CN101147070A - Reaction method - Google Patents

Abstract

A method for carrying out a multi-step reaction, said method comprising 1) adding one or more first reagents to a reaction vessel, said reaction vessel comprising an upper chamber capable of holding reagents, which is open to a lower chamber to which reagent flow is restricted, 2) subjecting said reaction vessel to a centrifugal force so as to drive the said one or more first reagents into the lower chamber; 3) adding a further reagent to the first chamber and closing said chamber; 4) subjecting at least one of the lower chamber or the upper chamber to conditions which cause said one or more first reagents or said further reagent to take part in a first reaction or reach a desired reaction condition; 5) subjecting said reaction vessel to a centrifugal force so as to drive the said further reagent into the lower chamber and allowing it to interact with the contents of the lower chamber; wherein at least steps (2) to (5) are carried out automatically . This method allows multiple steps to be carried out in vessels without opening the vessel. It is particularly suitable for use in connection with PCR reactions.

Description

Reaction method

The present invention relates to be used to carry out multistep reaction (for example chemistry or biochemical reaction, amplified reaction particularly, polymerase chain reaction for example) method, wherein realized subsequent step, for example detection of analytes or further amplification, and relate to for example reagent container that can in these methods, use and the element of reagent transfer device.

Much more very wherein chemistry or biochemical measurements are arranged or be reflected at the example that carries out in the multistep reaction, also promptly carry out first reaction, need then to add that one or more other reagent carry out second reaction or to the meaning that indication is provided of first reaction.Particularly need to remove from reaction vessel after first reaction under the situation that cap or lid allow to add this one or more other reagent, the introducing of described one or more other reagent can produce pollution problem.

Amplified reaction is introduced pollution especially easily, and this is because need the amount of raw material reagent very low.Even the product of small trace (for example aforesaid amplified production) can pollute, and becomes " introduction " of other reaction thus.

Under the situation that the needs reaction is carried out automatically, problem can be worsened, and this is because removing of lid etc. is not easy to realize in aut.eq. usually.Therefore, it may carry out in the opening reaction vessel, therefore can keep the risk of pollution.

Recently, some sealed tube assay methods have been developed.Yet use these assay methods, need amplification and probe in homogeneous system.Although this is feasible at present, there is the reason of preferred heterogeneous method sometimes, the probe that particularly needs to use can suppress under the situation of this amplified reaction on the either large or small degree.

The applicant has found a kind of improving one's methods of multistep reaction of carrying out.

According to a first aspect of the present invention, the method for carrying out multistep reaction is provided, described method comprises:

1) add one or more first reagent in reaction vessel, described reaction vessel comprises the upper chamber that can hold reagent, and this upper chamber is communicated with the lower chamber of restriction reagent stream,

2) make described reaction vessel bear centrifugal force, enter in the lower chamber to drive described one or more first reagent;

3) add other reagent at this first chamber, and close described chamber;

4) make upper chamber or lower chamber wherein stand one of at least the condition that makes described one or more first reagent or described other reagent participate in first reaction respectively or reach required reaction conditions;

5) make described reaction vessel bear centrifugal force, enter in the lower chamber, and the content in itself and the lower chamber is interacted to drive described other reagent;

Wherein carry out automatically step (2)～(5) at least.

By off-response container after adding described other reagent, can effectively eliminate subsequent external and pollute the possibility that takes place, for example when carrying out first reaction or reaching required reaction conditions.

Aptly, lower chamber comprises the limited pipe that enters, for example kapillary or other tubule, and reagent can be for example owing to surface tension enters this pipe under common environment.This Guan Zaiqi lower end will be closed.

For the reaction that material heats therein or cools off, preferably this chamber has high surface to volume ratio, makes the Rapid Thermal exchange can take place, and kapillary provides the good example of this chamber.These pipes can be used in the Fast Heating or the cooling of a little volume fluid sample.

Therefore in particularly preferred embodiments, in step (4), be that lower chamber stands required condition.

In above-mentioned steps (3), by the lid of suitable shape reaction vessel cuts out suitably, this lid can with the opening of reaction vessel be clasped (snap fit), or screw in its upper space, to form airtight sealing.Yet, also can use other method for closing and utensil, for example use the metal film of diaphragm seal, metal forming or lamination to be applied on the opening of reaction vessel.In addition, as described below, in some device, particularly therein sample etc. is delivered to automatically in the automation equipment in the reaction vessel, can use other parts, for example delivery nozzles, send rod (for example by using magnetic pearl and magnetism stick to realize the transfer of material) or cutter or puncture rod, as the utensil that is used to close this reaction vessel equally.

This one or more first reagent and this other reagent can be only just can a combination of agents that react when standing specified conditions, and these specified conditions for example heat and/or cool off or shine, UV rayed for example, and it can apply in above-mentioned steps (4).Selectively, these one or more first reagent can be used for reacting at preliminary step and the additive reagent that one or more have been assigned in the lower chamber, for example in the preparation centrifugation step.If suitable, any preallocated reagent can carry out freeze drying in lower chamber.

Yet described reagent needn't participate in reaction in the process of step (4).May only need to guarantee that before being mixed together this one or more first reagent or this other reagent reach required reaction conditions, for example reach required temperature, and randomly keep appropriate time under this temperature.In this case, in step (4), required condition can be used the required time interval.

This method can be widely used in the reaction of certain limit.For example, it can be used for inquiring at its destination county the polymerase chain reaction (PCR) of (interrogated).In this case, these one or more first reagent or this other reagent, but preferably these one or more first reagent comprises comprising or suspecting and comprises target nucleic acid (for example DNA) and carry out the required reagent of PCR, for example primer, damping fluid, magnesium salts and polymerase, sample.

If desired, the some or all of required reagent of PCR that carry out, particularly damping fluid, polymerase, salt and some stabilizing agents etc., can be included in the solid-state pearl, it is added in the top reaction compartment, when adding to fluid sample in the top reaction compartment just with its dissolving or disperse to discharge these components.The example of this pearl can be buied from for example Amersham BioSciences UK.

