CN107586700A - Digital PCR container - Google Patents
Digital PCR container Download PDFInfo
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- CN107586700A CN107586700A CN201710939961.2A CN201710939961A CN107586700A CN 107586700 A CN107586700 A CN 107586700A CN 201710939961 A CN201710939961 A CN 201710939961A CN 107586700 A CN107586700 A CN 107586700A
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- reaction
- reaction vessel
- cover plate
- reactive tank
- pcr
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Abstract
The invention discloses a digital PCR container, which comprises a base, a reaction container and a cover plate, wherein the reaction container is positioned in a closed reaction cavity formed by the base and the cover plate; the reaction vessel has reaction holes on the surface and the density of the reaction holes<170/mm2The area of the reaction holes is 0.5-5 times of the area of the non-reaction holes, and the surface of the reaction container is hydrophobic. The invention has the characteristics of good sealing performance, convenient operation, large sample loading amount, easy detection and the like. In the nucleic acid detection process, the invention can reflect nucleic acid expression substances in an original sample to a greater extent, can effectively avoid the evaporation of a reaction system and an oil phase, simplifies the operation process and reduces the false positive rate.
Description
Technical field
The present invention relates to molecular biology experiment field digital pcr container, more particularly to a kind of good airproof performance, operation are just
Profit, applied sample amount are big, a kind of digital pcr container easily detected.
Background technology
Digital pcr is a kind of new polymerase chain reaction technology, and its principle is to nucleic acid using Poisson distribution principle
Concentration carries out accurately quantitative.During digital pcr study and be actually detected, the sample size of digital pcr is small, from target
The purpose nucleic acid that PCR reactions are extracted and participated in biological specimen is less, it is more difficult to reacts the original sample feature of target organism sample.
Digital pcr shortcoming is that applied sample amount is smaller at present, and negative rate is higher.The present invention is designed and developed and be big according to the shortcomings that conventional
Size, high power capacity, new digital PCR containers simple to operate.
The content of the invention
In order to solve the above problems, present invention aims at provide a kind of large scale, high power capacity, numeral simple to operate
PCR containers.
The purpose of the present invention is achieved through the following technical solutions：A kind of digital pcr container, including base, reaction hold
Device and cover plate, the reaction vessel is located to be made up of in confined reaction chamber base and cover plate；The reaction vessel, which has, to be placed with
The reaction zone of reacting hole, in reaction zone, react hole density<170/mm2, reaction hole area is the 0.5~5 of non-reaction hole area
Times, reaction vessel surface hydrophobicity.
Further, pore volume is reacted>1nL/ holes, the reacting hole cross section >=3300um2。
Further, reaction vessel surface hydrophilic angle>100°.
Further, reaction vessel also has non-reaction zone, and non-reaction zone is a hydrophobic plane, neat with the reaction zone
It is flat.
Further, the base has the reactive tank for accommodating reaction vessel, and reactive tank top is formed by cover plate for sealing
Confined reaction chamber.
Further, the reaction vessel is arranged in the reactive tank by supporter so that reaction vessel and reaction
There is space between groove bottom, above support is fixed on reaction groove sidewall, or the independently installed side wall in reactive tank, or is installed on
The cover plate bottom surface.
Further, the cover plate central is transparent region, and transparent region is recessed, has between recessed area and reaction vessel
Gap.
A kind of digital pcr container, including base, reaction vessel and cover plate, the reaction vessel are located at by base and cover plate
Form in confined reaction chamber；The base has the reactive tank for accommodating reaction vessel, and cover plate has above reaction vessel
Central, transparent region, and the central, transparent region is recessed, and gap is left between reaction vessel；Reactive tank top passes through lid
Plate seals；The reaction vessel by react groove sidewall supporter be arranged on the reactive tank in, or by it is independently installed in
The supporter of the side wall of reactive tank is arranged in the reactive tank, or by being installed on the supporter of cover plate bottom surface installed in described
In reactive tank, there is gap between reaction vessel and reaction groove bottom and between cover plate；The oiling method of reaction chamber is, to
Oil phase is added in reactive tank, then covers cover plate, the oil phase gone out by the recessed central, transparent regional replacement of cover plate so that edge
The oil phase liquid level rise in region, in elevated oil phase liquid level filling reactive tank reaction vessel and reaction vessel and cover plate concave surface it
Between space, and form the reactiveness of Water-In-Oil.
