CN104673625B - Automatic reaction device and method for pretreating cells - Google Patents
Automatic reaction device and method for pretreating cells Download PDFInfo
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- CN104673625B CN104673625B CN201510080030.2A CN201510080030A CN104673625B CN 104673625 B CN104673625 B CN 104673625B CN 201510080030 A CN201510080030 A CN 201510080030A CN 104673625 B CN104673625 B CN 104673625B
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Abstract
The invention discloses automatic reaction device and method for pretreating cells. The device comprises a reaction box body, a base, a box cover and a piston, wherein a middle cavity is formed in the center of the reaction box body; seven cavities are formed in the periphery of the hollow cavity; seven square cavities are correspondingly formed between the middle cavity and the seven cavities in the periphery; a control valve is put into each square cavity; communication between an inner chamber and an outer chamber is controlled through up and down movement; the lower part of the reaction box body is tightly connected with the base; the box cover is arranged at the upper part of the reaction box body; a piston rod which is matched with the middle cavity penetrates through the box cover; and the outer side of a second PCR reagent cavity is connected with a PCR chip for detecting nucleic acid. According to the reaction device and method, crushing and cracking of original samples, and purifying and extracting of the nucleic acid and PCR amplification are integrated in a reaction box; the operation process is simplified; the pretreatment efficiency is greatly improved; meanwhile, the reaction volume is reduced; the portability of an instrument is improved; and a foundation is laid for rapid, safe and accurate nucleic acid detection.
Description
【Technical field】
The present invention relates to cell analysis technical field is and in particular to a kind of Automatic Reactor and side that cell is carried out with pretreatment
Method.
【Background technology】
Detection of nucleic acids is the field attracting most attention the genome times afterwards comprehensively, and hereditism, clinical medicine and molecular diagnostics all can
Produce immeasurable impact.And want to complete high-quality detection of nucleic acids, need cell is carried out with pretreatment rapidly and efficiently
To extract highly purified nucleic acid.Cell pretreatment mainly includes cell cracking, nucleic acid purification and PCR and expands three steps,
It is intended to by purification propagation being carried out to nucleic acid therein after smashing cell, to reach the required purity of detection and concentration.
Typically adopt extension set formula to operate for the pretreatment of cell in the market, each step operation completed on multiple stage instrument,
Whole system so not only can be made huge and heavy, the complexity of operation can be increased simultaneously, be unfavorable for nucleic acid rapidly and efficiently
Detection.
【Content of the invention】
It is an object of the invention to provide a kind of Automatic Reactor and method that cell is carried out with pretreatment, existing thin to solve
Born of the same parents' pretreatment system is complicated, huge and the problem of heaviness；The present invention on the basis of conventional process mode, by primary sample
Pulverizing cracking, the purification of nucleic acid and PCR amplification and being integrated in carries out it is achieved that cell pretreatment in a reaction box
Integration operation；Not only simplify operating process, substantially increase pretreatment efficiency；Reduce reaction volume simultaneously, improve
The portability of instrument, lays a good foundation for realizing quick, safety and accurate detection of nucleic acids.
In order to achieve the above object, the technical scheme that the present invention takes is：
A kind of Automatic Reactor that cell is carried out with pretreatment, including reaction box box body, base, lid and piston；Reaction
Box box body center is provided with intermediate cavity；It is provided with seven chambeies, this seven chambeies are respectively magnetic bead chamber, the first cleaning mixture around intermediate cavity
Chamber, the second washing sap cavity, eluting sap cavity, testing sample chamber, the first PCR reagent chamber, the second PCR reagent chamber；Middle
It is correspondingly arranged on seven rectangular cavities between seven chambeies of chamber and surrounding setting；Be equipped with each rectangular cavity control corresponding exocoel with
The control valve of intermediate cavity connection；Pre-packaged in magnetic bead chamber have magnetic bead；Reaction box box body bottom is connected with base fastening, and lid is pacified
It is loaded on reaction box box body top, the piston rod of the piston matching with intermediate cavity passes through lid.
It is correspondingly arranged on seven first through holes running through corresponding rectangular cavity between intermediate cavity and seven chambeies of surrounding setting；Seven chambeies
Bottom surface concordant with corresponding first through hole lower end；Control valve includes the square cylinder at center, and the upper end of square cylinder is connected with
Back-moving spring, lower end is connected with lead, and lead passes through base, and lead is located at and is fixed with valve on the end of base exterior
Lid；It is provided with the second through hole running through square cylinder in square cylinder.
When the second through hole of control valve and first through hole insertion, the chamber corresponding to this control valve is interconnected with intermediate cavity.
Lid bottom is provided with seven square column, and this seven square column are inserted in corresponding rectangular cavities, make reaction box box body top with
Lid fastening is connected.
