CN206298576U - A kind of reagent disc that pre-treatment is automated for detection of nucleic acids - Google Patents
A kind of reagent disc that pre-treatment is automated for detection of nucleic acids Download PDFInfo
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- CN206298576U CN206298576U CN201621267652.2U CN201621267652U CN206298576U CN 206298576 U CN206298576 U CN 206298576U CN 201621267652 U CN201621267652 U CN 201621267652U CN 206298576 U CN206298576 U CN 206298576U
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Abstract
The utility model discloses a kind of reagent disc that pre-treatment is automated for detection of nucleic acids, characterized in that, the reagent disc includes cover plate and reagent tray, reagent tray is provided with some reagent wells, packing has nucleic acid sequencing pre-treatment reagent in reagent wells, and aluminium foil sealer is set at reagent wells opening.Use the reagent disc that pre-treatment is automated for detection of nucleic acids of the present utility model, with reference to detection of nucleic acids pre-treatment automation equipment, the full-automatic of detection of nucleic acids pre-treatment, including extraction, library construction, product purification are capable of achieving, final product can be directly used for high-flux sequence, whole unattended.
Description
Technical field
The utility model belongs to nucleic acid molecules detection field, and in particular to a kind of to automate pre-treatment for detection of nucleic acids
Reagent disc.The utility model realizes detection of nucleic acids pre-treatment automatically, that is, before being automatically obtained nucleic acid extraction, amplification, purifying etc.
Treatment, the sample that is disposed can be detected that the utility model simplifies experiment process, reduces out through outside supporting sequenator
Wrong chance, improves the stability of whole detection.
Background technology
High throughput sequencing technologies are the novel molecular detection techniques that last decade starts development.The high flux of in the market main flow
Sequenator is mainly provided by Illumina, ThermoFisher etc., any high-flux sequence platform before being detected all
Can first require to extract DNA or RNA from biological specimen, DNA sample to be measured then be built sequencing library, i.e., in DNA to be measured
The general linker DNA sequence of the upper sequenator of two ends connection, many toolenzymes is with the addition of during entirely library is built and is eased up
System is rushed, is unfavorable for the reaction in later stage, so need to carry out the various enzymes of DNA purification process removal and buffer solution, so as to allow
The 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 purifying.
Nucleic acid extraction.Detection of nucleic acids first has to carry out sample nucleic acid extraction.Common method has ethanol precipitation, silicagel column
Combined techniques, glass bead method, magnetic-particle combination partition method.The methodical general principle of institute be exactly lysed sample --- with reference to ---
Cleaning --- four steps such as wash-out.Single sample whole process is about needed 40 minutes.
Library construction.The basic conception of library construction is connect plus the DNA supporting with follow-up sequencing instrument in DNA to be measured
Head.Basic procedure is：1. end-filling；2. base finally increases an A；3. jointing；4.PCR is expanded.Due to each step
There is independent enzyme and buffer system, so in conventional high-throughput sequencing library building process, in the middle of each secondary response all
Needs carry out the DNA purifying based on silicagel column or magnetic-particle.Also just say that the high-throughput sequencing library of routine builds flow and has：1.
End-filling；2. purify；3. base finally increases an A；4. purify；5. jointing；6. purify；7 steps such as 7.PCR amplifications
Suddenly.
Purifying.Various toolenzymes, DNA solution and magnetic-particle, pellosil, bead etc. are added in library construction process
Medium is combined, and cleaning fluid cleaning removes various impurity twice, finally uses elution DNA.
The all experiment links from original sample to final sequencing library are completed since then.Whole experiment process needs 5
~8 hours, it is required for mark test tube in order to avoid mistaking sample in each experiment link, wastes time and energy.
