CN103881902A - Multi-stage PCR reaction system and application thereof - Google Patents
Multi-stage PCR reaction system and application thereof Download PDFInfo
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- CN103881902A CN103881902A CN201210564539.0A CN201210564539A CN103881902A CN 103881902 A CN103881902 A CN 103881902A CN 201210564539 A CN201210564539 A CN 201210564539A CN 103881902 A CN103881902 A CN 103881902A
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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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- B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
Abstract
The present invention discloses a multi-stage PCR reaction system and an application thereof. Specifically the present invention discloses a multi-stage PCR reactor, which comprises two or a plurality of closeable PCR reaction tubes or chambers or compartments, wherein a closed or closeable connection channel is arranged between at least the two compartments, and is provided for transferring solid particles carrying a desired amount of a PCR amplification product to the downstream compartment from the upstream compartment so as to perform the multi-stage controllable PCR reaction with no cross interference. With the reactor, the multi-stage PCR reaction system with characteristics of high sensitivity and incapability of long term wide and practical application can become a conventional technology capable of being practically applied in a variety of fields.
Description
Technical field
The present invention relates to biology and detection field, particularly, the present invention relates to novel multistage PCR system and application thereof.
Background technology
PCR relates to by multiple circulations, the increase technology of certain polynucleotide sequence of index.Round pcr is well-known, and has been widely used.
Round pcr generally comprises following steps: sex change polynucleotide, are then annealed to pair of primers oligonucleotide at least on the polynucleotide of sex change (making the polynucleotide template hybridization of primer and sex change).After annealing steps, there is the enzyme of polymerase activity, use initial sex change polynucleotide as synthetic template, catalyze and synthesize a new polynucleotide chain that has mixed primer tasteless nucleotide.This series of steps (sex change, primer annealing and primer extension) forms a PCR circulation.
Along with the repetition of circulation, the volume index of new synthetic polynucleotide increases, and the new synthetic polynucleotide that early circulate because serving as reasons can be used as the synthetic template of follow-up circulation.
The sex change of DNA generally occurs in about 90-95 DEG C, and primer is generally annealed to the DNA of sex change at about 40-60 DEG C, as the step 1 of the primer of polymerase extension annealing, carries out at about 70-75 DEG C.Therefore, in PCR circulation, must change the temperature of reaction mixture (reaction system), in many cycle P CR experiment, will repeatedly change.
Round pcr has biological applications widely, for example comprise the contaminating microorganisms in DNA sequence analysis, probe generation, cloning nucleic acid sequences, site-directed mutagenesis, detection transgenation, judging viral infection, molecule " fingerprinting " and monitoring bio liquid and other sources.
Multiplex PCR (multiplex PCR, claim again Multiplex PCR or composite PCR), be to add two pairs or more primer in same PCR reaction system, amplify the PCR reaction of multiple nucleic acid fragments simultaneously, its reaction principle, reaction reagent and operating process are identical with general PCR.
Multiple PCR technique has been widely used in many fields of diagnostic nucleic acid, comprises gene knockout analysis, sudden change and polymorphism analysis, quantitative analysis and RNA detection etc.In infectious diseases field, multiple PCR technique has demonstrated its value, becomes identification virus, bacterium, fungi and parasitic effective ways.Utilize multi-PRC reaction one time, can detect simultaneously, identify multiple pathogens, in the differential diagnosis of clinical polyinfection, there is its unique advantage and very high practical value.
But in actual applications, conventional PCR reaction or multi-PRC reaction system no matter, pollutes if introduce the exogenous DNA of minute quantity in environment or operating process, just may occur false positive results.In order to prevent polluting, some experiments have to carry out at the operating equipment of high cleaning or operation room.
In PCR reaction process, improper being easy to of various test conditions control causes increasing unsuccessfully, reduces its sensitivity and specificity.
In addition, the sensitivity that existing round pcr can detect is also unsatisfactory, especially at the nucleic acid samples extremely low to content (as only contained the sample of 1-10 copy targeting sequence).Detect pathogenic agent (as HPV) in biopsy, postmortem sample time, because of the existence of irrelevant nucleic acid substances, detection sensitivity is difficult to improve always.
In sum, also lack at present in capping system, to pcr amplification product simple and effective carry out quantitatively or method that sxemiquantitative is shifted.
Therefore this area is in the urgent need to developing a kind of authentication method of highly sensitive, specificity good, immunity from interference is strong HPV type.
Summary of the invention
Object of the present invention is just to provide a kind of highly sensitive, specificity good, immunity from interference is strong PCR conversion unit and method.
A first aspect of the present invention, a kind of multistage PCR reaction tubes is provided, described reaction tubes comprises two or more closed PCR reaction chambers or compartment (10) (compartment), and between at least two compartments, be provided with sealing or closed connecting passage (40), described connecting passage is for allowing the solid particulate (50) that carries pcr amplification product be transferred to another compartment (or downstream compartment) from a compartment (or upstream compartment).
In another preference, described reaction tubes comprises n compartment, arbitrary positive integer that wherein n is 2-5000; Preferably, arbitrary positive integer that n is 2-500.
