acid can be removed from avoids nucleic acid from losing or pollute, can be realized the release of site nucleic acid using the reagent
And detection.
To achieve the goals above, the present invention provides following technical schemes:
A kind of nucleic acid releasing agent, the nucleic acid releasing agent are liquid preparation, and the nucleic acid releasing agent is liquid preparation, described
In nucleic acid releasing agent, the volumn concentration of nonionic surface active agent is 0.2~2%；The quality volume basis of trehalose
Content is 0.01~2%；The mass-volume concentration of Proteinase K is 0.1~5mg/ml；The concentration of potassium chloride is 50~200mmol/
L；The concentration of guanidinium isothiocyanate is 20~100mmol/L；The concentration of bovine serum albumin(BSA) is 0.5~10mmol/L；Ethylenediamine tetraacetic
The concentration of acetic acid is 1~10mmol/L；The concentration of Tris-HCl is 0.2~5mmol/L；The nucleic acid releasing agent further includes surplus
Sterile water.
Preferably, in the nucleic acid releasing agent, the volumn concentration of the nonionic surface active agent is 0.5~
1%；The quality volumn concentration of the trehalose is 0.02~0.5%；The mass-volume concentration of the Proteinase K is 0.5
~4.5mg/ml；The concentration of the potassium chloride is 55~100mmol/L；The concentration of the guanidinium isothiocyanate is 25~60mmol/
L；The concentration of the bovine serum albumin(BSA) is 1~9mmol/L；The concentration of the ethylenediamine tetra-acetic acid is 2~6mmol/L；It is described
The concentration of Tris-HCl is 0.3~3mmol/L.
Preferably, in the nucleic acid releasing agent, the volumn concentration of the nonionic surface active agent is 0.3~
1.5%；The quality volumn concentration of the trehalose is 0.1~1%；The mass-volume concentration of the Proteinase K be 0.6~
4mg/ml；The concentration of the potassium chloride is 60~80mmol/L；The concentration of the guanidinium isothiocyanate is 40~60mmol/L；It is described
The concentration of bovine serum albumin(BSA) is 3~10mmol/L；The concentration of the ethylenediamine tetra-acetic acid is 2~5mmol/L；The Tris-
The concentration of HCl is 0.5~4mmol/L.
Preferably, in the nucleic acid releasing agent, the volumn concentration of the nonionic surface active agent is 0.5~
1.4%；The quality volumn concentration of the trehalose is 0.01~0.3%；The mass-volume concentration of the Proteinase K is 1
~5mg/ml；The concentration of the potassium chloride is 100~150mmol/L；The concentration of the guanidinium isothiocyanate is 60~100mmol/
L；The concentration of the bovine serum albumin(BSA) is 5~10mmol/L；The concentration of the ethylenediamine tetra-acetic acid is 2~6mmol/L；It is described
The concentration of Tris-HCl is 0.2~5mmol/L.
Preferably, the pH value of the Tris-HCl is 7.2~7.7.
Preferably, the nonionic surface active agent is CA-630 class nonionic surfactant.
Preferably, the nonionic surface active agent is octyl phenyl-macrogol.
The present invention also provides the nucleic acid releasing agents to carry out nucleic acid on-site method for releasing, comprising the following steps:
1) the nucleic acid releasing agent is mixed with sample and obtains mixed liquor；
2) it after mixed liquor described in step 1) being stood 3~10min, is separated by solid-liquid separation, collects solid phase components and obtain purpose core
Acid.
Preferably, the volume ratio of nucleic acid releasing agent described in step 1) and sample is 1:(1~3).
Preferably, the method for separation of solid and liquid described in step 2) is centrifugation, the revolving speed of the centrifugation is 1500~
2500rpm, the time of the centrifugation are 5~10s.
Beneficial effects of the present invention: the protein structure in nucleic acid releasing agent energy rapid damage sample provided by the invention,
And the nucleic acid amplification inhibiting substances in variability, absorption and precipitating sample, while DNA can be protected.Nucleic acid provided by the invention
Releasing agent, which has, uses safe, easy to operate, the cheap advantage of manufacturing cost.Nucleic acid releasing agent of the present invention can be direct
DNA in throat swab, bacterial cultures, serum, blood plasma or whole blood sample is discharged, without heating, only needs step sample-adding, i.e.,
The release and extraction of achievable DNA.The present invention can be realized the detection of nucleic acids side such as DNA nucleic acid extraction and fluorescent PCR, isothermal duplication
The one-step method of method is used cooperatively, and is fundamentally solved multistep nucleic acid extraction method bring nucleic acid and is lost and pollution problem and nothing
Method really realizes the problem of nucleic acid on-site detection.
