CN104673625A - Automatic reaction device and method for pretreating cells - Google Patents
Automatic reaction device and method for pretreating cells Download PDFInfo
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- CN104673625A CN104673625A CN201510080030.2A CN201510080030A CN104673625A CN 104673625 A CN104673625 A CN 104673625A CN 201510080030 A CN201510080030 A CN 201510080030A CN 104673625 A CN104673625 A CN 104673625A
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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, be specifically related to one and pretreated Automatic Reactor and method are carried out to cell.
[background technology]
Detection of nucleic acids is the field attracted most attention the genome times afterwards comprehensively, all can produce immeasurable impact to genetics, clinical medicine and molecular diagnostics.And wanted high-quality detection of nucleic acids, need to carry out pre-treatment rapidly and efficiently to extract highly purified nucleic acid to cell.Cell pretreatment mainly comprises lysis, nucleic acid purification and pcr amplification three steps, is intended to carry out purifying propagation to nucleic acid wherein afterwards by being shattered by cell, to reach purity needed for detection and concentration.
Pre-treatment in the market for cell generally adopts extension set formula to operate, and on multiple stage instrument, namely completes the operation of each step, whole system so not only can be made huge and heavy, can increase the complexity of operation simultaneously, be unfavorable for the rapidly and efficiently detection of nucleic acid.
[summary of the invention]
The object of the present invention is to provide and a kind of pretreated Automatic Reactor and method are carried out to cell, to solve existing cell pretreatment system complex, the huge and problem of heaviness; The pulverizing cracking of primary sample, the purification of nucleic acid and pcr amplification, on the basis of conventional process mode, are integrated in a reaction box and carry out by the present invention, achieve the integration operation of cell pretreatment; Not only simplify operating process, substantially increase pre-treatment efficiency; Reduce reaction volume simultaneously, improve the portability of instrument, for realizing fast, safety and accurately detection of nucleic acids lay a good foundation.
In order to achieve the above object, the technical scheme that the present invention takes is:
One carries out pretreated Automatic Reactor to cell, comprises reaction box box body, base, lid and piston; Reaction box He Shen center is provided with intermediate cavity; Be provided with seven chambeies around intermediate cavity, these seven chambeies are respectively magnetic bead chamber, the first washing sap cavity, the second washing sap cavity, wash-out sap cavity, testing sample chamber, the first PCR reagent chamber, the second PCR reagent chamber; Intermediate cavity with around correspondingly between seven chambeies arranging be provided with seven rectangular cavities; The control valve controlling corresponding exocoel and be communicated with intermediate cavity is equipped with in each rectangular cavity; Pre-packaged in magnetic bead chamber have magnetic bead; Reaction box box body bottom is connected with base is fastening, and lid is installed on reaction box box body, and the piston rod of the piston matched with intermediate cavity is through lid.
Intermediate cavity with around correspondingly between seven chambeies arranging be provided with the first through hole that seven run through corresponding rectangular cavity; The bottom surface in seven chambeies is concordant with the first corresponding through hole lower end; Control valve comprises the square cylinder at center, and the upper end of square cylinder is connected with return spring, and lower end is connected with guidepost, and guidepost through base, the end that guidepost is positioned at base exterior is fixed with valve gap; The second through hole running through square cylinder is provided with in square cylinder.
Second through hole of control valve and the first through hole through time, being interconnected of the chamber corresponding to this control valve and intermediate cavity.
Lid bottom is provided with seven square column, and these seven square column are inserted in corresponding rectangular cavity, and reaction box box body is connected with lid is fastening.
Lid is provided with the aperture that seven are communicated with corresponding chamber.
Groove is provided with outside second PCR reagent chamber; The pcr chip in order to detect nucleic acid is provided with in this groove.
The diameter of valve gap is greater than the diameter of guidepost.
Control valve is when the un-compressed state of nature of return spring, and the second through hole and the first through hole misplace, and the chamber corresponding to control valve and intermediate cavity are completely cut off.
