CN106119242A - A kind of difficult trace forensic DNA extraction kit based on Automation workstation and method thereof - Google Patents
A kind of difficult trace forensic DNA extraction kit based on Automation workstation and method thereof Download PDFInfo
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- CN106119242A CN106119242A CN201610474321.4A CN201610474321A CN106119242A CN 106119242 A CN106119242 A CN 106119242A CN 201610474321 A CN201610474321 A CN 201610474321A CN 106119242 A CN106119242 A CN 106119242A
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Abstract
The present invention relates to a kind of difficult trace forensic DNA extraction kit based on Automation workstation and method thereof, test kit includes: DNA extraction magnetic bead, lysate, cleaning mixture, DNA eluent.The method that the invention still further relates to utilize this difficult trace forensic DNA extraction kit based on Automation workstation to extract DNA.DNA extraction method of the present invention uses magnetic bead absorption, by lysate, DNA extraction magnetic bead, cleaning mixture and the mutual synergism of DNA eluent, DNA is extracted from difficult trace sample, available existing Switzerland tecan Automation workstation is implemented, complete in 40 minutes once to test and can extract 4 93 parts of difficulties, trace biological evidence DNA, good stability, significantly improve inspection case success rate, and single part of sample extraction cost can be controlled in about 35 yuan, significantly reduce inspection cost.
Description
Technical field
The present invention relates to a kind of difficult trace forensic DNA extraction kit based on Automation workstation and side thereof
Method, belongs to forensic DNA extraction technical field.
Background technology
Along with the fast development of forensic dna inspection technology, the on-the-spot biological evidence category extracted is increasingly wider, is used for
The biological evidence scope of the demands such as criminal case detection, fixing evidence is often no longer limited to traditional blood stain, salivary stain, seminal stain etc.
Rule material evidence, various difficulties, trace biological evidence are included into the category of forensic DNA inspection, in real work also
Having quite a few successful story is by above-mentioned difficulty, the inspection of trace biological evidence are obtained detection clue, significantly
Improve cracking of cases rate.Corresponding be above-mentioned difficulty, trace biological evidence accept number and check number dramatically increase.
Such as 2013, Jinan Police Office's science and technology concerning criminal matters institute DNA control laboratory accepted different kind organism material evidence 1835 parts, wherein wiped
Rub difficulty, the trace biological evidences about 422 parts such as trace, fingerprint, glove print, outmoded blood stain, washing blood stain, account for 23%.2014
Year accepts different kind organism material evidence 2117 parts, wherein wipes and rubs trace, fingerprint, glove print, outmoded blood stain, to wash blood stain etc. difficult, micro-
Amount biological evidence about 818 parts, accounts for about 50%.By 2015, accept different kind organism material evidence 2854 parts, wherein wipe and rub trace, refer to
Difficulty, the trace biological evidences about 1725 parts such as escutcheon, glove print, outmoded blood stain, washing blood stain, account for more than 61%.2016
Accept different kind organism material evidence 840 parts, difficult trace material evidence 585 parts 1-3 month, account for 70%.Because being limited, at this by administration establishment
Among 3 years, this laboratory inspection appraiser maintains 7 people all the time.According to us in investigation across the entire province and and state
The exchange of interior other provinces and cities colleague finds: material evidence accepts number in geometric growth；Difficult, trace material evidence proportion dramatically increases；Inspection
Authenticate fix the number of workers's quantity and supplement deficiency.These 3 is current the whole province or even whole nation Public Security Organs criminal case biological evidence DNA inspection
Authenticate the principal contradiction determining laboratory institute facing.
Along with new criminal procedure law improving further and specification evidence system, Forensic Science especially have " evidence
King " the DNA identification technology of good reputation guiding investigations, collect evidence, will be strengthened further and play more in terms of asserting crime
Add important effect.The workload of whole nation Public Security Organs criminal case biological evidence DNA at different levels inspection will be heavier.?
" reduce finance and support personnel amount " and test sensitivity personnel education needs under the backgrounds such as certain cycle, by increasing surveyor
Quantity cannot solve above-mentioned contradiction in a short time.Therefore research and development one is economic and practical, extraction effect is stable, recall rate is high, speed is fast,
Be applicable to the difficulty of automaticity flux work station high, big, trace biological evidence DNA extraction reagent imperative.
The work station owner of automatic biological material evidence DNA inspection at present to be divided into two big classes: 1, is used for personnel's blood sample of breaking laws and commit crime
Automatization's blood sample DNA extraction work station of inspection.2, automatic for conventional and part difficult biological evidence DNA inspection primary flux
Change DNA extraction work station.Being limited by its supporting DNA extraction reagent place, the pluses and minuses of above two work station are fairly obvious:
The former, for automatization's blood sample DNA extraction work station of personnel's blood sample test of breaking laws and commit crime, advantage is:
Between 2008 to 2013, based on the experiment porch of the companies such as Switzerland tecan, by domestic Public Security Organs DNA laboratory
Introducing on a large scale, popularity rate is high.And such work station flux is big, 96 parts of even more blood sample DNA once can be extracted.But
Its shortcoming is the most fairly obvious, and its supporting DNA extraction reagent extracts sample scope and is only limitted to pure blood sample, is limited in scope.Reagent
Required experimental procedure is many, flow process is loaded down with trivial details, and during Automated inspection, stability is slightly worse.Limited by matched reagent performance, at present should
Class work station major part Public Security Organs DNA laboratory at home is substantially at idle state.
The latter, primary flux Automation workstation, the AutoMate Express etc. with American AB company of domestic introduction sets
Standby is main, and advantage is: it is more wide in range that its supporting DNA extraction reagent extracts the more above-mentioned Automation workstation of sample scope, portion
Point non-blood stain class material evidence such as material evidence such as seminal stain, skeleton can manually digest and later half automatically complete DNA extraction.But its shortcoming is
The flux of work station own is low, typically between 6-13 material evidence scope, is as good as relative to the material evidence amount of accepting of current Public Security Organs
In an utterly inadequate amount.Such work station technology and supporting DNA extraction reagent thereof are by the reason such as foreign patent and technical know-how, it is impossible to
The transformation carrying out increasing flux to it, and such work station and consumables associated therewith be import, the introduction expense of separate unit work station with
Million meters, the DNA extraction reagent cost of single material evidence is at about 120 yuan, costly.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, it is provided that a kind of difficult trace forensic based on Automation workstation
DNA extraction kit and method thereof.Use test kit of the present invention can extract material evidence DNA from difficulty, trace sample, significantly
Improve inspection case success rate, and complete once to test in being capable of 40 minutes and can extract that 4-93 part is difficult, trace biological evidence
DNA。
Term explanation
Sample: wipe and rub trace, fingerprint, glove print, outmoded blood stain, washing blood stain.