Selectively, these PCR reagent can be allocated in advance in the lower chamber, and with freeze-dried, or rotation descends in aforesaid preparation centrifugation step.

In step 4, with PCR reagent (this stage its preferably in lower chamber) stand to carry out the required thermal cycle step of PCR reaction.This can be incorporated in the thermal cycle control device by the lower chamber at least with reaction vessel and carry out, and described thermocirculator for example is the pillar well heater (solid block heater) by the whole bag of tricks heating and cooling.Present pillar well heater all heats by electrical equipment or thermal power unit etc.Other reaction vessel can heat by based on halogen bulb/turbulent air setting.This container can cool off by thermal power unit, Technology of Compressor Refrigeration, forced draft or cooling fluid.

Yet preferably, lower chamber and/or upper chamber comprise conducting polymer, or are adjacent, and this conducting polymer itself can be as electric resistance heater to realize heating or cooling.The example of this reaction vessel has been described in WO98/24548.

Especially, this lower chamber comprises the glass tube of sealing, is coated with conducting polymer around its sidewall at least.Can provide in the top and bottom of lower chamber and electrically contact, this lower chamber can be connected with electric supply installation by opertaing device (for example computing machine), and this opertaing device can be through programming to react required mode heated in sequence and cooling lower chamber according to carrying out PCR.Last contact guarantees that as heating radiator the heat protocol of carrying out can any other reagent that is stored in the upper chamber of superheated in this process in lower chamber.If this other reagent is thermal sensitivity, for example it is the reagent that is used to produce bioluminescence or chemiluminescence signal, and this can be a particular importance.

The special example of this reagent comprises signal system, and it detects DNA, preferably the DNA of the specific amplification in the remaining sample behind amplified reaction.The sort signal system can be based on various character, but especially for producing optical signal, it is fluorescence, chemiluminescence or noctilcent signal.

This method can be used in particular for adding otherwise may suppress the fluorescence probe (probe) of this amplified reaction.For example, the probe of some DNA bonds or high concentration, and use can be in some cases by to the inhibition of polymerase or by forming the probe of DNA analog (for example polypeptide-nucleic acid (the PNA)) preparation that highly stable complexing body suppresses pcr amplification.

If desired, lower chamber can stand some conditions of making described signal come into force required, for example temperature conditions.For example, probe may need to make the reaction mixture heating, so that the DNA sex change that exists is cooled to uniform temperature then, probe is annealed into the target sequence that comprises extension increasing sequence usually under this temperature.

This signal system preferably can be under the situation of not opening reaction vessel even detected signal system.

The sort signal system can comprise for example visible signal reagent, for example when with double-stranded DNA in conjunction with the time can discharge and DNA bond different when free in solution, diacritic visible signal.The example of this dyestuff is known, comprises ethidium bromide, and the reagent of selling with the trade name of SYBR (for example SYBRGreen I or SYBRGold), or other dyestuffs, for example YOPRO-1.Can represent that by existence amplified reaction carries out from remarkable or a large amount of DNA of the signal indicating of this reagent.

Selectively or additionally, this signal system can comprise the probe that has mark, its specificity is attached on the amplified production.Aptly, this is labeled as fluorescence labeling, can detect after with the light of proper wavelength irradiation, detects the emission that produces from this mark then.In commerce, can obtain the fluorescence labeling of relative broad range.The example is rhodamine, fluorescein or cyanine dye.The special example of dyestuff can Cy5, Cy5.5, TAMRA, ROX, FAM, HEX, TET and JOE buy.

In particularly preferred embodiments, this signal system comprises and has fluorescently-labeled probe and can be by absorbing probe emitted fluorescence energy or fluorescent energy being given with probe and the combination that has the interactional DNA bond of fluorescently-labeled probe.This known phenomenon is called as fluorescence energy transfer (FET) or the fluorescence resonant energy shifts (FRET).With this donor molecule of optical excitation of the specific wavelength that falls into the donor molecule excitation spectrum, it can be released in the light in its fluorescent emission wavelength coverage then.Acceptor molecule is being excited giving on the body emission wavelength, therefore by various apart from the energy of dependence NE BY ENERGY TRANSFER mechanism reception from donor molecule.Fluorescence energy transfer detect to be based on to the monitoring of giving body and acceptor variation in emission wavelength.

In this embodiment, add this agent combination as this other reagent, this lower chamber of heating and cooling is annealed into the subject matter that exists in the sample to guarantee this fluorescently-labeled probe.The DNA bond is inserted between the double helix (duplex) that is formed by probe and subject matter, therefore can interact with fluorescence labeling, perhaps (thus serve as fluorescence and give body to strengthen its signal by fluorescent energy being offered mark, simultaneously the mark on the probe is as fluorescent receptor or quencher), perhaps by quencher from fluorescently-labeled fluorescence signal.

This DNA bond can be under these conditions itself can fluorescence bond.Yet preferred, its be self be not fluorescence or do not discharge visible light under these conditions, and only as the reagent of the fluorescently-labeled quencher on the probe.Like this, the needs of complicated visible signal have been avoided decomposing.The special example of the compound that can operate in this mode comprises anthraquinone compounds, for example the DNA binding compounds of formula (I):

R wherein ¹, R ², R ³And R ⁴Independently be selected from hydrogen, X, NH-ANHR and NH-A-N (O) R ' R ", wherein X is hydroxyl, halogen, amino, C _1-4Alkoxy or C _2-8Alkanoyloxy, A are C _2-4Alkylidene, it is at NH and NHR or N (O) R ' R " between chain length be at least 2 carbon atoms, R, R ' and R " independently be selected from C separately _1-4Alkyl and C _2-4Hydroxy alkyl and C _2-4The dihydroxy alkyl, if with carbon atom that nitrogen-atoms links to each other on do not have hydroxyl and not by the carbon atom of two hydroxyls replacements; Perhaps R ' and R " be C together _2-6Alkylidene, its with R ' and R " nitrogen-atoms that links to each other forms the heterocycle with 3～7 atoms, as long as R ¹, R ², R ³And R ⁴At least one is NH-A-N (O) R ' R " get final product.

