CN105695450A - Magnetic-bead-process-based kit for extracting free DNAs (deoxyribonucleic acids) and application method thereof - Google Patents
Magnetic-bead-process-based kit for extracting free DNAs (deoxyribonucleic acids) and application method thereof Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
Abstract
The invention relates to a magnetic-bead-process-based kit for extracting free DNAs (deoxyribonucleic acids) and an application method thereof. The kit comprises a DNA fixation combination solution, a first magnetic bead suspension, a cleaning solution, a genome DNA removal solution, a second magnetic bead suspension and an elution solution which are respectively packaged independently. The kit can be used for directly extracting free DNAs from the whole blood sample. The object for extracting and purifying free DNAs is widened from the serum or plasma to the whole blood sample, and the free DNA extraction step is extended forward to the blood preliminary treatment, thereby greatly reducing the original steps for extracting free DNAs from blood. Besides, the kit can also be used for extracting and purifying free DNAs from a plasma or serum sample. The kit can be used for removing genome DNA interference. The kit can be used for removing trace genome DNAs mixed in the whole blood, plasma or serum, thereby lowering the subsequent testing interference. Different reagents in the kit are compounded, so that the free DNA extraction efficiency and purity are maximally ensured.
Description
Technical field
The invention belongs to biology field, be specifically related to a kind of test kit extracting dissociative DNA in whole blood based on paramagnetic particle method and using method thereof。
Background technology
DNA is the carrier of hereditary information, it it is one of main molecular biology research object, along with deepening continuously and the fast development of gene sequencing technology of molecular biology research, time needed for gene sequencing and cost rapid decrease, disease is diagnosed the basic fundamental means having become as clinical diagnosis by gene level, is increasingly becoming the important way in the fields such as major disease diagnosis, personalized medicine, fetus genetic disease credit analysis and individual identification based on the accurate medical treatment of gene sequencing。
After normal cell, tumor cell or fetal tissue's cell division, the DNA in cell can be discharged in human body fluid, is free in outside cell, this the part DNA wherein entering into blood is referred to as dissociative DNA in blood, also referred to as plasma DNA, i.e. cellfreeDNA, is called for short cfDNA。CfDNA fragment length is concentrated mainly between 100～240bp, and major part is between 160～200bp。
Being called ctDNA from the DNA of tumour cell division release in blood, for a part of cfDNA, for the tumor patient in different ill stages, ctDNA content and kind are not quite similar, and diagnosing tumor is had major clinical significance by this；For anemia of pregnant woman, the cfDNA coming from infant portion is extracted and is detected the important means being also increasingly becoming anemia of pregnant woman's prenatal diagnosis。But cfDNA content is extremely low, the total cfDNA content of blood is approximately in 10-200ng/ml, wherein anemia of pregnant woman is accounted for about the 10% of the total cfDNA content of blood from the cfDNA of baby cells, for tumor patient, the ctDNA from tumor cell only account for the one thousandth of the total cfDNA content of blood to ten thousand/；Additionally, cfDNA is small pieces segment DNA so that cfDNA extracts extremely difficult。
The extraction of conventional blood cfDNA mainly includes 2 steps: 1) multistage centrifugal obtains blood plasma；2) from blood plasma, extraction obtains cfDNA。The cfDNA of current main flow extracts test kit and is mainly column method, silicone hydroxyl post film used by column method, its aperture is typically in micron level, poor for small pieces segment DNA rejection effect, and need with centrifuge with the use of, in this extraction process, nucleic acid and silicone hydroxyl post film be combined into momentary action, action time is short so that utilize column method extraction purification cfDNA to seem particularly difficult。In recent years, paramagnetic particle method starts to be applied in the extraction work of DNA, in paramagnetic particle method, adopted magnetic bead is general all at Nano grade, have bigger serface, additionally, magnetic bead surfaces is modified with special chemical group, DNA molecular can be formed specific adsorption or desorption at different conditions, the interaction time of magnetic bead and small pieces segment DNA is controlled, add and itself have paramagnetic characteristic, after enrichment DNA very convenient with mother solution lock out operation, these features all make paramagnetic particle method can become cfDNA method of purification more easily。