Technical scheme is as follows:
A kind of difficult trace forensic DNA extraction kit based on Automation workstation, including:
DNA extraction magnetic bead, described DNA extraction magnetic bead be surface hydrophobicity, particle diameter be the spherical magnetic bead of 1～4 μm, nucleic acid is tied
Conjunction ability is more than 20 μ g DNA/mg magnetic beads, and saturation magnetization is more than 40emu/g；The polydispersity coefficient of magnetic bead is less than 0.2；
Lysate, in described lysate containing concentration be 0.3～3M guanidine hydrochloride, concentration be 1～4M guanidinium isothiocyanate,
Ethylenediaminetetraacetic acid (EDTA), concentration of volume percent that Tris-HCL that concentration is 10～50mM, concentration are 10～50mM are 3
～the Triton-X100 that the Tween-20 of 6%, concentration of volume percent are 5～20%, pH7.5～9.0；
Cleaning mixture, described cleaning mixture contains Tris-HCL that concentration is 5～20mM, concentration of volume percent 65～85%
Ethanol, pH 7.0～8.0；
DNA eluent, described DNA eluent contains Tris-HCL that concentration is 5～20mM, concentration is 0.5～2mM
Ethylenediaminetetraacetic acid (EDTA), pH7.5～9.0.
DNA extraction magnetic bead of the present invention is mainly used to specificity and combines genomic DNA, and not with protein, RNA with
And other amplification inhibitor combine；Lysate is for by during in sample, genomic DNA is discharged into lysate；Cleaning mixture can effectively go
Except the amplification mortifier of residual on magnetic bead, and ensure that DNA does not loses from magnetic bead；DNA eluent can be effectively by genomic DNA
Eluting from magnetic bead, each composition needs cooperation, thus ensures the DNA composition in high flux, completely extraction sample.
Preferably, described DNA extraction magnetic bead be surface hydrophobic, particle diameter be the spherical magnetic bead of 1 μm.
Preferably, described DNA extraction magnetic bead uses the castor nano-hydroxy of Beaver Nanotechnology (Suzhou) Inc.
Magnetic bead (Mag OH-1000 article No.: 70950).
DNA extraction magnetic bead is to have superparamagnetism, uniform hydrophobicity microballon, is uniformly distributed inside each microballon
Superparamagnetism material (γ Fe3O4), it is coated a thin layer lyophobic dust outside it to isolate magnetisable material.Superparamagnetism makes
Magnetic bead loses magnetism after showing magnetic in magnetic field and leaving magnetic field, size, shape, and homogeneity (the CV ＜ of surface area
3%) providing optimum response kinetics, ball, homogeneous surface decrease chemical and adhere to and non-specific binding.
Preferably, in described lysate containing concentration be the guanidine hydrochloride of 0.8M, concentration be the guanidinium isothiocyanate of 1.6M, concentration
For the Tris-HCL of 30mM, concentration be the ethylenediaminetetraacetic acid (EDTA) of 30mM, concentration of volume percent be the Tween-of 5%
20, concentration of volume percent is the Triton-X100 of 5%, pH 8.0.
Preferably, described cleaning mixture contain concentration be the Tris-HCL of 10mM, concentration of volume percent be the ethanol of 80%,
pH7.5。
Preferably, described DNA eluent contain concentration be the Tris-HCL of 10mM, concentration be the ethylenediamine tetrem of 1mM
Acid (EDTA), pH 8.0.
The preparation of above-mentioned lysate, cleaning mixture and DNA eluent is carried out preparing by the routine operation of this area.
Utilize the method that above-mentioned difficult trace forensic DNA extraction kit based on Automation workstation extracts DNA,
Comprise the steps:
(1), after sample being placed in centrifugal hand basket sleeve pipe, automatically working stands in addition 150～600 μ l in each centrifugal basket
Lysate after, the metal bath of 80～95 DEG C cracks, the time 20～50min, centrifugal segregation removing DNA after inspection
Material, is prepared DNA mixed liquor in sleeve pipe；
(2) the DNA mixed liquor that step (1) prepares is mixed by Automation workstation with DNA extraction magnetic bead, DNA extraction magnetic bead
With DNA mixed liquor mixed volume ratio for 1:(10～30), solid-liquid separation, take solid, prepare the magnetic bead being adsorbed with DNA；
(3) what step (2) was prepared by Automation workstation is adsorbed with the magnetic bead cleaning mixture washing of DNA, is adsorbed with DNA's
The mixed volume of magnetic bead and cleaning mixture ratio is for 1:(10～80), prepare the magnetic bead being adsorbed with DNA after washing；
(4) the magnetic bead eluent being adsorbed with DNA after the washing that step (3) is prepared by Automation workstation is 50～70
Eluting under the conditions of DEG C, is adsorbed with the magnetic bead of DNA and the mixed volume of eluent than for 1:(4～10 after washing), solid-liquid separation,
Take liquid, prepare DNA solution.
According to currently preferred, in described step (1), cracking condition is: 90 DEG C of cracking 30min.
According to currently preferred, in described step (1), centrifugal condition is: 9000～12000 turns/min be centrifuged 1～
3min；It is further preferred that in described step (1), centrifugal condition is: 12000 turns/min is centrifuged 1min.
According to currently preferred, in described step (2), mixing condition is: room temperature, 1000 turns/min concussion mixing 2～
10min。
According to currently preferred, in described step (3), wash conditions is: room temperature, 1000 turns/min shake cleaning 0.5
～2min.
According to currently preferred, in described step (4), elution requirement is: 65 DEG C, 1200 turns/min shake eluting 0.1
～1min.
According to currently preferred, in described step (2) and (4), solid-liquid separation is centrifugal, and condition is 9000～12000
Turn/min is centrifuged 1～3min；It is further preferred that in described step (2) and (4), centrifugal condition is: 12000 turns/min is centrifuged
1min。
Beneficial effect
1, DNA extraction method of the present invention use magnetic bead absorption, by lysate, DNA extraction magnetic bead, cleaning mixture and
The mutual synergism of DNA eluent, extracts DNA, available existing Switzerland tecan Automation workstation from difficult trace sample
Implement, complete in 40 minutes once to test and can extract 4-93 part difficulty, trace biological evidence DNA, good stability, significantly improve
Inspection case success rate, and single part of sample extraction cost can be controlled in about 35 yuan, significantly reduces inspection cost；
2, the DNA extraction magnetic bead in test kit of the present invention be surface hydrophobicity, particle diameter be the spherical magnetic bead of 1～4 μm, core
Acid binding ability is more than 40emu/g more than 20 μ g DNA/mg magnetic beads, saturation magnetization；The polydispersity coefficient of magnetic bead is less than
0.2；It is demonstrated experimentally that this magnetic bead specificity efficient absorption genomic DNA in lysate of the present invention, gone by cleaning mixture
Removal of residue, can be conveniently used for Automation workstation and realize high flux extraction.