In another preference, described reaction tubes comprises M multistage group, and each multistage group has respectively 2,3,4 or 5 compartments that are interconnected, arbitrary positive integer that wherein M is 1-1000.
In another preference, M is 192,96,48,24,12,10,9,8,7,6,5,4,3,2 or 1.
In another preference, the compartment of described PCR reaction tubes is linear configuration, chain configuration or loop configurations.Preferably, be the configurations such as " one " word configuration, " V " word configuration or " " or "○" word.
In another preference, described each compartment is series connection setting.
In another preference, described compartment is arranged, and described matrix is a × b matrix, the positive integer that wherein a is 2-100, the positive integer that b is 2-100.
In another preference, described matrix is 6 × 8,6 × 16,8 × 12,12 × 16 matrixes.
In another preference, described compartment has chamber lid (20), and for the multiple compartments that are interconnected, after chamber lid separately all covers, just forms an enclosed space.
In another preference, described chamber lid is sealing cover.
In another preference, described chamber lid is integrated with compartment; More preferably, above-mentioned chamber lid is connected with compartment cavity by web member (22).
In another preference, described chamber lid separates with compartment cavity.
In another preference, described multiple or whole chambeies lid is one.
In another preference, described connecting passage is positioned at reaction chamber or upper compartment.
In another preference, described connecting passage is higher than the liquid level of liquid phase P CR reaction system predetermined in compartment.
In another preference, the lower edge of described connecting passage entrance apart from the distance H 1 on edge on compartment and compartment along meeting with the ratio of the vertical height H of cell bottom: H1/H≤1/2, preferably≤1/3, more preferably≤1/4, preferably≤1/5.
In another preference, described PCR reaction tubes has 2 or 3 the PCR compartments that interconnect.
In another preference, the volume of described single PCR compartment is about 10-10000 microlitre, preferably 20-2000 microlitre, more preferably 30-1000 microlitre, best 40-500 microlitre.
In another preference, the internal diameter of described connecting passage is 0.1-20mm, is preferably 0.2-10mm, is more preferably 0.2-5mm.
In another preference, the length of described connecting passage is 0.1-100mm, preferably 0.2-50mm.
In another preference, described connecting passage seals.
In another preference, when after the compartment lid upper chamber cover of described upstream, described connecting passage is on the inlet compartment top of upstream compartment, and be positioned under the lid of chamber along below, thereby thereby the solid particulate that makes to carry pcr amplification product is lifted and leaves after the reaction system that is positioned at compartment below, enter described entrance, through connecting passage, the outlet in downstream compartment by described connecting passage again, enters downstream compartment.
In another preference, described upstream compartment is provided with directing plate, and the solid particulate that carries pcr amplification product for guiding enters connecting passage entrance from upstream compartment.
In a second aspect of the present invention, a kind of system of carrying out multistage PCR reaction is provided, described system comprises:
Multistage PCR reaction tubes described in first aspect present invention, and
Solid particulate, described solid particulate is arranged at least one upstream compartment of described multistage PCR reaction tubes, and in the compartment of described upstream, carries out PCR when reaction, and described solid particulate can be adsorbed on the amplified production forming in amplification procedure.
In another preference, described solid particulate is magnetic microsphere.
In another preference, the median size of described solid particulate is 0.01～10mm, is preferably 0.1-5mm, is 0.2-2mm best.
In another preference, the nucleic acid adsorptive power of described solid particulate is subject to the impact of the factor such as granule surface area and surface properties.Conventionally the approximately 1/200-1/10 of pcr amplification product in the whole PCR reaction system of each particle portability, preferably about 1/100-1/15, more preferably 1/50-1/20.
In another preference, described magnetic microsphere is nucleocapsid structure.
In another preference, described magnetic microsphere be surperficial unmodified, finishing or having coating layer on surface.
In a third aspect of the present invention, a kind of multistage PCR reaction method is provided, described method comprises step:
(a) provide the multistage PCR reaction tubes described in first aspect present invention;
(b) in the PCR of described multistage PCR reaction tubes reaction compartments, add and carry out PCR and react required reagent, form liquid phase P CR reaction system, and in the compartment of one or more upstreams, add pcr template material and for adsorbing the solid particulate of amplified production, but in downstream compartment, do not add pcr template material, and liquid phase P CR reaction system in each compartment is not communicated with mutually and does not contact mutually;
(c) seal upstream compartment and the downstream compartment of described multistage PCR reaction tubes, thereby for the multiple compartments that are interconnected, after chamber lid separately all covers, form an enclosed space;
(d) in the compartment R1 of upstream, carry out PCR reaction, forming the first pcr amplification product and absorption has the solid particulate of described the first pcr amplification product;
(e) described in lifting, adsorb the solid particulate of pcr amplification product, leave after the liquid phase P CR reaction system that is positioned at compartment below, described upstream, enter the entrance of described connecting passage, through connecting passage, enter another downstream compartment R2 that is positioned at compartment downstream, described upstream, as the pcr template material in described downstream compartment;
(f) in described downstream compartment R2, carry out PCR reaction, form the second pcr amplification product.