At present, paramagnetic particle method extracts cfDNA can only carry out mostly in plasma or serum, from whole blood, purify cfDNA still there is multiple difficulty, such as, blood freeze thawing can not must also can obtain being suitable for the blood plasma of extraction cfDNA through complex multistep treatment, needing 3-6 DEG C, 1600g initial centrifugation 8-12min, supernatant 3-6 DEG C again, 16000g just can obtain blood plasma after being centrifuged 8-12min afterwards, operate extremely complex；While it is true, the DNA of the genomic DNA of residual and some larger piece sections can not be removed by serum completely that obtain according to the method described above。Further, wherein can be mixed with the DNA fragmentation of 240-800bp, and fail to remove genomic interference。Although there being prior art can provide a kind of method extracted from serum less than 500bpDNA fragment, but still needing to obtain blood through complicated multistep treatment blood plasma or serum, process is complicated。
It is therefore desirable to develop a kind of more convenient test kit extracting cfDNA from blood plasma, serum or even whole blood and method, instrument for extracting nucleic acid and work station can be coordinated to realize high flux, automation mechanized operation simultaneously。
Summary of the invention
For the deficiency that prior art exists, it is an object of the invention to provide a kind of test kit and using method thereof extracting dissociative DNA based on paramagnetic particle method。The part of dissociative DNA extraction purification is worked forward integration in blood sample treatments step, directly from whole blood, dissociative DNA is carried out adsorption and enrichment, and so as to realize separating with other impurity in blood under the effect of externally-applied magnetic field, utilize specific genomic DNA to remove liquid afterwards to be removed by minigene group DNA, only retain dissociative DNA。
Need blood is carried out could for extracting dissociative DNA after multistage pretreatment obtains serum by traditional for this test kit and extracting method, it is directly extended to from whole blood sample, directly to extract dissociative DNA, the use of DNA secure bond liquid in this process, both ensured that genomic DNA was not degraded, also the dissociative DNA promoting content extremely low is effectively enriched on magnetic bead, is re-released in liquid subsequently under certain conditions again；Genomic DNA removes the specific removing minigene group DNA of liquid, only retains dissociative DNA, it is ensured that free high-purity and integrity, whole simple and convenient, effect stability, and extraction efficiency is high。Additionally, if needed can by genomic DNA output as a by-product。
For achieving the above object, the present invention is achieved through the following technical solutions:
A kind of test kit extracting dissociative DNA based on paramagnetic particle method, it includes the DNA secure bond liquid of independently subpackage, the first bead suspension, and cleanout fluid, genomic DNA remove liquid, the second bead suspension and eluent；
Wherein, described DNA secure bond liquid includes: carbamide, Tris alkali, sodium sulfite, chaotropic salt, chelating agen, detergent and acetate；Described chaotropic salt is selected from guanidine hydrochloride, guanidine thiocyanate, sodium iodide or its combination；Described chelating agen is selected from disodiumedetate, ethylenediamine tetraacetic acid,dipotassium salt or its combination；Described detergent is selected from TritonX-100, Tween-20, NP-40, NaTDC, sodium lauryl sulphate or its combination；Described acetate is selected from potassium acetate, sodium acetate or its combination；
Including the first magnetic bead of Surface coating silicone hydroxyl in described first bead suspension, the particle diameter of described first magnetic bead is 50-300nm；