One carries out pretreated method to cell, specifically comprises the following steps:
Step one, lysis: inject diluent in magnetic bead chamber, then suck in intermediate cavity, and push-and-pull piston dissolves repeatedly, after magnetic bead being adsorbed by the magnet bottom intermediate cavity, diluent is pushed back to magnetic bead chamber, then in testing sample chamber, inject lysate, testing sample and Proteinase K, then suck in intermediate cavity, release magnetic bead and repeatedly push-and-pull piston carry out hybrid reaction, afterwards magnetic bead is adsorbed to bottom, and reaction solution is pushed back to testing sample chamber;
Step 2, nucleic acid purification: inject the first washings in the first washing sap cavity, suck in intermediate cavity, release magnetic bead and repeatedly push-and-pull piston wash, then magnetic bead is adsorbed to bottom, and the first washings is pushed back to the first washing sap cavity, repeat aforesaid operations, with the second washings, magnetic bead is washed, then in wash-out sap cavity, inject elutriant, suck in intermediate cavity, release magnetic bead and repeatedly push-and-pull piston carry out wash-out, then push back to wash-out sap cavity, and by the magnet of bottom, magnetic bead is adsorbed, then suck back a part of nucleic acid solution to intermediate cavity;
Step 3, pcr amplification: inject the first PCR reagent in the first PCR reagent chamber, suck in intermediate cavity, and push-and-pull piston mixes repeatedly, then in the second chamber, PCR reagent chamber, injects the second PCR reagent, suck in intermediate cavity, and push-and-pull piston mixes repeatedly, finally push back in the 2nd PCR chamber, have partially liq to enter coupled pcr chip simultaneously, and then temperature cycle is carried out to pcr chip, complete amplified reaction.
Relative to prior art, the present invention has following beneficial effect: the present invention's one carries out pretreated Automatic Reactor and method to cell, by arranging the reaction unit of integral type, on the basis of conventional process mode, the pulverizing cracking of primary sample, the purification of nucleic acid and pcr amplification are integrated in a reaction box and carry out, achieve the integration operation of cell pretreatment; Not only simplify operating process, substantially increase pre-treatment efficiency; Reduce reaction volume simultaneously, improve the portability of instrument, for realizing fast, safety and accurately detection of nucleic acids lay a good foundation.
Based on this device cell pretreatment method adopt integral operational, improve pre-treatment efficiency, enhance portability simultaneously, for afterwards fast and accurately detection of nucleic acids provide good basis.
[accompanying drawing explanation]
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 is the schematic diagram that control valve control exterior chamber and intermediate cavity completely cut off; Fig. 3 b is the schematic diagram that control valve control exterior chamber is communicated with intermediate cavity;
Fig. 4 is the connection diagram in pcr chip and the second PCR reagent chamber.
[embodiment]
Below in conjunction with accompanying drawing, the present invention is described in detail.
Refer to shown in Fig. 1 to Fig. 4, the present invention's one carries out pretreated Automatic Reactor to cell, comprises reaction box box body 1, base 2, lid 3, piston 5.
Reaction box box body 1 center is provided with intermediate cavity 7, for cracking and purification reaction; Seven chambeies are provided with around intermediate cavity 7, these seven chambeies are respectively magnetic bead chamber 8, first and wash sap cavity 9, second washing sap cavity 10, wash-out sap cavity 11, PCR reagent chamber 14, PCR reagent chamber 13, second, testing sample chamber 12, first, are all connected by through hole 15 transfer carrying out liquid with intermediate cavity 7.