Detailed description of the invention
Fig. 1 is the result that nucleic acid and other method for extracting nucleic acid extraction nucleic acid are extracted using nucleic acid releasing agent of the present invention
Compare；
Fig. 2 is glimmering using carrying out after the nucleic acid in nucleic acid releasing agent of the present invention 9 hepatitis B serum samples of extraction
Light PCR amplification curve graph；
Fig. 3 is the fluorescent PCR amplification curve of the Salmonella cultures gradient dilution sample of the method for the invention test
Figure.
Specific embodiment
The present invention provides a kind of nucleic acid releasing agent, the nucleic acid releasing agent is liquid preparation, and the nucleic acid releasing agent is
Liquid preparation, in the nucleic acid releasing agent, the volumn concentration of nonionic surface active agent is 0.2~2%；Trehalose
Quality volumn concentration is 0.01~2%；The mass-volume concentration of Proteinase K is 0.1~5mg/ml；The concentration of potassium chloride is
50~200mmol/L；The concentration of guanidinium isothiocyanate is 20~100mmol/L；The concentration of bovine serum albumin(BSA) be 0.5~
10mmol/L；The concentration of ethylenediamine tetra-acetic acid is 1~10mmol/L；The concentration of Tris-HCl is 0.2~5mmol/L；The core
Sour releasing agent further includes the sterile water of surplus.
In the present invention, the nucleic acid releasing agent includes nonionic surface active agent, and the nonionic surfactant is excellent
It is selected as CA-630 class nonionic surfactant, more preferably octyl phenyl-macrogol.In the present invention, the nonionic
The volumn concentration of type surfactant is 0.2~2%, preferably 0.5~1.5%, more preferably 0.8~1.2%；This hair
The bright source to the nonionic surface active agent does not have particular/special requirement, commonly uses conventional commercial product.In the present invention, institute
It states nonionic surfactant to play the role of destroying cell membrane due to can be good at polarity and non-polar group, by core
Acid is released from envelope protein, while the nonionic surfactant of above-mentioned concentration range will not inhibit subsequent nucleic acid amplification
Reaction.
In the present invention, the nucleic acid releasing agent includes potassium chloride, and the concentration of the potassium chloride is 50~200mmol/L, excellent
It is selected as 60~180mmol/L, more preferably 100~150mmol/L.The present invention does not have special limit to the source of the potassium chloride
Fixed, using conventional commercial potassium chloride, preferably commercially available analysis is pure.In the present invention, the effect of the potassium chloride is low to provide
Salt environment, the potassium chloride of above-mentioned concentration range can increase the solubility of nucleic acid in the solution.
In the present invention, the nucleic acid releasing agent includes guanidinium isothiocyanate, and the concentration of the guanidinium isothiocyanate is 20~
100mmol/L, preferably 30~90mmol/L, more preferably 40~80mmol/L.The present invention carrys out the guanidinium isothiocyanate
Source is not particularly limited, using this field conventional commercial product.Heretofore described guanidinium isothiocyanate is as strength albumen
Matter denaturant, can rapid solubilising protein, cause eucaryotic cell structure broken, nucleoprotein due to being destroyed of its secondary structure, disappear
And separated with nucleic acid rapidly, promote being released effectively for nucleic acid.
In the present invention, the nucleic acid releasing agent includes the bovine serum albumin(BSA), and the concentration of the bovine serum albumin(BSA) is
0.5~10mmol/L, preferably 1~9mmol/L, more preferably 3~7mmol/L.The present invention is to the bovine serum albumin(BSA)
Source is not particularly limited, using conventional commercial product.In the present invention, the bovine serum albumin(BSA) is as a kind of stabilization
Agent can protect nucleic acid, prevent nucleic acid hydrolysis, while can inhibit the object of nucleic acid amplification reaction in conjunction with chelating agent, ferroheme etc.
Matter.