Described cleanout fluid includes isopropanol, chaotropic salt, chelating agen and detergent；Described chaotropic salt is selected from guanidine hydrochloride, guanidine thiocyanate, sodium iodide or its combination；Described chelating agen is selected from disodiumedetate, ethylenediamine tetraacetic acid,dipotassium salt or its combination；Described detergent is selected from TritonX-100, Tween-20, NP-40, NaTDC, sodium lauryl sulphate or its combination；
Described genome remove liquid include potassium carbonate, sodium acetate, ammonium hydrogen carbonate, disodiumedetate, sodium chloride, sodium sulfate, from saline solution and detergent；Described chaotropic salt is selected from guanidine hydrochloride, guanidine thiocyanate, sodium iodide or its combination；Described detergent is selected from TritonX-100, Tween-20, NP-40, NaTDC, sodium lauryl sulphate or its combination；
Including the second magnetic bead of Surface coating silicone hydroxyl in described second bead suspension, the particle diameter of described second magnetic bead is 100-500nm；
Described eluent is Tris alkali or deionized water。
Preferably, the described test kit extracting dissociative DNA based on paramagnetic particle method, wherein, in described DNA secure bond liquid, urea concentration is 0.05-0.2mol/L, Tris alkali concn is 20-100mmol/L, and concentration of sodium sulfite is 0.1-2wt%, chaotropic salt concentration is 2-8mol/L, chelating agent concentrations is 1-200mmol/L, and the volume fraction of detergent is 0.01-5%, and acetate concentration is 0.05-1mol/L。
Preferably, the described test kit extracting dissociative DNA based on paramagnetic particle method, wherein, in described first magnetic bead dispersion liquid, the concentration of the first magnetic bead is 25-65mg/mL。
Preferably, the described test kit extracting dissociative DNA based on paramagnetic particle method, wherein, in described cleanout fluid, the volume fraction of isopropanol is 45-65%, and the concentration of chaotropic salt is 0.1-0.5mol/L, the concentration of chelating agen is 10-100mmol/L, and the volume fraction of detergent is 0.01-1%。
Preferably, the described test kit extracting dissociative DNA based on paramagnetic particle method, wherein, it is 0.1-1mol/L that described genomic DNA removes concentration of potassium carbonate in liquid, and sodium acetate concentration is 0.05-1mol/L, the concentration of ammonium hydrogen carbonate is 0.5mmol/L, disodiumedetate concentration is 10-100mmol/L, and the concentration of sodium chloride is 3mol/L, and the concentration of sodium sulfate is 1mol/L, chaotropic salt concentration is 1-6mol/L, and the volume fraction of detergent is 0.01-3%。
Preferably, the described test kit extracting dissociative DNA based on paramagnetic particle method, wherein, in described second bead suspension, the concentration of the second magnetic bead is 10-40mg/mL。
Preferably, the described test kit extracting dissociative DNA based on paramagnetic particle method, wherein, when described eluent is Tris alkali, Tris paper mill wastewater is 0.9-1.1mmol/L。
A kind of using method of the test kit extracting dissociative DNA based on paramagnetic particle method as according to any one of such scheme, wherein, including:
Step 1) blood sample is transferred in EP pipe (centrifuge tube), add DNA secure bond liquid, be mixed；
Step 2) add the first bead suspension, vibration mixing to EP pipe；
Step 3) EP pipe is placed in magnetic frame device, Magnetic Isolation, after magnetic bead is adsorbed completely, topple over the liquid discarded in EP pipe；
Step 4) in EP pipe, add cleanout fluid, vibration EP pipe makes the first magnetic bead suspend uniformly, stands, Magnetic Isolation, after magnetic bead is adsorbed completely, topples over and discard cleanout fluid；
Step 5) in EP pipe, add genomic DNA removal liquid, vibration EP pipe makes the first magnetic bead suspend uniformly, and Magnetic Isolation stands, and Magnetic Isolation, after magnetic bead is adsorbed completely, Aspirate supernatant is transferred in new EP pipe；
Step 6) in the new EP pipe equipped with supernatant, add the second bead suspension, mixing, vibration combines；Stand, Magnetic Isolation, after magnetic bead is adsorbed completely, topple over the liquid discarded in EP pipe；
Step 7) in new EP pipe, add alcoholic solution, vibration EP pipe makes the second magnetic bead suspend uniformly, stands, Magnetic Isolation, removes supernatant；