Accompanying drawing explanation
Amplification rear electrophoresis figure is extracted in Fig. 1 trace bloodstains of the present invention 64 times dilution；
Amplification rear electrophoresis figure is extracted in Fig. 2 Promega company IQ test kit trace bloodstains 64 times dilution；
Fig. 3 China reflect again blue shield manual silica bead trace bloodstains 64 times dilution extract amplification rear electrophoresis figure；
Amplification rear electrophoresis figure is extracted in Fig. 4 AB company AutoMate Express automatic work station trace bloodstains 64 times dilution.
Detailed description of the invention
Below in conjunction with embodiment, technical scheme is described further, but institute of the present invention protection domain is not limited to
This.Medical law fields personnel can use for reference present disclosure, is transplanted in the Automation workstation of different brands or optimizes relevant ginseng
Number.Emphasizing especially, all similar Automation workstations are transplanted and optimized is all aobvious for medical law fields personnel
And be clear to, they are considered as being included in the invention.The test kit of the present invention extremely method verifies reality by relevant comparative
Testing and be described, related personnel substantially can be to method of the present invention in without departing from present invention, spirit and scope
Carry out suitable change or combination with application, realize and apply the technology of the present invention.
DNA extraction magnetic bead described in embodiment derives from the hydroxyl magnetic bead Mag of Beaver Nanotechnology (Suzhou) Inc.
OH-1000, article No.: 70950, this DNA extraction magnetic bead nucleic acid binding ability is more than 20 μ g DNA/mg magnetic beads, saturation magnetization
More than 40emu/g, the polydispersity coefficient of magnetic bead is less than 0.2.
Automation workstation described in embodiment is the EVO100-4 Automation workstation of tecan company of Switzerland.
Embodiment 1: trace bloodstains multiplication dilution experiment
A kind of extraction test kit of difficult trace forensic DNA based on Automation workstation, including:
DNA extraction magnetic bead, described DNA extraction magnetic bead be surface hydrophobicity, particle diameter be the spherical magnetic bead of 2 μm；
Lysate, in described lysate containing concentration be the guanidine hydrochloride of 0.8M, concentration be the guanidinium isothiocyanate of 1.6M, concentration
For Tris-HCL, the concentration of 30mM be the EDTA of 30mM, concentration of volume percent be 5% Tween-20, percent by volume dense
Degree is the Triton-X100 of 5%, pH8.0；
Cleaning mixture, described cleaning mixture contain concentration be the Tris-HCL of 10mM, the ethanol of concentration of volume percent 80%, pH
7.5；
DNA eluent, described DNA eluent contain concentration be the Tris-HCL of 10mM, concentration be the EDTA of 1mM,
pH8.0。
The preparation of sample
Taking blood count experiment is 10 × 109The Healthy Volunteers anticoagulation of known dna genotyping result with normal saline
Being diluted, extension rate is divided into 8 times, 16 times, 32 times, 64 times, 128 times；Each multiple dilutions blood is taken 1 μ l and joins centrifugal
In hand basket sleeve pipe, every multiple dilutions blood repeats to take 20 times, i.e. 20 pipes；
The test kit that extracts utilizing above-mentioned difficult trace forensic DNA based on Automation workstation extracts the side of DNA
Method, comprises the steps:
(1) preparation of DNA mixed liquor:
EVO100-4 Automation workstation is drawn lysate 300 μ l by liquid adding arm and is joined each centrifugal hand basket sleeve pipe
In；
Artificial by centrifugal hand basket sleeve pipe 95 DEG C of 30min on metal bath；
Centrifugal hand basket sleeve pipe is centrifugal 1min under 12000 turns/min, abandons hand basket；The solution containing DNA after being centrifuged
Sleeve pipe put back in work station laboratory table.
DNA solution is transferred to 96 hole depth orifice plates by liquid adding arm from 2ml centrifuge tube by EVO100-4 Automation workstation
In, prepare DNA mixed liquor；
(2) DNA in DNA mixed liquor is adsorbed on magnetic bead:
8 μ l DNA extraction magnetic beads are added in 96 hole depth orifice plates by EVO100-4 Automation workstation by liquid adding arm；
The 96 hole depth orifice plates that EVO100-4 Automation workstation will be equipped with DNA mixed liquor+magnetic bead by mechanical hand are transferred to
On agitator, room temperature, with 1000 turns/min concussion mixing 5min, makes the abundant adsorption of DNA of magnetic bead；
96 hole depth orifice plates are transferred on magnetic frame from agitator by EVO100-4 Automation workstation by mechanical hand, quiet
Put 30sec；
Solution sucking-off in 96 hole depth orifice plates is transferred in waste liquid tank by liquid adding arm by EVO100-4 Automation workstation,
Prepare the magnetic bead being adsorbed with DNA；
(3) washing of the magnetic bead of DNA it is adsorbed with:
350 μ l cleaning mixture are transferred in 96 hole depth orifice plates by EVO100-4 Automation workstation by liquid adding arm；
96 hole depth orifice plates are transferred on agitator by mechanical hand by EVO100-4 Automation workstation, and room temperature is with 1000
Turn/min concussion cleaning 30sec；
96 hole depth orifice plates are transferred on magnetic frame from agitator by EVO100-4 Automation workstation by mechanical hand, quiet
Put 30sec；
Cleaning mixture sucking-off in 96 hole depth orifice plates is transferred to waste liquid tank by liquid adding arm by EVO100-4 Automation workstation
In；
150ul cleaning mixture is transferred in 96 hole depth orifice plates by EVO100-4 Automation workstation by liquid adding arm；
96 hole depth orifice plates are transferred on agitator by mechanical hand by EVO100-4 Automation workstation, and room temperature is with 1000
Turn/min concussion cleaning 30sec；
96 hole depth orifice plates are transferred on magnetic frame from agitator by EVO100-4 Automation workstation by mechanical hand, quiet
Put
30sec；
Cleaning mixture sucking-off in 96 hole depth orifice plates is transferred to waste liquid tank by liquid adding arm by EVO100-4 Automation workstation
In；
96 hole depth orifice plates are transferred on deep-well plates heater by mechanical hand by EVO100-4 Automation workstation, at 85 DEG C
Lower drying 5min；After drying, 96 hole depth orifice plates are transferred on magnetic frame by mechanical hand by work station, prepare the absorption after washing
There is the magnetic bead of DNA；
(4) acquisition of DNA solution:
The DNA eluent of 40 μ l is transferred in 96 hole depth orifice plates by EVO100-4 Automation workstation by liquid adding arm, and
By mechanical hand, 96 hole depth orifice plates are transferred to 12000 turns/min on agitator and shake 15sec；
96 hole depth orifice plates are transferred on deep-well plates heater by mechanical hand by EVO100-4 Automation workstation, at 85 DEG C
Under hatch 6min；
96 hole depth orifice plates are transferred to magnetic force by mechanical hand from deep-well plates heater by EVO100-4 Automation workstation
On frame, stand 30sec；
EVO100-4 Automation workstation draws 30 μ l by liquid adding arm, i.e. obtains DNA solution.