In another preference, described PCR reaction comprises high temperature PCR, low temperature PCR, the reaction of two primer PCR, the reaction of many primer PCRs, RT-PCR reaction, archaeal dna polymerase PCR reaction, RNA polymerase PCR reaction.
In another preference, described carry out the reagent that PCR reacts required and be selected from lower group: PCR primer, damping fluid, dNTP, polysaccharase, magnesium ion etc.
In another preference, be arranged in described upstream compartment PCR primer or primer pair, be different or identical from being arranged in described downstream compartment PCR primer or primer pair.
In another preference, described method comprises:
(g) in the time that described multistage PCR reaction tubes has downstream compartment Ri, the positive integer that wherein i is >=2,
In downstream compartment Ri, carry out PCR reaction, forming absorption has the solid particulate of i level pcr amplification product; With
Described absorption there is is the solid particulate of i level pcr amplification product be transferred to the downstream compartment Ri+1 of time one-level by another connecting passage, and in described downstream compartment Ri+1, carry out PCR reaction, thereby formation i+1 level pcr amplification product and optional formation absorption have the solid particulate of i+1 level pcr amplification product;
(h) repeating step (g) one or many optionally.
In another preference, described method also comprises step: described the second pcr amplification product is detected; Or the detecting of amplified production to multistage PCR.
In another preference, described detection comprises probe hybridization, order-checking and/or electrophoresis.
In another preference, in described downstream compartment Ri, also place the solid particulate for adsorbing amplified production.
In another preference, described pcr template material comprises: biological tissue samples, organ samples, puncture sample, cell sample, nucleic acid extract, blood, serum, body fluid, saliva, hair, faecal samples, amniotic fluid samples, urine, secretory product (comprising cervical secretions, vaginal secretions etc.), nutrient solution, food samples, vaccine, pedotheque, water sample, air sample.
In another preference, described pcr template material source is from human body, animal, plant, microorganism, or is derived from the nucleic acid of synthetic.
In a fourth aspect of the present invention, a kind of pcr amplification equipment is provided, described equipment comprises:
For placing the reacting hole of PCR reaction tubes, wherein said PCR reaction tubes has upstream compartment and downstream compartment, and connects the connecting passage of described upstream compartment and downstream compartment;
For controlling the temperature regulating device of described reacting hole temperature, thereby make can carry out predetermined PCR reaction in the compartment of reaction tubes; With
The solid particulate that carries pcr amplification product for controlling is transferred to the control device of downstream compartment by described connecting passage from upstream compartment.
In another preference, described PCR reaction tubes is the multistage PCR reaction tubes described in first aspect present invention.
In another preference, described solid particulate is magnetic microsphere, and described control device is magnet or electro-magnet.
In another preference, described reacting hole is arranged, and described matrix is a × b matrix, the positive integer that wherein a is 2-100, the positive integer that b is 2-100.
In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and can combining mutually between specifically described each technical characterictic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tire out and state no longer one by one at this.
Brief description of the drawings
Fig. 1 has shown the schematic diagram of PCR reaction tubes in prior art.
Fig. 2 has shown the schematic diagram of a kind of multistage PCR reaction tubes of the present invention (having added liquid phase P CR reaction system).
Fig. 3 has shown the schematic diagram of a kind of multistage PCR reaction tubes of the present invention (not adding liquid phase P CR reaction system).
Fig. 4 has shown the schematic diagram of the another kind of multistage PCR reaction tubes of the present invention (triplet).
Embodiment
The inventor is through extensive and deep research, developed first a kind of highly sensitive, specificity good, immunity from interference is strong multistage PCR reaction tubes, equipment, system and method.Experiment shows, uses multistage PCR reaction tubes of the present invention not only can make detection sensitivity reach 1-2 copy, and possesses excellent antipollution immunity from interference.In addition, equipment of the present invention also can be realized the quantitative of pcr amplification product or sxemiquantitative transfer in capping system.Complete on this basis the present invention.
Multistage PCR reaction tubes
As used herein, term " reaction tubes of the present invention ", " multistage PCR reaction tubes ", " PCR reaction of high order pipe ", " multistage PCR reaction tubes ", " cascade PCR reaction tubes " are used interchangeably, refer to have at least two PCR reaction tubess relatively independent, PCR reaction compartments, and be provided with the transfering channel that allows the solid particulate that carries pcr amplification product to pass through between described PCR reaction compartments.
In the present invention, for any two the PCR reaction compartments that are communicated with by transfering channel, the reaction compartments of wherein advanced performing PCR reaction can be called to " upstream compartment ", " upstream reaction compartment " or " upstream PCR reaction compartments ", and another PCR reaction compartments is called to " downstream compartment ", " downstream reaction compartment " or " downstream PCR reaction compartments ".
Referring to Fig. 2 and Fig. 3.The multistage PCR reaction tubes of the present invention shown in figure comprises two closed PCR reaction chambers or compartment (10) (compartment).Each compartment 10 is provided with chamber lid (20).Preferably, described chamber lid is connected as a single entity by web member (22) and compartment body.
