CN106399055A - Reagent plate for automatic pretreatment of nucleic acid detection - Google Patents
Reagent plate for automatic pretreatment of nucleic acid detection Download PDFInfo
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- CN106399055A CN106399055A CN201611046472.6A CN201611046472A CN106399055A CN 106399055 A CN106399055 A CN 106399055A CN 201611046472 A CN201611046472 A CN 201611046472A CN 106399055 A CN106399055 A CN 106399055A
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- reagent
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Abstract
The invention discloses a reagent plate for automatic pretreatment of nucleic acid detection. The reagent plate is characterized by comprising a cover plate and a reagent tray, wherein a plurality of reagent holes are formed in the reagent tray; nucleic acid sequencing pretreatment reagents are contained in the reagent holes; aluminum foil sealing films are arranged at the opening of the reagent holes. The reagent plate for automatic pretreatment of nucleic acid detection, provided by the invention, in combination with an automatic pretreatment device for nucleic acid detection, can realize the full automatic pretreatment of the nucleic acid detection, including extraction, library construction and product purification; the final products can be directly applied to high-throughput sequencing; the whole process is unmanned.
Description
Technical field
The invention belongs to nucleic acid molecules detection field is and in particular to a kind of reagent for the pre-treatment of detection of nucleic acids automatization
Disk.Present invention achieves detection of nucleic acids pre-treatment is full-automatic, that is, it is automatically obtained the pre-treatments such as nucleic acid extraction, amplification, purification, process
Finish sample to be detected through outside supporting sequenator, this invention simplifies experiment process, decrease error chance, improve
The stability of whole detection.
Background technology
High throughput sequencing technologies are the novel molecular detection techniques that last decade starts to develop.The high flux of main flow on market
Sequenator is mainly provided by Illumina, ThermoFisher etc., any high-flux sequence platform before being detected all
Can require to extract DNA or RNA first from biological specimen, then DNA sample to be measured be built sequencing library, that is, in DNA to be measured
Two ends connect the general linker DNA sequence of upper sequenator, with the addition of many toolenzymes and ease up during whole structure library
Rush system, be unfavorable for the reaction in later stage, so needing to carry out the DNA purification process various enzymes of removal and buffer, thus allowing
This to be measured segment DNA can carry out sequencing reaction on the different chip of sequenator.
Conventional high-flux sequence detection pre-treatment includes nucleic acid extraction, library construction, three steps of purification.
Nucleic acid extraction.Detection of nucleic acids first has to carry out sample nucleic acid extraction.Common method has ethanol precipitation, silicagel column
Combined techniqueses, glass bead method, magnetic-particle combine partition method.The methodical ultimate principle of institute is exactly that lysed sample combines
Four steps such as cleaning eluting.Single sample whole process about needs 40 minutes.
Library construction.The basic conception of library construction is to connect in the DNA that DNA to be measured adds and follow-up sequencing instrument is supporting
Head.Basic procedure is：1. end-filling；2. base finally increases an A；3. jointing；4.PCR expands.Due to each step
There are independent enzyme and buffer system, so in conventional high-throughput sequencing library building process, in the middle of each secondary response all
Need to carry out the DNA purification based on silicagel column or magnetic-particle.Also just say that the high-throughput sequencing library of routine builds flow process and has：1.
End-filling；2. purification；3. base finally increases an A；4. purification；5. jointing；6. purification；7 steps such as 7.PCR amplification
Suddenly.
Purification.Various toolenzymes, DNA solution and magnetic-particle, pellosil, bead etc. is added in library construction process
Medium combines, and cleanout fluid cleaning removes various impurity twice, finally uses elution DNA.
Complete all test links from original sample to final sequencing library since then.Whole experiment process needs 5
～8 hours, being required for labelling test tube in order to avoid mistaking sample in each experiment link, wasting time and energy.
