CN103642919A - Sample conditioning fluid based on denaturation precipitation as well as application thereof and nucleic acid releasing method - Google Patents
Sample conditioning fluid based on denaturation precipitation as well as application thereof and nucleic acid releasing method Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
Abstract
The invention belongs to the biotechnology field, and in particular relates to sample conditioning fluid based on denaturation precipitation as well as application thereof and a nucleic acid releasing method. The sample conditioning fluid comprises the components in percentage by mass: 1-15 percent of PEG400, 0.1-2 percent of surfactant, 1-20 percent of solid phase resin chelex-100, 3-20 percent of polymerase chain reaction (PCR) enhancer, 10-40 mmol/L of potassium hydroxide and 100-400 mmol/L of potassium chloride (KCL), wherein the solvent of the conditioning fluid is sterile water. The sample conditioning fluid is used for removing substances inhibiting PCR amplification based on denaturation precipitation, and the treated product can be subjected to direct amplification so as to guarantee the amplification stability; because of non-selectivity of the treated product based on the principle on the amplification, the fluid is suitable for taq enzymes, chemically-decorated and antibody-decorated hot start enzymes and the like and is particularly suitable for performing amplification on long sections and gene sections with complicated structure.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of sample treatment solution precipitating based on sex change and application thereof, nucleic acid method for releasing.
Background technology
Along with going deep into of clinical gene diagnosis, gene diagnosis has been widely used in the detection of antenatal diagnosis, neonatal screening and heredity metabolize disease.
In the fields such as clinical, pathology, the gene diagnosis of take detects the PCR method that is widely used as molecules of interest.In general, PCR method be by a chain of the chain that dissociates of DNA double chain, be template, relatively the primer of the specific region of DNA chain makes the nucleic acid amplification method of the exponential amplification of target DNA fragment in conjunction with, the synthesis reaction of DNA of carrying out repetition by archaeal dna polymerase.In addition,, except pcr amplification method, also have the nucleic acid amplification methods such as RT-PCR, NASBA, LAMP, 3SR.
Biological specimen based in above-mentioned nucleic acid amplification method, easily be subject to the interference of the inhibitory substance such as oxyphorase, protoheme, Immunoglobulin IgD, these inhibitory substance suppress the amplification of PCR reaction conventionally, so while conventionally detecting with nucleic acid amplification, need to process biological specimen, the nucleic acid substances such as separation and purification DNA or RNA increase again, at present, method in order to separation and purification genomic dna is a lot, most methods all will be passed through white corpuscle separation, lysing cell, extraction nucleic acid and purification of nucleic acid etc., just can obtain high-quality DNA; But also some method only need lysing cell, without extracting, purification of nucleic acid, just can be directly used in pcr amplification, gene sequencing etc.Now some common methods of isolation of genomic DNA are compared as follows: classical organic solvent extraction method, non-organic solvent extraction method, solid phase vector and DNA extraction test kit method.
Above method can be removed the impact of above-mentioned inhibitory substance on amplification, but the method more complicated that biological specimen is processed is time-consuming; Also have at present and adopt the method for By Direct Pyrolysis to increase, conventional laboratory and clinical method for lysis mainly contain: (1) microwave method, boiling method, dilution method, ultrasonic method, freeze-thaw method based on mechanical effect, (2) the pH value degeneration methods based on chemical action, surfactant method (SDS, TritonX-100, Tween20, NP-40, CTAB, sarcosyl, Chelex-100), strong ion method (guanidinium isothiocyanate, Guanidinium hydrochloride, sodium iodide), (3) lysozyme Method based on enzymolysis, RNA extraction based on proteinase K digestion etc.
Now conventional cleavage method is compared as follows:
(1) mechanical effect: comprise the physics cleavage methods such as hypotonic cracking, ultrasonic degradation, microwave cracking, freezing-thawing and cracking and grain breakage.These methods make cytoclasis by mechanical force, but mechanical force also can cause nucleic acid chain break, thereby are not suitable for the separation of high molecular long-chain nucleic acid.Have the nucleic acid fragment length of report ultrasonic degradation method extraction between 500bp-20kb, and the nucleic acid that particle homogenate method is extracted is generally less than 10kb.