Lid is provided with the aperture that seven connections correspond to chamber.
It is provided with groove outside second PCR reagent chamber；It is provided with the pcr chip in order to be detected to nucleic acid in this groove.
The diameter with diameter greater than lead of valve gap.
In the un-compressed naturalness of back-moving spring, the second through hole is misplaced control valve with first through hole, makes control valve institute right
The chamber answered is completely cut off with intermediate cavity.
A kind of method carrying out pretreatment to cell, specifically includes following steps：
Step one, cell cracking：Inject diluent to magnetic bead intracavity, then suck in intermediate cavity, and push-and-pull piston enters repeatedly
Row dissolving, after adsorbing magnetic bead by the Magnet of intermediate cavity bottom, diluent is pushed back to magnetic bead chamber, then to testing sample
Intracavity injection lysate, testing sample and E.C. 3.4.21.64, then suck in intermediate cavity, release magnetic bead repeatedly push-and-pull piston enter
Magnetic bead is adsorbed to bottom by row hybrid reaction afterwards, and reactant liquor is pushed back to testing sample chamber；
Step 2, nucleic acid purification：Inject the first cleaning mixture to the first cleaning mixture intracavity, suck in intermediate cavity, release magnetic bead is simultaneously
Push-and-pull piston is washed repeatedly, then magnetic bead is adsorbed to bottom, and the first cleaning mixture is pushed back to the first washing sap cavity,
Repeat aforesaid operations, with the second cleaning mixture, magnetic bead is washed, then inject eluent to eluent intracavity, suck middle
In chamber, release magnetic bead repeatedly push-and-pull piston carry out eluting, then push back to eluting sap cavity, and pass through the Magnet of bottom by magnetic
Pearl is adsorbed, then sucks back a part of nucleic acid solution to intermediate cavity；
Step 3, PCR expands：Inject the first PCR reagent to the first PCR reagent intracavity, suck in intermediate cavity, and instead
Multiple push-and-pull piston is mixed, and then injects the second PCR reagent to the second PCR reagent chamber intracavity, sucks in intermediate cavity,
And push-and-pull piston is mixed repeatedly, finally push back the 2nd PCR intracavity, have partially liq to enter coupled PCR simultaneously
Chip, and then carries out temperature cycles to pcr chip, completes amplified reaction.
With respect to prior art, the invention has the advantages that：The present invention is a kind of to carry out the automatically anti-of pretreatment to cell
Answer device and method, by arranging the reaction unit of integral type, on the basis of conventional process mode, by the powder of primary sample
Fragmentation solution, the purification of nucleic acid and PCR amplification be integrated in carry out in a reaction box it is achieved that cell pretreatment one
Bodyization operates；Not only simplify operating process, substantially increase pretreatment efficiency；Reduce reaction volume simultaneously, improve
The portability of instrument, lays a good foundation for realizing quick, safety and accurate detection of nucleic acids.
Integral operational is adopted based on the cell pretreatment method of this device, improves pretreatment efficiency, enhance portable simultaneously
Property, for afterwards fast and accurately detection of nucleic acids provide good basis.
【Brief description】
Fig. 1 is the structural representation of apparatus of the present invention；
Fig. 2 is the sectional view of reaction box box body；
Fig. 3 a controls the schematic diagram of exterior chamber and intermediate cavity isolation for control valve；Fig. 3 b controls exterior chamber for control valve
The schematic diagram connecting with intermediate cavity；
Fig. 4 is the connection diagram in pcr chip and the second PCR reagent chamber.
【Specific embodiment】
Below in conjunction with the accompanying drawings the present invention is described in detail.
Refer to shown in Fig. 1 to Fig. 4, a kind of Automatic Reactor that cell is carried out with pretreatment of the present invention, including reaction box
Box body 1, base 2, lid 3, piston 5.
Reaction box box body 1 is provided centrally with intermediate cavity 7, for cracking and purification reaction；It is provided with seven around intermediate cavity 7
Chamber, this seven chambeies are respectively magnetic bead chamber 8, first washing sap cavity 9, second washing sap cavity 10, eluting sap cavity 11, treat test sample
Product chamber 12, the first PCR reagent chamber 13, the second PCR reagent chamber 14, all with intermediate cavity 7 by through hole 15 be connected into
The transfer of row liquid.