The particular instance of this dna double spiral bond be mitoxantrone (1,4-dihydroxy-5,8-two [[2-[(2-hydroxyethyl) amino] ethyl] amino]-9, the 10-amerantrone) or its salt, for example hydrochloride or dihydrochloride.

Other example that does not discharge the DNA bond of visible signal under these conditions comprises nogalamycin (2R-(2 α, 3 β, 4 α; 5 β, 6 α, 11 β; 13 α, 14 α)]-11-[6-deoxidation-3-C-methyl-2,3; 4-three-O-methyl-α-L-mannopyranose base) oxygen]-4-(dimethylamino)-3,4,5; 6,9,11; 12,13,14; 16-decahydro-3,5,8; 10,13-penta hydroxy group-6,13-dimethyl-9; 16-dioxo-2,6-epoxy-2H-aphthacene is [1,2-b] oxocin (oxocin)-14-carboxylate methyl ester also) or daunomycin ((8S;-cis)-and 8-acetyl group-10-[3-amino-2,3,6-three deoxidations-α-L-Lyxo-six pyrans glycosyls) oxygen]-7; 8,9,10-tetrahydrochysene-6; 8,11-trihydroxy-1-methoxyl-5,12-aphthacene diketone).

It will be apparent to one skilled in the art that or use conventional method can be identified for the DNA bond and the fluorescently-labeled appropriate combination of probe.

Yet, also can use chemiluminescence or bioluminescence signal system.

A kind of special example of bioluminescence signal system comprises the luciferase/luciferin system, wherein acts on the substrate fluorescein at luciferase in the presence of the ATP and produces light.Yet luciferase is a kind of enzyme with height thermal sensitivity, so it can not bear for example issuable temperature in the PCR process.The method of the application of the invention, when in lower chamber, under the rising temperature, reacting, described reagent can remain in the upper chamber, only at the convenient time, when the reaction ending, when the temperature in the chamber of bottom is reduced to the temperature levels of this luciferase maintenance activity, just can add this signal system.Described in the PCR process mensuration of the particular type of using the bioluminescence signal system in WO 02/090586, its content is included in that this is incorporated as a reference.This reaction can be specially adapted to use method described herein to operate automatically.

The signal that produces reads by the checkout equipment of routine, optical system for example, and such as spectrofluorimeter, the perhaps camera that under the situation of chemiluminescence or bioluminescence signal system, uses.This equipment can be included in this device.In the situation of optical detection system, preferably this fluorescence detector is the light sealing, to guarantee and can not detect under the interference from non-incident light.

Preferably, being arranged so that of this reaction vessel can directly be read signal by container, for example the clear bottom by lower chamber or transparent cover or seal element by container top.Yet can from reaction vessel, fluorescence be sent out if desired, by light transmitting fiber.

Yet this method also has much other application, if desired, can carry out the other stage of reaction afterwards in lower chamber in step (5).The example of this method is nested type or multi-PRC reaction.In this case, this other reagent can comprise other PCR reaction mixture, comprises different primers, and other damping fluid if desired, enzyme etc., to carry out the 2nd PCR reaction.In this case, should be other after reagent distributes,, make lower chamber through further thermal cycle in order to realize required the 2nd PCR reaction.

This high susceptibility for DNA cloning is advantageously.Under inhibitor may reside in situation in the specimen material, this was useful.It has improved relevant with nested PCR in actual applications important leftover problem.

This method also can be in every way and reverse transcription (RT-PCR) related application.For example, these one or more first reagent can comprise and carries out the required reagent of reverse transcriptase method, is used for preparing cDNA by RNA.So its rotation is descended, add the required PCR reagent of the corresponding cDNA of RNA sequence then for increasing and being concerned about as described other reagent.The lower chamber that will comprise the reverse transcriptase reaction potpourri in step (4) is cultivated, with the cDNA of preparation with the RNA complementation.

In above-mentioned steps (5), make to comprise the PCR reagent rotation decline that this cDNA is had specific primer, can carry out amplified reaction by making the required thermal cycle conditions of lower chamber experience then.

Selectively, PCR reagent can comprise this one or more first reagent, in step (2) its rotation is dropped in the lower chamber.This reverse transcriptase reaction potpourri is described other reagent, and it remains in the upper chamber.In step (4), upper chamber is cultivated, to carry out the RT reaction, produce cDNA.(for this reason, needing to use for example aforesaid conducting polymer to make upper chamber can independently heat/cool off).The potpourri rotation that comprises cDNA that will obtain thus in step (5) then drops in the lower chamber.Then, lower chamber is stood for carrying out the required thermal cycle of PCR reaction.

This method also can be applied in the measure of anti-leftover problem.For example, for can carry out the PCR reagent of homogeneous detection reaction, and in step 4, carry out under the situation of PCR at first reagent, described other reagent can comprise can analyze or the reading of content thing after make the reagent of product degradation.Selectively, under the situation that the independent signal system that use is added as described other reagent (as mentioned above) detects, can add the reagent that can make product degradation by multicompartmented cylindrical shell for example as described below or container in the following step in addition.

React in the ensuing stage at this then, can pierce through the compartment that this comprises degradation agent, with content rotation decline wherein.

Described reagent can comprise the uracil-n-glycosylase of the dUTP PCR product of can degrading, perhaps selectively suitable DNAse.The latter not only can reduce the risk of leaving over, and it can elimination can be present in potential harmful subject matter nucleic acid in the sample (for example known have communicable HBV DNA).

This method also can be fit to prepare the form of " warm start " amplified reaction.Amplified reaction (for example PCR reaction) depends on the order with the point-device order and the step (sex change, annealing and extension) of carrying out under the required accurate temperature of the operation of this step.When reagent is mixed (for example before the reaction beginning) in different temperatures, even very short time also can have problems.Primer may with nucleic acid-templated interaction, cause the primer extension of template.This can cause the reduction of required product overall yield, and the generation of non-specific product.