For above-mentioned sophisticated testing step, in the market has some instruments to replace artificial operation.Because high-flux sequence is
Technology brand-new in the market, there is the single equipment for each step, such as nucleic acid extraction can have polytype core
Sour extraction apparatus, library construction can be completed by automatic liquor removing workstation, and the purifying in library can be by having been manually done, it is also possible to logical
Cross automatic liquor removing workstation completion.Instrument for extracting nucleic acid, can once realize the 12-96 nucleic acid extraction not waited.Operator needs handle
Sample is put into instrument, and after 40 minutes, the sample that taking-up has been extracted is used after mark with treating subsequent experimental.Nucleic acid pre-treatment, instead
Should configure, temperature control, mixing can be realized by liquor removing workstation, start preceding operator and the matched reagent for needing is placed on work platform
On face, program is set, marked the developmental tube position of input and output, brought into operation.Because liquor removing workstation is open platform,
In whole multistep assays operating process, operator needs irregularly carry out 96 orifice plate sealers, open film, or irregularly carries out big
The lid opening and closing work of amount, although part work is completed by work station, but it is overall there is still a need for artificial a large amount of interventions, it is impossible to
The unattended of whole process is realized, multiple steps need manually to mark, sort, set.Simultaneously because experiment once,
Cannot temporarily increase or change experiment process again, there is the sample to need treatment once interim, or change experiment process, will be to entirety
The sample for participating in experiment produces influence.
The content of the invention
The purpose of this utility model is to overcome defect present in prior art, there is provided before being automated for detection of nucleic acids
The reagent disc for the treatment of, with reference to detection of nucleic acids pre-treatment automation equipment, is capable of achieving the full-automatic of detection of nucleic acids pre-treatment, including carry
Take, library construction, product purification, final product can be directly used for high-flux sequence, whole unattended.
It is a kind of for detection of nucleic acids automate pre-treatment reagent disc, it is characterised in that the reagent disc include cover plate and
Reagent tray, reagent tray is provided with packing in some reagent wells, reagent wells nucleic acid sequencing pre-treatment reagent, reagent wells opening
Place sets aluminium foil sealer.
Support of pipelines is set in aluminium foil sealer, layer of silica gel is provided between support of pipelines and cover plate.Carrying out detection of nucleic acids certainly
When dynamicization pre-treatment, the cover plate of reagent disc is first removed, the liquid-transfering device (such as liquid transfer gun head) of device can directly poke silicon
Glue and aluminium foil sealer, pipetting is carried out into chamber, and 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, reagent wells is still within sealing state, reagent will not volatilize in reagent wells influences whole
Body is tested.
The reagent disc is circular, square or rectangle, or other irregular shapes.Reagent wells can be according to reagent
The shape of disk makees optimization arrangement.
The nucleic acid sequencing pre-treatment reagent includes nucleic acid extracting reagent, library construction reagent and DNA purified reagents, including
But it is not limited to nucleic acid end-filling, adds the step reagents such as A, connection, purifying.
The reagent wells include cracking hole, magnetic bead hole, drawing holes, library construction hole, PCR reacting holes, purifying hole, final samples
This hole and waste liquid hole.
Reagent disc of the present utility model is supported the use with detection of nucleic acids pre-treatment automation equipment, and detection of nucleic acids pre-treatment is certainly
It is dynamic makeup put including：
Base for placing reagent disc, base is provided with temperature control module, rotary module and mixes module；
For realizing the magnetic separating module that magnetic-particle is separated, cleans and eluted；
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；
Moving guide rail platform for supporting liquid-transfering device；With
For the main frame for controlling temperature control module, rotary module, mixing the operation of module, magnetic separating module and liquid relief module.
Detection of nucleic acids pre-treatment automation equipment of the present utility model, the fully automatic operation nucleic acid in a separate agent disk
Extract, build the treatment such as storehouse, purifying, run as a unit autonomous closure per table apparatus, can be by series system infinite expanding
Quantity, realizes that multiple samples are detected simultaneously.
The utility model reagent disc is porous reagent disk, and nucleic acid sequencing pre-treatment reagent is preinstalled with hole.Porous reagent disk
It is preferred that using disposable separate agent disk, nucleic acid sequencing pre-treatment reagent to include but is not limited to contain nucleic acid extracting reagent, library
Build reagent and DNA purified reagents.
Sample to be detected is completed from before initial sample, nucleic acid extraction, detection of nucleic acids after adding reagent disc in the reagent disc
Whole flows for the treatment of.
The liquid relief module includes liquid relief pump.