In addition, between two compartments, be provided with sealing or closed connecting passage (40), described connecting passage is for allowing the solid particulate (50) that carries pcr amplification product from being transferred to another compartment (being downstream compartment) by a compartment (being upstream compartment).
In the time that described compartment is vertically placed, described connecting passage can be level or tilt.Conventionally, the angle of inclination of connecting passage (connecting passage and horizontal angle) is 0-30 degree, is preferably 0-15 degree, is more preferably 0-10 degree.
In the time that connecting passage presents certain angle of inclination, can, easily by gravity, roll to or slide to the exit end of connecting passage from the inlet end of connecting passage from solid particulate upstream compartment, that carry pcr amplification product, thereby enter downstream compartment.
In the present invention, internal diameter and the length of described connecting passage are not particularly limited, as long as can allow the solid particulate that carries pcr amplification product pass through.
Typically, the internal diameter of connecting passage is 0.1-20mm, is preferably 0.2-10mm, is more preferably 0.2-5mm.
Typically, the length of connecting passage is 0.1-100mm, preferably 0.2-50mm.
In the present invention, connecting passage can be that sealed or closed.For example, in the time that the chamber of multistage PCR reaction tubes lid is separate type with compartment cavity, chamber lid can be made to the upper cover of integral type, this upper cover not only has the chamber lid corresponding to compartment cavity, but also has the sealing cover corresponding to connecting passage.Like this, in the time covering described upper cover, not only seal each compartment, also sealed corresponding connecting passage, thereby formed the reaction compartment of sealing.
In the present invention, the upper cover of integral type is specially adapted to have when compartment the situation of a × b matrix structure.
The size of compartments of the multistage PCR reaction tubes of the present invention is not particularly limited.Conventionally, the volume of single PCR compartment is about 10-10000 microlitre, preferably 20-2000 microlitre, more preferably 30-1000 microlitre, best 40-500 microlitre.
The compartment shape of the multistage PCR reaction tubes of the present invention is not particularly limited, and can be cylindrical, conical or other analogous shapes.
The material of the multistage PCR reaction tubes of the present invention is not particularly limited, and can be the other materials that plastics, glass maybe can see through magnetic field.Preferably plastics, such as acrylic plastering etc.
Carry the solid particulate of pcr amplification product
In the present invention, in compartment, carry out among PCR reaction or afterwards, the part in the pcr amplification product of formation can be adsorbed or be adhered to the solid particles surface that is placed in described compartment, thereby forms the solid particulate that carries pcr amplification product.In the time that described solid particulate shifts downstream compartment from upstream compartment, entrained pcr amplification product just can be used as the pcr template in downstream compartment.
In the present invention, the shape of solid particulate is not particularly limited, and can be spherical, oval, cylindrical, taper, cube or other shapes.Solid particulate can be solid, hollow, latticed or other structures, as long as this solid particulate can adsorb or carry pcr amplification product.
The material of solid particulate is not particularly limited, but preferred solid particulate is magnetic-particle, as magnetic microsphere.In the present invention, described magnetic-particle comprises the magnetic particle of tool and the magnetic particle of tool under the action of a magnetic field.
In another preference, described magnetic microsphere is nucleocapsid structure.
In another preference, described magnetic microsphere is surperficial unmodified or finishing, having coating layer on surface.
In the present invention, the size of described solid particulate is not particularly limited.Conventionally, the median size of solid particulate is 0.01～10mm, is preferably 0.1-5mm, is 0.2-2mm best.
The nucleic acid adsorptive power of described solid particulate is subject to the impact of the factor such as granule surface area and surface properties.Technician can buy or prepare the various solid particulate that can carry pcr amplification product by ordinary method.
A kind of preferred solid particulate be surface hydrophilicity or with the particle of the hydrophilic radicals such as hydroxyl.Like this, not only can adsorb nucleic acid molecule, also can in the time shifting, carry partial reaction mixed solution, thereby a certain amount of pcr amplification product is transferred to downstream compartment.
After granular size and material are determined, by ordinary method, can determine the quantity of the entrained PCR product of each solid particulate.Conventionally the approximately 1/200-1/10 of pcr amplification product in the whole PCR reaction system of each particle portability, preferably about 1/100-1/15, more preferably 1/50-1/20.By volume, the reaction liquid of a common particle portability 0.1-2 microlitre, this depends on size and the surface property of particle.
Depend on the size of needs and solid particulate, can in the compartment of upstream, place one or more solid particulates.In addition, can in downstream compartment, place or not place one or more solid particulates.
In the time having multiple particle in a compartment, these particles can be transferred to one or more downstream compartment.
Multistage PCR reaction
In the present invention; for the upstream reaction compartment being communicated with by transfering channel and downstream PCR reaction compartments; conventionally described upstream PCR reaction compartments carried out PCR reaction (" first step PCR reaction ") afterwards or among, be placed in reaction system solid particulate and can adsorb a part of amplified production.By the solid particulate of described some pcr amplification product of absorption, be transferred to downstream compartment by described transfering channel from upstream reaction compartment, the template that just can react as follow-up PCR in downstream compartment, thus carry out the PCR reaction of next stage.