In the present invention, the nucleic acid releasing agent includes ethylenediamine tetra-acetic acid, and the concentration of the ethylenediamine tetra-acetic acid is 1~
10mmol/L, preferably 2~9mmol/L, more preferably 3~8mmol/L.Source of the present invention to the ethylenediamine tetra-acetic acid
It is not particularly limited, using conventional commercial product.In the present invention, the ethylenediamine tetra-acetic acid can effectively inhibit nucleic acid
Enzyme prevents DNA degradation.
In the present invention, the nucleic acid releasing agent includes Tris-HCl, and the concentration of the Tris-HCl is 0.2~5mmol/L,
Preferably 0.3~4.5mmol/L, more preferably 0.5~3mmol/L, the present invention in, the pH value of the Tris-HCl is preferred
It is 7.2~7.7, more preferably 7.5.In the present invention, the effect of the Tris-HCl is to provide alkaline buffer ring for nucleic acid release
Border, the pH value in effective maintenance system.
In the present invention, the nucleic acid releasing agent includes trehalose, and the quality volumn concentration of the trehalose is 0.01
~2%, preferably 0.02~1.9%, more preferably 0.5~1.5%.The present invention does not have special limit to the source of the trehalose
It is fixed, using conventional commercial product.The effect of heretofore described trehalose is the nucleic acid amplification of effective adsorption-condensation precipitating
Mortifier.
In the present invention, the nucleic acid releasing agent includes the Proteinase K, and the mass-volume concentration of the Proteinase K is 0.1
~5mg/ml, preferably 0.5~4.5mg/ml, more preferably 1~4mg/ml.The present invention does not have the source of the Proteinase K
Particular determination, using this field conventional commercial product.Heretofore described Proteinase K can be in higher ph 8.5-9.5
Under the conditions of dissociate nucleic acid binding protein, while Proteinase K does not influence subsequent detection of nucleic acids through high-temperature inactivation.
In the present invention, the nucleic acid releasing agent further includes the water of surplus, and heretofore described water is preferably sterile water.
Heretofore described nonionic surfactant merge with guanidinium isothiocyanate use can quickly, further destroy film
Albumen, the potassium chloride concentration in the present invention can effectively keep the active function of bovine serum albumin(BSA), and trehalose can be further
Nucleic acid amplification mortifier in removal system is not required to heat the influence that mortifier can be effectively reduced to realize.
Heretofore described nucleic acid releasing agent, according to the difference of sample type, can forming with each component of appropriate adjustment,
The following are a kind of optional scheme, in the nucleic acid releasing agent, the volumn concentration of the nonionic surface active agent is
0.5~1%；The quality volumn concentration of the trehalose is 0.02~0.5%；The mass-volume concentration of the Proteinase K
For 0.5~4.5mg/ml；The concentration of the potassium chloride is 55~100mmol/L；The concentration of the guanidinium isothiocyanate be 25~
60mmol/L；The concentration of the bovine serum albumin(BSA) is 1~9mmol/L；The concentration of the ethylenediamine tetra-acetic acid is 2~6mmol/
L；The concentration of the Tris-HCl is 0.3~3mmol/L.
Optional another kind scheme, in the nucleic acid releasing agent, the volume basis of the nonionic surface active agent contains
Amount is 0.3~1.5%；The quality volumn concentration of the trehalose is 0.1~1%；The quality volume of the Proteinase K is dense
Degree is 0.6~4mg/ml；The concentration of the potassium chloride is 60~80mmol/L；The concentration of the guanidinium isothiocyanate be 40~
60mmol/L；The concentration of the bovine serum albumin(BSA) is 3~10mmol/L；The concentration of the ethylenediamine tetra-acetic acid be 2~
5mmol/L；The concentration of the Tris-HCl is 0.5~4mmol/L.
Optional another kind scheme, in the nucleic acid releasing agent, the volume basis of the nonionic surface active agent contains
Amount is 0.5~1.4%；The quality volumn concentration of the trehalose is 0.01~0.3%；The mass body of the Proteinase K
Product concentration is 1~5mg/ml；The concentration of the potassium chloride is 100~150mmol/L；The concentration of the guanidinium isothiocyanate be 60~
100mmol/L；The concentration of the bovine serum albumin(BSA) is 5~10mmol/L；The concentration of the ethylenediamine tetra-acetic acid be 2~
6mmol/L；The concentration of the Tris-HCl is 0.2~5mmol/L.