For above-mentioned sophisticated testing step, market there are some instruments replace artificial operation.Because high-flux sequence is
Technology brand-new in the market, has the single equipment for each step, and such as nucleic acid extraction can have polytype core
Sour extraction apparatus, library construction can be completed by automatic liquor removing workstation, and the purification in library can be by having been manually done it is also possible to lead to
Cross automatic liquor removing workstation to complete.Instrument for extracting nucleic acid, once can achieve the 12-96 nucleic acid extraction not waited.Operator needs handle
Sample is put in instrument, after 40 minutes, takes out the sample having extracted, and is used with treating subsequent experimental after labelling.Nucleic acid pre-treatment, instead
Should configure, temperature control, mixing can be realized by liquor removing workstation, start front operator and the matched reagent needing is placed on work platform
On face, set program, the developmental tube position of the good input and output of labelling, bring into operation.Because liquor removing workstation is open platform,
In whole multistep assays operating process, operator needs irregularly to carry out 96 orifice plate sealers, opens film, or irregularly carries out big
The lid opening and closing work of amount although part work to be completed by work station, but overall there is still a need for manually a large amount of interventions it is impossible to
Realize the unmanned of whole process, multiple steps need manually to come labelling, sequence, setting.Simultaneously because test once,
Cannot increase again temporarily or change experiment process, once there is sample to need to process temporarily, or change experiment process, will be to entirety
The sample participating in test produces impact.
Content of the invention
It is an object of the invention to overcoming defect present in prior art, provide for the pre-treatment of detection of nucleic acids automatization
Reagent disc, bind nucleic acid detect pre-treatment automation equipment, achievable detection of nucleic acids pre-treatment full-automatic, include extraction,
Library construction, product purification, final product can be directly used for high-flux sequence, whole unmanned.
A kind of reagent disc for the pre-treatment of detection of nucleic acids automatization it is characterised in that described reagent disc include cover plate and
Reagent tray, reagent tray is provided with some reagent wells, and reagent in the hole subpackage has nucleic acid sequencing pre-treatment reagent, reagent wells opening
Place's setting aluminium foil sealer.
In aluminium foil sealer, support of pipelines is set, between support of pipelines and cover plate, is provided with layer of silica gel.Carrying out detection of nucleic acids certainly
When dynamicization pre-treatment, first remove the cover plate of reagent disc, the liquid-transfering device (such as liquid transfer gun head) of device can directly poke silicon
Glue and aluminium foil sealer, enter chamber and carry out pipetting, after operation terminates, liquid-transfering device exits aluminium foil and silica gel.Aluminium foil sealer
Through hole can be left, and after silica gel resilience, make reagent wells be still within sealing state, reagent in the hole reagent will not volatilize impact whole
Body is tested.
Described reagent disc is circular, square or rectangle or other are irregularly shaped.Reagent wells can be according to reagent
The shape of disk is made to optimize arrangement.
Described nucleic acid sequencing pre-treatment reagent includes nucleic acid extracting reagent, library construction reagent and DNA purified reagent, including
But it is not limited to nucleic acid end-filling, add the step reagent such as A, connection, purification.
Described reagent wells include cracking hole, magnetic bead hole, drawing holes, library construction hole, PCR reacting hole, purification hole, final samples
This hole and waste liquid hole.
The reagent disc of the present invention is supported the use with detection of nucleic acids pre-treatment automation equipment, detection of nucleic acids pre-treatment automatization
Device includes：
For placing the base of reagent disc, base is provided with temperature control module, rotary module and mixes module；
For realizing the magnetic separating module of magnetic-particle separation, cleaning and eluting；
For the liquid-transfering device of reagent in transfering reagent disk, liquid-transfering device is provided with liquid relief module and liquid transfer gun head；
For supporting the moving guide rail platform of liquid-transfering device；With
For controlling temperature control module, rotary module, mixing the main frame that module, magnetic separating module and liquid relief module are run.
The detection of nucleic acids pre-treatment automation equipment of the present invention, in a separate agent disk, fully automatic operation nucleic acid carries
Take, build the process such as storehouse, purification, every table apparatus run as a unit autonomous closure, can pass through series system infinite expanding number
Amount, realizes multiple samples and detects simultaneously.