Propose the various various measures that overcome this problem before, be called as " warm start " reaction.

By using according to method of the present invention, can keep one or more to carry out the required reagent of amplified reaction, for example magnesium ion, polymerase or primer are as described other reagent.When only the condition in lower chamber is suitable for carrying out suitable amplification (for example being heated the temperature that surpasses little dna molecular associating) then, this reagent is distributed.

Any heating in the top of this reaction vessel or the lower chamber or cooling all are suitable robotizations, for example control the electric current that is transported in the thermo cycler by using a computer.Aptly, this computing machine is through the temperature step order of programming to guarantee that realization is required.

This one or more first reagent and/or this other reagent can be assigned in the reaction vessel by any easy method respectively in step (1) and (3).For example, can reagent be assigned in the upper chamber, preferably use suitable distributor to carry out automatically by the injection technique of routine.

Selectively, this one or more first reagent and/or this other reagent can be placed in the frangible container (for example cylindrical shell), and this frangible container is arranged in the upper chamber on the opening that leads to lower chamber.

These can for example have the wall that can pierce through, for example metal or lamination metallic film surface on surface above and/or under the reagent chamber.Can make reagent discharge bottom by destroying chamber wall (for example piercing through the wall that rod or needle-penetration can pierce through) in the suitable stage then, thereby content is discharged by making by chamber.

This pierces through rod or pin can be provided on the lid of container, and it forms the upper surface of reagent chamber aptly, and can be by the introducing of exerting pressure on lid at the appropriate time.Selectively, it can be arranged on the machine that is used to react, and uses automatically in due course, and special itself is in the situation that can pierce through at this upper wall.

The upper surface of sealing bore or container can comprise for example film, plastic foil for example, and be combined with suitable rod or the pin of piercing through therein.In this embodiment, the upper surface of container itself can constitute the lid of container.Selectively, provide independent lid, can require described rod before the upper surface that arrives container, to pierce through this lid, so that pollution risk minimizes.

Special container is the tubular form that has more than a compartment.These compartments can comprise these one or more first reagent and/or this other reagent respectively.Yet, it also can bring following possibility: extra reagent can add in the upper chamber in the proper time in this procedure simultaneously or sequentially automatically, and then rotation is downward if desired, cause occurring to carry out the possibility of any amount of other reactions steps, and/or can not be stored in the possibility that reagent together just mixes when arriving reaction chamber.

This cylindrical shell will further make pollution risk minimize, because they guarantee that reagent is exposed to longer that time in this atmosphere can be than needs.

These compartments can mutual adjacent setting, for example circle or wheel shape setting, and perhaps it can be arranged on the top of each other.In arbitrary situation, one or more suitable rods are provided, make these compartments in this procedure, to destroy as required.

This container can regionally at an upper portion thereof have can make container be automatically moved to the utensil of the position in the reaction vessel.The example of this utensil can comprise one or more annular flanges, its arrange with the device of this method of enforcement on suitable extracting utensil interact.

By using this reagent container, can be only by sending suitable reagent with suitable order and appropriate time, order is carried out the combination of above-mentioned reaction, for example nested PCR, RT-PCR, non-homogeneous detection, anti-leaving over and/or heat start PCR.

This container has formed another aspect of the present invention.Therefore according to another aspect of the present invention, the frangible container that is used for storing reagent is provided, has reagent chamber in the described container, lower surface in this reagent chamber has the wall that at least one can pierce through, wherein its upper surface comprises the wall that another can pierce through or comprises the lid that pierces through utensil, and it is arranged so that piercing through the described wall that pierces through can cause reagent to discharge from chamber.

This reaction vessel can be installed in aptly can be with on the platform of centrifugal motion rotation etc.This reaction vessel is installed on the platform with pivoting aptly, makes in centrifugal process, and it can rotate so that lower chamber is positioned on the outer centrifugal pathway.This can for example realize that by one or more axles that are positioned on this container outer surface it can be installed in the socket that is arranged on the platform movably.

It also can move between each stage automatically.For example, it can be fit to reaction vessel is moved to the thermal cycle utensil that is used for procedure of processing.In this embodiment preferred, have at this reaction vessel under the situation of the heating appliances that constitutes by conducting polymer, use and to move equipment automatically it is moved in the socket suitable the power supply from hydro-extractor, thus can with reaction vessel on electrically contact and contact.Selectively, electrically contact and original position to make.

The providing of automatic utensil that is used to carry out this method means that sample manual operation amount minimizes, and this method is effectively carried out, and further reduces particularly the pollution risk from operator DNA.

Proposed to be used for the particularly suitable reaction vessel and the device of said method among the International Patent Application Publication No. WO2005019836 in common unsettled, its content is all introduced herein as a reference.Device described herein can carry out the very complicated multistep processing of sample.This device comprises:

(i) platform comprises:

(a) be applicable to the chamber that receives sample; With

(b) functional part;

The (ii) arm that can raise and reduce, it comprises and is used for the utensil that is connected with this functional part removeability, makes can raise and reduce described parts with this arm; And

(iii) be used for moving the utensil of this platform, make any chamber or function element to aim at respect to this arm.

This device has widely to be used, and can be fit to use widely.This term " functional part " is defined as through design and makes device element on its arm that can reversiblely be connected to this device.Can see that obviously this functional part can design to have purposes widely in from here open.According to the special application of this device, those skilled in the art can easily determine the special application of these one or more functional parts.For example, this functional part can comprise and is used for and the interactional utensil of fluid sample.This utensil can provide some physical treatments for sample, for example heats, cooling, optical processing, sonicated etc.Selectively, this functional part can comprise and be used for and the interactional utensil of this chamber itself, for example by being used as cutter to pierce through paper tinsel sealing, closed chamber, introducing filler etc.In addition, this functional part can be with acting on sample or being included in gatherer in another chamber that wherein analyte moves to device.