Further, the transparent material of the cover plate recessed area is the transparent materials such as PC, COC, COP, glass or PMMA.
Further, the fringe region of the cover plate is opaque, and fringe region is processed respectively with transparent region, or with transparent
Material is processed at one time, then peripheral region is carried out into nontransparentization.
Further, the transparent region of the cover plate and the transition face in nontransparent region are 45 degree of inclined-planes.
Further, the material of the reaction groove bottom is the good metal or metal alloy of heat conductivility such as copper, aluminium, also may be used
It is the small high temperature resistant easy processing flakiness thermoplastic of the coefficients of expansion such as PC, LCP, PTFE.
Further, the reaction vessel is that plate material punches to be formed, and described plate material is glass, crystal, gold
Category, thermoplastic.
The invention has the advantages that the present invention is applied to detection of nucleic acids process, its applied sample amount at least can be with 1nL/ holes, energy
Largely reflect original sample amplifying nucleic acid expression material, can effectively avoid reaction system and oil phase from evaporating, simplify operation stream
Journey, reduce false positive rate.
Brief description of the drawings
Fig. 1 is digital pcr structure of container overall pattern, shows container general structure schematic diagram.
Fig. 2 is digital pcr structure of container exploded view, shows container by base, reaction vessel, fixing glue, the part of cover plate four
Composition.
Fig. 3 is two kinds of concrete forms of reaction vessel, and (b) has non-reaction zone compared to (a).
Fig. 4 is container base figure, and for fixing reaction vessel, confined reaction chamber is formed together with cover plate.
Fig. 5 is container cover plate figure, and cover plate forms confined reaction chamber, protection and isolation PCR reactions with base by fixing glue.
Fig. 6 is digital pcr reservoir profiles figure, discloses confined reaction chamber and reaction chamber oil phase capacity.
Fig. 7 is the container fluoroscopic image that container reaction terminates collection.
Fig. 8 is the partial enlarged drawing for gathering image.
Fig. 9 is digital pcr container application schematic diagram, discloses digital pcr container application method.
Figure 10 is three kinds of concrete forms of supporter.
In figure, base 1, reactive tank 11, reaction vessel 2, reacting hole 21, non-reaction zone 22, fixing glue 3, cover plate 4, surrounding
Region 41, transparent region 42, transition face 43, supporter 5, lateral boss 51, positobe focus 61, feminine gender point 62, background bar 63, reaction
Plate 71, scraping blade 72, reaction system 73, observation interval I, temperature interval II.
Embodiment
Below with reference to the accompanying drawings this explanation is described further.
As illustrated in fig. 1 and 2, digital pcr container provided by the invention includes：By base 1, reaction vessel 2, fixing glue 3, lid
Plate 4.Reaction vessel 2 is between base 1 and cover plate 4, and cover plate 4 is connected with base 1 by mechanical means, between the two shape
Into closed reaction chamber, the including but not limited to glutinous fixing glue 3 of mechanical means in some specific embodiments.
As shown in Figure 3 a, there is reacting hole 21 on the surface of reaction vessel 2, and reacting hole 21 arranges to be formed instead according to certain rule
Answer area, in reaction zone, the density of reacting hole 21<170/mm2, the surface area of each reacting hole 21 is the table of the non-reacting hole of reaction zone
0.5~5 times of area, to ensure that the isolation strip between reacting hole 21 is sufficiently wide, be advantageous to the reactant for isolating each reacting hole
System, is also beneficial to when forming image, produces background bar, improves Signal-to-Noise, greatly reduces signal acquisition, separation and knowledge
Other difficulty.As the common knowledge of this area, reacting hole 11 is evenly distributed on the central area of reaction vessel 2, fringe region
Width be typically no less than 0.1mm.
