CN105400693A - Flat plate isothermal nucleic acid amplification chip - Google Patents
Flat plate isothermal nucleic acid amplification chip Download PDFInfo
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- CN105400693A CN105400693A CN201510934325.1A CN201510934325A CN105400693A CN 105400693 A CN105400693 A CN 105400693A CN 201510934325 A CN201510934325 A CN 201510934325A CN 105400693 A CN105400693 A CN 105400693A
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
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Abstract
The invention belongs to the biological technical field, and in particular, relates to a flat plate in-situ nucleic acid amplification chip. The chip is composed of an upper-layer carrier and a lower-layer carrier which are bonded mutually and can slide, and the upper-layer carrier and the lower-layer carrier are rectangles with same size; when the lower-layer carrier is fixed, the upper-layer carrier can be in relative friction sliding. An upper bonded face of the lower-layer carrier is provided with various groove runners with downward openings and various micro-cavities with downward openings, and a lower binding face of the upper-layer carrier is provided with various groove flow holes with upward openings, various micro-cavities with upward openings, and a plurality of inlet through holes and outlet through holes; through quantitative sample adding and different modes of detection, effective operation can be performed on different samples at the same time, and the experimental cost is reduced. The chip can be applied in forensic medicine detection, environmental detection, food detection and the like.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of dull and stereotyped original position isothermal nucleic acid amplification experimental installation and experimental technique.
Background technology
Microflow control technique is one of international high and new technology Disciplinary Frontiers now, and the application in nucleic acid extraction, amplification, detect delay achieves fast development, becomes one of most potential developing direction of nucleic acids research gradually.Its adopts micro-electromechanical technology to process the chip with micro-meter scale channel network configuration, receiving the upgrading manipulation of microfluid and control, realizing integrated analyzing and testing function that is biomedical and chemical laboratory by rising to skin in chip.Slip chip (SlipChip) technology is exactly the one of microflow control technique, be made up of two pieces of substrates up and down with microstructure, add primer premix, enzyme premix, DNA by upper substrate, realize liquid by slip upper substrate and to shift alternately at levels substrate thus to mix the amplification of all reaction reagent original positions in reaction chamber.
Ring mediated isothermal nucleic acid amplification (loop-mediatedisothermalamplification, LAMP) is a kind of constant temperature nucleic acid amplification method.This technology utilizes 4 species-specific primers to rely on a kind of high reactivity strand displacement archaeal dna polymerase (about 65 DEG C) when DNA is in running balance that strand displacement DNA is synthesized in ceaselessly oneself's circulation.Whole reaction without the need to thermal cycling repeatedly, and high specificity, highly sensitive, rapidly and efficiently, identify easy, for the fields such as medical jurisprudence, ecotope, food real-time diagnosis detect application create condition.
But domestic and international existing LAMP slip chip is still immature, not easily fixing and operation, and the system and device not having slip chip to mention can to realize original position amplification.Therefore, the present invention is based on above-mentioned research background, develop a kind of dull and stereotyped original position isothermal nucleic acid amplification chip.
Summary of the invention
The object of this invention is to provide dull and stereotyped original position isothermal nucleic acid amplification chip, this chip operation is easy, can realize the different sample that simultaneously increases, and reduces test operation intensity, and has saved the consumption of sample and reagent, reduces experimental cost.