Therefore in description to method of the present invention, this functional part can comprise and be used for and can be delivered in the reaction vessel at the pretreated sample of other local process of device that this reaction vessel is equal to " being applicable to the chamber that receives sample " in this case.Selectively, this function container can comprise the cutter that is used for discharging the reagent that is included in cylindrical shell or aforesaid frangible container or pierce through rod.

Yet in special embodiment, this platform is circular substantially, by the rotation campaign.This makes this platform to aim at chamber or functional part with respect to arm or other physics utensil.When this device comprised several different parts, this also had the minimized advantage of the device size of making.Randomly this platform can be furnished with sensing mechanism, when being used under arm above platform is being positioned at platform or other physical treatment utensil motion functional part or chamber is accurately located.Yet in the content of this method, another advantage is to make that the reaction vessel that remains on the platform can be easy to centrifugal motion.

In the special embodiment of another kind, this Design of device makes whole platform to take off and replaces easily.This makes after any given sample preparation program, can this device can be applied in the another program with taking off and replace with that cross and platform potentially contaminated.If this device so designs, then preferred this platform can for example adopt the twisting joint that is equipped with simple and easy lock also to be installed in the device safely easily.

The automatic processing of the reagent of Shi Yonging can further strengthen by using agent transfer equipment in the method for the invention.The applicant has further developed the method that is used for chemistry and biochemical measurement reagent treatment, and the equipment that is used for these methods.

In carrying out chemistry or biochemical measurement process, usually exist small amount of solid state reagent is transferred to needs another from a container.

More and more, as at method of the present invention and for example needs in the device described in the WO2005019836, these mensuration all are to use automated process and program to carry out, and therefore need to obtain and to shift the automatic equipment of a certain amount of reagent.

The special example of the reagent that can obtain with solid-state form is for carrying out the required reagent of amplified reaction (particularly polymerase chain reaction or PCR).Can obtain being used for the commercially available PCR instant pearl of having of DNA cloning, for example those of Amersham BioSciences UK sale.

They comprise carries out the required all components of Standard PC R, comprises buffering agent, salt and polymerase, with freeze drying or other solid-state form.Therefore it provides the convenient means that stable " homebrew " consumer goods are provided for the terminal user.But they will benefit from large-scale production that makes things convenient for storage form and the duplication of production prescription with long storage life.

Yet different with the hygrometric state reagent that can use pipette or transfer pipet to shift easily between well, pearl needs tweezers that it is transferred to another from consumer goods.This may be difficult to effective realization, particularly in automatic equipment.

The applicant has developed a kind of effective ways of valid function solid reagent.

Therefore on the other hand, the invention provides and be used for solid reagent is transferred to the method for second container or the second place from first container or primary importance, described method comprises

(i) rod that will comprise static electrification lotus material places near the described solid reagent of first container or primary importance, and described rod can electrostatic attraction and kept described solid reagent on its surface, extracting a certain amount of described solid reagent,

(ii) move this rod and/or this first or second container, make this rod be positioned at second container or the second place near;

(iii) remove this solid reagent, make it put into second container or the second place from described rod.

In a first aspect of the present invention, this second container or position comprise the upper chamber of reaction vessel.Yet this method can more be widely used for general agent transfer.

This method is particularly useful, because it can effectively transmit solid reagent, in addition, it is specially adapted to robotization, can be used for sensing equipment widely.

Term used herein " solid reagent " is meant reagent or the chemical substance that one or more are solid-state.For example, it can comprise powder, crystal, particle or pearl.Especially, it comprises combination of agents, and it combines with the pearl form, for example aforesaid PCR instant pearl.Usually exist in the situation about existing at reagent, for example, can pass through conventional method, for example the suitable solid form of freeze drying or spray drying method for preparation with the form of aqueous solution with liquid form.If desired, this pearl or particle can further comprise conventional reagent, and for example filler, spreading agent, surfactant etc. can dissolve or disperse when this particle or pearl are in adding liquid (for example water) to guaranteeing.

The person's character of this static electrification material can change according to the person's character of the solid reagent that is just shifting.Usually, this material dielectric material of being included as insulator or nonconductor or having negligible electric conductivity.This material should be a kind of material that maybe can keep the regular period static charge that carries usually.The special example of described material is polymkeric substance or plastics, for example polystyrene or latex.

In addition, can on this rod, increase or produce enough electric charges by the step that rubs in advance, wherein by should the rod one or many with insulator (for example be material or fabric) mechanical friction to produce or the increase static charge.The material that is fit to comprises synthetic textiles, for example nylon or polyester textile.

It carries out aptly automatically, preferably on the operator's console that suitably is provided with in automatic sensing equipment.

Be meant that at this used term " rod " any be fit to can be in the structure of introducing and moving between the reaction vessel etc.Usually its shape can be elongated, rod-shaped and tubular structure for example, but its side can tilt to form syringe-like or coniform profile.

It can be solid or hollow by nature.At this rod is under the situation of hollow, and it can hold other element, for example magnet.In this case, can make it be used to collect magnetic retention equally the magnet leading-in rod, magnetic beads for example, for example magnetic oxygenated silica bead, it randomly carries other reagent, binding reagents for example, for example antibody, or its binding fragment.This collection can be reclaimed in the identical operations at the static with solid reagent and be carried out, but preferably carries out as independent operation.

But whole rod can be made by the material of dielectric or static electrification, and perhaps it can comprise compound, if be used to attract the zone (particularly lower surface or lower area) of solid particle but comprise the material of dielectric or static electrification.

Aptly, the profile of Bang at least a portion outside surface can make reagent be contained in this profile.For example, the surface can have lacuna or groove, and it has the size of the holding solid of being suitable for reagent (for example amplifing reagent pearl, for example PCR pearl).