Reaction box box body 1 bottom is connected by draw-in groove is fastening with base 2, makes the bottom surface of each chamber just concordant with through hole 15 lower end, prevents the residual of reaction liquid.Intermediate cavity 7 with around correspondingly between seven chambeies arranging be provided with seven rectangular cavities 16; Lid 3 bottom is provided with 7 square column, and these 7 square column are inserted in corresponding rectangular cavity 16, and reaction box box body 1 top is connected with lid 3 is fastening; Lid 3 is provided with the aperture 19 that 7 are communicated with corresponding chamber, makes the air pressure in each chamber keep constant; Control valve 4 is placed with in rectangular cavity 16, control valve 4 comprises the square cylinder 40 at center, the upper end of square cylinder 40 is connected with spring 17 to control to reset, lower end is connected with circular guidepost 41, guidepost 41, through through hole corresponding on base 2, the end that guidepost 41 is positioned at base 2 outside is fixed with valve gap 18; Through hole 42 is provided with in square cylinder 40; As shown in Figure 3 a and Figure 3 b shows, moved up and down by the driving mechanism jacking valve gap 18 of outside, to control the upper-lower position of control valve 4 in the rectangular cavity of correspondence, make the through hole 42 of control valve 4 and through hole 15 through, to control being interconnected of each chamber corresponding to control valve 4 and intermediate cavity 7; Be combined with corresponding piston 5 in intermediate cavity 7, provide the power needed for liquid transfer by push-and-pull piston 5; Groove outside second PCR reagent chamber 14 is connected with pcr chip 6, in order to detect nucleic acid after completing the operation of each step.
The principle of work of this device is:
Being interconnected between interior outer chamber is controlled by moving up and down of control valve 4, then utilize the push-and-pull of piston 5 to provide the power needed for liquid transfer, thus make sample through the transfer between different chamber, complete the operations such as cracking, washing, wash-out, pcr amplification, reach the purity needed for detection of nucleic acids and concentration.
Below in conjunction with embodiment, cell pretreatment method of the present invention is described in detail.
Example one: based on the hemocyte pretreatment process of described Automatic Reactor, comprise the following steps:
Step one, lysis: inject 600 μ L diluents in magnetic bead chamber 8, then suck in intermediate cavity 7, and push-and-pull piston 5 dissolves repeatedly, after magnetic bead being adsorbed by the magnet bottom intermediate cavity 7, diluent is pushed back to magnetic bead chamber 8, then in testing sample chamber 12, inject 500 μ L lysates, 200 μ L blood samples and 20 μ L Proteinase Ks, then suck in intermediate cavity 7, release magnetic bead and repeatedly push-and-pull piston 5 carry out hybrid reaction, afterwards magnetic bead is adsorbed to bottom, and reaction solution is pushed back to testing sample chamber 12;
Step 2, nucleic acid purification: inject 700 μ L first washingss in the first washing sap cavity 9, suck in intermediate cavity 7, release magnetic bead and repeatedly push-and-pull piston 5 wash, then magnetic bead is adsorbed to bottom, and the first washings is pushed back to the first washing sap cavity 9, repeat aforesaid operations, with 700 μ L second washingss, magnetic bead is washed, then in wash-out sap cavity 11,100 μ L elutriants are injected, suck in intermediate cavity 7, release magnetic bead and repeatedly push-and-pull piston 5 carry out wash-out, then wash-out sap cavity 11 is pushed back to, and by the magnet of bottom, magnetic bead is adsorbed, suck back 20 μ L nucleic acid solutions again to intermediate cavity 7,
Step 3, pcr amplification: inject 17 μ L first PCR reagent in the first PCR reagent chamber 13, suck in intermediate cavity 7, and push-and-pull piston 5 mixes repeatedly, then in the 2nd PCR chamber 14, injects 3 μ L second PCR reagent, suck in intermediate cavity 7, and push-and-pull piston 5 mixes repeatedly, finally push back in the second PCR reagent chamber 14, have partially liq to enter coupled pcr chip 6 simultaneously, and then temperature cycle is carried out to pcr chip 6, complete amplified reaction.
Claims (9)
1. a pretreated Automatic Reactor is carried out to cell, it is characterized in that, comprise reaction box box body (1), base (2), lid (3) and piston (5);
Reaction box box body (1) center is provided with intermediate cavity (7); Intermediate cavity (7) is provided with seven chambeies around, and these seven chambeies are respectively magnetic bead chamber (8), the first washing sap cavity (9), the second washing sap cavity (10), wash-out sap cavity (11), testing sample chamber (12), the first PCR reagent chamber (13), the second PCR reagent chamber (14); Intermediate cavity (7) with around correspondingly between seven chambeies arranging be provided with seven rectangular cavities (16); The control valve (4) controlling corresponding exocoel and be communicated with intermediate cavity (7) is equipped with 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) is fastening, lid (3) is installed on reaction box box body (1) top, and the piston rod of the piston (5) matched with intermediate cavity (7) is through lid (3).