The magnetic separating module includes the movable magnet beside base and reagent disc.
Detection of nucleic acids pre-treatment automation equipment has temperature control module, liquid relief module, mixes module and magnetic separating module,
It is capable of achieving temperature control, liquid relief, mixing, magnetic separating function.Mixing module makes device inside be capable of achieving level vortex, vertically mixes up and down
Even sample.Temperature control module can be heated in one or more steps to reagent in reagent disc, low temperature, rise intermittent warming.Move
Liquid module sees the transfer for realizing the liquid between reagent disc difference reagent wells, can be built-in by instrument or reagent disc includes one or many
Individual liquid transfer gun head is realized.Magnetic separating module can be by setting electromagnet or permanent magnet realizes that magnetic-particle is separated, cleans, washed
It is de-.
In being embodied at one, detection of nucleic acids pre-treatment automation equipment is using the disposable reagent disc for abandoning, reagent
Disk is contained within extracts reagent, library construction reagent, DNA purified reagents.Sample is added after reagent disc sample aperture, and instrument internal is moved
Liquid adds lysate；Carry out mixing 10 minutes using instrument；Liquid relief is added and combines liquid and magnetic bead, is mixed 10 minutes；Using magnetic force
Sorting module absorption magnetic bead, liquid relief module removal supernatant adds 500ul cleaning fluids, mixes magnetic bead 2 minutes, magnetic separating removal
Cleaning fluid, in triplicate；30ul eluents and magnetic bead mixing are added, 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 joints, DNA connections
Enzyme, 20 degree 30 minutes, 75 degree 20 minutes inactivate；Liquid relief goes to purify hole, adds magnetic bead and purifying combination liquid, mixes 10 minutes；Plus
Magnetic separating, removes supernatant, and 500ul cleaning fluids clean magnetic bead twice；50ul eluents add magnetic bead, and 60 degree are heated 5 minutes, are moved
To final sample room, sample nucleic acid pre-treatment terminates liquid DNA.
The utility model is realized in a separate agent disk for closing, full-automatic unmanned detection of nucleic acids pre-treatment on duty
Automation, operator is put into sample and can allow instrument automatic running after reagent disc, and product can be directly used for later stage sequencing point
Analysis.Enormously simplify experiment process, it is to avoid conventional manual operation it is cumbersome, it also avoid using instrument for extracting nucleic acid, liquid relief work
When the instrument such as stand is supported the use, it is necessary to repeatedly mark test tube, the links such as test tube manually are moved, prevented from experiment process
The possibility that sample is obscured.
Reagent disc combination detection of nucleic acids pre-treatment automation equipment of the present utility model, can carry out automating preceding place to nucleic acid
Reason, with advantages below：
(1) it is full-automatic.It is full-automatic that instrument automatically realizes 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 no more than 5 minutes, sample
Be put into reagent disc, reagent disc be put into instrument by fully automatic operation.
(2) close.Reagent disc runs in a closed system, it is ensured that sample will not be obscured, also ensure that sample it
Between will not pollute mutually.
(3) it is quick.Due to being directly connected between the different tests step realized by instrument, reducing multiple links needs
The sample labeling wanted works, and greatly accelerates bulk testing speed.
(4) may be programmed.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 one operating unit of single sample, but can infinitely be amplified by cascade, so that clever
Work realizes that high flux is operated.
Brief description of the drawings
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 that the method that provides the utility model and technical scheme are further illustrated, but should not managed
Solution limitation of the present utility model in pairs.
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 moved liquid transfer gun head 5 along moving guide rail platform 1, and
Rotated by rotary module.Moving guide rail platform 1 is moved and rotated and realized by liquid relief pump and liquid transfer gun head on reagent disc top
Liquid relief.The corresponding reagent disc position of base 2 has temperature control module.Base 2 and reagent disc are fitted, there is provided temperature control.Reagent disc can
Rotated by the rotary module on base 2, moved.