The size of these grooves or lacuna depends on the size of the solid reagent (for example amplified reaction or PCR pearl) that is about to use this rod to move.Yet usually, any lacuna can be the diameter and the degree of depth of 0.5～2mm, and similarly, groove can be the 0.5～2mm width and the degree of depth.

Any desired contour all is positioned on this excellent lower surface aptly.

Rod with suitable profile can be novel, and it has formed another aspect of the present invention.

The motion that realizes in (ii) in above-mentioned steps can be carried out in any mode that is fit to specific mensuration and used sensing equipment.After collecting this solid reagent, can will should manually move to the another location by rod, but this operation is carried out automatically suitably from a position.This can comprise that for example rod moves vertically upward, for example it is taken out from first container fully, and tangential movement makes it aim at second container then, then if desired, then moves downward, and makes the end regions of rod of holding solid reagent be arranged in second container.Selectively, after rod being taken out, can move this container itself from first container by moving upward of for example rod, make second container be positioned at excellent below.Then if desired, just it is entered in second container rod reduction.

The transmission utensil that is fit to is well known in the art, can comprise travelling belt or transmission dish etc.

The container that is fit to can be any reaction vessel, comprises independent reaction vessel and at plate and the reaction well in other, if desired, this transmission utensil will be fit to make its motion.

Aptly, from rod solid reagent being removed in (iii) in step is that machinery carries out.Especially, rod is impregnated in the liquid, this solution has the effect that solid is removed from rod.Aptly, this liquid is required solvent or solution in the next stage of measuring.For example, comprise in the situation of PCR pearl at this solid reagent, can be by rod being impregnated into again in the buffer suspension liquid (being DNA/RNA extract etc.) with its distribution, to remove pearl from rod.

Preferably should after use, can handle arbitrarily by rod.

Can moulding or makes it be fit to be installed in the required accessory or annex on the automatic sensing equipment in the upper end of rod.

Therefore in this manual, the rod that is used for method of the present invention can comprise the feature of aforesaid International Patent Application Publication No. WO2005019836 equipment.Especially, it can comprise and a kind ofly is suitable for extracting PCR instant pearl and it is moved to the parts of the reaction vessel that is used for automatic PCR technology.

In the preferred embodiment of the equipment of WO2005019836, this functional part comprises sheath, and this sheath provides the interface between magnet that is used to attract magnetic reagent beads and pearl itself, and this magnetic reagent beads can be fixed with analyte or reactant in the above.Preferably, this sheath is positioned on the platform, can be made by the material that sheath attracts by this complex when magnet is arranged in sheath.In this embodiment, preferably this device comprises and the arm magnet together of device, and it can be reduced to and apply magnetic field in the sheath, rises to the sheath outside to remove this magnetic field.Then this sheath is put into the chamber that device comprises magnetic reagent beads.Magnet is reduced in the sheath, and this magnetic reagent beads and sheath combine.Mention this sheath and magnet then, mobile platform makes and aims at new chamber, reduces sheath and magnet then, removes magnet.When removing magnet, magnetic reagent beads will fall from sheath and enter in second chamber.Make sheath carry out slight moving up and down by arm, guarantee not have magnetic reagent beads still to combine, also be used for reagent or the solution of magnetic reagent beads with new chamber is mixed mutually with guard shield.Selectively, can analyte elution from pearl be come out by any suitable mode.

The design of magnet and arm makes it interact with each other under the situation that does not influence operation each other, makes no matter whether magnet is in place, and this sheath can promote separately and reduce.

In specific embodiment, if become bar-shapedly according to this jacket-shaped of the present invention, it also can be used for by the electrostatic transfer solid reagent as described here.

The equipment that is specially adapted to carry out aforesaid agent transfer method has constituted another aspect of the present invention.

Therefore especially, the present invention further provides device, but it comprises the rod that contains aforesaid static electrification material, and the utensil that is used for described rod is transferred to from first container second container.

The present invention is described especially by the mode of embodiment now with reference to accompanying drawing, wherein:

Fig. 1 is the synoptic diagram of describing according to method of the present invention;

Fig. 2 shows the skeleton view of the device that can be used for this method;

Fig. 3 shows the device transverse section from the side among Fig. 2;

Fig. 4 shows the top view of the platform that is used for Fig. 2 and 3 devices;

Fig. 5 shows functional part, is cutter herein, Operation Profile figure, pierce through the laminated film on the chamber that is positioned at this device;

Fig. 6 shows herein the view of the details that is connected between the fork spare for the functional part of cutter and arm;

Fig. 7 show have be used for from sample cavity take out combination analyte magnet be the viewgraph of cross-section of the functional part operation of sheath herein;

Fig. 8 show be used for heating this certain volume solution that installs one of them chamber be the viewgraph of cross-section of the physical treatment utensil that is used for heating appliances herein;

Fig. 9 show have the analyte that is used for combination discharge into reaction vessel magnet be the viewgraph of cross-section of the functional part of sheath herein;

Figure 10 shows this and is in the position that is used for sealed reaction vessel and has herein viewgraph of cross-section for the reaction chamber of the functional part of cutter;

Figure 11 shows a kind of alternative form of the container that is used for method of the present invention, it can carry out a plurality of operations, wherein (A) described the synoptic diagram of the reaction vessel with reagent container in place, (B) is by the xsect of the container of (A), (C) is the schematic side elevation of this container;

Figure 12 shows the container that can be used for method of the present invention;

Figure 13 shows a kind of alternative form of this container; With

Figure 14 shows the side view of the rod that can be used for the agent transfer method;

Figure 15 shows the backplan of rod among Figure 14; With

Figure 16 has schematically described the agent transfer method.

Fig. 1 schematically describes the operation of method of the present invention.This method is carried out in reaction vessel 68, and this reaction vessel 68 comprises the upper chamber 685 with opening or openable mouth 686, and this above-mentioned chamber 685 leads to the lower chamber that comprises glass capillary 680.In this special embodiment, the side of kapillary 680 is coated with electric conductive polymer 681 and electrically contacts, and comprises contact 683 in the lower end of pipe 680.Second contact (682) is positioned at the bottom periphery (Figure 1B) of upper chamber (685).