The present invention also provides a kind of preparation methods of nucleic acid rapid releasing agent, comprising the following steps: by non-ionic surface
Activating agent, potassium chloride, guanidinium isothiocyanate, bovine serum albumin(BSA), ethylenediamine tetra-acetic acid, trehalose, Tris alkali, Proteinase K and water,
Solution after pH value is transferred to 7.2~7.7 with HCl, is filtered degerming up to the nucleic acid releasing agent by mixed configuration solution.
The preparation method of heretofore described nucleic acid releasing agent is simple, meets the requirement for facilitating detection.
The present invention also provides the nucleic acid releasing agents to carry out nucleic acid on-site method for releasing, comprising the following steps: 1) by institute
It states nucleic acid releasing agent and mixes acquisition mixed liquor with sample；2) after mixed liquor described in step 1) being stood 3~10min, solid-liquid point
From collection solid phase components obtain purpose nucleic acid.
The present invention nucleic acid releasing agent is mixed with sample obtain mixed liquor, the sample be bacterial cultures, whole blood,
Blood plasma, serum, throat swab eluent or secretion.The volume ratio of heretofore described nucleic acid releasing agent and sample is preferably 1:(1
~3), more preferably 1:(1.5~2.5).The currently preferred mixing that nucleic acid releasing agent and sample are carried out in PCR pipe, this
The method of mixing described in invention preferably uses to be blown and beaten repeatedly repeatedly in liquid-transfering gun insertion mixed solution.
The present invention stands 3~10min after obtaining the mixed liquor, by the mixed liquor, is separated by solid-liquid separation, and collects solid phase
Component obtains purpose nucleic acid.In the present invention, the time of the standing is preferably 4~7min, more preferably 5min；Institute in the present invention
The method for the separation of solid and liquid stated is centrifugation, and the revolving speed of the centrifugation is preferably 1500~2500rpm, more preferably 1800~
The time of 2200rpm, most preferably 2000rpm, the centrifugation are preferably 5~10s, more preferably 6~9s.In the present invention
The method of the separation of solid and liquid can also be artificial whipping PCR pipe, and the present invention is to the number and dynamics of the whipping without spy
Different to limit, solid phase components, which are sunken to tube bottom, to be advisable.Heretofore described purpose nucleic acid is DNA DNA.
Heretofore described nucleic acid releasing agent and using the nucleic acid releasing agent scene release nucleic acid method solve often
Advise method for extracting nucleic acid recovery of nucleic acid it is low, it is complex for operation step, it is at high cost and can not on-site test the problems such as.Using this
Invention reagent and method, without individually extracting nucleic acid, it is only necessary to after mixing sample to be tested in proportion with the nucleic acid releasing agent
The release for the nucleic acid that can achieve the goal, the step such as extracting, centrifugation and tube without previous methods are handled according to the method for releasing
Suddenly.Operating procedure is largely reduced, reduces nucleic acid loss, increases the sensitivity of detection and reduce costs,
And without the nucleic acid releasing agent for removing sample addition, it is used directly for detection of nucleic acids, will not influence testing result
Stability and sensitivity.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
Embodiment 1
Method of the invention, boiling method, centrifugal column formulation, magnetic bead extraction method is respectively adopted to a Salmonella cultures
DNA extraction is carried out, measures nucleic acid OD value, and carry out electrophoresis detection simultaneously.
The each group distribution ratio of nucleic acid releasing agent described in the present embodiment are as follows: the non-ionic surface of percent by volume 0.6%
Activating agent, the potassium chloride of 100mM, the guanidinium isothiocyanate of 20mM, the bovine serum albumin(BSA) of 1.5mM, 3mM ethylenediamine tetra-acetic acid,
The trehalose of quality percent by volume 0.25%, the Proteinase K of 3mg/ml, 1.5mM Tris.HCl (PH7.5), surplus is nothing
Bacterium water.Extraction step: 10 μ L of nucleic acid releasing agent and 20 μ L of Salmonella cultures sample is drawn respectively, is put into
0.5mLEppendorf centrifuge tube is blown and beaten for several times repeatedly with pipettor, is uniformly mixed, then stands the mixed solution
5min, 2000rpm are centrifuged to obtain purpose nucleic acid.