The detection of carried DNA
DNA after extraction carries out expanding with Identifiler plus test kit, with 3500xl genetic analyzer electrophoresis, with
GeneMapper software is analyzed.Take 12 and the above site assay consistent with volunteer's DNA typing result for having
Effect result.
Carried DNA extracts reagent/method with other and carries out contrast experiment
Promega company of the U.S. manual magnetic bead DNA extraction reagent, China reflect blue shield manual silica bead DNA extraction reagent, U.S. again
AutouMate Express automatization of AB company of state DNA extraction work station takes DNA sample prepared by above-mentioned experiment, according to each
Flow process extracts DNA, and contrasts with this experimental result, and comparing result is as shown in table 1.
Table 1
Trace bloodstains multiplication dilution experiment, 1 μ l anticoagulation extracts DNA after diluting 64 times, 12 and above site and aspiration
The assay number that person's DNA typing result is consistent, Automation workstation of the present invention detection number is 20, Promega company IQ
Test kit manually detects several 20, and mirror blue shield silica bead test kit manually detects number again is 7, AB company AutoMate Express
Automatically work station detection number is 16.
More than experiment shows, the present invention is compared with the Promeg company IQ magnetic bead kit of manual extraction, to trace bloodstains
And the test effect of exfoliative cyte class trace sample without significant difference but has the high-throughout advantage of automatization.
Embodiment 2: the extraction of the relevant difficult trace sample of case
A kind of extraction test kit of difficult trace forensic DNA based on Automation workstation, including:
DNA extraction magnetic bead, described DNA extraction magnetic bead be surface hydrophobicity, particle diameter be the spherical magnetic bead of 1 μm；
Lysate, in described lysate containing concentration be the guanidine hydrochloride of 3M, concentration be that the guanidinium isothiocyanate of 4M, concentration are
Tween-20, the concentration of volume percent that the Tris-HCL of 50mM, concentration are the EDTA of 50mM, concentration of volume percent is 5%
It is the Triton-X100 of 20%, pH8.0；
Cleaning mixture, described cleaning mixture contain concentration be the Tris-HCL of 20mM, the ethanol of concentration of volume percent 85%, pH
7.5；
DNA eluent, described DNA eluent contain concentration be the Tris-HCL of 20mM, concentration be the EDTA of 2mM,
pH8.0。
The preparation of sample
The sample choosing known dna assay is as follows:
Fingerprint trace swab 10 pieces, wiping rub trace swab 10 pieces, glove print swab 10 pieces, the placement outmoded blood stain of more than 5 years filter
10 parts of paper.
By above-mentioned swab class sample clip about 3mm × 3mm size, blood stain filter paper clip about 2mm × 2mm size.Will preparation
Good sample joins in centrifugal hand basket sleeve pipe.
The test kit that extracts utilizing above-mentioned difficult trace forensic DNA based on Automation workstation extracts the side of DNA
Method, comprises the steps:
(1) preparation of DNA mixed liquor:
EVO100-4 Automation workstation is drawn lysate 300 μ l by liquid adding arm and is joined each centrifugal hand basket sleeve pipe
In；
Artificial by centrifugal hand basket sleeve pipe 95 DEG C of 30min on metal bath；
Centrifugal hand basket sleeve pipe is centrifugal 1min under 12000 turns/min, abandons hand basket；The solution containing DNA after being centrifuged
Sleeve pipe put back in work station laboratory table.
DNA solution is transferred to 96 hole depth orifice plates by liquid adding arm from 2ml centrifuge tube by EVO100-4 Automation workstation
In, prepare DNA mixed liquor；
(2) DNA in DNA mixed liquor is adsorbed on magnetic bead:
8 μ l DNA extraction magnetic beads are added in 96 hole depth orifice plates by EVO100-4 Automation workstation by liquid adding arm；
The 96 hole depth orifice plates that EVO100-4 Automation workstation will be equipped with DNA mixed liquor+magnetic bead by mechanical hand are transferred to
On agitator, room temperature, with 1000 turns/min concussion mixing 5min, makes the abundant adsorption of DNA of magnetic bead；
96 hole depth orifice plates are transferred on magnetic frame from agitator by EVO100-4 Automation workstation by mechanical hand, quiet
Put 30sec；
Solution sucking-off in 96 hole depth orifice plates is transferred in waste liquid tank by liquid adding arm by EVO100-4 Automation workstation,
Prepare the magnetic bead being adsorbed with DNA；
(3) washing of the magnetic bead of DNA it is adsorbed with:
350 μ l cleaning mixture are transferred in 96 hole depth orifice plates by EVO100-4 Automation workstation by liquid adding arm；
96 hole depth orifice plates are transferred on agitator by mechanical hand by EVO100-4 Automation workstation, and room temperature is with 1000
Turn/min concussion cleaning 30sec；
96 hole depth orifice plates are transferred on magnetic frame from agitator by EVO100-4 Automation workstation by mechanical hand, quiet
Put 30sec；
Cleaning mixture sucking-off in 96 hole depth orifice plates is transferred to waste liquid tank by liquid adding arm by EVO100-4 Automation workstation
In；
150 μ l cleaning mixture are transferred in 96 hole depth orifice plates by EVO100-4 Automation workstation by liquid adding arm；
96 hole depth orifice plates are transferred on agitator by mechanical hand by EVO100-4 Automation workstation, and room temperature is with 1000
Turn/min concussion cleaning 30sec；
96 hole depth orifice plates are transferred on magnetic frame from agitator by EVO100-4 Automation workstation by mechanical hand, quiet
Put 30sec；
Cleaning mixture sucking-off in 96 hole depth orifice plates is transferred to waste liquid tank by liquid adding arm by EVO100-4 Automation workstation
In；
96 hole depth orifice plates are transferred on deep-well plates heater by mechanical hand by EVO100-4 Automation workstation, at 85 DEG C
Lower drying 5min；After drying, 96 hole depth orifice plates are transferred on magnetic frame by mechanical hand by work station, prepare the absorption after washing
There is the magnetic bead of DNA；
(4) acquisition of DNA solution:
The DNA eluent of 40 μ l is transferred in 96 hole depth orifice plates by EVO100-4 Automation workstation by liquid adding arm, and
By mechanical hand, 96 hole depth orifice plates are transferred to 12000 turns/min on agitator and shake 15sec；
96 hole depth orifice plates are transferred on deep-well plates heater by mechanical hand by EVO100-4 Automation workstation, at 85 DEG C
Under hatch 6min；
96 hole depth orifice plates are transferred to magnetic force by mechanical hand from deep-well plates heater by EVO100-4 Automation workstation
On frame, stand 30sec；
EVO100-4 Automation workstation draws 30 μ l by liquid adding arm, i.e. obtains DNA solution.