Reaction box box body 1 bottom is connected by draw-in groove fastening with base 2, so that the bottom surface of each chamber is just held level with both hands with through hole 15
Together, prevent the residual of reaction liquid.It is correspondingly arranged on seven rectangular cavities 16 between seven chambeies of intermediate cavity 7 and surrounding setting；
Lid 3 bottom is provided with 7 square column, and this 7 square column are inserted in corresponding rectangular cavity 16, make reaction box box body 1 top
It is connected with lid 3 fastening；Lid 3 is provided with the aperture 19 that 7 connections correspond to chamber, makes the air pressure of each within the chamber keep permanent
Fixed；It is placed with control valve 4, control valve 4 includes the square cylinder 40 at center, the upper end of square cylinder 40 in rectangular cavity 16
It is connected with spring 17 to control reset, lower end is connected with circular lead 41, lead 41 passes through corresponding logical on base 2
Hole, lead 41 is located at and is fixed with valve gap 18 on the end outside base 2；It is provided with through hole 42 in square cylinder 40；As figure
Shown in 3a and Fig. 3 b, moved up and down by outside drive mechanism jacking valve gap 18, to control control valve 4 in corresponding side
Upper-lower position in shape chamber, makes through hole 42 and through hole 15 insertion of control valve 4, to control each chamber corresponding to control valve 4
Room is interconnected with intermediate cavity 7；It is combined with corresponding piston 5 in intermediate cavity 7, provide liquid by push-and-pull piston 5
The required power of transfer；The groove in the second PCR reagent chamber 14 outside is connected with pcr chip 6, in order to complete each step behaviour
After work, nucleic acid is detected.
The operation principle of this device is：
Being interconnected between interior exocoel is controlled by moving up and down of control valve 4, is then carried using the push-and-pull of piston 5
Shift required power for liquid so that sample is through the transfer between different chamber, complete cracking, washing, eluting,
The operations such as PCR amplification, reach the purity needed for detection of nucleic acids and concentration.
With reference to embodiment, the cell pretreatment method of the present invention is described in detail.
Example one：Based on the hemocyte preprocess method of described Automatic Reactor, comprise the following steps：
Step one, cell cracking：Inject 600 μ L diluents into magnetic bead chamber 8, then suck in intermediate cavity 7, and repeatedly
Push-and-pull piston 5 is dissolved, and after adsorbing magnetic bead by the Magnet of intermediate cavity 7 bottom, diluent is pushed back to magnetic bead chamber 8,
Then 500 μ L lysates, 200 μ L blood samples and 20 μ L E.C. 3.4.21.64s are injected into testing sample chamber 12, in then sucking
Between in chamber 7, release magnetic bead repeatedly push-and-pull piston 5 carry out hybrid reaction, afterwards magnetic bead are adsorbed to bottom, and reacting
Liquid pushes back to testing sample chamber 12；
Step 2, nucleic acid purification：Inject 700 μ L the first cleaning mixture into the first washing sap cavity 9, suck in intermediate cavity 7,
Release magnetic bead repeatedly push-and-pull piston 5 are washed, and then magnetic bead are adsorbed to bottom, and the first cleaning mixture is pushed back to the
One washing sap cavity 9, repeats aforesaid operations, with 700 μ L the second cleaning mixture, magnetic bead is washed, then to eluting sap cavity 11
Interior injection 100 μ L eluents, suck in intermediate cavity 7, release magnetic bead repeatedly push-and-pull piston 5 carry out eluting, then push back
To eluting sap cavity 11, and by the Magnet of bottom, magnetic bead is adsorbed, then suck back 20 μ L nucleic acid solutions to intermediate cavity 7；
Step 3, PCR expands：Inject 17 μ L the first PCR reagent into the first PCR reagent chamber 13, suck intermediate cavity 7
In, and push-and-pull piston 5 is mixed repeatedly, then injects 3 μ L the second PCR reagent into the 2nd PCR chamber 14, sucks
In intermediate cavity 7, and push-and-pull piston 5 is mixed repeatedly, finally pushes back in the second PCR reagent chamber 14, has part simultaneously
Liquid enters coupled pcr chip 6, and then carries out temperature cycles to pcr chip 6, completes amplified reaction.