(2) chemical action: under certain pH environment and Denaturing, cell rupture, protein denaturation precipitation, nucleic acid is released to water.Or by heating, add tensio-active agent (SDS, TritonX-100, Tween20, NP-40, CTAB, sarcosyl, Chelex-100 etc.) or strong ionic agent (guanidinium isothiocyanate, Guanidinium hydrochloride, creatine guanidine) to obtain.PH environment is provided by the highly basic adding (NaOH) or damping fluid (TE, STE etc.).Under certain pH environment; tensio-active agent or strong ionic agent can make lysis, protein and polysaccharide precipitation; some metal ion chelation agents (EDTA etc.) in damping fluid can chelating to the necessary metal ions M g2+ of nuclease, Ca2+; thereby the activity that suppresses nuclease, protection nucleic acid is not degraded.
(3) enzyme effect: be mainly by adding N,O-Diacetylmuramidase or proteolytic enzyme (Proteinase K, plant protease or pronase) so that cell rupture, nucleic acid discharges.The protein that proteolytic enzyme can also be degraded and is combined with nucleic acid, promotes the separation of nucleic acid.The wherein protein-polysaccharide N-acetyl-glucosamine of N,O-Diacetylmuramidase energy catalysis bacteria cell wall and the β between-acetylmuramic acid residue-(Isosorbide-5-Nitrae) key hydrolysis.Proteinase K energy catalytic hydrolysis multiple polypeptides key, it is at 65 ℃ and still retain enzymic activity while having EDTA, urea (1-4mol/L) and stain remover (0.5%SDS or 1%TritonX-100) to exist, and this is conducive to improve the extraction efficiency to high molecular nucleic acid.
In real work, the sample of employing aforesaid method cracking is not suitable for the amplification of long segment, because its inhibitory substance is not effectively removed.Publication number is that the Chinese invention patent of 101712954A is mentioned the quick release reagent of a kind of nucleic acid and method, and the Chinese invention patent that application number is 200910157536.3 discloses cell nucleic acid releasing agent, the Chinese invention patent that application number is 200310116699.X discloses micro-nucleic acid releasing agent.And non-patent article Evaluation of an Alkaline Lysis Method for the Extraction of DNA from Whole Blood and Forensic Stains for STR Analysis; All mention reagent and the method for nucleic acid being carried out to rapid amplifying, the fragment that amplification method based on above-mentioned is generally all only applicable to below <500bp increases, poor to fragment amplification effect more than >1kb, even without amplification.
Publication number be 101712954A Chinese disclosure of the invention the quick release reagent of a kind of nucleic acid and method, it is a kind of tensio-active agent based on biologically active peptides, the lysing cell in the situation that of high salt, discharge nucleic acid, but reagent itself is not processed the inhibitory substance of biological specimen, after adopting the method to process to the PCR inhibitory substance containing in blood sample, can not effectively increase, therefore the nucleic acid releasing agent based on this method invention is to the release of nucleic acid and follow-up amplification, can not directly carry out, need to coordinate the amplification polysaccharase of himself, expensive, be not easy to apply.
Application number is that 201210319905.6 Chinese invention patent discloses a kind of nucleic acid release reagent and method for releasing, the method is carried out cracking by Proteinase K and alkaline denaturation to cell, discharge nucleic acid DNA, DNA after release need to digest deactivation to Proteinase K, operate more loaded down with trivial details, at least need two methods just can carry out, and only for the amplification of serum, blood plasma etc., be not suitable for the direct amplification of blood.
Non-patent article Evaluation of an Alkaline Lysis Method for the Extraction of DNA from Whole Blood and Forensic Stains for STR Analysis directly increases after adopting alkaline denaturation to process biological specimen, generally be only applicable to STR and analyze, be not suitable for the amplification of long segment.
Also have in addition article to adopt the bonding agent BSA that changes pH value, additional blood sample inhibition, improve the amplification efficiency of Taq enzyme, biological material is directly increased, but these methods all can not be from basic solution biological material to the restraining effect increasing.