Reagent disc as Figure 2-3, including cover plate 6 and reagent tray 7, reagent tray 7 are provided with some reagent wells 8,
Packing has nucleic acid sequencing pre-treatment reagent in reagent wells 8, and aluminium foil sealer 9 is set at reagent wells opening.Pipe is set 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 hydrochlorides, 20% different
Propyl alcohol)；2-magnetic bead hole (40ul silanizations magnetic bead)；3-cleaning fluid (80% ethanol)；4-combine liquid (60% isopropanol)；5—
Eluent (pure water pH8.0)；6-drawing holes；7-library construction hole；(Klenow DNA's 8-end-filling enzyme mix aperture are polymerized
Enzyme I, T4 polynueleotide kinase)；9-coupled reaction mix aperture (T4DNA ligases)；10-purifying hole (40ul silanization magnetic
Pearl)；11-preformed hole；12-final sample hole；13-waste liquid hole；14-preformed hole；15-preformed hole.
It is as shown in Figure 44 detection of nucleic acids pre-treatment automation equipment series connection schematic diagrames, can arbitrarily connects as needed,
Realize that multiple samples are detected simultaneously.
Embodiment 2
Plasma DNA is small fragment, fragmentation DNA, and between 100~300bp of fragment length, peak value is in 160bp or so.
1997, Lu Yu penetrating judgments awarded discovery dissociative DNA containing fetus in maternal blood slurry, are inspection so as to start with dissociative DNA
Source is surveyed, whether the pregnant youngster of analysis pregnant woman institute has chromosome abnormality.The circulating tumor from tumour cell is there is also in blood plasma
DNA (ctDNA), ctDNA are widely used for doing tumour liquid biopsy, can be with the early diagnosis of non-invasive, with diagnosis, prognosis
Follow-up etc..
Conventional detection method is to be included extracting DNA in blood plasma first by clinical sample, carries out detection of nucleic acids treatment, detection
Three steps.Completing whole pattern detection needs 6~13 hours.Wherein sample nucleic acid is extracted and detection pre-treatment link work
It is cumbersome.
One separate agent disk of closing may be implemented in using detection of nucleic acids pre-treatment automation equipment of the present utility model,
The automation of full-automatic unmanned detection of nucleic acids pre-treatment on duty, operator is put into sample after reagent disc can allow instrument automatic
Operation, product can be directly used for later stage sequencing analysis.Concrete operation method is as follows：
600ul plasma samples, being put into detection of nucleic acids pre-treatment automation equipment carries out full-automatic high flux library construction.
1. 600ul blood plasma adds reagent disc cracking hole, cracking hole to contain 1.2ml blood plasma lysate (6M guanidine hydrochlorides, 20%
Isopropanol)；
2. mixing module mix 5 minutes, liquid relief module, and 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 cleaning fluids (80% ethanol) cleaning magnetic bead three times, supernatant moves to waste liquid hole；
5. 60ul eluents move to drawing holes, 56 degree of 10 minutes eluted dnas；
6. DNA moves to Jian Ku holes, adds filling-in enzyme (DNA polymerase i, T4 polynueleotide kinases) mixing 10ul；
7. 37 degree 30 minutes, 75 degree inactivate for 20 minutes；
8. DNA joint 5ul are added, DNA ligase (T4 nucleotides ligase) mixture 20ul is added；
9. 16 degree 20 minutes, 75 degree inactivate for 20 minutes；
10. liquid relief module, pipettes all of product to purified reagent hole, purifies the magnetic required for purification reaction is contained in 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；
Twice, upper movable magnet, supernatant moves to waste liquid hole to 12. 500ul cleaning fluids cleaning magnetic bead；
13. 40ul eluents go to purify hole, 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 research object of transcript profile sequencing to be transcribed out for specific cells under a certain functional status
Summation, mainly includes mRNA and non-coding RNA.Transcript profile research is basis and the starting point of gene function and structural research, is led to
High-flux sequence of new generation is crossed, a certain species particular organization or organ can be rapidly obtained comprehensively under a certain state almost
All transcript sequence information, are widely used to the fields such as basic research, clinical diagnosis and medicament research and development.
Conventional detection method is to include extracting RNA in whole blood, blood plasma by clinical sample first, is carried out at detection of nucleic acids
Reason, three steps of detection.Completing whole pattern detection needs 6~30 hours.Wherein sample nucleic acid is extracted and detection pre-treatment
Link intricate operation.