Claims (8)
1. a kind of Automatic Reactor that cell is carried out with pretreatment is it is characterised in that include reaction box box body (1), base (2), lid (3) and piston (5)；
Reaction box box body (1) center is provided with intermediate cavity (7)；It is provided with seven chambeies, this seven chambeies are respectively magnetic bead chamber (8), the first washing sap cavity (9), the second washing sap cavity (10), eluting sap cavity (11), testing sample chamber (12), the first PCR reagent chamber (13), the second PCR reagent chamber (14) around intermediate cavity (7)；It is correspondingly arranged on seven rectangular cavities (16) between seven chambeies of intermediate cavity (7) and surrounding setting；It is equipped with the control valve (4) controlling corresponding exocoel to connect with intermediate cavity (7) in each rectangular cavity (16)；Pre-packaged in magnetic bead chamber (8) have magnetic bead；
Reaction box box body (1) bottom is connected with base (2) fastening, lid (3) is installed on reaction box box body (1) top, and the piston rod of the piston (5) matching with intermediate cavity (7) passes through lid (3)；
It is correspondingly arranged on seven first through holes (15) running through corresponding rectangular cavity (16) between intermediate cavity (7) and seven chambeies of surrounding setting；The bottom surface in seven chambeies is concordant with corresponding first through hole (15) lower end；Control valve (4) includes the square cylinder (40) at center, the upper end of square cylinder (40) is connected with back-moving spring (17), lower end is connected with lead (41), lead (41) passes through base (2), and lead (41) is located at and is fixed with valve gap on the outside end of base (2)；It is provided with the second through hole (42) running through square cylinder (40) in square cylinder (40).
2. a kind of Automatic Reactor that cell is carried out with pretreatment according to claim 1, it is characterized in that, when second through hole (42) of control valve (4) and first through hole (15) insertion, the chamber corresponding to this control valve (4) is interconnected with intermediate cavity (7).
3. a kind of Automatic Reactor that cell is carried out with pretreatment according to claim 1, it is characterized in that, lid (3) bottom is provided with seven square column, this seven square column are inserted in corresponding rectangular cavity (16), so that reaction box box body (1) top is connected with lid (3) fastening.
4. a kind of Automatic Reactor that cell is carried out with pretreatment according to claim 1 is it is characterised in that lid (3) is provided with the aperture (19) that seven connections correspond to chamber.
5. a kind of Automatic Reactor that cell is carried out with pretreatment according to claim 1, it is characterised in that being provided with groove outside the second PCR reagent chamber (14), is provided with the pcr chip (6) in order to be detected to nucleic acid in this groove.
6. a kind of Automatic Reactor that cell is carried out with pretreatment according to claim 1 is it is characterised in that the diameter with diameter greater than lead (41) of valve gap.
7. a kind of Automatic Reactor that cell is carried out with pretreatment according to claim 1, it is characterized in that, control valve (4) is in back-moving spring (17) un-compressed naturalness, second through hole (42) is misplaced with first through hole (15), so that the chamber corresponding to control valve (4) is completely cut off with intermediate cavity (7).
8. a kind of method that cell is carried out with pretreatment is it is characterised in that based on a kind of Automatic Reactor that cell is carried out with pretreatment any one of claim 1 to 7, specifically include following steps：
Step one, cell cracking：The injection diluent into magnetic bead chamber (8), then suck in intermediate cavity (7), and push-and-pull piston (5) is dissolved repeatedly, after magnetic bead being adsorbed by the Magnet of intermediate cavity (7) bottom, diluent is pushed back to magnetic bead chamber (8), then injection lysate, testing sample and E.C. 3.4.21.64 into testing sample chamber (12), then suck in intermediate cavity (7), release magnetic bead repeatedly push-and-pull piston (5) carry out hybrid reaction, afterwards magnetic bead is adsorbed to bottom, and reactant liquor is pushed back to testing sample chamber (12)；
Step 2, nucleic acid purification：Injection the first cleaning mixture into the first washing sap cavity (9), suck in intermediate cavity (7), release magnetic bead repeatedly push-and-pull piston (5) are washed, then magnetic bead is adsorbed to bottom, and the first cleaning mixture is pushed back to the first washing sap cavity (9), repeat aforesaid operations, with the second cleaning mixture, magnetic bead is washed, then the injection eluent into eluting sap cavity (11), suck in intermediate cavity (7), release magnetic bead repeatedly push-and-pull piston (5) carry out eluting, then push back to eluting sap cavity (11), and by the Magnet of bottom, magnetic bead is adsorbed, suck back a part of nucleic acid solution again to intermediate cavity (7)；
Step 3, PCR expands：Injection the first PCR reagent into the first PCR reagent chamber (13), suck in intermediate cavity (1), and push-and-pull piston (5) is mixed repeatedly, then to injection the second PCR reagent in the second PCR reagent chamber (14), suck in intermediate cavity (7), and push-and-pull piston (5) is mixed repeatedly, finally push back in the second PCR reagent chamber (14), there is partially liq to enter coupled pcr chip (6) simultaneously, and then temperature cycles are carried out to pcr chip (6), complete amplified reaction.
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| CN112175787A (en) * | 2019-07-01 | 2021-01-05 | 申翌生物科技(杭州)有限公司 | Method for performing PCR reaction by using PCR reaction system |
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| CN112175787A (en) * | 2019-07-01 | 2021-01-05 | 申翌生物科技(杭州)有限公司 | Method for performing PCR reaction by using PCR reaction system |
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