Step 8) new EP pipe put in drying baker dry, remove ethanol, new EP pipe adds eluent, vibration EP pipe makes the second magnetic bead suspend uniformly, standing, Magnetic Isolation removes the second magnetic bead, namely contains target dissociative DNA in the eluent in new EP pipe。
The invention has the beneficial effects as follows:
1, this case can realize directly extracting from whole blood sample dissociative DNA；Being expanded the object of extraction purification dissociative DNA to whole blood sample by serum or blood plasma, the early stage that the step extracting dissociative DNA extends forwardly to blood processes, and greatly reduces blood and originally extracted the step of dissociative DNA；It addition, this test kit equally can also compatible extraction purification dissociative DNA from blood plasma or serum sample。
2, genomic DNA interference can be removed。The minigene group DNA being mixed in whole blood, blood plasma or serum can be removed, reduce follow-up test interference；The cooperation of the different reagent in this test kit so that extraction efficiency and the purity of dissociative DNA are farthest ensured。
3, simple and convenient, required matching requirements is low, obtains the eluting dissociative DNA started to last from initial blood, and whole process does not need centrifugally operated, saves time and cost。
4, provide a kind of efficiently, the dissociative DNA extraction scheme of low cost, it is possible to coordinate instrument for extracting nucleic acid and work station to complete automatization and high flux operation。
Accompanying drawing explanation
Fig. 1 adopts the sepharose electrophoresis of the dissociative DNA that test kit extracts from blood sample in embodiment 1 to identify glue figure。(in Fig. 1, magnetic bead 1 represents the magnetic bead in the first bead suspension, and magnetic bead 2 represents the magnetic bead in the second bead suspension)
Fig. 2 adopts the sepharose electrophoresis of the dissociative DNA that test kit extracts from blood sample in embodiment 2 to identify glue figure。
Detailed description of the invention
Below in conjunction with accompanying drawing, the present invention is described in further detail, to make those skilled in the art can implement according to this with reference to description word。
Embodiment 1:
A kind of paramagnetic particle method dissociative DNA in blood extracts test kit and includes independently subpackage:
DNA secure bond liquid: 0.05mol/L carbamide, Tris alkali concn is 100mmol/L, concentration of sodium sulfite is 1%, 1mol/L guanidine hydrochloride, 3mol/L guanidine thiocyanate, 2mol/L sodium perchlorate, 100mmol/L disodiumedetate, 0.01%TritonX-100,1%NP-40,0.5% sodium lauryl sulphate, 0.1mol/L potassium acetate and 0.1mol/L sodium acetate。
First bead suspension: magnetic bead concentration is the magnetic bead suspension of 40mg/mL, and magnetic bead kernel is ferroso-ferric oxide (Fe3O4), Surface coating silicone hydroxyl layer。Concrete compound method: weigh 4g magnetic bead, joins in 100mL deionized water, and ultrasonic disperse is uniform。
Cleanout fluid: 60% isopropanol, 50mmol/L disodiumedetate, 0.2mol/L guanidine hydrochloride and 0.5mol/L guanidinium isothiocyanate, 0.01% sodium lauryl sulphate。
Genomic DNA removes liquid: 1mol/L potassium carbonate, 1mol/L sodium acetate, 1mol/L ammonium hydrogen carbonate, 50mmol/L disodiumedetate, 3mol/L sodium chloride, 1mol/L sodium sulfate, 3mol/L guanidine hydrochloride, 1mol/L guanidinium isothiocyanate, 0.01%NP-40 and 1% sodium lauryl sulphate。
Second bead suspension: magnetic bead concentration is the magnetic bead suspension of 20mg/mL, and magnetic bead kernel is ferroso-ferric oxide (Fe3O4), Surface coating silicone hydroxyl layer。Concrete compound method: weigh 2g magnetic bead, joins in 100mL deionized water, and ultrasonic disperse is uniform。
The Tris aqueous alkali of eluent: 1mM, pH value is 8.0。
The present embodiment is for 2 parts of analog samples (in blood, the artificial concentration that adds is 80ng/mL200bp sheet segment DNA simulation dissociative DNA), utilize simple magnetic means, in EP pipe, dissociative DNA therein (i.e. 200bp sheet segment DNA therein) is extracted in the operation of manual method, comprises the steps:
Step 1) draw 0.2mL blood sample in 1.5mlEP pipe, add DNA secure bond liquid 200ul and E.C. 3.4.21.64 20ul, mixing of turning upside down, 58 DEG C of temperature bath 10min；
Described Proteinase K Solution is 20mg/ml。
Step 2) to step 1) in EP pipe add bead suspension 150ul and isopropanol 400ul, reverse mixing for several times, and is vibrated in conjunction with 5min；