The volume of reacting hole 21>1nL/ holes, cross section >=3300um2, be advantageous to load more reaction systems and reaction letter
Number collection.The shape of cross section of reacting hole 21 is the figures such as circle, square, rectangle, hexagon.
Sample size and enough support strengths in order to ensure reaction vessel, reaction vessel 2 are that plate material punches to be formed,
Plate material thickness is general>0.2mm.The plate material of reaction vessel 2 is including but not limited to being plastics (PC, LCP), metal
(stainless steel, aluminium alloy), glass, crystal (monocrystalline silicon, polysilicon) etc..Monocrystalline silicon is used in some specific embodiments.
The size of reaction vessel 2 is 12mm*12mm*0.4mm in some specific embodiments, and the quantity of reacting hole 21 is 15000, reacting hole 21
Sectional area is 5000um2, the volume of reacting hole 21 is 2nL, and every square millimeter of the number of reacting hole 21 is 104.
As shown in Figure 3 b, reaction vessel also has non-reaction zone, is generally located on reaction zone side, and non-reaction zone is one thin
Horizontal plane, flushed with the reaction zone.Non-reaction zone length is typically no less than 0.1mm, and the width of non-reaction zone is no more than sample-adding
The width of head moving direction.By the setting of non-reaction zone, reaction solution first can be loaded into non-reaction zone, load reaction solution
Reaction solution can be loaded into reacting hole by feed head from non-reaction zone.
In some specific embodiments, when reaction vessel 2 is by the use of monocrystalline silicon as plate material, pass through ion(ic) etching work
Skill completes the processing of reacting hole 21.After the shape of reacting hole 21 machines, pass through 1200 DEG C of surface high-temperature oxydation, oxide-film typical case
Thickness control is in 110nm, and at 110 °~120 °, hydrophobic property is presented in plate material surface for hydrophilic angle control.Using other materials
When material is used as plate material, the general surface modification such as silanization, bulk doped can be used, which to be allowed to surface, has hydrophobic layer, hydrophilic
Angle is preferably>100°.
Base 1 has the reactive tank 11 for accommodating reaction vessel, and the top of reactive tank 11 is sealed by cover plate 4, is formed closed anti-
Answer chamber.The reaction vessel 2 is arranged in the reactive tank 11 by supporter 5 so that reaction vessel 2 and the bottom surface of reactive tank 11
Between leave space, the space is generally 0.1mm~5mm, is 0.2mm in some specific embodiments.Above support 5 is solid
Due to the side wall of reaction vessel 22, and do not contacted substantially in the bottom surface of reactive tank 11, as shown in Figure 10 a, or independently of reactive tank 11,
As shown in fig. lob, or it is installed on the cover plate bottom surface.In some specific embodiments, supporter 5 is relative with the upper table of base 1
Face is rich property, and reaction vessel 2 can be made to have certain elasticity in the above-below direction of reactive tank 11, avoid the large area of reaction vessel 2 with it is anti-
Answer the upper surface Hard link injury response container 2 of groove 11.In some specific implementation cases, supporter 5 also has lateral boss 51,
As shown in Figure 5.Supporter 5 is distributed along base edge, to determine reaction vessel 2 according to the distance of base edge 21, this
Distance needs be 2-10mm according to rich property.In Figure 10 a, supporter 5 is to be connected to the L-shaped fastener for reacting groove sidewall, Figure 10 b
In T-shaped fastener independently of reactive tank 11 is shown, the contact area contacted with the bottom surface of reactive tank 11 is few.In Figure 10 c
It is shown that being fixed on the fastener of the bottom surface of cover plate 4, do not contacted completely with the bottom surface of reactive tank 11.Pass through the setting of supporter 5 so that
Reaction vessel 2 is almost suspended in reactive tank, and the temperature of isolation reaction base and reaction vessel 2 is directly transmitted by supporter 5, made
Reaction vessel 2 can only enter trip temperature transmission by the oil phase being wrapped in around reaction vessel so that the temperature that reaction vessel 2 receives
More uniformly, avoid because the influence that reaction vessel is directly connected to caused trickle temperature and directly transmitted with reacting base,
So that the biological respinse environment of reaction vessel is closer to preferable state.Wherein, the supporter 5 in Figure 10 b typically uses
Thermoplastic or rubber injection mold or Metal Cutting, burn into seaming operation are completed, it is generally preferable to the relatively low thermoplasticity modeling of thermal capacity
Material, thermal capacity is reduced, improve the speed of temperature transmission.In some specific implementation cases, supporter 5 is carried out using PC, LCP etc.
