CN103881902B - Multistage PCR response system and application thereof - Google Patents
Multistage PCR response system and application thereof Download PDFInfo
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- CN103881902B CN103881902B CN201210564539.0A CN201210564539A CN103881902B CN 103881902 B CN103881902 B CN 103881902B CN 201210564539 A CN201210564539 A CN 201210564539A CN 103881902 B CN103881902 B CN 103881902B
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- 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 discloses a kind of multistage PCR response system and application thereof.Specifically, the invention discloses a kind of multistage PCR reactor, described reactor includes two or more closed PCR reaction tubes or chamber or compartment, and be provided with between at least two compartment closing or closed interface channel, described interface channel, for allowing the solid particle carrying the desired amount of pcr amplification product be transferred to downstream compartment from upstream compartment, is carried out the multistage controlled PCR without cross interference and is reacted.The reactor of the present invention, but make the sensitiveest but for a long time cannot the multistage PCR reaction system of broad practice become one can be at the routine techniques of the actual application of multiple wide spectrum.
Description
Technical field
The present invention relates to biology and detection field, in particular it relates to the multistage PCR system of novelty and application thereof.
Background technology
PCR relates to by multiple circulations, the technology of certain polynucleotide sequence of exponential amplification.Round pcr is many institute's weeks
Know, and be widely used.
Round pcr generally comprises following steps: denatured polynucleotide, is then annealed to by least pair of primers oligonucleotide
(the polynucleotide template hybridization of primer and degeneration is made) on the polynucleotide of degeneration.After annealing steps, there is polymerase activity
Enzyme, use initial denatured polynucleotide as synthesis template, catalyze and synthesize a new primer tasteless nucleotide that is mixed with
Polynucleotide chain.This series of steps (degeneration, primer annealing and primer extension) constitutes a PCR cycle.
Along with the repetition of circulation, the volume index of newly synthesized polynucleotide increases, because of newly synthesized many by relatively early circulating
Nucleotide can be used as the synthesis template of following cycle.
The degeneration of DNA typically occurs in about 90-95 DEG C, and primer is typically annealed to the DNA of degeneration at about 40-60 DEG C, with poly-
The step of the primer that synthase extends annealing is typically carried out at about 70-75 DEG C.Therefore, in PCR cycle, it is necessary to change reaction mixing
The temperature of thing (reaction system), to be varied multiple times in multi cycle PCR experiment.
Round pcr has biological applications widely, and including such as, DNA sequence analysis, probe produce, cloning nucleic acid sequence
Row, site-directed mutagenesis, detection gene mutation, judging viral infection, molecule " fingerprint analysis " and monitoring biofluid and other sources
In contaminating microorganisms.
Multiplex PCR (multiplex PCR, also known as Multiplex PCR or composite PCR), is in same PCR reaction system
Add two pairs or more primer, amplify simultaneously multiple nucleic acid fragment PCR reaction, its reaction principle, reaction reagent and
Operating process is identical with general PCR.
Multiple PCR technique has been widely used in many fields of diagnostic nucleic acid, including gene knockout analysis, sudden change and many
State property analysis, quantitative analysis and RNA detection etc..In infectious disease field, multiple PCR technique has shown that its value,
Become identification virus, antibacterial, fungus and the effective ways of parasite.Utilize a multi-PRC reaction, can detect simultaneously, differentiate
Go out multiple pathogens, the Differential Diagnosis of clinical mixed infection has the advantage of its uniqueness and the highest practical value.
But, in actual applications, Standard PCR reaction no matter or multi-PRC reaction system, if environment or operation
During introduce the exogenous DNA pollution of very small amount, it is possible to false positive results occurs.In order to prevent polluting, some must not test
Operation equipment or operating room at high cleaning are not carried out.
In PCR course of reaction, various experimental conditions control improper to easily lead to expand unsuccessfully, reduce its sensitivity with
Specificity.
Additionally, the sensitivity that existing round pcr can detect is the most unsatisfactory, especially at the core extremely low to content
Acid sample (as only contained the sample of 1-10 copy targeting sequence).When detecting pathogen (such as HPV) in biopsy, autopsy samples,
Because of the existence of unrelated nucleic acid material, 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
The method of sxemiquantitative transfer.
Therefore this area is in the urgent need to a kind of highly sensitive, mirror of specificity good, capacity of resisting disturbance is strong HPV type of exploitation
Determine method.
Summary of the invention
It is an object of the invention to provide a kind of highly sensitive, specificity good, capacity of resisting disturbance is strong PCR consersion unit and
Method.
A first aspect of the present invention, it is provided that a kind of multistage PCR reaction tube, described reaction tube includes that two or more can seal
The PCR reaction chamber closed or compartment (10) (compartment), and between at least two compartment, it is provided with maybe can closing of closing
Interface channel (40), described interface channel for allow the solid particle (50) carrying pcr amplification product from a compartment (or
Trip compartment) it is transferred to another compartment (or downstream compartment).
In another preference, described reaction tube includes n compartment, and wherein n is arbitrary positive integer of 2-5000；Preferably
Ground, n is arbitrary positive integer of 2-500.