Step 3) EP pipe is placed in simple and easy magnetic frame device, Magnetic Isolation 30～90s, after magnetic bead is adsorbed completely, topples over and discards other blood impurities；
Step 4) in step 3) after EP pipe in add cleaning buffer solution 800ul, shake 10～60s, Magnetic Isolation 30～90s with have gentle hands, after magnetic bead is adsorbed completely, topple over and discard cleanout fluid；
Step 5) to step 4) remove liquid 200ul equipped with the EP pipe of magnetic bead adds genome, shake 10～30s with have gentle hands, stand 5-10min；Magnetic Isolation 30～90s, after magnetic bead is adsorbed completely, draws supernatant and transfers in new EP pipe；
Step 6) to step 5) in equipped with the EP pipe of supernatant adds bead suspension 2100ul and isopropanol 200ul, for several times, vibration is in conjunction with 5～10min in reverse mixing；
Step 7) clean: in EP pipe, add 700uL80% ethanol water, 10～60s, Magnetic Isolation will be shaken with have gentle hands, and remove supernatant, by 80% ethanol water repeated washing once。
Step 8) except alcohol and eluting: the vacuum drying oven that the above-mentioned pipe containing magnetic bead puts into 45 DEG C is dried 5～10min, remove ethanol, EP pipe adds the eluent of 20～200uL, 1min is vibrated under 800～1200rpm, Magnetic Isolation removes magnetic bead, namely obtains target 200bp sheet segment DNA in EP pipe eluent。
Embodiment 2:
The present embodiment uses test kit identical in embodiment 1, carries out the parallel laboratory test of many points of samples in 48 orifice plates simultaneously。
The present embodiment is simultaneous for 8 parts of analog samples (the artificial concentration that adds is 100ng/mL200bp sheet segment DNA simulation dissociative DNA in blood) and extracts, and comprises the steps:
Step 1): in 48 orifice plates, add 200 μ L sample of blood, 20 μ L protease k solution and 200 μ LDNA secure bond liquid, parallel 8 parts of samples, vortex oscillation fully mixing in 30 seconds with multichannel automatic pipettor, be placed in water-bath and digest 10 minutes。The frequency of vibration of vortex mixed instrument is set as 1150rpm；
Step 2): take out 48 orifice plates, with multichannel automatic pipettor to after 8 sample wells are separately added into 50 μ L the first magnetic bead dispersion liquids, 400 μ L isopropanols, 48 orifice plates are placed in vortex mixed instrument vibrate 5 minutes, 48 orifice plates are placed on 48 orifice plate magnetic sheet framves and stand 20 seconds to magnetic bead absorption completely, 48 orifice plates are kept to be fixed on magnetic sheet frame, direct back-off abandoning supernatant, then back-off shape state is placed in absorbent paper, thoroughly removes supernatant residual；
Step 3): 48 orifice plates are taken off from magnetic sheet base, in sample well, it is separately added into 800 μ L cleanout fluid with multichannel automatic pipettor, afterwards 48 orifice plates are placed on vortex mixed instrument and vibrate 60 seconds, 48 orifice plates are reinstalled on magnetic sheet frame and stand 20 seconds to magnetic bead absorption completely, 48 orifice plates are kept to be fixed on magnetic sheet frame, direct back-off abandoning supernatant, then back-off shape is placed in absorbent paper, thorough abandoning supernatant；
Step 4): 48 orifice plates are taken off from magnetic sheet base, in sample well, it is separately added into 200 μ L genomic DNAs removal liquid with multichannel automatic pipettor, afterwards 48 orifice plates are placed on vortex mixed instrument and vibrate 60 seconds, after 48 orifice plates are stood 5 minutes, it is placed on magnetic frame to magnetic bead absorption completely, keeping 48 orifice plates to be fixed on magnetic sheet frame, Aspirate supernatant, to 48 orifice plates one piece new, thoroughly discards magnetic bead；
Step 5): with multichannel automatic pipettor to after 8 sample wells on 48 new orifice plates are separately added into 100 μ L the second magnetic bead dispersion liquids, 200 μ L isopropanols, 48 orifice plates are placed in vortex mixed instrument vibrate 5 minutes, 48 orifice plates are placed on 48 orifice plate magnetic sheet framves and stand 20 seconds to magnetic bead absorption completely, 48 orifice plates are kept to be fixed on magnetic sheet frame, direct back-off abandoning supernatant, then back-off shape state is placed in absorbent paper, thoroughly removes supernatant residual；