Summary of the invention
The invention solves in nucleic acid amplification sample that sample prepares that organic efficiency is low, complicated operation and high in cost of production problem, provide a kind of without centrifugal, without protease K digesting, be applicable to long segment and multiplex PCR sample treatment solution and nucleic acid method for releasing based on sex change precipitation that amplification efficiency is high and stable.
A technical scheme of the present invention is for providing a kind of sample treatment solution based on sex change precipitation, the solid-phase resin chelex-100 of the PEG400 of the component that comprises following mass percent: 1-15%, the tensio-active agent of 0.1-2%, 1-20% and the PCR toughener of 3-20%, described sample treatment solution also comprises: the potassium hydroxide of 10-40mmol/L and the KCL of 100-400mmol/L, described sample treatment solution solvent is sterilized water.
Preferably, the PCR toughener that above-mentioned sample treatment solution comprises the PEG400 of the component of following mass percent: 3-10%, the solid-phase resin chelex-100 of the tensio-active agent of 0.5-1.2%, 5-15% and mass percent are 5-8%, described sample treatment solution also comprises: the potassium hydroxide of 20-30mmol/L and the KCL of 150-300mmol/L, described sample treatment solution solvent is sterilized water.
Preferably, in above-mentioned sample treatment solution, described PCR toughener is glycerine or DMSO, and the amplification tougheners such as trimethyl-glycine, BSA, benzamide type are based on reducing melting temperature (Tm), stability influence to multiplex amplification is larger, especially larger on the homogeneity impact of GC content and the amplification of baroque sample.Described tensio-active agent the best is Teween-20 or TritonX-100, SDS and ionogenic surfactant poor effect in this programme.
Preferably, above-mentioned sample treatment solution comprises the component of following mass percent: 6% PEG400,1.2% Teween-20,5% glycerine and 8% solid-phase resin chelex-100, described sample treatment solution also comprises: the KOH of 30mmol/L, described sample treatment solution solvent is sterilized water.
Another technical scheme of the present invention, for a kind of nucleic acid method for releasing is provided, comprises the steps:
Step 1: prepare sample treatment solution as claimed in claim 1, and particle is suspended evenly;
Step 2: sample is joined in sample treatment solution, and vortex mixes, 56 ℃ of temperature are bathed 10min, obtain processing product.
Preferably, in above-mentioned nucleic acid method for releasing, described sample is blood, serum, blood plasma, dry blood point, tissue, Stomatocyte, legal medical expert's sample or environmental samples.
Preferably, in above-mentioned nucleic acid method for releasing, in described step 2, the volume ratio of sample and sample treatment solution is 1:1.5-7.5.
Preferably, above-mentioned nucleic acid method for releasing comprises: step 1: prepare sample treatment solution as claimed in claim 4;
Step 2: the thalassemic blood sample of 20 μ L is joined in the sample treatment solution of 100 μ L, and vortex mixes, 56 ℃ of temperature are bathed 10min, obtain processing product.
Another technical scheme of the present invention is for providing the application of a kind of above-mentioned sample treatment solution at sample nucleic acid dispose procedure.
Beneficial effect of the present invention: sample treatment solution of the present invention is removed processing based on sex change precipitation to the inhibitory substance of pcr amplification, processing product is added to after PCR reaction solution, the change of PEG400 final concentration causes the change of solution acid-basicity, add the pH environment of PCR system after PCR reaction system without considerable change, the product of processing like this can be directly used in augmentation detection, guarantees the stability of amplification; Processing product based on present principles, to amplification non-selectivity, is applicable to the warm start enzyme of taq enzyme, chemically modified and antibody modification etc., is especially adapted to long segment and baroque gene fragment to increase.
Stable high GC, long-fragment nucleic acid and baroque sample are increased of sample treatment solution energy of the present invention, can be applied to the biological specimen of the different sourcess such as serum, blood plasma, dry blood point, tissue, Stomatocyte, legal medical expert's sample and environmental samples, improve greatly clinical detection efficiency and reduce the source of pollution that operate simultaneously.