Should be understood that in the present invention, although can carry out PCR reaction at least two upstream PCR reaction compartments, but, also can not carry out any reaction in some PCR reaction compartments, or not carry out PCR reaction.For example, only carry out probe hybridization, order-checking, electrophoresis or other detection reaction.
In the present invention, be not particularly limited for carrying out the reagent that PCR reacts required, can adopt the various PCR of this area routine to react required reagent.Representational reagent comprises (but being not limited to): polysaccharase; DNTP, damping fluid, magnesium ion etc.
In another preference, the length of described PCR primer is 12-100bp, preferably 15-50bp, more preferably 18～30bp.
In another preference, described reaction system comprises following component: 10 × amplification buffer, 4 kinds of dNTP mixtures, all kinds of archaeal dna polymerase, Mg
2+.
In another preference, each component concentration of described reaction system is as follows: 10 × amplification buffer, 5～20 μ l, 4 kinds of dNTP mixture 50～500 μ l, PCR primer 10～100 μ l, template DNA 0.1～2 μ g, Taq archaeal dna polymerase 1～5 μ l, Mg
2+0.5～3mmol/L, water 50～200 μ l.
In the present invention, the PCR primer in each compartment can be identical or different.For example, for three PCR reaction compartments that are communicated with by two connecting passages, a PCR primer pair, the 2nd PCR primer pair and the 3rd PCR primer pair lay respectively in different PCR compartments.A kind of representational three grades of PCR reaction tubess (triplet) as shown in Figure 4.
Now, in conjunction with Fig. 2, representational multistage PCR method of the present invention is described.The method comprises:
First in the PCR of described multistage PCR reaction tubes reaction compartments, add and carry out PCR and react required reagent, form liquid phase P CR reaction system 31 and 32, and in the compartment of upstream, add pcr template material and for adsorbing the solid particulate (as magnetic-particle) of amplified production, but in downstream compartment, do not add pcr template material, and liquid phase P CR reaction system 31 in two compartments is not communicated with mutually and does not contact mutually with 32;
Seal upstream compartment and the downstream compartment of described multistage PCR reaction tubes, form an enclosed space;
In the compartment R1 of upstream, carry out PCR reaction, forming the first pcr amplification product and absorption has the solid particulate 50 of described the first pcr amplification product;
The solid particulate 50 of absorption pcr amplification product as described in lifting (as by magnetic force or magnetic field), leave after the liquid phase P CR reaction system that is positioned at compartment below, described upstream, enter the entrance of described connecting passage, through connecting passage, enter another downstream compartment R2 that is positioned at compartment downstream, described upstream, as the pcr template material in described downstream compartment;
In described downstream compartment R2, carry out PCR reaction, form the second pcr amplification product;
If need, can repeat above-mentioned steps: the absorption forming in downstream compartment Ri (positive integer that i is>=2) is had to the solid particulate of i level pcr amplification product, be transferred to the downstream compartment R of time one-level by another connecting passage
i+1, and at described downstream compartment R
i+1in carry out PCR reaction, thereby form i+1 level pcr amplification product.
To detect HPV as example, the genomic dna of a kind of HPV that increases, for use in differentiating human papillomavirus HPV type method for distinguishing, comprises step:
(a) in the first step PCR reaction chamber or compartment of sealing, taking HPV genomic dna as template, increase by specific the first primer pair of HPV, thereby obtain the first amplified production P1;
(b) the first amplified production is transferred to the PCR compartment of the second stage from first step PCR compartment, as the template of second stage PCR, and in the second stage PCR compartment of sealing, increase by specific the second primer pair of HPV, thereby obtain the second amplified production P2;
(c) optionally, the amplified production Pi of previous step is transferred to i+1 level PCR compartment from i level PCR compartment, as the template of i+1 level PCR, and in the i+1 level PCR compartment of sealing, increase by the specific i+1 primer pair of HPV, thereby obtain i+1 amplified production P
i+1, the positive integer that wherein i is>=2; This step can be carried out one or many;
(d) to described the second amplified production P2 or the described i+1 amplified production P that obtain in previous step
i+1, check order, thus the sequence of acquisition HPV; With
(e) the HPV sequence recording and HPV standard sequence are compared, thereby identify the type of HPV.
Compared with the PCR reaction tubes (Fig. 1) of prior art, multistage PCR reaction tubes of the present invention can be at reaction compartment in the situation that sealing, PCR reaction product is adsorbed in solid particulate (as magnetic microsphere) for the first time, then easily it is transferred to downstream compartment from upstream compartment, thereby in downstream compartment, carry out PCR for the second time using the amplified production of transfer as template, thereby keeping high specific in the situation that, by twice or repeatedly PCR reaction significantly improved detection sensitivity.