The detection of carried DNA
DNA after extraction carries out expanding with Identifiler plus test kit, with 3500xl genetic analyzer electrophoresis, with
GeneMapper software is analyzed.Take 12 and the above site assay consistent with known dna genotyping result is effective
Result.
Carried DNA extracts reagent/method with other and carries out contrast experiment
Promega company of the U.S. manual magnetic bead DNA extraction reagent, China reflect blue shield manual silica bead DNA extraction reagent, U.S. again
AutouMate Express automatization of AB company of state DNA extraction work station takes DNA sample prepared by above-mentioned experiment, according to each
Flow process extracts DNA, and contrasts with this experimental result, and comparing result is as shown in table 2.
Table 2
Total recall rate of three classes common vestige swab (fingerprint trace swab, wiping rub vestige swab, glove print swab) is respectively
For:
Embodiment 2 76%.6；Promega magnetic bead: 63.3%；Mirror blue shield silica bead again: 23.3%；AutouMate
Express:76%.6.
Above-mentioned experiment shows have higher recall rate for the vestige swab class sample present invention common in actual case,
It is better than two kinds of conventional manual test kits, equally matched with AB company AutoMate Express automatic work station assay.
Recall rate of the present invention for outmoded blood stain is 100%, is better than the AB company automatic work station of AutoMate Express.
Embodiment 3: trace exfoliative cyte DNA extraction efficiency is tested
A kind of extraction test kit of difficult trace forensic DNA based on Automation workstation, including:
DNA extraction magnetic bead, described DNA extraction magnetic bead be surface hydrophobicity, particle diameter be the spherical magnetic bead of 1 μm；
Lysate, in described lysate containing concentration be the guanidine hydrochloride of 0.3M, concentration be that the guanidinium isothiocyanate of 1M, concentration are
Tween-20, the concentration of volume percent that the Tris-HCL of 10mM, concentration are the EDTA of 10mM, concentration of volume percent is 5%
It is the Triton-X100 of 5%, pH8.0；
Cleaning mixture, described cleaning mixture contain concentration be the Tris-HCL of 5mM, the ethanol of concentration of volume percent 65%, pH
7.5；
DNA eluent, described DNA eluent contain concentration be the Tris-HCL of 5mM, concentration be the EDTA of 0.5mM, pH
8.0。
The preparation of sample
Cleanser Rubber gloves, normal activity more than 4 hours is worn by the Healthy Volunteers of known dna genotyping result.By this breast
Soak with pure water after Rubber gloves medial surface cotton swab, carry out under 400 power microscopes after taking 50 μ l soak eosin stains
Observe and result is diluted according to the observation；Finally it is prepared as 30-50 nucleated cell/full the exfoliative cyte solution in the visual field, 10-
Below 20 nucleated cell/full the exfoliative cyte solution in the visual field, 10 nucleated cell/the exfoliative cyte solution in the full visual field.Take
State exfoliative cyte solution 100 μ l to join in centrifugal hand basket sleeve pipe, be repeated 5 times, i.e. 5 pipes；
The test kit that extracts utilizing above-mentioned difficult trace forensic DNA based on Automation workstation extracts the side of DNA
Method, comprises the steps:
(1) preparation of DNA mixed liquor:
EVO100-4 Automation workstation is drawn lysate 300 μ l by liquid adding arm and is joined each centrifugal hand basket sleeve pipe
In；
Artificial by centrifugal hand basket sleeve pipe 95 DEG C of 30min on metal bath；
Centrifugal hand basket sleeve pipe is centrifugal 1min under 12000 turns/min, abandons hand basket；The solution containing DNA after being centrifuged
Sleeve pipe put back in work station laboratory table.
DNA solution is transferred to 96 hole depth orifice plates by liquid adding arm from 2ml centrifuge tube by EVO100-4 Automation workstation
In, prepare DNA mixed liquor；
(2) DNA in DNA mixed liquor is adsorbed on magnetic bead:
8 μ l DNA extraction magnetic beads are added in 96 hole depth orifice plates by EVO100-4 Automation workstation by liquid adding arm；
The 96 hole depth orifice plates that EVO100-4 Automation workstation will be equipped with DNA mixed liquor+magnetic bead by mechanical hand are transferred to
On agitator, room temperature, with 1000 turns/min concussion mixing 5min, makes the abundant adsorption of DNA of magnetic bead；
96 hole depth orifice plates are transferred on magnetic frame from agitator by EVO100-4 Automation workstation by mechanical hand, quiet
Put 30sec；
Solution sucking-off in 96 hole depth orifice plates is transferred in waste liquid tank by liquid adding arm by EVO100-4 Automation workstation,
Prepare the magnetic bead being adsorbed with DNA；
(3) washing of the magnetic bead of DNA it is adsorbed with:
350 μ l cleaning mixture are transferred in 96 hole depth orifice plates by EVO100-4 Automation workstation by liquid adding arm；
96 hole depth orifice plates are transferred on agitator by mechanical hand by EVO100-4 Automation workstation, and room temperature is with 1000
Turn/min concussion cleaning 30sec；
96 hole depth orifice plates are transferred on magnetic frame from agitator by EVO100-4 Automation workstation by mechanical hand, quiet
Put 30sec；
Cleaning mixture sucking-off in 96 hole depth orifice plates is transferred to waste liquid tank by liquid adding arm by EVO100-4 Automation workstation
In；
150 μ l cleaning mixture are transferred in 96 hole depth orifice plates by EVO100-4 Automation workstation by liquid adding arm；
96 hole depth orifice plates are transferred on agitator by mechanical hand by EVO100-4 Automation workstation, and room temperature is with 1000
Turn/min concussion cleaning 30sec；
96 hole depth orifice plates are transferred on magnetic frame from agitator by EVO100-4 Automation workstation by mechanical hand, quiet
Put 30sec；
Cleaning mixture sucking-off in 96 hole depth orifice plates is transferred to waste liquid tank by liquid adding arm by EVO100-4 Automation workstation
In；
96 hole depth orifice plates are transferred on deep-well plates heater by mechanical hand by EVO100-4 Automation workstation, at 85 DEG C
Lower drying 5min；After drying, 96 hole depth orifice plates are transferred on magnetic frame by mechanical hand by work station, prepare the absorption after washing
There is the magnetic bead of DNA；
(4) acquisition of DNA solution:
The DNA eluent of 40 μ l is transferred in 96 hole depth orifice plates by EVO100-4 Automation workstation by liquid adding arm, and
By mechanical hand, 96 hole depth orifice plates are transferred to 12000 turns/min on agitator and shake 15sec；
96 hole depth orifice plates are transferred on deep-well plates heater by mechanical hand by EVO100-4 Automation workstation, at 85 DEG C
Under hatch 6min；
96 hole depth orifice plates are transferred to magnetic force by mechanical hand from deep-well plates heater by EVO100-4 Automation workstation
On frame, stand 30sec；
EVO100-4 Automation workstation draws 30 μ l by liquid adding arm, i.e. obtains DNA solution.