Injection.Oil phase is loaded in reactive tank 11, oil phase covers reaction vessel 2, to the aqueous phase PCR reaction system shapes in reaction vessel 2
Into parcel, in actual mechanical process, base 1 is placed on reaction plate 71 and heated, and passes through the setting of supporter 5 so that anti-
Container 2 is answered almost to be suspended in reactive tank, the temperature of isolation reaction base is directly transmitted, and makes reaction vessel 2 can only be by being wrapped in
Oil phase around reaction vessel enters trip temperature transmission, and each tropism of temperature is good when oil phase transmits temperature, and temperature is uniform, transmission speed
Hurry up, be advantageous to PCR reactions temperature is accurate and uniform conductive so that the temperature that reaction vessel 2 receives is more uniform, avoid because
Reaction vessel 2 and base 1 are directly connected to the influence that caused trickle temperature is directly transmitted so that the biology of reaction vessel 2
Reaction environment is closer to preferable state.In certain embodiments, oil phase is chosen for and PCR reaction systems not phase
Hold, the transparency is good, heat conduction is good, and not volatile fluorinated oil, mineral oil, vegetable oil etc. be single or blending constituent oil phase.
As shown in figure 5, the cover plate 4 includes opaque peripheral region 41 and the clear area above reaction vessel 2
Domain 42.Reaction vessel can be advantageous to comprehensive observation or detection reaction vessel installation site and surface etc. by transparent region
Production and Quality Control.As shown in fig. 6, the side wall of peripheral region 41 and reactive tank 11 is relatively fixed, confined reaction chamber, reaction are formed
Intracavitary fills oil phase.Transparent region 42 is up concave type, and keeps minute surface and smooth in favor of observing response hole 21.Reaction vessel 2
Observation interval I, shape between the lower surface of reaction vessel 2 and the bottom surface of reactive tank 11 are formed between upper surface and the transparent region 42 of cover plate
Into temperature interval II, observation interval I and temperature interval II can carry out independent setting by supporter 5, and observation interval I is isolation
PCR reacts and the reaction result of observing response hole 21, temperature interval II are to transmit PCR reaction temperatures, makes reaction temperature more equal
It is even.Observation interval I and temperature interval II is generally 0.1mm~5mm, is that observation interval I is in some specific embodiments
0.2mm, temperature interval II are 0.4mm.
As shown in fig. 6, the thickness of transparent region 42 is generally 0.1~5mm, it is 0.5mm in some specific embodiments.
As shown in fig. 6, transparent region 42 and the transition face 43 in nontransparent region 41 are 45 degree of inclined-planes, clear area is favorably reduced
The reflection of the light source caused by material curved surface changes of domain 42, improves transparent region signal to noise ratio.
The invention also discloses a kind of oiling method of reaction chamber：Oil phase is added into reactive tank, then covers cover plate, is led to
Cross the oil phase that the recessed central, transparent regional replacement of cover plate goes out in reactive tank so that oil phase liquid in the reactive tank in side edge thereof region
Face raises, oil phase filling reaction chamber, and forms the reactiveness of Water-In-Oil.This method operation facility, oil phase filling speed is fast,
Good airproof performance, avoids oil phase bubble caused by prior art cover plate perforate oiling, and tank filler sleeve poor sealing causes oil phase fast
Speed volatilization, and tank filler sleeve band are cumbersome.In addition, oil phase may volatilize in PCR courses of reaction, it is closed
Reaction chamber, which helps to reduce, to volatilize.Different oil phases has different boiling characteristics, with it, it is quantitative to calculate addition
Oil phase so that liquid level is higher by the volume of the lower surface of 24 transparent region of cover plate 42 and waved more than oil phase in PCR reaction tests after rising
Volume is sent out, is terminated in reaction, the liquid level of oil phase in the reactor chamber is not less than the lower surface of cover plate clear area 42.