In order to realize above object, the invention provides a kind of dull and stereotyped in-situ nucleic acid amplification chip, comprising upper strata carrier bonded to each other and lower floor's carrier; Upper strata carrier can horizontal slip on lower floor's carrier;
Upper strata carrier is provided with sample feeding mouth group, polysaccharase injection port group, primer injection port group, the first outlet through hole, the second outlet through hole, the first groove discharge orifice group, the second groove discharge orifice group, first flow;
Lower floor's carrier is provided with sample introduction tank, the second runner group, reaction chamber group, comb teeth shape passage, the 3rd groove discharge orifice group; Reaction chamber is mounted between comb teeth shape passage increment or side, and the increment of the inflow end of reaction chamber group and comb teeth shape passage is located along the same line;
When upper strata carrier and lower floor's carrier are positioned at starting position, sample feeding mouth group circulates mutually with the position of reaction chamber group; Polysaccharase injection port group circulates mutually with comb teeth shape passage, the 3rd groove discharge orifice group, the second outlet through hole; Primer injection port group circulates mutually with sample introduction tank, the second runner group, the first groove discharge orifice group, first flow, the first outlet through hole;
When adding primer to reaction chamber group, translation upper strata carrier, makes the first groove discharge orifice group circulate mutually with reaction chamber group, and primer flows in reaction chamber group from the first groove discharge orifice;
When adding polysaccharase to reaction chamber group, translation upper strata carrier, makes reaction chamber group circulate mutually with the second groove discharge orifice group, and polysaccharase flows in reaction chamber group from the second groove discharge orifice group.
As preferably, upper strata carrier and lower floor's carrier are rectangle carrier.
As preferably, the first groove discharge orifice group comprises the first groove discharge orifice, and the second groove discharge orifice group comprises the second groove discharge orifice, and the 3rd groove discharge orifice group comprises the 3rd groove discharge orifice; Reaction chamber group comprises reaction chamber; Sample feeding mouth group comprises sample feeding mouth;
Sample feeding mouth is arranged on same straight line equably; First groove discharge orifice is arranged on same straight line equably, primer injection port and the first groove discharge orifice are arranged on same straight line, second groove discharge orifice is arranged on same straight line equably, sample feeding mouth is corresponding with the position of the first groove discharge orifice, and the second groove discharge orifice and sample feeding mouth are staggered; 3rd groove discharge orifice is arranged on same straight line equably, and reaction chamber is arranged on same straight line equably; Second runner group comprises the second runner be positioned on straight line and the second runner be located on the same line with the 3rd groove discharge orifice group;
Sample feeding mouth, the first groove discharge orifice, the second groove discharge orifice parallel with the central horizontal axle of upper strata carrier; Reaction chamber, the 3rd groove discharge orifice parallel with the central horizontal axle of lower floor's carrier.
As preferably, upper strata carrier is provided with two groups of sample feeding mouth groups, polysaccharase injection port group, primer injection port group, the first outlet through hole, the second outlet through hole, the first groove discharge orifice group, the second groove discharge orifice group, first flow;
Two groups of sample feeding mouth groups, polysaccharase injection port group, primer injection port group, the first outlet through hole, the second outlet through hole, the first groove discharge orifice group, the second groove discharge orifice group, first flow are relative to the central horizontal axle rotational symmetry of upper strata carrier;
Lower floor's carrier is provided with two groups of sample introduction tank, the second runner group, reaction chamber group, comb teeth shape passage, the 3rd groove discharge orifice group;
Two groups of sample introduction tank, the second runner group, reaction chamber group, comb teeth shape passage, the 3rd groove discharge orifice group are relative to the central horizontal axle rotational symmetry of lower floor's carrier.
As preferably, primer injection port and the first groove discharge orifice are arranged on the central horizontal axle of upper strata carrier, and sample introduction tank is positioned on the central horizontal axle of lower floor's carrier.
As preferably, when overlooking the first groove discharge orifice, the second groove discharge orifice, the 3rd groove discharge orifice, the first groove discharge orifice is cruciform; Second groove discharge orifice, the 3rd groove discharge orifice are rectangle.
As preferably, the volume of each the first groove discharge orifice is identical; The volume of each the second groove discharge orifice is identical; The volume of each reaction chamber is identical.
As preferably, the volumetric ratio between reaction chamber, the first groove discharge orifice, the second groove discharge orifice is 10:6:3.
As preferably, the diameter of sample feeding mouth group, polysaccharase injection port group, primer injection port group, the first outlet through hole, the second outlet through hole is 1mm.
As preferably, first flow is T-shaped runner.
Present invention also offers a kind of isothermal nucleic acid amplification device, comprise the dull and stereotyped original position isothermal nucleic acid amplification chip of square heat transfer platform and above-mentioned any one; Heat transfer platform is provided with heating unit; Lower floor's carrier is fixed on heat transfer platform, and heating unit is to lower floor's carrier homogeneous heating.