In another preference, described reaction tube includes M multistage group, and each multistage group is respectively provided with 2,3,4 or 5 phases
Intercommunicated compartment, wherein M is arbitrary positive integer of 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 tube is linear configuration, branched configuration or loop configurations.Relatively
Goodly, in " one " word configuration, " V " word configuration or the configuration such as " " or "○" word.
In another preference, described each compartment is for being arranged in series.
In another preference, described compartment is arranged in arrays, and described matrix is a × b matrix, and wherein a is 2-100's
Positive integer, b is the positive integer of 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 being interconnected,
After respective chamber lid all covers, just constitute one and close space.
In another preference, described chamber lid is for sealing lid.
In another preference, described chamber lid is integrated with compartment；More preferably, above-mentioned chamber lid passes through connector (22)
It is connected with compartment cavity.
In another preference, described chamber lid separates with compartment cavity.
In another preference, the plurality of or whole chambeies lid is one.
In another preference, described interface channel is positioned at reaction chamber or upper compartment.
In another preference, the liquid level of the liquid phase P CR reaction system that described interface channel is predetermined higher than in compartment.
In another preference, lower distance H1 on edge and the edge on compartment along distance compartment of described interface channel entrance
Meet with the ratio of 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 tube has 2 or 3 the PCR compartments interconnected.
In another preference, the volume of described single PCR compartment is about 10-10000 microlitre, and preferably 20-2000 is micro-
Rise, more preferably 30-1000 microlitre, most preferably 40-500 microlitre.
In another preference, the internal diameter of described interface channel is 0.1-20mm, preferably 0.2-10mm, is more preferably
0.2-5mm。
In another preference, a length of 0.1-100mm of described interface channel, preferably 0.2-50mm.
In another preference, described interface channel is to close.
In another preference, when, after described upstream compartment lid upper chamber cover, described interface channel is at the entrance of upstream compartment
Upper compartment, and be positioned at along lower section under the lid of chamber, so that the solid particle carrying pcr amplification product is lifted thus leaves
After being positioned at the reaction system below compartment, enter described entrance, through interface channel, then by described interface channel in downstream every
The outlet of room, enters downstream compartment.
In another preference, described upstream compartment is provided with guided plate, for guiding the solid carrying pcr amplification product
Granule enters interface channel entrance from upstream compartment.
In a second aspect of the present invention, it is provided that a kind of system carrying out multistage PCR reaction, described system includes:
Multistage PCR reaction tube described in first aspect present invention, and
Solid particle, described solid particle is positioned at least one upstream compartment of described multistage PCR reaction tube, and
When carrying out PCR reaction in described upstream compartment, described solid particle can adsorb the amplified production formed in amplification procedure.
In another preference, described solid particle is magnetic microsphere.
In another preference, the mean diameter of described solid particle is 0.01～10mm, preferably 0.1-5mm,
Goodly for 0.2-2mm.
In another preference, the nucleic acid absorption power of described solid particle is by the factor such as granule surface area and surface nature
Impact.Generally, in each granule portability whole PCR reaction system, the about 1/200-1/10 of pcr amplification product, is 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 surface unmodified, surface modify or surface with painting
Layer.
In a third aspect of the present invention, it is provided that a kind of multistage PCR reaction method, described method includes step:
A () provides the multistage PCR reaction tube described in first aspect present invention；
B () adds the reagent carried out needed for PCR reaction in the PCR reaction compartments of described multistage PCR reaction tube, form liquid
Phase PCR reaction system, and in one or more upstream compartment, add pcr template material and for adsorbing consolidating of amplified production
Body granule, but the liquid phase P CR reaction system being added without in downstream compartment in pcr template material, and each compartment does not connects
And be not in contact with each other；
C () closes upstream compartment and the downstream compartment of described multistage PCR reaction tube, hence for be interconnected multiple every
For room, after respective chamber lid all covers, constitute one and close space；
D () carries out PCR reaction in upstream compartment R1, form the first pcr amplification product and be adsorbed with a described PCR
The solid particle of amplified production；
Adsorb the solid particle of pcr amplification product described in (e) lifting, leave the liquid phase being positioned at below described upstream compartment
After PCR reaction system, enter the entrance of described interface channel, through interface channel, enter and be positioned at described upstream compartment downstream
Another downstream compartment R2, as the pcr template material in described downstream compartment；
F () carries out PCR reaction in described downstream compartment R2, form the second pcr amplification product.
In another preference, described PCR reaction includes high temperature PCR, low temperature PCR, decoding for DTMF PCR reaction, many primers
PCR reaction, RT-PCR reaction, archaeal dna polymerase PCR reaction, RNA polymerase PCR reaction.
In another preference, described carry out PCR reaction needed for reagent selected from lower group: PCR primer, buffer,
DNTP, polymerase, magnesium ion etc..
In another preference, be positioned at the PCR primer in described upstream compartment or primer to be positioned at described downstream compartment
Middle PCR primer or primer are to being different or identical.
In another preference, described method includes:
G (), when described multistage PCR reaction tube has downstream compartment Ri, wherein i is the positive integer of >=2,
In downstream compartment Ri, then carry out PCR reaction, form the solid particle being adsorbed with i-stage pcr amplification product；With
The described solid particle being adsorbed with i-stage pcr amplification product is transferred to time one-level by another interface channel
Downstream compartment Ri+1, and in described downstream compartment Ri+1, carry out PCR reaction, thus form i+1 level pcr amplification product and appoint
The formation of choosing is adsorbed with the solid particle of i+1 level pcr amplification product；
H () optionally repeats step (g) one or many.