The present invention is explained further below by way of example explanation.
PCR reaction systems 73 are loaded into the upper surface of reaction vessel 2 or non-reaction zone 22, and will by the motion of scraping blade 72
Reaction solution is coated uniformly on the surface of reaction vessel 2, because the surface hydrophobicity of reaction vessel 2, in the motion stress of scraping blade 72 and reacting hole
In the presence of capillary effectiveness, partial reaction system is added into reacting hole 21 and removes the PCR reactants of isolation strip remained on surface
It is 73.
Oil phase will be loaded in the reactive tank 11 for being loaded with reaction vessel 2, the reaction system 73 that oil phase parcel has loaded
Reaction vessel 2.
Cover plate 4 is fixed on formation confined reaction chamber in the side wall of base 1, base 1, reaction vessel 2, cover plate 4 are built into
PCR containers.
PCR containers, which are placed on the reaction plate 71 of PCR amplification instrument, carries out amplified reaction.
Imaging function and image store function equipment are carried by band imaging function microscope or other optics, by transparent
Window 42 can be imaged or record the result reacted in the reacting hole 21 of reaction vessel 2, and the reacting hole 21 for having nucleic acid substances is exciting
Bright spot is presented under light irradiation, as shown in Figure 7.
Fig. 7 is amplified, can clearly observe the positobe focus 61 formed after reacting hole 31 reacts, negative point 62,
The bar 63 that has powerful connections that isolation strip is formed, background bar 63 is as the separation marking for detecting reflecting point in image, if reacting hole 21 is close
Two reacting holes 21 can be imaged to possible UNICOM, after being unfavorable for the reaction of PCR reacting holes 21 after the too small imaging of Du Taigao or isolation strip
Signal detection.
The volume of reacting hole 21 is bigger, and the reaction oil phase into reacting hole 21 accumulates bigger, the signal of reacting hole 21 in the picture
Stronger, reaction pore volume is not less than 1nL/ holes.
By taking the detection of HBV carrying capacity in blood plasma as an example, the volume 2nL of reacting hole 21, reacting hole in some specific embodiments
21 numbers are 15000, and it is 30 μ L once to load reaction solution, by taking blood plasma as an example, is extracted by nucleic acid purification process from 1mL blood plasma
DNA is soluble in 20 μ L buffer solution, plus corresponding PCR reacted constituents, that is, forms 30 μ L PCR reaction systems, with
This is that reaction system carries out digital pcr reaction.Digital pcr sensitivity of the present invention is 1copy, is detected equivalent to from 1mL blood plasma
To 1copy nucleic acid, i.e., the concentration of HBV nucleic acid is 0.2IU/mL in blood plasma.
Claims (13)
1. a kind of digital pcr container, including base, reaction vessel and cover plate, it is characterised in that the reaction vessel is located at the bottom of by
Seat and cover plate are formed in confined reaction chamber；The reaction vessel, which has, is placed with the reaction zone of reacting hole, in reaction zone, reacting hole
Density<170/mm2, reaction hole area is 0.5~5 times of non-reaction hole area, reaction vessel surface hydrophobicity.
2. PCR containers as claimed in claim 1, it is characterised in that reaction pore volume>1nL/ holes, the reacting hole cross section
≥3300um2。
3. PCR containers as claimed in claim 1, it is characterised in that reaction vessel surface hydrophilic angle>100°.
4. PCR containers as claimed in claim 1, it is characterised in that reaction vessel can also have non-reaction zone, and non-reaction zone is
Hydrophobic plane, flushed with the reaction zone.
5. PCR containers as claimed in claim 1, it is characterised in that the base has the reactive tank for accommodating reaction vessel, instead
Groove top is answered to form confined reaction chamber by cover plate for sealing.