Step 6): 48 orifice plates are taken off from magnetic sheet base, in sample well, it is separately added into 700 μ L80% ethanol with multichannel automatic pipettor, afterwards 48 orifice plates are placed on vortex mixed instrument and vibrate 25 seconds, 48 orifice plates are reinstalled on magnetic sheet frame and stand 20 seconds to magnetic bead absorption completely, 48 orifice plates are kept to be fixed on magnetic sheet frame, direct back-off abandoning supernatant, then back-off shape is placed in absorbent paper, thorough abandoning supernatant；And reuse step 6) once；
Step 7): it is placed in 45 DEG C of vacuum drying ovens vacuum drying 3 minutes by cleaning complete 48 orifice plates together with magnetic sheet frame, take out 48 orifice plates and take off from magnetic sheet base, in sample well, it is separately added into 100 μ L eluents with multichannel automatic pipettor, 48 orifice plates are placed on vortex mixed instrument and vibrate after 1 minute, 48 orifice plates are put in 55 DEG C of water-baths and stand 5 minutes；
Step 8): 48 orifice plates are positioned on magnetic sheet frame and stand 20 seconds, 48 orifice plates are kept to be fixed on magnetic sheet frame, with in multichannel automatic pipettor transfer eluent to PCR plate, it is thus achieved that target dna product is (Fig. 2 is the sample extracted glue figure after pcr amplification) in the eluent of clarification。
Although embodiment of the present invention are disclosed as above, but listed utilization that it is not restricted in description and embodiment, it can be applied to various applicable the field of the invention completely, for those skilled in the art, it is easily achieved other amendment, therefore, under the general concept limited without departing substantially from claim and equivalency range, the present invention is not limited to specific details and shown here as the legend with description。
Claims (8)
1. the test kit extracting dissociative DNA based on paramagnetic particle method, it is characterised in that include the DNA secure bond liquid of independently subpackage, the first bead suspension, cleanout fluid, genomic DNA remove liquid, the second bead suspension and eluent；
Wherein, described DNA secure bond liquid includes: carbamide, Tris alkali, sodium sulfite, chaotropic salt, chelating agen, detergent and acetate；Described chaotropic salt is selected from guanidine hydrochloride, guanidine thiocyanate, sodium iodide or its combination；Described chelating agen is selected from disodiumedetate, ethylenediamine tetraacetic acid,dipotassium salt or its combination；Described detergent is selected from TritonX-100, Tween-20, NP-40, NaTDC, sodium lauryl sulphate or its combination；Described acetate is selected from potassium acetate, sodium acetate or its combination；
Including the first magnetic bead of Surface coating silicone hydroxyl in described first bead suspension, the particle diameter of described first magnetic bead is 50-300nm；
Described cleanout fluid includes isopropanol, chaotropic salt, chelating agen and detergent；Described chaotropic salt is selected from guanidine hydrochloride, guanidine thiocyanate, sodium iodide or its combination；Described chelating agen is selected from disodiumedetate, ethylenediamine tetraacetic acid,dipotassium salt or its combination；Described detergent is selected from TritonX-100, Tween-20, NP-40, NaTDC, sodium lauryl sulphate or its combination；
Described genome remove liquid include potassium carbonate, sodium acetate, ammonium hydrogen carbonate, disodiumedetate, sodium chloride, sodium sulfate, from saline solution and detergent；Described chaotropic salt is selected from guanidine hydrochloride, guanidine thiocyanate, sodium iodide or its combination；Described detergent is selected from TritonX-100, Tween-20, NP-40, NaTDC, sodium lauryl sulphate or its combination；
Including the second magnetic bead of Surface coating silicone hydroxyl in described second bead suspension, the particle diameter of described second magnetic bead is 100-500nm；
Described eluent is Tris alkali or deionized water。
2. the test kit extracting dissociative DNA based on paramagnetic particle method as claimed in claim 1, it is characterized in that, in described DNA secure bond liquid, urea concentration is 0.05-0.2mol/L, Tris alkali concn is 20-100mmol/L, concentration of sodium sulfite is 0.1-2wt%, and chaotropic salt concentration is 2-8mol/L, and chelating agent concentrations is 1-200mmol/L, the volume fraction of detergent is 0.01-5%, and acetate concentration is 0.05-1mol/L。