One separate agent disk of closing may be implemented in using detection of nucleic acids pre-treatment automation equipment of the present utility model,
The automation of full-automatic unmanned detection of nucleic acids pre-treatment on duty, operator is put into sample after reagent disc can allow instrument automatic
Operation, product can be directly used for later stage sequencing analysis.Concrete operation method is as follows：
200ul whole blood samples, are put into detection of nucleic acids pre-treatment automation equipment and realize that storehouse full automatic treatment is set up in transcription.
1. after 200ul samples add reagent disc sample aperture, liquid relief adds 300ul lysates；
2. mixing module mix 10 minutes；
3. liquid relief adds 200ul combinations liquid and 10ul magnetic beads, mixes 10 minutes；
4. using magnetic separating module absorption magnetic bead, liquid relief module removal supernatant；
5. 500ul cleaning fluids are added, magnetic bead is mixed 2 minutes, magnetic separating removal cleaning fluid, in triplicate；
6. 30ul eluents and magnetic bead mixing are added, and 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, the cleaning of 500ul cleaning fluids, magnetic separating removal supernatant；
10. 30ul eluents add 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 enzyme and dATP, 37 degree 30 minutes, 65 degree fire extinguishing 5 minutes；
12. liquid reliefs add DNA joints, ligase, 20 degree 30 minutes, 65 degree 5 minutes inactivations；
13. addition 10ul magnetic beads and 200ul purifying combine liquid, mix 10 minutes；
14. add magnetic separating, remove supernatant, and 500ul cleaning fluids clean magnetic bead twice；
15. 50ul eluents add magnetic bead, and 60 degree are heated 5 minutes；
16. liquid relief 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. it is a kind of for detection of nucleic acids automate pre-treatment reagent disc, it is characterised in that the reagent disc include cover plate and examination
Agent pallet, reagent tray is provided with packing in some reagent wells, reagent wells nucleic acid sequencing pre-treatment reagent, at reagent wells opening
Aluminium foil sealer is set.
2. it is as claimed in claim 1 to be used for the reagent disc that detection of nucleic acids automates pre-treatment, it is characterised in that in aluminium foil sealer
Support of pipelines is set, layer of silica gel is provided between support of pipelines and cover plate.
3. it is as claimed in claim 1 to be used for the reagent disc that detection of nucleic acids automates pre-treatment, it is characterised in that the reagent disc
It is circular, square or rectangle.
4. it is as claimed in claim 1 to be used for the reagent disc that detection of nucleic acids automates pre-treatment, it is characterised in that the nucleic acid is surveyed
Sequence pre-treatment reagent includes nucleic acid extracting reagent, library construction reagent and DNA purified reagents.
5. it is as claimed in claim 1 to be used for the reagent disc that detection of nucleic acids automates pre-treatment, it is characterised in that the reagent wells
Including cracking hole, magnetic bead hole, drawing holes, library construction hole, PCR reacting holes, purifying hole, final sample hole and waste liquid hole.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201621267652.2U CN206298576U (en) | 2016-11-23 | 2016-11-23 | A kind of reagent disc that pre-treatment is automated for detection of nucleic acids |
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| CN201621267652.2U CN206298576U (en) | 2016-11-23 | 2016-11-23 | A kind of reagent disc that pre-treatment is automated for detection of nucleic acids |
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| CN206298576U true CN206298576U (en) | 2017-07-04 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019192595A1 (en) * | 2018-04-04 | 2019-10-10 | 美林美邦（厦门）生物科技有限公司 | Reagent cup box for sample processing and detection provided with material transfer structure |
-
2016
- 2016-11-23 CN CN201621267652.2U patent/CN206298576U/en active Active
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019192595A1 (en) * | 2018-04-04 | 2019-10-10 | 美林美邦（厦门）生物科技有限公司 | Reagent cup box for sample processing and detection provided with material transfer structure |
| CN110869517A (en) * | 2018-04-04 | 2020-03-06 | 美林美邦（厦门）生物科技有限公司 | Sample treatment and detection reagent cup box with material transfer structure |
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