In the first step, first group reagent (for example PCR reaction mixture) is assigned in the upper chamber 685 of reaction vessel.Surface tension can stop these reagent to enter kapillary 680.Yet reaction vessel 68 is installed on platform or the garden dish gearing by axle 72 pivotings, process centrifugation step shown in curve arrow.This drives the PCR reaction mixture and enters in the kapillary, shown in Fig. 1 (c).

Here, one or more other reagent (Fig. 1 (D)) are assigned in the upper chamber 685.Surface tension makes it can not enter in the kapillary 680 again.Yet they are allowed to be retained in the upper chamber 685.Use lid 687 closing containers 68 (Fig. 1 E).

After this, handle this reaction vessel, make in pipe 680 to react, for example PCR reaction.In this special embodiment, make suitable electric current by polymkeric substance 681 by contacting 682,683, make the reagent in the kapillary 680 carry out thermal cycle.Can not participate in this reaction at described other reagent of this stage.In addition, contact 682 makes it isolated with the thermal cycle of carrying out in pipe 680 as heat sink.

In case yet this reaction finish, can under the situation of not taking off lid 687, add described other reagent by carrying out second centrifugation step.

Fig. 2 has described the skeleton view of the device 1 that is specially adapted to carry out method of the present invention.This device comprises the platform 2 by twist lock 4 fix in position.This platform comprises a plurality of chambers and functional part (also showing) in Fig. 4.This platform drives by stepper motor 6 and driving-belt (not shown).Use the indexing sensor (not shown) also by the motion of monitoring stepper motor 6, come the position of monitor supervision platform.

Arm 10 is positioned on the platform 2, and it comprises and is used for movably the fork spare 12 that is connected with functional part (not being shown specifically) on the platform 2.Show this arm 10 in the position that raises, container 68 is remained on the platform 2.This device also comprises the magnet 14 on the fork spare that is located immediately at arm 12.Show the position that this magnet 14 is in rising.

This device comprises that also a plurality of samples (biological example sample) that make can carry out equipment and the utensil of pre-service therefrom to extract DNA.These comprise and also are positioned on the platform 2 and are illustrated in the heating appliances 16 of raised position and are positioned on the platform 2 equally and are illustrated in the utensil 18 that is used for sample is carried out sonicated of raised position.The linear movement of arm 10 and magnet 14 drives and controls by linear actuators 24 by the motor 20 that is connected with driving-belt 22.Similarly, heating appliances 16 drives and controls respectively by linear actuators 26 and 28 by the motor that is connected with driving-belt 22 20 with the linear movement that is used for sample is carried out the utensil 18 of sonicated.This device also comprises control panel 30 and power supply 32.

Fig. 3 shows the transverse section of installing among Fig. 2.Identical among the parts that illustrate and Fig. 2, except in this view, can't see linear actuators.This view has been described driving-belt that is connected with motor 6 40 that is used for rotatable platform and the sensor 42 that is used to respond to the position of platform in addition.

Fig. 4 shows the top view of the platform 2 that is designed for the device of handling of fluids sample before nucleic acid amplification.This platform uses rotating lock mechanism 4 to be installed on this device.This platform comprises two functional parts: cutter 50 and sheath 52.Each functional part all comprises protuberance 54 in a side separately, and this functional part can be interacted with the arm (not shown) of this device.The orientation of this protuberance makes that the forked part of arm can be slided along with smooth rotation under the protuberance of functional part.This device also comprises several chambers 56,58,60,62,64 and 66.In these chambers or the reaction vessel each all has with the processing of biological sample (for example urine samples) makes it can carry out the relevant different task of amplified reaction therefrom to extract DNA.Chamber 56,58,60,64 and 66 xsect are ellipticalness, at bottom 560,580,600 and 640 each self-contained circular well shape recess of chamber.The xsect of chamber 62 is circular.Any or all chamber can cover with the metal level press mold before use, to keep the reagent cleaning.

This device further comprises reaction vessel 68, and it is to be applicable to the reaction vessel that carries out method of the present invention.This container has upper chamber 685, and its xsect is circular, and it narrows to lower chamber gradually, this lower chamber be included in container bottom with the kapillary shown in 680.Reaction vessel 68 further comprises the interactional protuberance 70 of the arm (not shown) that makes this chamber and device.Reaction vessel 68 has axle 72, and its pivoting is installed in the socket 74 on the platform 2.Reaction vessel 68 also is coated with the metal level press mold.

Aptly, as above about described in Fig. 1, this kapillary 680 is coated with metallic conduction polymkeric substance 681 (referring to Fig. 8), and just has top on pipe 680 top and electrically contact 682, has to place outside bottom to electrically contact 683 on the bottom of pipe 680.

Chamber 60 and 62 is installed in the single container 76 jointly.This container 76 can disassemble from platform.This platform also comprises notch portion 78.

Below with reference to above accompanying drawing and other 5～10 pairs of these devices of accompanying drawing and platform be used for the handling of fluids sample and then the method according to this invention application of carrying out nucleic acid amplification reaction be described.

According to the mensuration of selecting, select to comprise the container 76 of sample chamber 60.In chamber 62, add in advance and be used for the required several agents of this mensuration.Sample chamber 60 is collected and placed to the fluid sample that will comprise the DNA analysis thing.Can add in advance in this sample chamber can sample dissolution chaotropic salt (for example guanidine hydrochloride, urea or sodium iodide).Then magnetic is added in the sample in conjunction with pearl 100, close the lid of sampling receptacle.This container 76 that comprises sample chamber 60, sample and reagent chamber 62 is contained on the platform 2.Then platform 2 is arranged in the device 1, uses twist lock 4 locks in place.Arm 10 is reduced, and platform 2 rotations make fork part 12 be engaged under the protuberance 52 of cutter 50.Then arm 12 is raise, rotation platform 2 then, and chamber 56 is positioned under the cutter 50.Reduce arm, cutter 50 pierces through the lamination metal film (not shown) that covers chamber 56.Repetitive operation makes this cutter 50 pierce through continuously to cover chamber 58,60,62,64 and 66 and the film of reaction vessel 68.