In another preference, described method further comprises the steps of: and detects described second pcr amplification product；Or it is right
The carrying out of the amplified production of multistage PCR detects.
In another preference, described detection includes probe hybridization, order-checking and/or electrophoresis.
In another preference, described downstream compartment Ri is also placed the solid particle for adsorbing amplified production.
In another preference, described pcr template material includes: biological tissue samples, organ samples, puncture sample,
Cell sample, nucleic acid extraction thing, blood, serum, body fluid, saliva, hair, fecal specimens, amniotic fluid samples, urine, secretions (bag
Include cervical secretions, vaginal secretions etc.), culture fluid, food samples, vaccine, pedotheque, water sample, air sample.
In another preference, described pcr template material is derived from human body, animal, plant, microorganism, or is derived from artificial
The nucleic acid of synthesis.
In a fourth aspect of the present invention, it is provided that a kind of PCR amplification device, described equipment includes:
For placing the reacting hole of PCR reaction tube, wherein said PCR reaction tube has upstream compartment and downstream compartment, with
And connect the interface channel of described upstream compartment and downstream compartment；
For controlling the temperature regulating device of described reacting hole temperature, so that can carry out predetermined in the compartment of reaction tube
PCR reacts；With
It is transferred to downstream from upstream compartment by described interface channel for controlling to carry the solid particle of pcr amplification product
The control device of compartment.
In another preference, described PCR reaction tube is the multistage PCR reaction tube described in first aspect present invention.
In another preference, described solid particle is magnetic microsphere, and described control device is Magnet or electromagnetism
Ferrum.
In another preference, described reacting hole is arranged in arrays, and described matrix is a × b matrix, and wherein a is 2-100
Positive integer, b is the positive integer of 2-100.
In should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and having in below (eg embodiment)
Can be combined with each other between each technical characteristic that body describes, thus constitute new or preferred technical scheme.As space is limited, exist
This tires out the most one by one states.
Accompanying drawing explanation
Fig. 1 shows the schematic diagram of PCR reaction tube in prior art.
Fig. 2 shows the schematic diagram of a kind of multistage PCR reaction tube (having added liquid phase P CR reaction system) of the present invention.
Fig. 3 shows the schematic diagram of a kind of multistage PCR reaction tube (not adding liquid phase P CR reaction system) of the present invention.
Fig. 4 shows the schematic diagram of the another kind of multistage PCR reaction tube (triplet) of the present invention.
Detailed description of the invention
The present inventor through extensively in-depth study, develop first a kind of highly sensitive, specificity good, anti-interference energy
Multistage PCR reaction tube that power is strong, equipment, system and method.Experiment shows, uses the multistage PCR reaction tube of the present invention not only may be used
So that detection sensitivity reaches 1-2 copy, and possesses the antipollution capacity of resisting disturbance of excellence.Additionally, the present invention's sets
The standby quantitatively or semi-quantitatively transfer that also can realize pcr amplification product in capping system.Complete this on this basis
Bright.
Multistage PCR reaction tube
As used herein, term " reaction tube of the present invention ", " multistage PCR reaction tube ", " PCR reaction of high order pipe ", " multistage
PCR reaction tube ", " cascade PCR reaction tube " be used interchangeably, refer to have at least two relatively independent, PCR reaction compartments
PCR reaction tube, and turning of being provided with between described PCR reaction compartments that the solid particle allowing to carry pcr amplification product passes through
Mobile Communication road.
In the present invention, for any two PCR reaction compartments carrying out connecting by transfering channel, can be by wherein advanced
The reaction compartments of performing PCR reaction is referred to as " upstream compartment ", " upstream reaction compartment " or " upstream PCR reaction compartments ", and by another
PCR reaction compartments is referred to as " downstream compartment ", " downstream reaction compartment " or " downstream PCR reaction compartments ".
See Fig. 2 and Fig. 3.The present invention multistage PCR reaction tube shown in figure include two closed PCR reaction chambers or
Compartment (10) (compartment).Each compartment 10 is provided with chamber lid (20).It is preferred that described chamber lid by connector (22) with every
Room body is connected as a single entity.
Additionally, that be provided with closing between two compartments or closed interface channel (40), described interface channel is used for
The solid particle (50) carrying pcr amplification product is allowed to be transferred to another compartment (i.e. from by a compartment (i.e. upstream compartment)
Trip compartment).
When described compartment is vertically placed, described interface channel can be level or tilt.Generally, interface channel
Angle of inclination (interface channel with horizontal angle) be 0-30 degree, preferably 0-15 degree, is more preferably 0-10 degree.
When interface channel presents certain angle of inclination, from upstream compartment, the solid particle that carries pcr amplification product
The port of export of interface channel can be rolled to or slide to from the arrival end of interface channel easily by gravity, hence into downstream every
Room.
In the present invention, internal diameter and the length of described interface channel are not particularly limited, and carry PCR amplification as long as can allow
The solid particle of product passes through.