As preferably, heat transfer platform comprises four movable blocks, limit assembly and underframe; Limit assembly comprises slide shaft portion and limiting section;
Lower floor's carrier is fixed on underframe, and the one end in slide shaft portion is run through movable block and is fixedly connected with underframe; Movable block can in the horizontal slip of slide shaft portion; Limiting section is located at the other end in slide shaft portion, and cross-sectional area is greater than the cross-sectional area in slide shaft portion.
As preferably, each movable block is provided with at least 2 limit assemblies.
As preferably, limit assembly is iron nail.A kind of isothermal nucleic acid amplification method, make use of the isothermal nucleic acid amplification device of above-mentioned any one, comprises the following steps:
S0, upper strata carrier is moved to predeterminated position;
S1, add primer from primer injection port group, and make primer be full of the first groove discharge orifice group;
S2, translation upper strata carrier, make the first groove discharge orifice group circulate mutually with reaction chamber group, and primer flows in reaction chamber group from the first groove discharge orifice;
S3, upper strata carrier is moved back to predeterminated position;
S4, add polysaccharase from polysaccharase injection port group, and make polysaccharase be full of the second groove discharge orifice group;
S5, translation upper strata carrier, make reaction chamber group circulate mutually with the second groove discharge orifice group, and polysaccharase flows in reaction chamber group from the second groove discharge orifice group;
S6, upper strata carrier is moved back to predeterminated position;
S7, add sample from sample feeding mouth group;
S8, control heating unit make reaction solution keep at the reaction temperatures.
As preferably, at two groups of sample feeding mouth groups, polysaccharase injection port group, primer injection port group, the first outlet through hole, the second outlet through hole, the first groove discharge orifice group, the second groove discharge orifice group, first flow relative to the central horizontal axle rotational symmetry of upper strata carrier; And during central horizontal axle rotational symmetry relative to lower floor's carrier of two groups of sample introduction tank, the second runner group, reaction chamber group, comb teeth shape passage, the 3rd groove discharge orifice group, after step S3, again primer injection port group adds primer, and translation upper strata carrier, first groove discharge orifice group is circulated mutually with the reaction chamber group of opposite side, and primer flows in the reaction chamber group of opposite side from the first groove discharge orifice.
Compared with prior art, beneficial effect of the present invention is:
1, dull and stereotyped original position isothermal nucleic acid amplification chip of the present invention can carry out multiple sample and carry out amplification operation simultaneously, greatly saves the reaction times, has saved the consumption of sample and reagent, reduced experimental cost.
2, dull and stereotyped original position isothermal nucleic acid amplification chip of the present invention can realize smooth sliding and accurate temperature controller by Simple heater, effectively can carry out original position amplification to nucleic acid.
3, the volume size of this chip reaction chamber, discharge orifice, runner designs by a certain percentage, effectively can control reaction system, improve amplification efficiency.
4, for part Experiment, can be detected by an unaided eye reaction by this chip.
Accompanying drawing explanation
Fig. 1 is the chip structure vertical view of the first embodiment of the present invention.
Fig. 2 is the chip structure vertical view of the second embodiment of the present invention.
Fig. 3 is the movable block of platform structural representation when being positioned at starting position that conducts heat in the third embodiment of the present invention.
Fig. 4 be conduct heat in the third embodiment of the present invention platform movable block slide after structural representation.
Fig. 5 be the present invention at the middle and upper levels carrier be positioned at the chip structure vertical view of starting position.
Fig. 6 is chip structure vertical view when adding primer in the present invention.
Fig. 7 is chip structure vertical view when adding polysaccharase in the present invention.
Fig. 8 adds the chip structure vertical view during polysaccharase when being and utilizing chip of the present invention to carry out controlled trial.