Compare with current existing method for extracting nucleic acid, the present invention without centrifugal, without protease K digesting, without the transfer between sample, almost approach automatization, error and the pollution of avoiding manual operation to cause, can be widely used in the diagnostic detection of clinical gene, neonatal examination detection, the detection of mutant and the detection of pathogenic micro-organism etc., it is suitable that the amplification efficiency after sample treatment solution processing blood sample and market test kit extract DNA cloning efficiency.
Accompanying drawing explanation
Fig. 1 is gene of alpha thalassemia detection kit amplification, and wherein 1:QIANGEN Qiamp DNA Blood mini Kit extracts DNA cloning result; 2: sample treatment solution of the present invention is processed product amplification;
Fig. 2 is β-thalassemia detection kit amplification, and wherein 1:QIANGEN Qiamp DNA Blood mini Kit extracts DNA cloning result; 2: sample treatment solution of the present invention is processed product amplification.
Embodiment
By describing technology contents of the present invention, structural attitude in detail, being realized object and effect, below in conjunction with embodiment and coordinate accompanying drawing to be explained in detail.
Polyoxyethylene glycol can be induced macromolecular gathering in the aqueous solution, under the effect of polyoxyethylene glycol, can increase the wetting ability of solution, promote solution O H-ionic concn, the strong basicity environment providing contributes to the quick cracking of cell and to nucleoprotein structure deteriorate, and add in PCR reaction solution when it, its pH value can be reduced to normal level, can amplification not produced and be suppressed.
High saline solution is conducive to the precipitation of nucleoprotein in cell, solid phase resin can be integrated polyvalent metal ion, especially selectivity is integrated divalent ion, than conventional ion exchanger, there is higher metalloform-selective and stronger bonding force, can may affect other allogenic materials (as ferrous ion) that a step is analyzed in conjunction with many, by centrifugal removal solid phase particles resin and impurity, reach the object of separated allogenic material.
The hydroxyl value of polyoxyethylene glycol is larger on the pH impact of solution, and the change by comparative studies PEG200-PEG20000 to solution acid-basicity is as shown in table 1.Polyoxyethylene glycol PEG200 is generally that the change based on solution acid-basicity increases the cracking intensity to cell, conventionally the PEG200 that needs high density, volume ratio is more than 60%, can realize the change to pH value of solution environment, but to PcR amplification inhibition, can not effectively remove, cause the less stable of amplification; And macromolecular polyoxyethylene glycol series is stronger to the adsorption of DNA fragmentation, its hydroxyl value is lower, the acid-basicity of solution is changed little, is mainly by causing that the gathering of DNA molecular strengthens the efficiency of amplification, to study this cracking ability a little less than; The preferred polyoxyethylene glycol of the present invention is PEG400, and larger hydroxyl value can be larger to the pH environment change of solution, increases the cracking intensity to cell, and its strong viscosity is stronger to the adsorption of macromole inhibition, can effectively remove the inhibition of pcr amplification.