Typically, the internal diameter of interface channel is 0.1-20mm, preferably 0.2-10mm, is more preferably 0.2-5mm.
Typically, a length of 0.1-100mm of interface channel, preferably 0.2-50mm.
In the present invention, interface channel can be that closed or closed.Such as, when the chamber of multistage PCR reaction tube
When lid and compartment cavity are separate types, chamber lid can be formed integrally the upper cover of formula, this upper cover not only has corresponding to compartment cavity
Chamber lid, but also there is the sealing lid corresponding to interface channel.So, when covering described upper cover, not only enclose respectively every
Room, also encloses corresponding interface channel, thus forms the reaction compartment of closing.
In the present invention, the upper cover of integral type is particularly well-suited to when compartment has the situation of a × b matrix structure.
The size of compartments of the present invention multistage PCR reaction tube is not particularly limited.Generally, the volume of single PCR compartment is about
10-10000 microlitre, preferably 20-2000 microlitre, more preferably 30-1000 microlitre, most preferably 40-500 microlitre.
The compartment shape of the present invention multistage PCR reaction tube is not particularly limited, and can be cylindrical, conical or other
Analogous shape.
The material of the present invention multistage PCR reaction tube is not particularly limited, and can be its of plastics, glass or permeable magnetic field
His material.Preferably plastics, such as polypropylene plastics etc..
Carry the solid particle of pcr amplification product
In the present invention, carry out in compartment among PCR reaction or afterwards, the part in the pcr amplification product of formation
Can adsorb or adhere to the solid particles surface being placed in described compartment, thus form the solid particle carrying pcr amplification product.When
Described solid particle is when upstream compartment transfer downstream compartment, and entrained pcr amplification product just can be as in downstream compartment
Pcr template.
In the present invention, the shape of solid particle is not particularly limited, can be spherical, oval, cylinder, taper,
Cube or other shapes.Solid particle can be solid, hollow, latticed or other structures, if this solid
Grain can adsorb or carry pcr amplification product.
The material of solid particle is not particularly limited, but preferably solid particle is magnetic-particle, such as magnetic microsphere.?
In the present invention, described magnetic-particle includes having magnetic granule and have magnetic granule under the action of a magnetic field.
In another preference, described magnetic microsphere is nucleocapsid structure.
In another preference, described magnetic microsphere is surface unmodified or surface modification, having coating layer on surface.
In the present invention, the size of described solid particle is not particularly limited.Generally, the mean diameter of solid particle is
0.01～10mm, preferably 0.1-5mm, be most preferably 0.2-2mm.
The nucleic acid absorption power of described solid particle is affected by the factor such as granule surface area and surface nature.Technical staff
Commercially available or prepare the various different solid particle that can carry pcr amplification product by conventional method.
A kind of preferred solid particle is surface hydrophilicity or the granule with hydrophilic radicals such as hydroxyls.So, no
It is only capable of absorption nucleic acid molecules, also can carry partial reaction mixed liquor when transfer, thus a certain amount of pcr amplification product is turned
Move to downstream compartment.
After granular size and material determine, by conventional method, the PCR entrained by each solid particle can be determined
The quantity of product.Generally, the about 1/200-1/10 of pcr amplification product in each granule portability whole PCR reaction system, preferably
Ground about 1/100-1/15, more preferably 1/50-1/20.By volume, the reactant liquor of a usual granule portability 0.1-2 microlitre
Body, this size depending on granule and surface characteristic.
Depend on the size of needs and solid particle, one or more solid particle can be placed in upstream compartment.
Furthermore, it is possible to place in downstream compartment or do not place one or more solid particle.
When having multiple granule in a compartment, these granules can be transferred to one or more downstream compartment.
Multistage PCR reacts
In the present invention, for the upstream reaction compartment connected by transfering channel and downstream PCR reaction compartments, logical
Often after described upstream PCR reaction compartments has carried out PCR reaction (" reaction of first order PCR ") or among, be placed in reaction system
Middle solid particle can adsorb a part of amplified production.By the described solid particle being adsorbed with a part of pcr amplification product, by institute
State transfering channel and be transferred to downstream compartment from upstream reaction compartment, so that it may the template reacted 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 PCR reaction can be carried out at least two upstream PCR reaction compartments, but,
Some PCR reaction compartments also can not carry out any reaction, or does not carry out PCR reaction.Such as, probe hybridization, order-checking, electricity are only carried out
Swimming or other detection reactions.
In the present invention, the reagent needed for carrying out PCR reaction is not particularly limited, this area routine can be used
Reagent needed for various different PCR reactions.Representational reagent includes (but being not limited to): polymerase；DNTP, buffer,
Magnesium ion etc..
In another preference, a length of 12-100bp of described PCR primer, preferably 15-50bp, more preferably 18～
30bp。
In another preference, described reaction system includes following components: 10 × amplification buffer, 4 kinds of dNTP mixture,
All kinds of archaeal dna polymerases, Mg2+。
In another preference, each constituent content of described reaction system is as follows: 10 × amplification buffer 5～20 μ l, 4 kinds
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, Mg2+
0.5～3mmol/L, water 50～200 μ l.