The detection of carried DNA
DNA after extraction carries out expanding with Identifiler plus test kit, with 3500xl genetic analyzer electrophoresis, with
GeneMapper software is analyzed.Take 12 and the above site assay consistent with volunteer's DNA typing result for having
Effect result.
Carried DNA extracts reagent/method with other and carries out contrast experiment
Promega company of the U.S. manual magnetic bead DNA extraction reagent, China reflect blue shield manual silica bead DNA extraction reagent, U.S. again
AutouMate Express automatization of AB company of state DNA extraction work station takes DNA sample prepared by above-mentioned experiment, according to each
Flow process extracts DNA, and contrasts with this experimental result, and comparing result is as shown in table 3.
Table 3
Trace exfoliative cyte DNA extraction efficiency is tested, when under mirror, cell number is 10-20, and 12 and above site and will
The assay number that hope person's DNA typing result is consistent, Automation workstation of the present invention detection number is 18, Promega company
IQ test kit manually detects several 19, and mirror blue shield silica bead test kit manually detects number again is 8, AB company AutoMate
Express automatic work station detection number is 13.
More than experiment shows, the present invention is better than multiple mirror blue shield silica bead reagent to the test effect of exfoliative cyte class trace sample
Box, the automatic work station of AB company AutoMate Express, and there is the high-throughout advantage of automatization.
Embodiment 4: trace bloodstains dilution experiment limit DNA extraction stability repeats experiment and carries with trace exfoliative cyte DNA
Take stabilised efficiency and repeat experiment
A kind of extraction test kit of difficult trace forensic DNA based on Automation workstation, including:
DNA extraction magnetic bead, described DNA extraction magnetic bead be surface hydrophobicity, particle diameter be the spherical magnetic bead of 1 μm；
Lysate, in described lysate containing concentration be the guanidine hydrochloride of 2M, concentration be that the guanidinium isothiocyanate of 2.5M, concentration are
Tween-20, the concentration of volume percent that the Tris-HCL of 20mM, concentration are the EDTA of 30mM, concentration of volume percent is 5%
It is the Triton-X100 of 10%, pH8.0；
Cleaning mixture, described cleaning mixture contain concentration be the Tris-HCL of 15mM, the ethanol of concentration of volume percent 75%,
pH7.5；
DNA eluent, described DNA eluent contain concentration be the Tris-HCL of 10mM, concentration be the EDTA of 1.5mM,
pH 8.0。
The preparation of sample
During the blood 1 μ l of 64 times of dilutions instills centrifugal hand basket sleeve pipe in Example 1, do 93 and do Repeatability checking；Treating excess syndrome
Execute in example 3 the exfoliative cyte solution in 10-20 nucleated cell/full visual field and take 100 μ l in centrifugal hand basket sleeve pipe, do 93 and do weight
Renaturation is checked.
The test kit that extracts utilizing above-mentioned difficult trace forensic DNA based on Automation workstation extracts the side of DNA
Method, comprises the steps:
(1) preparation of DNA mixed liquor:
EVO100-4 Automation workstation is drawn lysate 300 μ l by liquid adding arm and is joined each centrifugal hand basket sleeve pipe
In；
Artificial by centrifugal hand basket sleeve pipe 95 DEG C of 30min on metal bath；
Centrifugal hand basket sleeve pipe is centrifugal 1min under 12000 turns/min, abandons hand basket；The solution containing DNA after being centrifuged
Sleeve pipe put back in work station laboratory table.
DNA solution is transferred to 96 hole depth orifice plates by liquid adding arm from 2ml centrifuge tube by EVO100-4 Automation workstation
In, prepare DNA mixed liquor；
(2) DNA in DNA mixed liquor is adsorbed on magnetic bead:
8 μ l DNA extraction magnetic beads are added in 96 hole depth orifice plates by EVO100-4 Automation workstation by liquid adding arm；
The 96 hole depth orifice plates that EVO100-4 Automation workstation will be equipped with DNA mixed liquor+magnetic bead by mechanical hand are transferred to
On agitator, room temperature, with 1000 turns/min concussion mixing 5min, makes the abundant adsorption of DNA of magnetic bead；
96 hole depth orifice plates are transferred on magnetic frame from agitator by EVO100-4 Automation workstation by mechanical hand, quiet
Put 30sec；
Solution sucking-off in 96 hole depth orifice plates is transferred in waste liquid tank by liquid adding arm by EVO100-4 Automation workstation,
Prepare the magnetic bead being adsorbed with DNA；
(3) washing of the magnetic bead of DNA it is adsorbed with:
350 μ l cleaning mixture are transferred in 96 hole depth orifice plates by EVO100-4 Automation workstation by liquid adding arm；
96 hole depth orifice plates are transferred on agitator by mechanical hand by EVO100-4 Automation workstation, and room temperature is with 1000
Turn/min concussion cleaning 30sec；
96 hole depth orifice plates are transferred on magnetic frame from agitator by EVO100-4 Automation workstation by mechanical hand, quiet
Put 30sec；
Cleaning mixture sucking-off in 96 hole depth orifice plates is transferred to waste liquid tank by liquid adding arm by EVO100-4 Automation workstation
In；
150 μ l cleaning mixture are transferred in 96 hole depth orifice plates by EVO100-4 Automation workstation by liquid adding arm；
96 hole depth orifice plates are transferred on agitator by mechanical hand by EVO100-4 Automation workstation, and room temperature is with 1000
Turn/min concussion cleaning 30sec；
96 hole depth orifice plates are transferred on magnetic frame from agitator by EVO100-4 Automation workstation by mechanical hand, quiet
Put 30sec；
Cleaning mixture sucking-off in 96 hole depth orifice plates is transferred to waste liquid tank by liquid adding arm by EVO100-4 Automation workstation
In；
96 hole depth orifice plates are transferred on deep-well plates heater by mechanical hand by EVO100-4 Automation workstation, at 85 DEG C
Lower drying 5min；After drying, 96 hole depth orifice plates are transferred on magnetic frame by mechanical hand by work station, prepare the absorption after washing
There is the magnetic bead of DNA；
(4) acquisition of DNA solution:
The DNA eluent of 40 μ l is transferred in 96 hole depth orifice plates by EVO100-4 Automation workstation by liquid adding arm, and
By mechanical hand, 96 hole depth orifice plates are transferred to 12000 turns/min on agitator and shake 15sec；
96 hole depth orifice plates are transferred on deep-well plates heater by mechanical hand by EVO100-4 Automation workstation, at 85 DEG C
Under hatch 6min；
96 hole depth orifice plates are transferred to magnetic force by mechanical hand from deep-well plates heater by EVO100-4 Automation workstation
On frame, stand 30sec；
EVO100-4 Automation workstation draws 30 μ l by liquid adding arm, i.e. obtains DNA solution.