Reference numeral: 1, upper strata carrier; 11, sample feeding mouth group; 12, polysaccharase injection port group; 13, primer injection port group; 14, the second groove discharge orifice group; 15, the first groove discharge orifice group; 16, first flow; 17, the first outlet through hole; 18, the second outlet through hole; 2, lower floor's carrier; 21, reaction chamber group; 22, comb teeth shape passage; 23, sample introduction tank; 24, the second runner group; 25, the 3rd groove discharge orifice group; 3, conduct heat platform; 31, limit assembly; 32, underframe; 33, movable block.
Embodiment
For making the object, technical solutions and advantages of the present invention clearly, below in conjunction with accompanying drawing, the embodiments of the present invention are explained in detail.
For solving the problems of the technologies described above, the first embodiment of the present invention provides a kind of dull and stereotyped in-situ nucleic acid amplification chip, as shown in Figure 1, comprises upper strata carrier 1 bonded to each other and lower floor's carrier 2; Upper strata carrier 1 can horizontal slip on lower floor's carrier 2;
Upper strata carrier 1 is provided with sample feeding mouth group 11, polysaccharase injection port group 12, primer injection port group 13, first outlet through hole 17, second outlet through hole 18, first groove discharge orifice group 15, second groove discharge orifice group 14, first flow 16;
Lower floor's carrier 2 is provided with sample introduction tank 23, second runner group 24, reaction chamber group 21, comb teeth shape passage 22, the 3rd groove discharge orifice group 25; Reaction chamber group 21 is located between comb teeth shape passage 22 increment or side, and the increment of the inflow end of reaction chamber group 21 and comb teeth shape passage 22 is located along the same line;
When upper strata carrier 1 and lower floor's carrier 2 are positioned at starting position, sample feeding mouth group 11 circulates mutually with the position of reaction chamber group 21; Polysaccharase injection port group 12 circulates with comb teeth shape passage 22, the 3rd groove discharge orifice group 25, second outlet through hole 18 phase; Primer injection port group 13 circulates with sample introduction tank 23, second runner group 24, first groove discharge orifice group 15, first flow 16, first outlet through hole 17 phase;
When adding primer to reaction chamber group 21, translation upper strata carrier 1, the first groove discharge orifice group 15 is circulated with reaction chamber group 21 phase, and primer flows in reaction chamber group 21 from the first groove discharge orifice 15;
When adding polysaccharase to reaction chamber group 21, translation upper strata carrier 1, reaction chamber group 21 is circulated with the second groove discharge orifice group 14 phase, and polysaccharase flows in reaction chamber group 21 from the second groove discharge orifice group 14.
In the present embodiment, the second runner group 24 comprises 6 sections of runners, and the first groove discharge orifice group 15 comprises 5 the first groove discharge orifices, and the second groove discharge orifice group 14 comprises 5 the second groove discharge orifices; 3rd groove discharge orifice group 25 comprises 10 the 3rd groove discharge orifices; Reaction chamber group 21 comprises 5 reaction chambers, and reaction chamber is set to bulb-shaped; The volume of each the first groove discharge orifice is identical; The volume of each the second groove discharge orifice is identical; The volume of each reaction chamber is identical, and the volumetric ratio between reaction chamber, the first groove discharge orifice, the second groove discharge orifice is 10:6:3.
In the present embodiment, when overlooking the first groove discharge orifice, the second groove discharge orifice, the 3rd groove discharge orifice, the first groove discharge orifice is cruciform; Second groove discharge orifice, the 3rd groove discharge orifice are rectangle.Wherein, the first groove discharge orifice is designed to cruciform, there is following technique effect:
1, when adding primer in the first groove discharge orifice, the first groove discharge orifice and the second runner group 24 circulate, and during mobile upper strata glass, can isolate quickly between the first groove discharge orifice and the second runner group 24.
2, when adding polysaccharase in reaction chamber, the first groove discharge orifice and reaction chamber, the 3rd groove discharge orifice circulate, and during mobile upper strata glass, if there is skew, polysaccharase is also not easy to flow in adjacent 3rd groove discharge orifice.
In the present embodiment, the diameter of sample feeding mouth group 11, polysaccharase injection port group 12, primer injection port group 13, first outlet through hole 17, second outlet through hole 18 is 1mm.