In the present invention, the PCR primer in each compartment may be the same or different.Such as, for by two interface channels even
For three logical PCR reaction compartments, the first PCR primer to, the second PCR primer to and the 3rd PCR primer to laying respectively at not
In same PCR compartment.A kind of representational three grades of PCR reaction tubes (triplet) are as shown in Figure 4.
In conjunction with Fig. 2, the representational multistage PCR method of the present invention is described.The method includes:
In the PCR reaction compartments of described multistage PCR reaction tube, first add the reagent carried out needed for PCR reaction, form liquid
Phase PCR reaction system 31 and 32, and in upstream compartment, add pcr template material and for adsorbing the solid of amplified production
Grain (such as magnetic-particle), but in downstream compartment, it is added without pcr template material, and the liquid phase P CR reactant in two compartments
It is 31 not connect with 32 and be not in contact with each other mutually；
Close upstream compartment and the downstream compartment of described multistage PCR reaction tube, constitute one and close space；
In upstream compartment R1, carry out PCR reaction, form the first pcr amplification product and be adsorbed with a described PCR expansion
The solid particle 50 of volume increase thing；
As described in lifting (by magnetic force or magnetic field), the solid particle 50 of absorption pcr amplification product, leaves and is positioned on described
After liquid phase P CR reaction system below trip compartment, enter the entrance of described interface channel, through interface channel, enter and be positioned at institute
State another downstream compartment R2 in upstream compartment downstream, 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 it is required, repeatable above-mentioned steps: it is adsorbed with i-th by what downstream compartment Ri (i is the positive integer of >=2) was formed
The solid particle of level pcr amplification product, is transferred to downstream compartment R of time one-level by another interface channeli+1, and under described
Trip compartment Ri+1In carry out PCR reaction, thus form i+1 level pcr amplification product.
As a example by detection HPV, a kind of genomic DNA of HPV that expands is for use in differentiating human papillomavirus's HPV type
Method, including step:
(a) close first order PCR reaction chamber or compartment in, with HPV genomic DNA as template, by HPV specificity
The first primer to expanding, thus obtain the first amplified production P1；
B first amplified production is transferred to the PCR compartment of the second level by () from first order PCR compartment, as second level PCR's
Template, and in the second level PCR compartment closed, by specific second primer of HPV to expanding, thus obtain second
Amplified production P2；
C the amplified production Pi of previous step optionally, is transferred to i+1 level PCR compartment by () from i-stage PCR compartment,
As the template of i+1 level PCR, and in the i+1 level PCR compartment closed, by HPV specific i+1 primer to entering
Row amplification, thus obtain i+1 amplified production Pi+1, wherein i is the positive integer of >=2；This step can carry out one or many；
(d) described second amplified production P2 or described i+1 amplified production P to obtaining in previous stepi+1, survey
Sequence, thus obtain the sequence of HPV；With
E the HPV sequence recorded is compared by () with HPV standard sequence, thus identify the type of HPV.
Compared with the PCR reaction tube (Fig. 1) of prior art, the multistage PCR reaction tube of the present invention can be at reaction compartment
In the case of closing, first time PCR product is adsorbed in solid particle (such as magnetic microsphere), the most easily by it from upper
Trip compartment is transferred to downstream compartment, thus in downstream compartment, as template, the amplified production of transfer is carried out second time PCR, from
And keeping in the case of high specific, by twice or repeatedly PCR reaction significantly improve detection sensitivity.
It addition, another distinguishing feature of the present invention is that whole multistage PCR reacts at twice or repeatedly in PCR course of reaction
Pipe or each multistage group connected are in closed state, therefore effectively prevent introducing polluter in environment and operating process, also prevent
Stop the pollution to environment, thus without the need for the high operation equipment of clean level or operating room.
In the present invention, although whole multistage PCR reaction tube or each multistage group connected are in closed state, but still can be non-
Often utilize easily solid particle by product (such as pcr amplification product) from upstream compartment quantitatively or semi-quantitatively shifts
Trip compartment, thus the PCR reaction of next stage is carried out in downstream.The size of nucleic acid transfer amount, can be by regulating the big of solid particle
Little and quantity realizes.
PCR amplification device
Present invention also offers a kind of PCR amplification device that can be used for the inventive method.A kind of preferred equipment includes:
For placing the reacting hole of PCR reaction tube, wherein said PCR reaction tube has upstream compartment and downstream compartment, with
And connect the interface channel of described upstream compartment and downstream compartment；
For controlling the temperature regulating device of described reacting hole temperature, so that can carry out predetermined in the compartment of reaction tube
PCR reacts；With
It is transferred to downstream from upstream compartment by described interface channel for controlling to carry the solid particle of pcr amplification product
The control device of compartment.
Generally, described solid particle is magnetic microsphere, and described control device is Magnet or electric magnet.This magnet one
As be positioned at the top of reacting hole, its magnetic field can cover all or part of of reacting hole region, thus magnetic microsphere is disposable
Or it is transferred to downstream compartment from upstream compartment in batches.
In another preference, described reacting hole is arranged in arrays, and described matrix is a × b matrix, and wherein a is 2-100
Positive integer, b is the positive integer of 2-100, in order to 96 conventional orifice plates etc. with the use of.