6. PCR containers as claimed in claim 5, it is characterised in that the reaction vessel is arranged on described anti-by supporter
Answer in groove so that have space between reaction vessel and reaction groove bottom, above support is fixed on reaction groove sidewall, or independent peace
Side wall loaded on reactive tank, or it is installed on the cover plate bottom surface.
7. PCR containers as claimed in claim 5, it is characterised in that the cover plate central is transparent region, under transparent region
It is recessed, there is gap between recessed area and reaction vessel.
8. a kind of digital pcr container, including base, reaction vessel and cover plate, it is characterised in that the reaction vessel is located at the bottom of by
Seat and cover plate are formed in confined reaction chamber；The base has the reactive tank for accommodating reaction vessel, and cover plate has to be held positioned at reaction
Central, transparent region above device, and the central, transparent region is recessed, and gap is left between reaction vessel；On reactive tank
Portion passes through cover plate for sealing；The reaction vessel is arranged in the reactive tank by reacting the supporter of groove sidewall, or by only
The supporter of the vertical side wall for being installed on reactive tank is arranged in the reactive tank, or the supporter by being installed on cover plate bottom surface is pacified
In the reactive tank, there is gap between reaction vessel and reaction groove bottom and between cover plate；The oiling of reaction chamber
Method is that oil phase is added into reactive tank, then covers cover plate, the oil gone out by the recessed central, transparent regional replacement of cover plate
Phase so that the oil phase liquid level rise of fringe region, in elevated oil phase liquid level filling reactive tank reaction vessel and reaction vessel with
Space between cover plate concave surface, and form the reactiveness of Water-In-Oil.
9. PCR containers as claimed in claim 7 or 8, it is characterised in that the transparent material of the cover plate recessed area be PC,
The transparent materials such as COC, COP, glass or PMMA.
10. PCR containers as claimed in claim 7 or 8, it is characterised in that the fringe region of the cover plate is opaque, marginal zone
Domain is processed respectively with transparent region, or is processed at one time with transparent material, then peripheral region is carried out into nontransparentization.
11. PCR containers as claimed in claim 7, it is characterised in that the transparent region of the cover plate and the mistake in nontransparent region
It is 45 degree of inclined-planes to cross face.
12. the PCR containers as described in claim any one of 1-11, it is characterised in that it is described reaction groove bottom material for copper,
The good metal or metal alloy of the heat conductivilitys such as aluminium, also but the small high temperature resistant easy processing of the coefficient of expansion such as PC, LCP, PTFE is into thin
Piece thermoplastic.
13. the PCR containers as described in claim any one of 1-11, it is characterised in that the reaction vessel is beaten for plate material
Hole is formed, and described plate material is glass, crystal, metal, thermoplastic.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710939961.2A CN107586700A (en) | 2017-10-11 | 2017-10-11 | Digital PCR container |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710939961.2A CN107586700A (en) | 2017-10-11 | 2017-10-11 | Digital PCR container |
Publications (1)
| Publication Number | Publication Date |
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| CN107586700A true CN107586700A (en) | 2018-01-16 |
Family
ID=61052738
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710939961.2A Pending CN107586700A (en) | 2017-10-11 | 2017-10-11 | Digital PCR container |
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Cited By (4)
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| CN110068558A (en) * | 2018-01-24 | 2019-07-30 | 思纳福(北京)医疗科技有限公司 | Microlayer model container |
| WO2019144905A1 (en) * | 2018-01-24 | 2019-08-01 | 北京光阱管理咨询合伙企业（有限合伙） | Microdroplet container, method for preparing microdroplet container, microdroplet spreading method, microdroplet formation kit, temperature control device, oil phase composition for microdroplet formation and treatment method therefor |
| CN110541023A (en) * | 2018-05-28 | 2019-12-06 | 北京中科生仪科技有限公司 | digital PCR chip |
| US11666900B2 (en) | 2018-01-24 | 2023-06-06 | Sniper (Suzhou) Life Technology Co. | Motion controlling mechanism, liquid discharging nozzle, microdroplet generating device and method, liquid driving mechanism and method, microdroplet generating method, and surface processing method of liquid discharging nozzle |
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