Reagent disc of the present invention is porous reagent disk, and in the hole is preinstalled with nucleic acid sequencing pre-treatment reagent.Porous reagent disk is preferred
Using disposable separate agent disk, nucleic acid sequencing pre-treatment reagent including but not limited to contains nucleic acid extracting reagent, library construction
Reagent and DNA purified reagent.
Sample to be detected completes before initial sample, nucleic acid extraction, detection of nucleic acids after adding reagent disc in this reagent disc
The whole flow processs processing.
Described liquid relief module includes liquid relief pump.
Described magnetic separating module includes the movable magnet located at base and reagent disc side.
Detection of nucleic acids pre-treatment automation equipment has temperature control module, liquid relief module, mixes module and magnetic separating module,
Achievable temperature control, liquid relief, mixing, magnetic separating function.Mixing module makes to can achieve inside device that level is vortexed, vertically mixes up and down
Even sample.Temperature control module can carry out to reagent in reagent disc heating in one or more steps, low temperature, rises intermittent warming.Move
Liquid module sees the transfer realizing liquid between reagent disc difference reagent wells, can be built-in or reagent disc includes one or many by instrument
Individual liquid transfer gun head is realized.Magnetic separating module can be realized magnetic-particle and separate, cleans, washes by arranging electric magnet or permanent magnet
De-.
In being embodied as at one, detection of nucleic acids pre-treatment automation equipment is using the disposable reagent disc abandoning, reagent
Disk is contained within extracts reagent, library construction reagent, DNA purified reagent.After sample adds reagent disc sample aperture, instrument internal is moved
Liquid adds lysate；Carry out mixing 10 minutes using instrument；Liquid relief adds with reference to liquid and magnetic bead, mixes 10 minutes；Using magnetic force
Sorting module absorption magnetic bead, liquid relief module removes supernatant, adds 500ul cleanout fluid, mixes magnetic bead 2 minutes, magnetic separating removes
Cleanout fluid, in triplicate；Add 30ul eluent and magnetic bead mixing, 60 degree are heated 5 minutes；Library is cleaned in liquid relief module liquid relief
Build hole；Liquid relief add end-filling enzyme, 37 degree 30 minutes, 75 degree inactivation 20 minutes；Liquid relief adds DNA joint, and DNA connects
Enzyme, 20 degree 30 minutes, 75 degree 20 minutes inactivation；Liquid relief goes to purification hole, adds magnetic bead and purification to combine liquid, mixes 10 minutes；Plus
Magnetic separating, removes supernatant, 500ul cleanout fluid cleaning magnetic bead twice；50ul eluent adds magnetic bead, and 60 degree are heated 5 minutes, move
To final sample room, sample nucleic acid pre-treatment terminates liquid DNA.
Present invention achieves one closing separate agent disk, full-automatic unmanned detection of nucleic acids pre-treatment on duty from
Dynamicization, operator puts into sample and can allow instrument automatic running after reagent disc, and product can be directly used for later stage sequencing analysis.Greatly
Simplify greatly experiment process, it is to avoid conventional manual operation loaded down with trivial details, it also avoid using instrument for extracting nucleic acid, liquor removing workstation etc.
When instrument supports the use, need multiple labelling test tube, manually to move the links such as test tube, sample has been prevented from experiment process
The possibility obscured.
The reagent disc bind nucleic acid detection pre-treatment automation equipment of the present invention, can carry out automatization's pre-treatment to nucleic acid,
There is advantages below：
(1) full-automatic.It is full-automatic that instrument automatically achieves detection of nucleic acids pre-treatment, including nucleic acid extraction, end-filling,
Plus the step such as A, jointing, product purification.Full-automatic unmanned on duty, whole process manual operations is less than 5 minutes, sample
Put into reagent disc, reagent disc is put into and got final product fully automatic operation in instrument.
(2) close.Reagent disc runs it is ensured that sample will not be obscured in a closed system, also ensure that sample it
Between will not pollute mutually.
(3) quick.Due to being directly connected between the different tests step realized by instrument, decreasing multiple links needs
The sample labeling work wanted, greatly accelerates bulk testing speed.