Boiling method extracts reagent (Shanghai Zhijiang River company) using DNA, is operated to specifications.
Centrifugal column formulation uses bacterial genomes DNA extraction kit (Beijing Tiangeng company), is grasped to specifications
Make.
Magnetic bead extraction method uses bacterial genomes DNA extraction kit (omega company), is operated to specifications.
Four kinds of methods use microplate reader to measure OD260/280 value after extracting, the OD260/280 value of this method is 1.5, boil
Method is 1.6, and centrifugal column formulation is 1.8, and magnetic bead extraction method is 1.9.
Four kinds of methods all carry out RNA electrophoresis after extracting nucleic acid, and applied sample amount is 5 μ L, and electrophoretogram is shown in attached drawing 1, wherein 1 be from
Stem formulation, 2 be boiling method, and 3 be magnetic bead extraction method, and 4 be the method for the present invention, and NTC is blank control.As can be seen from Fig., from
Stem formulation extraction efficiency highest, band is clearest, and boiling method has impurity, the magnetic bead extraction methods such as certain other albumen and goes to clean
Matter effect is preferable, and the method for the present invention removal impurity effect is also preferable, but yield is lower, but for subsequent nucleic acid amplification method
Have no influence.
Embodiment 2
The each group distribution ratio of nucleic acid releasing agent described in the present embodiment are as follows: the non-ionic surface of percent by volume 0.3%
Activating agent, the potassium chloride of 80mM, the guanidinium isothiocyanate of 20mM, the bovine serum albumin(BSA) of 2mM, the ethylenediamine tetra-acetic acid of 5mM, quality
The trehalose of percent by volume 0.5%, the Proteinase K of 0.5mg/ml, 1mM Tris.HCl (PH7.5), surplus is sterile water.
Extraction step: drawing 5 μ L of nucleic acid releasing agent and 5 μ L of hepatitis B sample respectively, be put into 0.5mLEppendorf centrifuge tube, uses
Pipettor is blown and beaten for several times repeatedly, is uniformly mixed, and the mixed solution is then stood 5min, and 2000rpm is centrifuged to obtain target DNA
Nucleic acid.
The present embodiment extracts 4 hepatitis B virus positive serum samples using nucleic acid releasing agent respectively, after extracting nucleic acid, respectively
2 μ L nucleic acid extraction liquid are drawn, hepatitis B fluorescent PCR commercialization detection kit (Shanghai Zhijiang River company) is added to and carries out fluorescence
RT-PCR detection, the results are shown in attached figure 2, the Ct value of 4 hepatitis B virus positive samples be respectively as follows: 31.56,34.89,37.43 and
38.32.4 hepatitis B sample standard deviations have positive amplification curve, illustrate that this method can effectively extract DNA virus nucleic acid.
Embodiment 3
The each group distribution ratio of nucleic acid releasing agent described in the present embodiment are as follows: the non-ionic surface of percent by volume 0.6%
Activating agent, the potassium chloride of 100mM, the guanidinium isothiocyanate of 20mM, the bovine serum albumin(BSA) of 1.5mM, 3mM ethylenediamine tetra-acetic acid,
The trehalose of quality percent by volume 0.25%, the Proteinase K of 3mg/ml, 1.5mM Tris.HCl (PH7.5), surplus is nothing
Bacterium water.Extraction step: nucleic acid releasing agent 10uL and Salmonella cultures sample 20uL is drawn respectively, is put into
0.5mLEppendorf centrifuge tube is blown and beaten for several times repeatedly with pipettor, is uniformly mixed, then stands the mixed solution
5min, 2000rpm are centrifuged to obtain target DNA nucleic acid.