In addition, another distinguishing feature of the present invention is at twice or repeatedly in PCR reaction process, whole multistage PCR reaction tubes or each multistage group of being communicated with are in closed state, therefore effectively prevented from introducing source of pollution in environment and operating process, also prevent the pollution to environment, thereby needn't need the high operating equipment of clean level or operation room.
In the present invention, although whole multistage PCR reaction tubes or each multistage group of being communicated with are in closed state, but still can utilize very easily solid particulate by reaction product (as pcr amplification product) from upstream compartment quantitatively or sxemiquantitative shift downstream compartment, thereby carry out the PCR reaction of next stage in downstream.The size of nucleic acid transfer amount, can realize by the size and number that regulates solid particulate.
Pcr amplification equipment
The present invention also provides a kind of pcr amplification equipment that can be used for the inventive method.A kind of preferred equipment comprises:
For placing the reacting hole of PCR reaction tubes, wherein said PCR reaction tubes has upstream compartment and downstream compartment, and connects the connecting passage of described upstream compartment and downstream compartment;
For controlling the temperature regulating device of described reacting hole temperature, thereby make can carry out predetermined PCR reaction in the compartment of reaction tubes; With
The solid particulate that carries pcr amplification product for controlling is transferred to the control device of downstream compartment by described connecting passage from upstream compartment.
Conventionally, described solid particulate is magnetic microsphere, and described control device is magnet or electro-magnet.This magnet is generally positioned at the top of reacting hole, and its magnetic field can cover all or part of of reacting hole region, thereby by disposable magnetic microsphere or be transferred to downstream compartment from upstream compartment in batches.
In another preference, described reacting hole is arranged, and described matrix is a × b matrix, the positive integer that wherein a is 2-100, and the positive integer that b is 2-100, to be used in conjunction with 96 orifice plates of routine etc.
In another preference, described equipment is also provided with the device of automatic controlling magnetic field, for the movement of controlling magnet or control unlatching/movement of electro-magnet, thereby realize magnetic microsphere synchronously with automatic controlled transfer.
Use pcr amplification equipment of the present invention, can realize extensive, high-throughput and automated operation.
Major advantage of the present invention comprises:
(a) highly sensitive;
(b) specificity is good;
(c) immunity from interference is strong;
(d) easy and simple to handle.
(e) can realize the transfer of quantitative and semi-quantitative.
(f) reduce or eliminate the pollution to environment.
PCR reaction tubes of the present invention and system, but make multistage PCR reaction system extremely sensitive but for a long time cannot broad practice become the routine techniques of an energy in multiple wide spectrum practical application.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Embodiment 1
Multistage PCR reaction
In the present embodiment, adopt the multistage PCR reaction tubes of diad shown in Fig. 2,
Primer is as follows:
MY09：5’-CGTCCMARRGGAWACTGATC-3’(SEQ?ID?NO.:1)
MY11：5’-GCMCAGGGWCATAAYAATGG-3’(SEQ?ID?NO.:2)
GP5：5’-TTTGTTACTGTGGTAGATACYAC-3’(SEQ?ID?NO.:3)
GP6：5’-GAAAAATAAACTGTAAATCATATTC-3’(SEQ?ID?NO.:4)
Wherein, the primer in the compartment of upstream is MY09/MY11, and the primer in downstream compartment is GP6/MY11 or GP5/MY9.
Will be containing the secretory product of HPV (human papillomavirus) with after ordinary method extracting nucleic acid, after 5 times or 10 times of serial dilutions, make HPV copy number and be about 1,2,5,10,25,50,100,200 sample.Add upstream compartment using described sample as template.In the compartment R1 of upstream, add respectively conventional PCR reaction system (, polysaccharase nucleic acid-templated comprising DNA, dNTP, primer, the ddH of 25 microlitres
2o) magnetic microsphere (literalness steel ball) of and 2 diameter 200nm.
First carry out PCR reaction for the first time, 95 DEG C of denaturations, 3 minutes, 95 DEG C, 30S; 60 DEG C of annealing 30s, 72 DEG C are extended 1 minute, totally 30 circulations.Then, 75 DEG C are extended 10 minutes again.
Then by magnetic field, 2 magnetic microspheres are transferred to downstream compartment by connecting passage, carry out PCR reaction for the second time.Condition is 95 DEG C, 30S; 60 DEG C of annealing 30s, 72 DEG C are extended 1 minute, totally 30 circulations.
The second pcr amplification product obtaining in downstream compartment R2 is detected by order-checking.Then the standard sequence database of detected result and HPV is compared, thereby whether and kind the existence that draws HPV.
Detected result shows, the method can detect the HPV virus of trace (in sample only containing 5-10 copy).In addition, not only sensitivity significantly improves, and false negative and false positive rate are all significantly lower than conventional PCR method (reduce approximately 10 times, improve approximately 1 order of magnitude) or HC2 method (reducing approximately 50%).
Embodiment 2
Multistage PCR reaction
Repeat embodiment 1, difference is: the multistage PCR reaction tubes of use comprises 2 multistage group, and each multistage group has respectively 2 compartments that are interconnected.4 rectangular arrangements of compartment.Like this, can carry out multistage PCR reaction to two samples simultaneously.