In the present embodiment, upper strata carrier 1, lower floor's carrier 2 select sheet glass, and other can certainly be selected to can be used in carrying out the carrier board of Bioexperiment.Groove runner, discharge orifice and the microcavity be positioned on carrier can be offered by laser sculpture mode.The shape of sheet glass, size are not particularly limited, however for the ease of operation and fixing, in the present embodiment, the rectangular sheet glass that preferred dimension is identical.
In the present embodiment, the first groove discharge orifice group 15 comprises several the first groove discharge orifices, and the second groove discharge orifice group 14 comprises several the second groove discharge orifices, and the 3rd groove discharge orifice group 25 comprises several the 3rd groove discharge orifices; Reaction chamber group 21 comprises several reaction chambers; Sample feeding mouth group 11 comprises several sample feeding mouths;
Sample feeding mouth is arranged on same straight line equably; First groove discharge orifice is arranged on same straight line equably, primer injection port 13 and the first groove discharge orifice are arranged on same straight line, second groove discharge orifice is arranged on same straight line equably, sample feeding mouth is corresponding with the position of the first groove discharge orifice, and the second groove discharge orifice and sample feeding mouth are staggered; 3rd groove discharge orifice is arranged on same straight line equably, and reaction chamber is arranged on same straight line equably; Second runner group 24 comprises the second runner be positioned on straight line and the second runner be located on the same line with the 3rd groove discharge orifice group 25;
Sample feeding mouth, the first groove discharge orifice, the second groove discharge orifice parallel with the central horizontal axle of upper strata carrier 1; Reaction chamber, the 3rd groove discharge orifice parallel with the central horizontal axle of lower floor carrier 2.
In the present embodiment, by sample feeding mouth, the first groove discharge orifice, the second groove discharge orifice and upper strata carrier 1 be set to parallel with central horizontal axle; Simultaneous reactions chamber, the 3rd groove discharge orifice parallel with the central horizontal axle of lower floor carrier 2, make dull and stereotyped in-situ nucleic acid amplification chip outward appearance provided by the invention seem more neat.
Second embodiment of the present invention relates to a kind of dull and stereotyped in-situ nucleic acid amplification chip, the second embodiment is the improvement of the first embodiment, as shown in Figure 2, its improvements are, upper strata carrier 1 is provided with two groups of sample feeding mouth groups 11, polysaccharase injection port group 12, first outlet through hole 17, second outlet through hole 18, second groove discharge orifice group 14; Each group sample feeding mouth group 11, polysaccharase injection port group 12, first outlet through hole 17, second outlet through hole 18, second groove discharge orifice group 14 are positioned at the side of the central horizontal axle of upper strata carrier 1;
Two groups of sample feeding mouth groups 11, polysaccharase injection port group 12, first outlet through hole 17, second outlet through hole 18, second groove discharge orifice group 14, central horizontal axle rotational symmetry relative to upper strata carrier 1;
Lower floor's carrier 2 is provided with two group reaction chamber groups 21, comb teeth shape passage 22, the 3rd groove discharge orifice group 25; Each group reaction chamber group 21, comb teeth shape passage 22, the 3rd groove discharge orifice group 25 are positioned at the side of the central horizontal axle of lower floor's carrier 2;
Two group reaction chamber groups 21, comb teeth shape passage 22, the 3rd groove discharge orifice group 25 are relative to the central horizontal axle rotational symmetry of lower floor's carrier 2.
In the present embodiment, by groove, reaction chamber, runner are arranged to relative to the axisymmetric mode of upper and lower layer glass central horizontal axle, the dull and stereotyped in-situ nucleic acid amplification chip that the present embodiment is provided possesses more reaction chamber group, make it possible to carry out more group reactions experiment simultaneously, save time.Preferably, can sample introduction tank, the second runner group, the first groove discharge orifice group, primer injection port group be arranged on central horizontal axle, the quantity of Pocket Machining can be reduced like this.