Table 1
| The name of an article (specification) | Outward appearance | Fusing point | PH value | Molecular-weight average | Viscosity | Hydroxyl value |
| PEG-200 | Water white transparency | -50±2 | 6.0-8.0 | 190-210 | 22-23 | 534-590 |
| PEG-400 | Water white transparency | 5±2 | 6.0-8.0 | 380-420 | 37-45 | 268-294 |
| PEG-600 | Water white transparency | 20±2 | 6.0-8.0 | 570-630 | 1.9-2.1 | 178-196 |
| PEG-800 | White lotion | 28±2 | 6.0-8.0 | 760-840 | 2.2-2.4 | 133-147 |
| PEG-1000 | White is wax-like | 37±2 | 6.0-8.0 | 950-1050 | 2.4-3.0 | 107-118 |
| PEG-2000 | White solid | 51±2 | 6.0-8.0 | 1800-2200 | 5.0-6.7 | 51-62 |
| PEG-4000 | White solid | 55±2 | 6.0-8.0 | 3600-4400 | 8.0-11 | 25-32 |
| PEG-6000 | White solid | 57±2 | 6.0-8.0 | 5500-7500 | 12-16 | 15-20 |
| PEG-8000 | White solid | 60±2 | 6.0-8.0 | 7500-8500 | 16-18 | 12-15 |
| PEG-10000 | White solid | 61±2 | 6.0-8.0 | 8600-10500 | 19-21 | 8-11 |
| PEG-20000 | White solid | 62±2 | 6.0-8.0 | 18500-2200 | 30-35 | --- |
The present invention is based on sex change precipitation principle, the inhibitory substance such as the inhibition protoheme that biological specimen is existed when the nucleic acid amplification, oxyphorase, IgD are carried out combination, and be adsorbed on solid-phase resin surface by centrifugation, optionally in conjunction with removing the inhibition of amplification, the nucleic acid discharging, in supernatant, is got the amplification that supernatant carries out nucleic acid.Sample treatment solution of the present invention is to utilize under PEG400 (polyoxyethylene glycol) and KOH acting in conjunction, By Direct Pyrolysis cell, and further destroy nucleoprotein structure, in high salt ion situation, selective precipitation albumen, is adsorbed on solid-phase media surface; Nucleic acid is released in supernatant solution, gets supernatant and directly increases; The strong basicity environmental energy forming suppresses the biological enzyme in sample, has avoided sample degraded, chemically modified that traditional method occurs, with protein, the rough sledding such as molecule crosslinked has occurred.
Processing product is added in PCR reaction solution, the change of PEG400 final concentration causes the change of solution acid-basicity, add the pH environment of PCR system after PCR reaction system without considerable change, the product of processing like this can be directly used in augmentation detection, be widely used on the market the amplification of common Taq enzyme, hot-star Taq etc., by PCR synergistic agent such as outer glycerol addings, maintain efficiency and the stability of amplification.
Sample treatment solution of the present invention is comprised of (accounting for the mass percent of sample treatment solution) 1-15%PEG400,10-40mmol/L potassium hydroxide (KOH), 0.1-2% tensio-active agent (Teween-20, TritonX-100), 1-20% solid-phase resin chelex-100,3-20%PCR toughener (glycerine, DMSO) and the KCL of 100-400mmol/L.
Collocation method: with sterilized water, add 1-15%PEG400 and 10-40mmol/L potassium hydroxide (KOH), the KCL that adds 0.1-2% tensio-active agent (Teween-20, TritonX-100) and 100-400mmol/L, then add 3-20%PCR synergistic agent (glycerine, DMSO), finally add 1-20% solid-phase resin chelex-100 to mix and obtain sample treatment solution
Sample treatment solution and the blood sample of configuration add and mix according to a certain percentage, the blood sample that preferred blood sample additional proportion is 20 μ L joins in the lysate of 20-500 μ L, vortex mixes, the blood sample that is more preferably 20 μ L joins in the lysate of 50-150 μ L, after vortex mixes, in 10% ratio, adds and in PCR reaction liquid, carries out follow-up amplification.
The sample process liquid proportional that optimization of the present invention obtains is (accounting for the mass percent of sample treatment solution): the PCR synergistic agent of solid-phase resin chelex-100, the 5-8% of the PEG400 of 3-10%, 20-30mmol/LKOH, 0.5-1.2% tensio-active agent, 5-15% and 150-300mmol/LKCL form.
Embodiment 3
Preferred sample treatment solution of the present invention forms (mass percent that accounts for sample treatment solution) by following component: the PCR synergistic agent of solid-phase resin chelex-100, the 5-8% of 3% PEG400,0.5-1.2% tensio-active agent, 5-15%, 150-300mmol/LKCL and 20mmol/LKOH.Described sample treatment solution solvent is sterilized water.
The use of the above-mentioned sample treatment solution of the present invention:
1, take out sample treatment solution (this reagent is suspension liquid, will fully shake up before use, and particle is suspended), in 1.5mL centrifuge tube, add 300 μ L sample treatment solutions, in the process of absorption sample treatment solution, should frequently with pipettor, blow and beat, particle is suspended evenly.