The present embodiment extracts Salmonella cultures gradient dilution sample using nucleic acid releasing agent respectively, after extracting nucleic acid,
2 μ L nucleic acid extraction liquid are drawn respectively, are added to salmonella fluorescent PCR commercialization detection kit (Shanghai Zhijiang River company) and are carried out
Fluorescent PCR detection, the results are shown in attached figure 3, the Ct value of the Salmonella cultures sample of 5 concentration gradients are as follows: 14.21,19.13,
24.35,28 and 32.87.Salmonella gradient sample standard deviation has positive amplification curve, illustrates that this method can be extracted effectively carefully
Bacterium DNA.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of nucleic acid releasing agent, which is characterized in that the nucleic acid releasing agent is liquid preparation, non-in the nucleic acid releasing agent
The volumn concentration of ionic surfactant is 0.2~2%；The quality volumn concentration of trehalose is 0.01~2%；
The mass-volume concentration of Proteinase K is 0.1~5mg/ml；The concentration of potassium chloride is 50~200mmol/L；Guanidinium isothiocyanate it is dense
Degree is 20~100mmol/L；The concentration of bovine serum albumin(BSA) is 0.5~10mmol/L；The concentration of ethylenediamine tetra-acetic acid be 1~
10mmol/L；The concentration of Tris-HCl is 0.2~5mmol/L；The nucleic acid releasing agent further includes the sterile water of surplus.
2. nucleic acid releasing agent according to claim 1, which is characterized in that described non-ionic in the nucleic acid releasing agent
The volumn concentration of surfactant is 0.5~1%；The quality volumn concentration of the trehalose is 0.02~0.5%；
The mass-volume concentration of the Proteinase K is 0.5~4.5mg/ml；The concentration of the potassium chloride is 55~100mmol/L；It is described
The concentration of guanidinium isothiocyanate is 25~60mmol/L；The concentration of the bovine serum albumin(BSA) is 1~9mmol/L；The ethylenediamine
The concentration of tetraacethyl is 2~6mmol/L；The concentration of the Tris-HCl is 0.3~3mmol/L.
3. nucleic acid releasing agent according to claim 1, which is characterized in that described non-ionic in the nucleic acid releasing agent
The volumn concentration of surfactant is 0.3~1.5%；The quality volumn concentration of the trehalose is 0.1~1%；
The mass-volume concentration of the Proteinase K is 0.6~4mg/ml；The concentration of the potassium chloride is 60~80mmol/L；It is described different
The concentration of guanidine thiocyanate is 40~60mmol/L；The concentration of the bovine serum albumin(BSA) is 3~10mmol/L；The ethylenediamine tetraacetic
The concentration of acetic acid is 2~5mmol/L；The concentration of the Tris-HCl is 0.5~4mmol/L.
4. nucleic acid releasing agent according to claim 1, which is characterized in that described non-ionic in the nucleic acid releasing agent
The volumn concentration of surfactant is 0.5~1.4%；The quality volumn concentration of the trehalose be 0.01~
0.3%；The mass-volume concentration of the Proteinase K is 1~5mg/ml；The concentration of the potassium chloride is 100~150mmol/L；
The concentration of the guanidinium isothiocyanate is 60~100mmol/L；The concentration of the bovine serum albumin(BSA) is 5~10mmol/L；It is described
The concentration of ethylenediamine tetra-acetic acid is 2~6mmol/L；The concentration of the Tris-HCl is 0.2~5mmol/L.
5. nucleic acid releasing agent described in any one according to claim 1~4, which is characterized in that the pH value of the Tris-HCl
It is 7.2~7.7.
6. nucleic acid releasing agent according to claim 1, which is characterized in that the nonionic surface active agent is CA-
630 class nonionic surfactants.
7. nucleic acid releasing agent according to claim 6, which is characterized in that the nonionic surface active agent is octyl
Phenyl-macrogol.
8. carrying out nucleic acid on-site method for releasing, including following step using nucleic acid releasing agent described in claim 1~7 any one
It is rapid:
1) the nucleic acid releasing agent is mixed with sample and obtains mixed liquor；
2) it after mixed liquor described in step 1) being stood 3~10min, is separated by solid-liquid separation, collects solid phase components and obtain purpose nucleic acid.
9. according to the method described in claim 8, it is characterized in that, the volume ratio of nucleic acid releasing agent described in step 1) and sample
For 1:(1~3).
10. method according to claim 8 or claim 9, which is characterized in that the method for separation of solid and liquid described in step 2) be from
The heart, the revolving speed of the centrifugation are 1500~2500rpm, and the time of the centrifugation is 5~10s.
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| CN112522370A (en) * | 2020-12-09 | 2021-03-19 | 陕西师范大学 | Nucleic acid hand-free reagent and application and use method thereof in nucleic acid detection |
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