(4) programmable.Because instrument has the modules such as mixing, liquid relief, temperature control, magnetic separating, therefore equipment can be according to reality
Border needs to be developed again, and autgmentability is big.
(5) flux is high.Instrument is single one operating unit of sample, but infinitely can be amplified by cascade, thus clever
High flux operation is realized in work.
Brief description
Fig. 1 is detection of nucleic acids pre-treatment automation equipment structural representation.
Fig. 2 is reagent disc schematic diagram.
Fig. 3 is reagent disc partial sectional view.
Fig. 4 is that multiple detection of nucleic acids pre-treatment automation equipments realize series connection extension schematic diagram.
Specific embodiment
Specific examples below is to the method for present invention offer and further illustrating of technical scheme, but is not construed as
Limitation of the present invention.
Embodiment 1
Detection of nucleic acids pre-treatment automation equipment as shown in Figure 1, by moving guide rail platform 1, base 2, movable magnet 3,
Liquid relief pump 4 and liquid transfer gun head 5 are constituted., on liquid relief pump 4, liquid relief pump can be mobile along moving guide rail platform 1 for liquid transfer gun head 5, and
Rotated by rotary module.Moving guide rail platform 1 passes through liquid relief pump and liquid transfer gun head and moves on reagent disc top and rotate realization
Liquid relief.The corresponding reagent disc position of base 2 has temperature control module.Base 2 and reagent disc laminating, provide temperature control.Reagent disc can
Carry out rotating, move by the rotary module on base 2.
Reagent disc as Figure 2-3, including cover plate 6 and reagent tray 7, reagent tray 7 is provided with some reagent wells 8,
In reagent wells 8, subpackage has nucleic acid sequencing pre-treatment reagent, arranges aluminium foil sealer 9 at reagent wells opening.Setting pipe in aluminium foil sealer 9
Road support 10, is provided with layer of silica gel 11 between support of pipelines and cover plate.Reagent wells include：1 cracking hole is (6M guanidine hydrochloride, 20% different
Propanol)；2 magnetic bead holes (40ul silanization magnetic bead)；3 cleanout fluid (80% ethanol)；4 combine liquid (60% isopropanol)；5—
Eluent (pure water pH8.0)；6 drawing holes；7 library construction holes；(Klenow DNA is polymerized 8 end-filling enzyme mix aperture
Enzyme I, T4 polynueleotide kinase)；9 coupled reaction mix aperture (T4DNA ligase)；10 purification holes (40ul silanization magnetic
Pearl)；11 preformed holes；12 final sample holes；13 waste liquid holes；14 preformed holes；15 preformed holes.
It is 4 detection of nucleic acids pre-treatment automation equipment series connection schematic diagrams as shown in Figure 4, can arbitrarily connect as needed,
Realize multiple samples to detect simultaneously.
Embodiment 2
Plasma DNA is small fragment, fragmentation DNA, and between fragment length 100～300bp, peak value is in 160bp.
1997, Lu Yu penetrating judgment awarded discovery dissociative DNA containing fetus in maternal blood slurry, thus starting with dissociative DNA for inspection
Survey source, whether the pregnant youngster of analysis anemia of pregnant woman institute has chromosomal abnormality.The circulating tumor from tumor cell is there is also in blood plasma
DNA (ctDNA), ctDNA are widely used for doing tumor liquid biopsy, can be with the early diagnosiss of non-invasive, with diagnosis, prognosis
Follow-up etc..
Conventional detection method is to include extracting DNA in blood plasma by clinical sample first, carries out detection of nucleic acids process, detection
Three steps.Completing whole pattern detection needs 6～13 hours.Wherein sample nucleic acid extracts and detection pre-treatment link work
Loaded down with trivial details.
May be implemented in the separate agent disk of a closing using the detection of nucleic acids pre-treatment automation equipment of the present invention, entirely certainly
Move the automatization of unattended detection of nucleic acids pre-treatment, operator puts into sample and instrument can be allowed after reagent disc automatically to transport
OK, product can be directly used for later stage sequencing analysis.Concrete operation method is as follows：
600ul plasma sample, puts into detection of nucleic acids pre-treatment automation equipment and carries out full-automatic high flux library construction.