The detection of carried DNA
DNA after extraction carries out expanding with Identifiler plus test kit, with 3500xl genetic analyzer electrophoresis, with
GeneMapper software is analyzed, and takes 12 and the above site assay consistent with volunteer's DNA typing result for having
Effect result；
Recall rate computational methods: 12 and the above site assay number consistent with volunteer's DNA typing result/
93；
Table 4
This experiment shows: on the premise of high flux, has more for trace bloodstains, the exfoliative cyte class sample present invention
Stable experimental result repeatability.And consumables cost is the most important considerations when big flux is checked, single of the present invention
Inspection cost is about 30 yuan, the IQ test kit of substantially less than Promega company, the automatic chemical industry of AB company Automate Express
Make station test kit, the reagent cost of China's mirror blue shield manual silica bead DNA extraction reagent three again.
Comparative example
Contrast groups 1, test kit as described in Example 1, difference is:
DNA extraction magnetic bead uses the IQ test kit magnetic bead of Promega company.
Contrast groups 2, test kit as described in Example 1, difference is:
Lysate uses the IQ test kit lysate of Promega company.
Contrast groups 3, test kit as described in Example 1, difference is:
Cleaning mixture uses the IQ test kit cleaning mixture of Promega company.
Contrast groups 4, test kit as described in Example 1, difference is:
DNA eluent uses the IQ test kit eluent of Promega company.
The DNA extraction method described in embodiment 1 is used to use the test kit of contrast groups 1～4 to carry out DNA extraction operation, knot
Fruit is shown in Table 5,6.
Table 5, trace bloodstains stability repeat to test 64 times of dilutions
Table 6, trace exfoliative cyte stability repeat 10-20 cell of experiment/full visual field detection number and recall rate
By this comparative example it can be seen that magnetic bead, lysate, cleaning mixture, four kinds of component especially magnetic beads of eluent with split
Solving liquid the most obvious for the impact of experimental result, lysate for the cracking of cell, the efficiency of released dna and is specifically splitting
Solving magnetic bead in pendular ring border and combining the efficiency of DNA is the key factor affecting experimental result.The lysate that the present invention chooses with
Magnetic bead cooperates and could obtain ideal experimental result.Cleaning mixture and DNA eluent are on the impact of experimental result earlier above
Both are slightly smaller but such as preferable cooperation still can not can affect experimental result with the above two.Amid all these factors, selected by the present invention
Magnetic bead, lysate, cleaning mixture and DNA eluent jointly act on the pass being to ensure that the present invention has higher inspection success rate
Key.
The above is only the preferred embodiment of the present invention, it is noted that for medical law fields personnel, is not taking off
On the premise of the principle of the invention, it is also possible to be transplanted in the Automation workstation of different brands or optimize relevant parameter, this
Transplanting and optimization between a little different brands Automation workstations also should be considered as protection scope of the present invention.
Claims (10)
1. a difficult trace forensic DNA extraction kit based on Automation workstation, it is characterised in that including:
DNA extraction magnetic bead, described DNA extraction magnetic bead be surface hydrophobicity, particle diameter be the spherical magnetic bead of 1～4 μm, nucleic acid combines energy
Power is more than 20 μ g DNA/mg magnetic beads, and saturation magnetization is more than 40emu/g；The polydispersity coefficient of magnetic bead is less than 0.2；
Lysate, in described lysate containing concentration be 0.3～3M guanidine hydrochloride, concentration be 1～4M guanidinium isothiocyanate, concentration
Be 10～50mM Tris-HCL, concentration be 10～50mM ethylenediaminetetraacetic acid (EDTA), concentration of volume percent be 3～
The Tween-20 of 6%, concentration of volume percent are the Triton-X100, pH7.5～9.0 of 5～20%；
Cleaning mixture, described cleaning mixture contains Tris-HCL, concentration of volume percent 65～the second of 85% that concentration is 5～20mM
Alcohol, pH 7.0～8.0；
DNA eluent, the second two that described DNA eluent contains Tris-HCL that concentration is 5～20mM, concentration is 0.5～2mM
Amine tetraacethyl (EDTA), pH7.5～9.0.
2. difficult trace forensic DNA extraction kit based on Automation workstation as claimed in claim 1, its feature
Be, described DNA extraction magnetic bead be surface hydrophobic, particle diameter be the spherical magnetic bead of 1 μm；
Preferably, described DNA extraction magnetic bead uses the castor nano-hydroxy magnetic bead of Beaver Nanotechnology (Suzhou) Inc.
Mag OH-1000 article No.: 70950.
3. difficult trace forensic DNA extraction kit based on Automation workstation as claimed in claim 1, its feature
Be, in described lysate containing concentration be the guanidine hydrochloride of 0.8M, concentration be the guanidinium isothiocyanate of 1.6M, concentration be 30mM's
Tween-20 that ethylenediaminetetraacetic acid (EDTA) that Tris-HCL, concentration are 30mM, concentration of volume percent are 5%, volume hundred
Proportion by subtraction concentration is the Triton-X100 of 5%, pH 8.0.