Embodiment 3
Multistage PCR reaction
Repeat embodiment 1, difference is: the multistage PCR reaction tubes of use comprises 48 multistage group, and each multistage group has respectively 2 compartments that are interconnected; Or use multistage PCR reaction tubes comprise 32 multistage group, each multistage group has respectively 3 compartments that are interconnected.
96 compartments present rectangular arranged, corresponding with 96 orifice plates of routine, and this multistage PCR reaction tubes can be positioned on 96 orifice plates.Like this, can carry out the multistage PCR reaction of secondary to 48 samples simultaneously; Or can carry out the multistage PCR reaction of three grades to 32 samples simultaneously.
Embodiment 4
The multistage PCR reaction of three grades
Repeat embodiment 1, difference is: the primer in downstream compartment R3 is GP6/GP5.
First step PCR: with embodiment 1.
Second stage PCR: with embodiment 1.
Third stage PCR: the magnetic-particle that carries the second pcr amplification product in downstream compartment R2 is transferred to downstream compartment R3, then carries out PCR for the third time, thereby obtain the 3rd pcr amplification product.
The 3rd pcr amplification product obtaining in downstream compartment R3 is detected by order-checking.Then the standard sequence database of detected result and HPV is compared, thereby whether and kind the existence that draws HPV.
Detected result shows, this detected result can detect in sample only containing 1-2 the HPV viruses molecule copying.In addition, not only sensitivity significantly improves, and false negative and false positive rate are all significantly lower than conventional PCR method or HC2 method (reducing approximately 50%).
Embodiment 5
The qualification of HPV type
5.1. uterine neck epidermis sampling:
(1) clinician, in the time getting detection sample, with sampling brush, after scraping patient's sample, first inserts in an aseptic 5ml stopple coupon, and sampling brush pastes tube wall and gently revolves half cycle, and small portion patient's sample is bonded in stopple coupon;
(2) guarantee to have enough samples to carry out multistage round pcr and detect after HPV, sampling brush is preserved to liquid from extracting out to put into detect in 5ml stopple coupon;
(3) in the 5ml of step 1 stopple coupon, add 2ml95% ethanol, cover lid, vibrates stopple coupon for several times up and down, makes to be attached to Sample preservation on tube wall in liquid;
(4) will on the stopple coupon of step 3, carry out mark, make it corresponding one by one with corresponding test experience;
(5) stopple coupon of step 4 is put to 4 DEG C of Refrigerator stores, to be measured.
5.2 multistage PCR
(1) first step PCR
(2) second stage PCR
(3) third stage PCR
The result that detects PCR reaction for the third time with 2% Agarose, positive products is for order-checking.
The 5.3 β oxyphorase PCR that detect for sample quality
B1：5’-ACACAACTGTGTTCACTAGC(SEQ?ID?NO.:5)
Β2：5’-CAACTTCATCCACGTTCACC(SEQ?ID?NO.:6)
5.4 order-checking
Water 13.5 μ L, 5 times of reaction buffer 3.5 μ L, BigDye1.11 μ L, sequencing primer 1 μ, PCR product 1 μ L carries out sequencing reaction on PCR instrument for the second time.PCR product is purified with Centri-Sep strip purification column, after purifying, in PCR pipe, draw 10 μ L purified products to the plate that checks order, sequencing primer can be selected from last PCR primer, upper machine sequencing, then analyzes sequencing result and HPV standard sequence (can available from published common data base).The DNA sequence dna that obtains conform to gene pool Plays HPV genotype DNA using 100% as HPV shaping standard (being the highest gold standard of the relevant HPV of U.S. FDA sizing).
Result
The inventive method, shows 3320 detected results that detect sample:
1319 samples are that non-HPV infects, and 1352 samples are that high-risk HPV infects, and 377 samples are that low risk HPV infects and 272 samples are polyinfection.
The result comparison of two kinds of methods of table 1
In 3320 samples, have an appointment 60% ([1352+377+272]/3320) are infected and are had HPV virus, and approximately 40% (1319/3320) do not infect HPV virus.
Conclusion: multistage PCR reaction tubes and reactive system have highly sensitive, accurate, antipollution and jamproof feature.
All documents of mentioning in the present invention are all quoted as a reference in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (16)
1. a multistage PCR reaction tubes, it is characterized in that, described reaction tubes comprises PCR reaction chamber or the compartment (10) that two or more are closed, and between at least two compartments, be provided with sealing or closed connecting passage (40), described connecting passage is for allowing the solid particulate (50) that carries pcr amplification product be transferred to another compartment (or downstream compartment) from a compartment (or upstream compartment).
2. PCR reaction tubes as claimed in claim 1, is characterized in that, described reaction tubes comprises n compartment, arbitrary positive integer that wherein n is 2-5000.
3. PCR reaction tubes as claimed in claim 1, is characterized in that, described reaction tubes comprises M multistage group, and each multistage group has respectively 2,3,4 or 5 compartments that are interconnected, arbitrary positive integer that wherein M is 1-1000.