The third embodiment of the present invention relates to a kind of isothermal nucleic acid amplification device, comprises the dull and stereotyped original position isothermal nucleic acid amplification chip of square heat transfer platform 3 and above-mentioned any one; Heat transfer platform 3 is provided with heating unit; Lower floor's carrier 2 is fixed on heat transfer platform 3, and heating unit is to lower floor's carrier 2 homogeneous heating.
In the present embodiment, the bottom of lower floor's glass is fixed on metal fixing heat transfer platform, and heat transfer platform 3 comprises four movable blocks 33, limit assembly 31 and underframe 32; Limit assembly 31 comprises slide shaft portion and limiting section; Lower floor's carrier 2 is fixed on underframe 32, and the one end in slide shaft portion is run through movable block 33 and is fixedly connected with underframe 32; Movable block 33 can in the horizontal slip of slide shaft portion; Limiting section is located at the other end in slide shaft portion, and cross-sectional area is greater than the cross-sectional area in slide shaft portion.Best, this movable block 33 is provided with four, as shown in Figure 3 and Figure 4.Namely the distance that movable block can slide determines the distance that upper strata glass can slide.In the present embodiment, limit assembly can be iron nail.In Fig. 4, give upper strata glass direction state of sliding wherein, when upper strata glass slides to the right, upper strata glass promotes movable block 33 and moves laterally, and when movable block 33 arrives head of a nail position, movable block 33 cannot continue outside slip, and then limit the moving range of upper strata glass, can prevent from when excessive mobile upper strata glass, occurring that fluid seepage is in reaction chamber, the amount of primer or polysaccharase in reaction chamber be changed, affects experiment effect.Preferably, each movable block is fixed on underframe by two iron nails, enable movable block when mobile with underframe edge keeping parallelism.
Meanwhile, in the present embodiment, on the heat transfer platform 3 of uniformly transfer heat, heating unit can be realized homogeneous heating carried out to this lower floor's carrier 2, make the temperature in each reaction chamber identical, realize the equivalents of experimental situation by lower floor's glass is arranged on.
Utilize above-mentioned isothermal nucleic acid amplification device, a kind of isothermal nucleic acid amplification method can also be realized, comprise the following steps:
S0, upper strata carrier 1 is moved to predeterminated position.
Before experiment starts, need the sheet glass used of experiment needs to be placed into starting position, now, the position of upper strata glass and lower floor's glass as shown in Figure 4, for follow-up interpolation primer is prepared.
S1, add primer from primer injection port group 13, and make primer be full of the first groove discharge orifice group 15.
When adding primer premix, add from primer injection port group 13, primer premix is successively by primer injection port group 13, sample introduction tank 23, second runner group 24, first groove discharge orifice group 15, first flow 16, and the air in runner and unnecessary primer premix are discharged from the first outlet through hole 17.Identical in order to ensure the primer pre-mixing liquid measure be added in reaction chamber, when adding, need to guarantee that primer premix is full of each first groove discharge orifice.
S2, when adding primer premix to reaction chamber group 21, translation upper strata carrier 1, makes the first groove discharge orifice group 15 circulate with reaction chamber group 21 phase, and now, primer premix flows in reaction chamber group 21 and the 3rd groove discharge orifice group 25 from the first groove discharge orifice 15.
S3, upper strata carrier 1 is moved back to predeterminated position, prepare to add polysaccharase premix.
S4, add polysaccharase from polysaccharase injection port group 12, and make polysaccharase be full of the second groove discharge orifice group 14.
Add polysaccharase premix from polysaccharase injection port group 12, flowed in the 3rd groove discharge orifice group 25 by comb teeth shape passage 22.Air in comb teeth shape passage 22 and unnecessary polysaccharase premix flow out from the second outlet through hole 18.Identical in order to ensure polysaccharase premix addition, guarantee to be full of polymerase mix in the 3rd groove discharge orifice group.Described polysaccharase premix comprises polysaccharase, dNTP, Mg2+, reaction buffer.
S5, when adding polysaccharase in reaction chamber group 21, translation upper strata carrier 1, makes reaction chamber group 21 circulate with the second groove discharge orifice group 14 phase, and as shown in Figure 5, polysaccharase flows in reaction chamber group 21 and the 3rd groove discharge orifice group 25 from the second groove discharge orifice group 14.