4. difficult trace forensic DNA extraction kit based on Automation workstation as claimed in claim 1, its feature
Be, described cleaning mixture contain concentration be the Tris-HCL of 10mM, concentration of volume percent be the ethanol of 80%, pH 7.5.
5. difficult trace forensic DNA extraction kit based on Automation workstation as claimed in claim 1, its feature
Be, described DNA eluent contain concentration be the Tris-HCL of 10mM, concentration be the ethylenediaminetetraacetic acid (EDTA) of 1mM, pH
8.0。
6. utilize difficult trace forensic DNA extraction kit based on Automation workstation described in claim 1 to extract DNA
Method, it is characterised in that comprise the steps:
(1), after sample being placed in centrifugal hand basket sleeve pipe, automatically working stands in and adds splitting of 150～600 μ l in each centrifugal basket
After solving liquid, the metal bath of 80～95 DEG C cracks, the time 20～50min, the sample after centrifugal segregation removing DNA, set
Pipe is prepared DNA mixed liquor；
(2) the DNA mixed liquor that step (1) prepares is mixed by Automation workstation with DNA extraction magnetic bead, DNA extraction magnetic bead and DNA
Mixed liquor mixed volume ratio is for 1:(10～30), solid-liquid separation, take solid, prepare the magnetic bead being adsorbed with DNA；
(3) what step (2) was prepared by Automation workstation is adsorbed with the magnetic bead cleaning mixture washing of DNA, is adsorbed with the magnetic bead of DNA
With the mixed volume of cleaning mixture ratio for 1:(10～80), prepare the magnetic bead being adsorbed with DNA after washing；
(4) the magnetic bead eluent being adsorbed with DNA after the washing that step (3) is prepared by Automation workstation is at 50～70 DEG C of bars
Eluting under part, is adsorbed with the magnetic bead of DNA and the mixed volume of eluent than for 1:(4～10 after washing), solid-liquid separation, take liquid
Body, prepares DNA solution.
7. method as claimed in claim 6, it is characterised in that in described step (1), cracking condition is: 90 DEG C of cracking
30min；
Preferably, in described step (1), centrifugal condition is: 9000～12000 turns/min is centrifuged 1～3min；Further preferably
, in described step (1), centrifugal condition is: 12000 turns/min is centrifuged 1min.
8. method as claimed in claim 6, it is characterised in that in described step (2), mixing condition is: room temperature, 1000 turns/
Min concussion mixing 2～10min；
Preferably, in described step (3), wash conditions is: room temperature, 1000 turns/min concussion cleans 0.5～2min.
9. method as claimed in claim 6, it is characterised in that in described step (4), elution requirement is: 65 DEG C, 1200 turns/
Min shakes eluting 0.1～1min.
10. method as claimed in claim 6, it is characterised in that in described step (2) and (4), solid-liquid separation is centrifugal, bar
Part is that 9000～12000 turns/min is centrifuged 1～3min；It is further preferred that in described step (2) and (4), centrifugal condition is:
12000 turns/min is centrifuged 1min.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107354149A (en) * | 2017-08-30 | 2017-11-17 | 广州奇辉生物科技有限公司 | A kind of kit and extracting method for extracting trace amount DNA |
| CN109055365A (en) * | 2018-09-29 | 2018-12-21 | 江苏齐耀生物科技有限公司 | A kind of Full-automatic magnetic beads method DNA extraction platform and operating method |
| CN109280662A (en) * | 2018-09-03 | 2019-01-29 | 海宁市公安局 | One kind being based on silicon class medium microsphere ultramicron DNA extraction purification kit |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101323852A (en) * | 2007-06-11 | 2008-12-17 | 北京东胜创新生物科技有限公司 | Reagent box and method for genome extraction on automatic nucleic acid extraction workstation |
| CN102352351A (en) * | 2011-10-17 | 2012-02-15 | 西安金磁纳米生物技术有限公司 | Method utilizing magnetic particles to purify deoxyribonucleic acid (DNA) from samples containing trace nucleic acid |
| CN105349532A (en) * | 2015-12-15 | 2016-02-24 | 杭州千基生物科技有限公司 | Method and kit for extracting free nucleic acid by using paramagnetic particle method |
-
2016
- 2016-06-24 CN CN201610474321.4A patent/CN106119242A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101323852A (en) * | 2007-06-11 | 2008-12-17 | 北京东胜创新生物科技有限公司 | Reagent box and method for genome extraction on automatic nucleic acid extraction workstation |
| CN102352351A (en) * | 2011-10-17 | 2012-02-15 | 西安金磁纳米生物技术有限公司 | Method utilizing magnetic particles to purify deoxyribonucleic acid (DNA) from samples containing trace nucleic acid |
| CN105349532A (en) * | 2015-12-15 | 2016-02-24 | 杭州千基生物科技有限公司 | Method and kit for extracting free nucleic acid by using paramagnetic particle method |
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|---|---|---|---|---|
| US11753687B2 (en) | 2008-05-14 | 2023-09-12 | Dermtech, Inc. | Diagnosis of melanoma and solar lentigo by nucleic acid analysis |
| CN107354149A (en) * | 2017-08-30 | 2017-11-17 | 广州奇辉生物科技有限公司 | A kind of kit and extracting method for extracting trace amount DNA |
| US11976332B2 (en) | 2018-02-14 | 2024-05-07 | Dermtech, Inc. | Gene classifiers and uses thereof in non-melanoma skin cancers |
| CN109280662A (en) * | 2018-09-03 | 2019-01-29 | 海宁市公安局 | One kind being based on silicon class medium microsphere ultramicron DNA extraction purification kit |
| CN109055365A (en) * | 2018-09-29 | 2018-12-21 | 江苏齐耀生物科技有限公司 | A kind of Full-automatic magnetic beads method DNA extraction platform and operating method |
| US11578373B2 (en) | 2019-03-26 | 2023-02-14 | Dermtech, Inc. | Gene classifiers and uses thereof in skin cancers |
| CN110208063A (en) * | 2019-06-27 | 2019-09-06 | 中国农业科学院农业质量标准与检测技术研究所 | A kind of on-line automaticization magnetic Nano material concussion-desorption apparatus |
| CN113088514A (en) * | 2021-05-06 | 2021-07-09 | 广州万淋潍生物科技有限公司 | Novel efficient trace detection material nucleic acid extraction reagent, kit and use method thereof |
| CN113604558A (en) * | 2021-08-09 | 2021-11-05 | 成都诺森医学检验有限公司 | Vitamin D receptor gene SNP locus detection reagent |
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