2, the anticoagulation of 20.0ul is added in sample treatment solution, vortex concussion 25sec, the of short duration centrifugal drop of inside pipe wall of avoiding is residual.
3, put into 56 ℃ of temperature and bathe 10min, the sample room temperature (15-25 ℃) of processing is preserved or 2-8 ℃ of preservation.
4, getting 1-2 μ L processes product and adds in 25 μ LPCR reaction systems and increase.
The use limitation of above-mentioned sample treatment solution: as sample fast processing amplifing reagent, can not be directly used in the extraction of nucleic acid.
The product performance index of above-mentioned sample treatment solution:
1, solution acid-basicity: fixed by PH instrumentation, the pH value of solution is 12.5 ± 0.2.
2, lysis rate: carry out cell counting by microscope, more than 90% cell is all cleaved, strong to the cracking ability of cell.
3, stability: by three batches of reagent are carried out to real-time stabilization experiment, result show product normal temperature (15-25 ℃) preserve 15 months up-to-standard, determine that its effect phase is 12 months;
By uncork stability study, result shows in each normal temperature uncork 2min, uncork every day 1 time, uncork 30 times on result without impact.
Embodiment 4
Pcr amplification for thalassemia sample detects
1, the configuration of sample treatment solution (accounting for the mass percent of sample treatment solution): in 6% the water-soluble solution of PEG400, the KOH, 1.2% Teween-20,5% glycerine, the 200mM KCl that add 30mM, mix the solid-phase resin chelex-100 that then adds 8%.
2, the release of nucleic acid: 20 μ L blood join in the sample treatment solution of 100 μ L, and vortex mixes, enters 56 ℃ of temperature and bathes 10min.
3, process the amplification of product: adopt 2.0 μ L to process product and carry out α-thalassemia, β-thalassemia augmentation detection as template, gene of alpha thalassemia detection kit, β-thalassemia detection kit are Yaneng Biotechnology's product (Guangdong medicine food prison tool is produced and permitted No. 20020515), the sample DNA that adopts therewith German QIANGEN Qiamp DNA Blood mini Kit to extract test kit extraction directly increases, and detects expanding effect; Concrete operations are undertaken by test kit specification sheets.
3, the detection of amplified production: the agarose gel electrophoresis of race 1.5% detects,
The results are shown in Figure shown in 1,2.Fig. 1 is gene of alpha thalassemia detection kit amplification, wherein 1:Qiamp DNA Blood Mini Kit(Germany QIAGEN) extraction DNA cloning result; 2: sample treatment solution of the present invention is processed product amplification; Fig. 2 is β-thalassemia detection kit amplification, wherein 1:Qiamp DNA Blood Mini Kit(Germany QIAGEN) extraction DNA cloning result; 2: sample treatment solution of the present invention is processed product amplification.
Embodiment 5
Augmentation detection for human papillomavirus HPV gene parting detecting reagent
1, the configuration of sample treatment solution (accounting for the mass percent of sample treatment solution): in 10% the water-soluble solution of PEG400, the KOH, 1.2% Teween-20,8% glycerine, the 150mM KCl that add 30mM, mix the solid-phase resin chelex-100 that then adds 15%.
2, sample is prepared: the sample on abundant wash-out Uterine neck bush, and after extracting, abandon Uterine neck bush on tube wall, and whole elutriant specialties, to 1.5ml centrifuge tube, are abandoned to supernatant, retain the pipe cell lump at the end.
3, the release of nucleic acid: 20 μ L blood sample treatment solutions are joined in above-mentioned pipe, and vortex mixes, enters 56 ℃ of temperature and bathes 10min.
4, the amplification of processing product: adopt 2.0 μ L to process products and carry out augmentation detection as template, human papillomavirus HPV gene type adopts the human papillomavirus HPV gene parting detecting reagent of sub-energy biotechnology.
5, hybridization check amplified production, result demonstration, hybridization signal, hybridization specificity and homogeneity show consistent with conventional viral extracting method.