S6, upper strata carrier 1 is moved back to predeterminated position.
S7, add sample from sample feeding mouth group 11, sample is directly entered in reaction chamber.
S8, control heating unit make reaction solution keep at the reaction temperatures.In the present embodiment, General reactions temperature is at 60 DEG C ~ 65 DEG C.
When carrying out feminine gender, positive control experiment, mobile upper strata glass is to the position of Fig. 8, and now, the reaction chamber group being positioned at comb teeth shape passage 22 side just can not add polysaccharase premix.Chip is moved back to starting position, add positive DNA sample, after the reaction times, the negative control group without polysaccharase is formed in the reaction chamber not adding polysaccharase premix, in the second runner 24 be located on the same line with the 3rd groove discharge orifice group 25, form the negative control group without DNA, the reaction chamber adding positive DNA sample is positive controls.
In addition, if arrange axisymmetric through hole, groove or runner respectively on upper strata glass, lower floor's glass, after step S3, again primer injection port group 13 adds primer, and translation upper strata carrier 1, first groove discharge orifice group 15 is circulated with reaction chamber group 21 phase of opposite side, and primer flows in the reaction chamber group 21 of opposite side from the first groove discharge orifice 15.
After the reaction times of 40 minutes, observe the turbidity of each reaction chamber, can direct qualitative reaction result.In this example, isothermal duplication is carried out to 8 different DNA sample, avoided loaded down with trivial details temperature cycle, substantially reduce the reaction times, reduced test operation intensity.
The respective embodiments described above realize specific embodiments of the invention, persons of ordinary skill in the art may appreciate that and in actual applications, can do various change in the form and details to it, and without departing from the spirit and scope of the present invention.
Claims (10)
1. a dull and stereotyped in-situ nucleic acid amplification chip, is characterized in that, comprises upper strata carrier (1) bonded to each other and lower floor's carrier (2); Can slide by relative level between described upper strata carrier (1) and described lower floor carrier (2);
Described upper strata carrier (1) is provided with sample feeding mouth group (11), polysaccharase injection port group (12), primer injection port group (13), the first outlet through hole (17), the second outlet through hole (18), the first groove discharge orifice group (15), the second groove discharge orifice group (14), first flow (16);
Described lower floor carrier (2) is provided with sample introduction tank (23), the second runner group (24), reaction chamber group (21), comb teeth shape passage (22), the 3rd groove discharge orifice group (25); Described reaction chamber group (21) is located between described comb teeth shape passage (22) increment or side, and the increment of the inflow end of described reaction chamber group (21) and described comb teeth shape passage (22) is located along the same line;
When described upper strata carrier (1) and lower floor's carrier (2) are positioned at starting position, described sample feeding mouth group (11) is circulated mutually with the position of described reaction chamber group (21); Described polysaccharase injection port group (12) is circulated mutually with described comb teeth shape passage (22), the 3rd groove discharge orifice group (25), the second outlet through hole (18); Described primer injection port group (13) is circulated mutually with described sample introduction tank (23), the second runner group (24), the first groove discharge orifice group (15), first flow (16), the first outlet through hole (17);
When adding primer to described reaction chamber group (21), upper strata carrier (1) described in translation, described first groove discharge orifice group (15) is circulated mutually with described reaction chamber group (21), and primer flows in described reaction chamber group (21) from described first groove discharge orifice (15);
When adding polysaccharase to described reaction chamber group (21), upper strata carrier (1) described in translation, described reaction chamber group (21) is circulated mutually with described second groove discharge orifice group (14), and polysaccharase flows in described reaction chamber group (21) from described second groove discharge orifice group (14).
2. dull and stereotyped in-situ nucleic acid amplification chip according to claim 1, is characterized in that, described upper strata carrier and lower floor's carrier are rectangle carrier.