4. PCR reaction tubes as claimed in claim 1, is characterized in that, the compartment of described PCR reaction tubes is linear configuration, chain configuration or loop configurations; And/or
Described compartment is arranged, and described matrix is a × b matrix, the positive integer that wherein a is 2-100, the positive integer that b is 2-100.
5. PCR reaction tubes as claimed in claim 1, is characterized in that, described compartment has chamber lid (20), and for the multiple compartments that are interconnected, after chamber lid separately all covers, just forms an enclosed space.
6. PCR reaction tubes as claimed in claim 1, is characterized in that, described connecting passage is positioned at reaction chamber or upper compartment.
7. PCR reaction tubes as claimed in claim 6, it is characterized in that, when after the compartment lid upper chamber cover of described upstream, described connecting passage is on the inlet compartment top of upstream compartment, and is positioned under the lid of chamber along below, thereby thereby the solid particulate that makes to carry pcr amplification product is lifted and leaves after the reaction system that is positioned at compartment below, enter described entrance, through connecting passage, then the outlet in downstream compartment by described connecting passage, downstream compartment entered.
8. a system of carrying out multistage PCR reaction, described system comprises:
Multistage PCR reaction tubes claimed in claim 1, and
Solid particulate, described solid particulate is arranged at least one upstream compartment of described multistage PCR reaction tubes, and in the compartment of described upstream, carries out PCR when reaction, and described solid particulate can be adsorbed on the amplified production forming in amplification procedure.
9. system as claimed in claim 8, is characterized in that, described solid particulate is magnetic microsphere.
10. a multistage PCR reaction method, is characterized in that, described method comprises step:
(a) provide multistage PCR reaction tubes claimed in claim 1;
(b) in the PCR of described multistage PCR reaction tubes reaction compartments, add and carry out PCR and react required reagent, form liquid phase P CR reaction system, and in the compartment of one or more upstreams, add pcr template material and for adsorbing the solid particulate of amplified production, but in downstream compartment, do not add pcr template material, and liquid phase P CR reaction system in each compartment is not communicated with mutually and does not contact mutually;
(c) seal upstream compartment and the downstream compartment of described multistage PCR reaction tubes, thereby for the multiple compartments that are interconnected, after chamber lid separately all covers, form an enclosed space;
(d) in the compartment R1 of upstream, carry out PCR reaction, forming the first pcr amplification product and absorption has the solid particulate of described the first pcr amplification product;
(e) described in lifting, adsorb the solid particulate of pcr amplification product, leave after the liquid phase P CR reaction system that is positioned at compartment below, described upstream, enter the entrance of described connecting passage, through connecting passage, enter another downstream compartment R2 that is positioned at compartment downstream, described upstream, as the pcr template material in described downstream compartment; h
(f) in described downstream compartment R2, carry out PCR reaction, form the second pcr amplification product.
11. methods as claimed in claim 10, is characterized in that, described PCR reaction comprises high temperature PCR, low temperature PCR, the reaction of two primer PCR, the reaction of many primer PCRs, RT-PCR reaction, archaeal dna polymerase PCR reaction, RNA polymerase PCR reaction.
12. methods as claimed in claim 10, is characterized in that, described method also comprises:
(g) in the time that described multistage PCR reaction tubes has downstream compartment Ri, the positive integer that wherein i is >=2,
In downstream compartment Ri, carry out PCR reaction, forming absorption has the solid particulate of i level pcr amplification product; With
Described absorption there is is the solid particulate of i level pcr amplification product be transferred to the downstream compartment R (i+1) of time one-level by another connecting passage, and carry out PCR reaction in described downstream compartment R (i+1), thereby formation i+1 level pcr amplification product and optional formation absorption have the solid particulate of i+1 level pcr amplification product; With
(h) repeating step (g) one or many optionally.
13. methods as claimed in claim 10, it is characterized in that, described pcr template material comprises: biological tissue samples, organ samples, puncture sample, cell sample, nucleic acid extract, blood, serum, body fluid, saliva, hair, faecal samples, amniotic fluid samples, urine, secretory product, nutrient solution, food samples, vaccine, pedotheque, water sample, air sample.
14. 1 kinds of pcr amplification equipment, is characterized in that, described equipment comprises:
For placing the reacting hole of PCR reaction tubes, wherein said PCR reaction tubes has upstream compartment and downstream compartment, and connects the connecting passage of described upstream compartment and downstream compartment;
For controlling the temperature regulating device of described reacting hole temperature, thereby make can carry out predetermined PCR reaction in the compartment of reaction tubes; With
The solid particulate that carries pcr amplification product for controlling is transferred to the control device of downstream compartment by described connecting passage from upstream compartment.
15. equipment as claimed in claim 14, it is characterized in that, described solid particulate comprises magnetic microsphere, and described control device are magnet or electro-magnet.
16. equipment as claimed in claim 14, is characterized in that, described reacting hole is arranged, and described matrix is a × b matrix, the positive integer that wherein a is 2-100, the positive integer that b is 2-100.
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