The foregoing is only embodiments of the invention; not thereby limit the scope of the claims of the present invention; every equivalent structure or conversion of equivalent flow process that utilizes specification sheets of the present invention and accompanying drawing content to do; or be directly or indirectly used in other relevant technical fields, be all in like manner included in scope of patent protection of the present invention.
Claims (9)
1. the sample treatment solution based on sex change precipitation, it is characterized in that, the solid-phase resin chelex-100 of the PEG400 of the component that comprises following mass percent: 1-15%, the tensio-active agent of 0.1-2%, 1-20% and the PCR toughener of 3-20%, described sample treatment solution also comprises: the potassium hydroxide of 10-40mmol/L and the KCL of 100-400mmol/L, the solvent of described sample treatment solution is sterilized water.
2. the sample treatment solution based on sex change precipitation according to claim 1, it is characterized in that, the PCR toughener that the solid-phase resin chelex-100 of the PEG400 of the component that comprises following mass percent: 3-10%, the tensio-active agent of 0.5-1.2%, 5-15% and mass percent are 5-8%, described sample treatment solution also comprises: the potassium hydroxide of 20-30mmol/L and the KCL of 150-300mmol/L, the solvent of described sample treatment solution is sterilized water.
3. the sample treatment solution based on sex change precipitation according to claim 1, is characterized in that, described PCR toughener is glycerine or DMSO, and described tensio-active agent is Teween-20 or TritonX-100.
4. the sample treatment solution based on sex change precipitation according to claim 1, it is characterized in that, the component that comprises following mass percent: 6% PEG400,1.2% Teween-20,5% glycerine and 8% solid-phase resin chelex-100, described sample treatment solution also comprises: the KOH of 30mmol/L, described sample treatment solution solvent is sterilized water.
5. a nucleic acid method for releasing, is characterized in that, comprises the steps:
Step 1: prepare sample treatment solution as claimed in claim 1, and the particle of sample treatment solution is suspended evenly;
Step 2: sample is joined in the sample treatment solution of step 1 gained, vortex mixes, 56 ℃ of temperature are bathed 10min, obtain processing product.
6. nucleic acid method for releasing according to claim 5, is characterized in that, described sample is blood, serum, blood plasma, dry blood point, tissue, Stomatocyte, legal medical expert's sample or environmental samples.
7. nucleic acid method for releasing according to claim 5, is characterized in that, in described step 2, the volume ratio of sample and sample treatment solution is 1:1.5-7.5.
8. nucleic acid method for releasing according to claim 5, is characterized in that, comprises the steps:
Step 1: prepare sample treatment solution as claimed in claim 4, with pipettor piping and druming, the particle of sample treatment solution is suspended evenly;
Step 2: the thalassemic blood sample of 20 μ L is joined in the sample treatment solution of 100 μ L step 1 gained, vortex mixes, 56 ℃ of temperature are bathed 10min, obtain processing product.
9. the sample treatment solution based on sex change precipitation as claimed in claim 1 is in the application of sample nucleic acid dispose procedure.
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| CN107586734A (en) * | 2016-07-08 | 2018-01-16 | 潍坊盛泉生物科技有限公司 | It is a kind of to be used for the alkaline decomposed and microbial bacterial agent of soil improvement of string |
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| CN108342463A (en) * | 2018-02-10 | 2018-07-31 | 杭州泰领生物技术有限公司 | A kind of gene detecting kit from nucleic acid purification |
| CN112921071A (en) * | 2021-02-24 | 2021-06-08 | 上海基灵生物科技有限公司 | Cat body fluid sample treatment reagent, detection kit and application |
| CN112921075A (en) * | 2021-03-31 | 2021-06-08 | 武汉友芝友医疗科技股份有限公司 | Hand-taking-free reagent for blood sample fluorescence PCR and application thereof |
| CN114019160A (en) * | 2022-01-05 | 2022-02-08 | 广州科方生物技术股份有限公司 | Release agent for releasing N protein from coronavirus, and preparation method and application thereof |
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