3. the test kit extracting dissociative DNA based on paramagnetic particle method as claimed in claim 1, it is characterised in that in described first magnetic bead dispersion liquid, the concentration of the first magnetic bead is 25-65mg/mL。
4. the test kit extracting dissociative DNA based on paramagnetic particle method as claimed in claim 1, it is characterised in that in described cleanout fluid, the volume fraction of isopropanol is 45-65%, the concentration of chaotropic salt is 0.1-0.5mol/L, and the concentration of chelating agen is 10-100mmol/L, and the volume fraction of detergent is 0.01-1%。
5. the test kit extracting dissociative DNA based on paramagnetic particle method as claimed in claim 1, it is characterized in that, it is 0.1-1mol/L that described genomic DNA removes concentration of potassium carbonate in liquid, sodium acetate concentration is 0.05-1mol/L, and the concentration of ammonium hydrogen carbonate is 0.5mmol/L, and disodiumedetate concentration is 10-100mmol/L, the concentration of sodium chloride is 3mol/L, the concentration of sodium sulfate is 1mol/L, and chaotropic salt concentration is 1-6mol/L, and the volume fraction of detergent is 0.01-3%。
6. the test kit extracting dissociative DNA based on paramagnetic particle method as claimed in claim 1, it is characterised in that in described second bead suspension, the concentration of the second magnetic bead is 10-40mg/mL。
7. the test kit extracting dissociative DNA based on paramagnetic particle method as claimed in claim 1, it is characterised in that when described eluent is Tris alkali, Tris paper mill wastewater is 0.9-1.1mmol/L。
8. the using method of the test kit extracting dissociative DNA based on paramagnetic particle method as according to any one of claim 1-7, it is characterised in that including:
Step 1) blood sample is transferred in EP pipe, add DNA secure bond liquid, be mixed；
Step 2) add the first bead suspension, vibration mixing to EP pipe；
Step 3) EP pipe is placed in magnetic frame device, Magnetic Isolation, after magnetic bead is adsorbed completely, topple over the liquid discarded in EP pipe；
Step 4) in EP pipe, add cleanout fluid, vibration EP pipe makes the first magnetic bead suspend uniformly, stands, Magnetic Isolation, after magnetic bead is adsorbed completely, topples over and discard cleanout fluid；
Step 5) in EP pipe, add genomic DNA removal liquid, vibration EP pipe makes the first magnetic bead suspend uniformly, and Magnetic Isolation stands, and Magnetic Isolation, after magnetic bead is adsorbed completely, Aspirate supernatant is transferred in new EP pipe；
Step 6) in the new EP pipe equipped with supernatant, add the second bead suspension, mixing, vibration combines；Stand, Magnetic Isolation, after magnetic bead is adsorbed completely, topple over the liquid discarded in EP pipe；
Step 7) in new EP pipe, add alcoholic solution, vibration EP pipe makes the second magnetic bead suspend uniformly, stands, Magnetic Isolation, removes supernatant；
Step 8) new EP pipe put in drying baker dry, remove ethanol, new EP pipe adds eluent, vibration EP pipe makes the second magnetic bead suspend uniformly, standing, Magnetic Isolation removes the second magnetic bead, namely contains target dissociative DNA in the eluent in new EP pipe。
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| CN108531472A (en) * | 2017-03-05 | 2018-09-14 | 北京天健惠康生物科技有限公司 | A kind of lysate for nucleic acid extraction |
| CN108531472B (en) * | 2017-03-05 | 2021-07-16 | 北京新羿生物科技有限公司 | Lysis solution for nucleic acid extraction |
| CN109613234A (en) * | 2018-12-29 | 2019-04-12 | 郑州安图生物工程股份有限公司 | Magnetic particle agglutination dissociation agent |
| CN110257370B (en) * | 2019-06-21 | 2020-08-28 | 海南晨海水产有限公司 | Method for rapidly extracting rockfish mitochondrial genome DNA |
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| WO2022252212A1 (en) * | 2021-06-04 | 2022-12-08 | 京东方科技集团股份有限公司 | Magnetic bead suspension reagent, nucleic acid purification method, and nucleic acid sorting method |
| CN113528507A (en) * | 2021-07-12 | 2021-10-22 | 中国农业科学院作物科学研究所 | Kit for extracting chicken blood genome DNA by high-throughput rapid paramagnetic particle method and extraction method |
| WO2023050156A1 (en) * | 2021-09-29 | 2023-04-06 | 京东方科技集团股份有限公司 | Nucleic acid extraction composition, nucleic acid extraction device and nucleic acid extraction method |
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