In another preference, described equipment is additionally provided with the device automatically controlling magnetic field, for controlling the movement of Magnet
Or the unlatching/movement of control electric magnet, thus realize magnetic microsphere and synchronize and automatic controlled transfer.
Use the PCR amplification device of the present invention, it is possible to achieve extensive, high flux and automation mechanized operation.
Main advantages of the present invention include:
A () is highly sensitive；
B () specificity is good；
C () capacity of resisting disturbance is strong；
D () is easy and simple to handle.
E () can realize the transfer of quantitative and semi-quantitative.
F () is reduced or eliminated the pollution to environment.
The PCR reaction tube of the present invention and system, but make the sensitiveest but for a long time cannot broad practice multistage
PCR reaction system becomes one can be at the routine techniques of the actual application of multiple wide spectrum.
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention
Rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to conventional strip
Part, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage ratio and number are calculated by weight.
Embodiment 1
Multistage PCR reacts
In the present embodiment, use the dyad multistage PCR reaction tube 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 upstream compartment is MY09/MY11, and the primer in downstream compartment is GP6/MY11 or GP5/
MY9。
After containing the secretions conventional method extracting nucleic acid of HPV (human papillomavirus), through 5 times or 10 times of serial dilutions
After, prepare the sample that HPV copy number is about 1,2,5,10,25,50,100,200.Using described sample as template add upstream every
Room.Upstream compartment R1 is separately added into 25 microlitres conventional PCR reaction system (, polymerase nucleic acid-templated including DNA,
DNTP, primer, ddH2The magnetic microsphere (literalness steel ball) of O) and 2 diameter about 200nm.
First carry out PCR for the first time to react, denaturation 95 DEG C, 3 minutes, 95 DEG C, 30S；60 DEG C of annealing 30s, 72 DEG C extend 1 point
Clock, totally 30 circulations.Then, 75 DEG C re-extend 10 minutes.
Then by magnetic field, 2 magnetic microspheres are transferred to downstream compartment by interface channel, carry out second time PCR anti-
Should.Condition is 95 DEG C, 30S；60 DEG C of annealing 30s, 72 DEG C extend 1 minute, totally 30 circulations.
The second pcr amplification product obtained in downstream compartment R2 is detected by order-checking.Then by testing result with
The standard sequence data base of HPV compares, thus draws presence or absence and the kind of HPV.
Testing result shows, the method can detect the HPV virus of trace (only containing 5-10 copy in sample).This
Outward, not only sensitivity significantly improves, and false negative and false positive rate are substantially lower than Standard PCR method and (reduce about 10 times, i.e. carry
High about 1 order of magnitude) or HC2 method (reducing about 50%).
Embodiment 2
Multistage PCR reacts
Repeating embodiment 1, difference is: the multistage PCR reaction tube of use includes 2 multistage group, each multistage group of difference
There are 2 compartments being interconnected.4 rectangular arrangements of compartment.So, two samples can be carried out multistage PCR reaction simultaneously.
Embodiment 3
Multistage PCR reacts
Repeating embodiment 1, difference is: the multistage PCR reaction tube of use includes 48 multistage group, each multistage group of difference
There are 2 compartments being interconnected；Or the multistage PCR reaction tube used includes 32 multistage group, each multistage group is respectively provided with 3
The compartment being interconnected.
96 compartments present rectangular arranged, and corresponding with 96 conventional orifice plates, this multistage PCR reaction tube can be positioned over 96 orifice plates
On.So, can 48 samples be carried out the multistage PCR reaction of two grades simultaneously；Or can 32 samples be carried out more than three grades simultaneously
Level PCR reaction.
Embodiment 4
The multistage PCR reaction of three grades
Repeating embodiment 1, difference is: the primer in downstream compartment R3 is GP6/GP5.
First order PCR: with embodiment 1.
Second level PCR: with embodiment 1.
Third level PCR: the magnetic-particle carrying the second pcr amplification product in downstream compartment R2 is transferred to downstream compartment
R3, then carries out third time PCR, thus obtains the 3rd pcr amplification product.
The 3rd pcr amplification product obtained in downstream compartment R3 is detected by order-checking.Then by testing result with
The standard sequence data base of HPV compares, thus draws presence or absence and the kind of HPV.
Testing result shows, this testing result can detect in sample only containing the HPV viruses molecule of 1-2 copy.Additionally,
Not only sensitivity significantly improves, and false negative and false positive rate are substantially lower than Standard PCR method or HC2 method (reduces about
50%)。
Embodiment 5
HPV type is identified
5.1. cervix uteri epidermis sampling:
(1) clinician is when taking detection sample, with sampling brush, after scraping patient's sample, first inserts an aseptic 5ml
In probe tube, sampling brush patch tube wall gently revolves half cycle so that fraction patient's sample is bonded in probe tube；
(2), after guaranteeing to have enough samples to carry out multistage round pcr detection HPV, sampling brush is extracted out in 5ml probe tube and puts
Enter detection and preserve liquid；
(3) adding 2ml95% ethanol in the 5ml probe tube of step 1, close the lid, vibration probe tube is for several times up and down, makes
The Sample preservation on tube wall must be attached in liquid；
(4) labelling will be carried out on the probe tube of step 3 so that it is with corresponding test experience one_to_one corresponding；
(5) probe tube of step 4 is put 4 DEG C of Refrigerator stores, to be measured.