1. 600ul blood plasma add reagent disc cracking hole, cracking hole contain 1.2ml blood plasma lysate (6M guanidine hydrochloride, 20%
Isopropanol)；
2. mix module to carry out mixing 5 minutes, liquid relief module, liquid relief 600ul is to drawing holes and magnetic bead (40ul silane successively
Change magnetic bead) combine；
3. movable magnet realizes magnetic separating, and supernatant moves to waste liquid hole；
4. 500ul cleanout fluid (80% ethanol) cleaning magnetic bead three times, supernatant moves to waste liquid hole；
5. 60ul eluent moves to drawing holes, 56 degree of 10 minutes eluted dnas；
6. DNA moves to Jian Ku hole, adds filling-in enzyme (DNA polymerase i, T4 polynueleotide kinase) mixing 10ul；
7. 37 degree 30 minutes, 75 degree of inactivations in 20 minutes；
8. add DNA joint 5ul, add DNA ligase (T4 nucleotide ligase) mixture 20ul；
9. 16 degree 20 minutes, 75 degree of inactivations in 20 minutes；
10. liquid relief module, pipettes all of product to purified reagent hole, the magnetic required for purification reaction is contained in purification hole
Pearl；
11. mix sample and magnetic bead up and down using liquid relief module, stand 5 minutes, and upper movable magnet stands 5 minutes, liquid relief
Module moves supernatant to waste liquid hole；
12. 500ul cleanout fluid clean magnetic beads twice, upper movable magnet, and supernatant moves to waste liquid hole；
13. 40ul eluents go to purification hole, and eluted dna, using liquid relief module liquid relief to final sample hole；
14. products can be directly used for follow-up high-flux sequence detection.
Embodiment 3
All RNA's that the object of study of transcript profile sequencing can transcribe out under a certain functional statuses for specific cells
Summation, main inclusion mRNA and non-coding RNA.It is basis and the starting point of gene function and structural research that transcript profile is studied, and leads to
Cross high-flux sequence of new generation, can rapidly obtain a certain species particular organization or organ under a certain state almost comprehensively
All transcript sequence information, are widely used to the fields such as basic research, clinical diagnosises and medicament research and development.
Conventional detection method is to include whole blood, extracting RNA in blood plasma by clinical sample first, carries out at detection of nucleic acids
Reason, three steps of detection.Completing whole pattern detection needs 6～30 hours.Wherein sample nucleic acid extracts and detection pre-treatment
Link intricate operation.
May be implemented in the separate agent disk of a closing using the detection of nucleic acids pre-treatment automation equipment of the present invention, entirely certainly
Move the automatization of unattended detection of nucleic acids pre-treatment, operator puts into sample and instrument can be allowed after reagent disc automatically to transport
OK, product can be directly used for later stage sequencing analysis.Concrete operation method is as follows：
200ul whole blood sample, puts into detection of nucleic acids pre-treatment automation equipment and realizes transcription establishment storehouse full automatic treatment.
1., after 200ul sample adds reagent disc sample aperture, liquid relief adds 300ul lysate；
2. mix module to carry out mixing 10 minutes；
3. liquid relief adds 200ul to combine liquid and 10ul magnetic bead, mixes 10 minutes；
4. utilize magnetic separating module to adsorb magnetic bead, liquid relief module removes supernatant；
5. add 500ul cleanout fluid, mix magnetic bead 2 minutes, magnetic separating removes cleanout fluid, in triplicate；
6. add 30ul eluent and magnetic bead mixing, 60 degree are heated 5 minutes；
7. liquid relief module liquid relief supernatant is to drawing holes；
8. drawing holes contains the magnetic bead of 30ul oligo dT, 65 degree 30 minutes；
9. magnetic separating, removes supernatant, and 500ul cleanout fluid cleans, and magnetic separating removes supernatant；
10. 30ul eluent adds drawing holes, and 60 degree are heated 5 minutes, and liquid relief supernatant cDNA goes to library construction hole；
11. liquid reliefs add end-filling enzymes and dATP, 37 degree 30 minutes, 65 degree of fire extinguishings 5 minutes；
12. liquid reliefs add DNA joints, ligase, 20 degree 30 minutes, 65 degree 5 minutes inactivate；
13. addition 10ul magnetic beads and 200ul purification combine liquid, mix 10 minutes；
14. add magnetic separating, remove supernatant, 500ul cleanout fluid cleaning magnetic bead twice；
15. 50ul eluents add magnetic bead, and 60 degree are heated 5 minutes；
16. liquid reliefs DNA are to final sample room, the off-test of this automatic processing device；
17. final DNA are used directly for subsequent high pass and measure sequence detection.