2. one according to claim 1 carries out pretreated Automatic Reactor to cell, it is characterized in that, intermediate cavity (7) with around correspondingly between seven chambeies arranging be provided with the first through hole (15) that seven run through corresponding rectangular cavity (16); The bottom surface in seven chambeies is concordant with corresponding the first through hole (15) lower end; Control valve (4) comprises the square cylinder (40) at center, the upper end of square cylinder (40) is connected with return spring (17), lower end is connected with guidepost (41), guidepost (41) is through base (2), and guidepost (41) is positioned on the outside end of base (2) and is fixed with valve gap; The second through hole (42) running through square cylinder (40) is provided with in square cylinder (40).
3. one according to claim 2 carries out pretreated Automatic Reactor to cell, it is characterized in that, when second through hole (42) of control valve (4) is through with the first through hole (15), the chamber corresponding to this control valve (4) and intermediate cavity (7) are interconnected.
4. one according to claim 1 carries out pretreated Automatic Reactor to cell, it is characterized in that, lid (3) bottom is provided with seven square column, these seven square column are inserted in corresponding rectangular cavity (16), and reaction box box body (1) top is connected with lid (3) is fastening.
5. one according to claim 1 carries out pretreated Automatic Reactor to cell, it is characterized in that, lid (3) is provided with the aperture (19) that seven are communicated with corresponding chamber.
6. one according to claim 1 carries out pretreated Automatic Reactor to cell, it is characterized in that, the second outside, PCR reagent chamber (14) is provided with groove, is provided with the pcr chip (6) in order to detect nucleic acid in this groove.
7. one according to claim 2 carries out pretreated Automatic Reactor to cell, it is characterized in that, the diameter of valve gap is greater than the diameter of guidepost (41).
8. one according to claim 2 carries out pretreated Automatic Reactor to cell, it is characterized in that, control valve (4) is when return spring (17) un-compressed state of nature, second through hole (42) and the first through hole (15) misplace, and the chamber corresponding to control valve (4) and intermediate cavity (7) are completely cut off.
9. a pretreated method is carried out to cell, it is characterized in that, based on the one according to any one of claim 1 to 8, pretreated Automatic Reactor is carried out to cell, specifically comprise the following steps:
Step one, lysis: inject diluent in magnetic bead chamber (8), then suck in intermediate cavity (7), and push-and-pull piston (5) dissolves repeatedly, after magnetic bead being adsorbed by the magnet of intermediate cavity (7) bottom, diluent is pushed back to magnetic bead chamber (8), then in testing sample chamber (12), lysate is injected, testing sample and Proteinase K, then suck in intermediate cavity (7), release magnetic bead and repeatedly push-and-pull piston (5) carry out hybrid reaction, afterwards magnetic bead is adsorbed to bottom, and reaction solution is pushed back to testing sample chamber (12),
Step 2, nucleic acid purification: inject the first washings in the first washing sap cavity (9), suck in intermediate cavity (7), release magnetic bead and repeatedly push-and-pull piston (5) wash, then magnetic bead is adsorbed to bottom, and the first washings is pushed back to the first washing sap cavity (9), repeat aforesaid operations, with the second washings, magnetic bead is washed, then in wash-out sap cavity (11), elutriant is injected, suck in intermediate cavity (7), release magnetic bead and repeatedly push-and-pull piston (5) carry out wash-out, then wash-out sap cavity (11) is pushed back to, 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 amplification: inject the first PCR reagent in the first PCR reagent chamber (13), suck in intermediate cavity (1), and push-and-pull piston (5) mixes repeatedly, then the second PCR reagent is injected in the second PCR reagent chamber (14), suck in intermediate cavity (7), and push-and-pull piston (5) mixes 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 cycle is carried out to pcr chip (6), complete amplified reaction.
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