5.2 multistage PCR
(1) first order PCR
Reaction system 20 μ L, primer MY091 μ L, primer MY111 μ L, patient's sample DNA1 μ L and water 2 μ L, amount to 25 μ L；
(2) second level PCR
Reaction system 20 μ L, primer GP61 μ L, primer MY111 μ L, the first PCR primer (about 1 μ L) shifted by magnetic bead,
Water 2 μ L, amounts to 25 μ L.
(3) third level PCR
Reaction system 20 μ L, primer GP51 μ L, primer GP61 μ L, PCR primer (about 1 μ L), water 2 μ L, amount to 25 μ for the second time
L。
Agarose with 2% detects the result of third time PCR reaction, and positive products is used for checking order.
5.3 β hemoglobin PCR detected for sample quality
Reaction system 20 μ L, primer Β 11 μ L, primer Β 21 μ L, patient's sample DNA1 μ L, water 2 μ L, amount to 25 μ L.
B1:5 '-ACACAACTGTGTTCACTAGC (SEQ ID NO.:5)
Β 2:5 '-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, for the second time PCR primer 1 μ L,
PCR instrument carries out sequencing reaction.PCR primer Centri-Sep strip purification column is purified, and draws after purification in PCR pipe
10 μ L purified products can select to the plate that checks order, sequencing primer from last PCR primer, and upper machine sequencing, then to order-checking
Result is analyzed with HPV standard sequence (available from published common data base).Obtained DNA sequence is with 100% and gene bank
Plays HPV genotype DNA is consistent as HPV shaping standard (i.e. the highest goldstandard of U.S. FDA relevant HPV sizing).
Result
The inventive method, shows the testing result of 3320 detection samples:
1319 samples are non-HPV infection, and 1352 samples are that high-risk HPV infects, and 377 samples are low risks
HPV infection and 272 samples are mixed infection.
The results contrast of 1 two kinds of methods of table
In 3320 samples, there are about 60% ([1352+377+272]/3320) to infect and have HPV virus, and about 40%
(1319/3320) HPV virus is not infected.
Conclusion: multistage PCR reaction tube and response system have highly sensitive, accurate, antipollution and jamproof feature.
The all documents mentioned in the present invention are incorporated as reference the most in this application, just as each document by individually
It is incorporated as with reference to like that.In addition, it is to be understood that after the above-mentioned teachings having read the present invention, those skilled in the art can
To make various changes or modifications the present invention, these equivalent form of values fall within the model that the application appended claims is limited equally
Enclose.
Claims (20)
1. a multistage PCR reaction tube, it is characterised in that described reaction tube by two or more closed PCR reaction chambers or
Compartment (10), and the closing being provided with between at least two compartment or closed interface channel (40) formed, described
Interface channel is for allowing the solid particle (50) carrying pcr amplification product be transferred to another compartment from a compartment or upstream compartment
Or downstream compartment, wherein start to multistage PCR reaction to terminate period in multistage PCR reaction, whole described multistage PCR reaction tube
It is in closed state；
And described compartment has chamber lid (20), and for the multiple compartments being interconnected, when respective chamber, lid is whole
After covering, just constitute one and close space, and described chamber lid is for sealing lid.
2. PCR reaction tube as claimed in claim 1, it is characterised in that described reaction tube includes n compartment, and wherein n is 2-
Arbitrary positive integer of 5000.
3. PCR reaction tube as claimed in claim 1, it is characterised in that described reaction tube includes M multistage group, each multistage
Group is respectively provided with 2,3,4 or 5 compartments being interconnected, and wherein M is arbitrary positive integer of 1-1000.
4. PCR reaction tube as claimed in claim 1, it is characterised in that the compartment of described PCR reaction tube be linear configuration,
Branched configuration or loop configurations；And/or
Described compartment is arranged in arrays, and described matrix is a × b matrix, and wherein a is the positive integer of 2-100, b be 2-100 just
Integer.
5. PCR reaction tube as claimed in claim 1, it is characterised in that described chamber lid is integrated with compartment.
6. PCR reaction tube as claimed in claim 1, it is characterised in that described interface channel is positioned at reaction chamber or upper compartment.
7. PCR reaction tube as claimed in claim 6, it is characterised in that when after described upstream compartment lid upper chamber cover, described connection
Passage is on the inlet compartment top of upstream compartment, and is positioned at along lower section under the lid of chamber, so that carry consolidating of pcr amplification product
After body granule is lifted thus leaves the reaction system being positioned at below compartment, enter described entrance, through interface channel, then pass through
Described interface channel, in the outlet of downstream compartment, enters downstream compartment.
8. PCR reaction tube as claimed in claim 1, it is characterised in that described upstream compartment is provided with guided plate, is used for guiding
The solid particle carrying pcr amplification product enters interface channel entrance from upstream compartment.
9. carrying out a device for multistage PCR reaction, described device includes:
Multistage PCR reaction tube described in claim 1, and
Solid particle, described solid particle is positioned at least one upstream compartment of described multistage PCR reaction tube, and described
When carrying out PCR reaction in upstream compartment, described solid particle can adsorb the amplified production formed in amplification procedure.