3. dull and stereotyped in-situ nucleic acid amplification chip according to claim 2, it is characterized in that, described first groove discharge orifice group (15) comprises several the first groove discharge orifices, described second groove discharge orifice group (14) comprises several the second groove discharge orifices, and described 3rd groove discharge orifice group (25) comprises several the 3rd groove discharge orifices; Described reaction chamber group (21) comprises several reaction chambers; Described sample feeding mouth group (11) comprises several sample feeding mouths;
Described sample feeding mouth is arranged on same straight line equably; Described first groove discharge orifice is arranged on same straight line equably, described primer injection port (13) and described first groove discharge orifice are arranged on same straight line, described second groove discharge orifice is arranged on same straight line equably, described sample feeding mouth is corresponding with the position of described first groove discharge orifice, and described second groove discharge orifice and described sample feeding mouth are staggered; Described 3rd groove discharge orifice is arranged on same straight line equably, and described reaction chamber is arranged on same straight line equably; Described second runner group (24) comprises the second runner be positioned on straight line and the second runner be located on the same line with described 3rd groove discharge orifice group (25);
The straight line at the straight line at described sample feeding mouth place, the straight line at the first groove discharge orifice place, the second groove discharge orifice place parallels with the central horizontal axle on described upper strata carrier (1); The straight line at described reaction chamber place, the straight line at the 3rd groove discharge orifice place parallel with the central horizontal axle of described lower floor carrier (2).
4. dull and stereotyped in-situ nucleic acid amplification chip according to claim 3, it is characterized in that, described upper strata carrier (1) is provided with sample feeding mouth group (11) described in two groups, polysaccharase injection port group (12), the first outlet through hole (17), the second outlet through hole (18), the second groove discharge orifice group (14); Each group sample feeding mouth group (11), polysaccharase injection port group (12), the first outlet through hole (17), the second outlet through hole (18), the second groove discharge orifice group (14) are positioned at the side of the central horizontal axle on described upper strata carrier (1);
Sample feeding mouth group (11) described in two groups, polysaccharase injection port group (12), the first outlet through hole (17), the second outlet through hole (18), the second groove discharge orifice group (14), central horizontal axle rotational symmetry relative to described upper strata carrier (1);
Described lower floor carrier (2) is provided with reaction chamber group (21) described in two groups, comb teeth shape passage (22), the 3rd groove discharge orifice group (25); The side that each organizes described reaction chamber group (21), comb teeth shape passage (22), the 3rd groove discharge orifice group (25) are positioned at the central horizontal axle of described lower floor carrier (2);
Reaction chamber group (21) described in two groups, comb teeth shape passage (22), the 3rd groove discharge orifice group (25) are relative to the central horizontal axle rotational symmetry of described lower floor carrier (2).
5. dull and stereotyped in-situ nucleic acid amplification chip according to claim 4, it is characterized in that, described primer injection port (13) and described first groove discharge orifice are arranged on the central horizontal axle on described upper strata carrier (1), and described sample introduction tank is positioned on the central horizontal axle of described lower floor carrier (2).
6. dull and stereotyped in-situ nucleic acid amplification chip according to claim 4, is characterized in that, described first flow is T-shaped runner.
7. dull and stereotyped in-situ nucleic acid amplification chip according to claim 3, is characterized in that, when overlooking described first groove discharge orifice, the second groove discharge orifice, the 3rd groove discharge orifice, described first groove discharge orifice is cruciform; Described second groove discharge orifice, the 3rd groove discharge orifice are rectangle.
8. dull and stereotyped in-situ nucleic acid amplification chip according to claim 3, is characterized in that, described in each, the volume of the first groove discharge orifice is identical; Described in each, the volume of the second groove discharge orifice is identical; Described in each, the volume of reaction chamber is identical.
9. dull and stereotyped in-situ nucleic acid amplification chip according to claim 8, is characterized in that, the volumetric ratio between described reaction chamber, the first groove discharge orifice, the second groove discharge orifice is 10:6:3.
10. dull and stereotyped in-situ nucleic acid amplification chip according to claim 1, it is characterized in that, the diameter of described sample feeding mouth group (11), polysaccharase injection port group (12), primer injection port group (13), the first outlet through hole (17), the second outlet through hole (18) is 1mm.
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