Claims (5)
1. a kind of reagent disc for the pre-treatment of detection of nucleic acids automatization is it is characterised in that described reagent disc includes cover plate and examination
Agent pallet, reagent tray is provided with some reagent wells, and reagent in the hole subpackage has nucleic acid sequencing pre-treatment reagent, at reagent wells opening
Setting aluminium foil sealer.
2. it is used for as claimed in claim 1 the reagent disc of detection of nucleic acids automatization pre-treatment it is characterised in that aluminium foil sealer
Setting support of pipelines, is provided with layer of silica gel between support of pipelines and cover plate.
3. it is used for as claimed in claim 1 the reagent disc of detection of nucleic acids automatization pre-treatment it is characterised in that described reagent disc
For circular, square or rectangle.
4. it is used for the reagent disc of detection of nucleic acids automatization pre-treatment as claimed in claim 1 it is characterised in that described nucleic acid is surveyed
Sequence pre-treatment reagent includes nucleic acid extracting reagent, library construction reagent and DNA purified reagent.
5. it is used for as claimed in claim 1 the reagent disc of detection of nucleic acids automatization pre-treatment it is characterised in that described reagent wells
Including cracking hole, magnetic bead hole, drawing holes, library construction hole, PCR reacting hole, purification hole, final sample hole and waste liquid hole.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754339A (en) * | 2016-12-14 | 2017-05-31 | 杭州杰毅麦特医疗器械有限公司 | Detection of nucleic acids pre-treatment automatic processing device |
| CN108196076A (en) * | 2017-07-11 | 2018-06-22 | 赛纳生物科技(北京)有限公司 | A kind of kit and its reagent extraction mechanism |
| CN109957492A (en) * | 2017-12-26 | 2019-07-02 | 安诺优达基因科技(北京)有限公司 | A kind of automatic fluid processing workstation for two generations sequencing DNA library building |
| CN111902527A (en) * | 2018-03-29 | 2020-11-06 | 荣研化学株式会社 | Full-automatic gene detection device |
| CN112608813A (en) * | 2020-12-21 | 2021-04-06 | 上海思路迪生物医学科技有限公司 | Multi-degree-of-freedom library preparation card box with external power source and method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754339A (en) * | 2016-12-14 | 2017-05-31 | 杭州杰毅麦特医疗器械有限公司 | Detection of nucleic acids pre-treatment automatic processing device |
| CN108196076A (en) * | 2017-07-11 | 2018-06-22 | 赛纳生物科技(北京)有限公司 | A kind of kit and its reagent extraction mechanism |
| CN109957492A (en) * | 2017-12-26 | 2019-07-02 | 安诺优达基因科技(北京)有限公司 | A kind of automatic fluid processing workstation for two generations sequencing DNA library building |
| CN111902527A (en) * | 2018-03-29 | 2020-11-06 | 荣研化学株式会社 | Full-automatic gene detection device |
| CN112608813A (en) * | 2020-12-21 | 2021-04-06 | 上海思路迪生物医学科技有限公司 | Multi-degree-of-freedom library preparation card box with external power source and method |
| CN112608813B (en) * | 2020-12-21 | 2022-07-01 | 上海思路迪生物医学科技有限公司 | Multi-degree-of-freedom library preparation card box with external power source and method |
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Application publication date: 20170215 |