10. device as claimed in claim 9, it is characterised in that described solid particle is magnetic microsphere.
11. 1 kinds of non-diagnostic and the multistage PCR reaction method of non-therapeutic, it is characterised in that described method includes step:
A () provides the multistage PCR reaction tube described in claim 1；
B () adds the reagent carried out needed for PCR reaction in the PCR reaction compartments of described multistage PCR reaction tube, form liquid phase
PCR reaction system, and in one or more upstream compartment, add pcr template material and for adsorbing the solid of amplified production
Granule, but the liquid phase P CR reaction system being added without in downstream compartment in pcr template material, and each compartment does not connects and
It is not in contact with each other；
C () closes upstream compartment and the downstream compartment of described multistage PCR reaction tube, hence for the multiple compartments being interconnected
Speech, after respective chamber lid all covers, constitutes one and closes space；
D () carries out PCR reaction in upstream compartment R1, form the first pcr amplification product and be adsorbed with a described PCR amplification
The solid particle of product；
Adsorb the solid particle of pcr amplification product described in (e) lifting, leave that to be positioned at liquid phase P CR below described upstream compartment anti-
After answering system, enter the entrance of described interface channel, through interface channel, enter be positioned at described upstream compartment downstream another under
Trip compartment R2, as the pcr template material in described downstream compartment；
F () carries out PCR reaction in described downstream compartment R2, form the second pcr amplification product；
And described solid particle is magnetic microsphere.
12. methods as claimed in claim 11, it is characterised in that described PCR reaction includes high temperature PCR, low temperature PCR, double
Primer PCR reaction, the reaction of many primer PCRs, RT-PCR reaction, archaeal dna polymerase PCR reaction, RNA polymerase PCR reaction.
13. methods as claimed in claim 11, it is characterised in that described method also includes:
G (), when described multistage PCR reaction tube has downstream compartment Ri, wherein i is the positive integer of >=2,
In downstream compartment Ri, then carry out PCR reaction, form the solid particle being adsorbed with i-stage pcr amplification product；With
The described solid particle being adsorbed with i-stage pcr amplification product is transferred to by another interface channel the downstream of time one-level
Compartment R (i+1), and in described downstream compartment R (i+1), carry out PCR reaction, thus form i+1 level pcr amplification product and appoint
The formation of choosing is adsorbed with the solid particle of i+1 level pcr amplification product；With
H () optionally repeats step (g) one or many.
14. methods as claimed in claim 11, it is characterised in that described pcr template material includes: puncture sample, cell
Sample, nucleic acid extraction thing, blood, serum, body fluid, saliva, hair, fecal specimens, amniotic fluid samples, urine, culture fluid, food sample
Product, vaccine, pedotheque, water sample, air sample.
15. methods as claimed in claim 11, it is characterised in that described pcr template material is biological tissue samples.
16. methods as claimed in claim 11, it is characterised in that described pcr template material is organ samples.
17. methods as claimed in claim 11, it is characterised in that described pcr template material is secretions.
18. 1 kinds of PCR amplification device, described augmentation apparatus uses the multistage PCR reaction tube described in claim 1 to carry out PCR expansion
Increase, it is characterised in that described equipment includes:
For placing the reacting hole of PCR reaction tube, wherein said PCR reaction tube has upstream compartment and downstream compartment, Yi Jilian
Connect the interface channel of described upstream compartment and downstream compartment；
For controlling the temperature regulating device of described reacting hole temperature, so that predetermined PCR can be carried out in the compartment of reaction tube
Reaction；With
It is transferred to downstream compartment from upstream compartment by described interface channel for controlling to carry the solid particle of pcr amplification product
Control device；
And described solid particle includes magnetic microsphere, and described control device is Magnet or electric magnet.
19. equipment as claimed in claim 18, it is characterised in that described equipment is additionally provided with the device automatically controlling magnetic field,
For controlling the movement of Magnet or controlling the unlatching/movement of electric magnet, thus realize magnetic microsphere and synchronize and automatic controlled transfer.
20. equipment as claimed in claim 18, it is characterised in that described reacting hole is arranged in arrays, described matrix be a ×
B matrix, wherein a is the positive integer of 2-100, and b is the positive integer of 2-100.
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|---|---|---|---|---|
| WO2008074023A2 (en) * | 2006-12-13 | 2008-06-19 | Luminex Corporation | Systems and methods for multiplex analysis of pcr in real time |
| CN101675170A (en) * | 2007-03-02 | 2010-03-17 | 考贝特研究控股公司 | The apparatus and method that are used for nucleic acid amplification |
| CN101802164A (en) * | 2007-07-13 | 2010-08-11 | 汉迪实验室公司 | Intergrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008074023A2 (en) * | 2006-12-13 | 2008-06-19 | Luminex Corporation | Systems and methods for multiplex analysis of pcr in real time |
| CN101675170A (en) * | 2007-03-02 | 2010-03-17 | 考贝特研究控股公司 | The apparatus and method that are used for nucleic acid amplification |
| CN101802164A (en) * | 2007-07-13 | 2010-08-11 | 汉迪实验室公司 | Intergrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples |
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