Fig. 5 shows herein the cross-sectional view for the operation of the functional part of the cutter 50 of the laminated film (not shown) that pierces through the chamber that is positioned at this device (for example 56) top.This chamber 56 combines with platform 2.This accompanying drawing has been described the protuberance that is used for the functional part 52 that the fork spare (not shown) with arm is meshed.

Fig. 6 shows the view of the details that description is connected between the fork spare 12 for the functional part of cutter 50 and arm 10 herein.The fork spare 12 of this arm 10 is meshed with cutter under being positioned at protuberance 52.

In case all laminated films of this device are all pierced through, then by rotation platform 2, reduce arm 10 and platform rotated to reverse direction and make the fork spare 12 of this arm and the protuberance 52 of cutter be separated from, thereby this cutter 50 is turned back to original position on platform 2.

Rotation platform makes sample chamber 60 be positioned at and is used to sample to carry out under the utensil 18 of sonicated then.The utensil 18 that is used to sample to carry out sonicated drops in the sample chamber 60, begins sample is carried out sonicated.This is provided for dissolving and anyly is present in spore in the sample to discharge the physical dissolution step of any DNA.Simultaneously, playing promotion DNA from liquid reagent such as guanidine hydrochloride liquid combines with the magnetic bond material to form the effect of complexing body.When finish ultrasonic after, from sample chamber 60, take out and be used to sample to carry out the utensil 18 of sonicated.Before storing, at first this is used for sample is carried out the utensil 18 of sonicated in two washing chambers (chamber 56 and 58) washing.Add the damping fluid that is fit to, for example 50% hydrous ethanol solution 80 in these chambers in advance.This is used for sample is carried out the utensil 18 of sonicated from sample chamber 60 liftings, rotation platform 2 is positioned at damping fluid chamber 56 to be used to sample to carry out under the utensil 18 of sonicated, this utensil that is used to sample to carry out sonicated drops in the damping fluid chamber 56, of short duration activation and lifting.Chamber 58 is repeated this operation.After washing for the second time, the utensil 18 that is used to sample to carry out sonicated is promoted and stores.

Arm 10 is reduced, and rotation platform 2 makes 12 to be engaged under the protuberance 52 of sheath 54.Mention arm 10 then, thus sheath 54 is risen on the platform 2.Rotation platform 2 makes sample chamber 60 be located immediately under the sheath 54 then.Reduce arm 10, thus sheath 54 is reduced in the sample chamber 60.Then magnet 14 is reduced in the sheath 54, will be attracted on the sheath 54 in conjunction with the magnetic beads 100 of DNA.Promote arm 10 then, thus sheath 54 is risen to beyond the sample chamber 60.Magnet 14 and arm 10 are raised simultaneously, make it be retained in sheath 54 inside.

Fig. 7 show have be used for from sample chamber 60 take out the analyte that adheres to magnet 14 be the viewgraph of cross-section of the operation of the functional part of sheath 54 herein.This chamber 60 is connected on the platform 2.By the arm (not shown) sheath is reduced in the sample 102 that is included in the sample chamber 60.Magnet 14 is inserted in the sheath 54, have the magnetic beads 100 of DNA to be attracted on the sheath 54 complexing.

Can clean the DNA that is attached on the magnetic beads 100 then, for example in damping fluid (for example ethanol damping fluid, for example 50% ethanol damping fluid), clean twice.Rotation platform 2 is located immediately at the first damping fluid chamber 64 that comprises suitable buffer solution to attract to be magnetic under the sheath 54 of pearl 100.Arm 10 is reduced, thus sheath 54 is reduced under the damping fluid chamber 64.But do not reduce magnet 14.This means that pearl 100 no longer attracts on sheath 54, but reduce and fall into buffer solution.Fast lifting and reduction arm 10, the small-scale vertical movement of sheath 54 guarantees that all pearls 100 all come off from sheath 54 thus, and is mixed well with buffer solution.Sheath 54 is reduced in first buffer chamber 64 then, magnet 14 is dropped in the sheath 54, the magnetic beads 100 that still is combined with DNA adheres again on the sheath 54.In the second damping fluid chamber 66, in second damping fluid that comprises 50% hydrous ethanol solution, repeat this process, washing pearl 100.After in the second damping fluid chamber 66, the magnetic beads 100 that is combined with DNA being cleaned, promote arm 10, promote sheath 54 thus, magnetic beads 100 is retained in the damping fluid chamber 66.Yet this sheath 54 does not turn back on the platform 2, but keeps being connected with arm 10.

Present rotation platform is under the utensil 16 that reaction vessel 68 is located immediately at be used to heat.This reaction vessel 68 comprises lower area 90 and the upper area 92 that contains kapillary 680.By complete laminated film 94 lower area 90 and upper area 92 are separated.Upper area comprises a little volume, the water 96 of about 100 μ l.The utensil 16 that is used for heating is reduced to the upper area 92 of reaction vessel 68, starts water 96 is heated to about 90 ℃ temperature.In case water 96 is heated, the utensil 16 that raises and be used to heat takes out it from reaction vessel 68.The utensil 16 that will be used to heat then is stored into and is provided with the back use on the device 1.

What Fig. 8 showed the certain volume solution 96 that is used in the top part of upper chamber of reaction vessel 68 that heat packs is contained in device 1 is the viewgraph of cross-section of the physical treatment utensil of the utensil 16 that is used to heat herein.Reaction chamber is connected on the platform 2.The utensil heating that is used for heating is kept at the water 96 of the top part 92 of reaction vessel 68.The top part 92 and the bottom part 90 of reaction vessel 68 are separated by complete film 94.

Rotation platform is located immediately under the sheath 54 the second damping fluid chamber 66 that comprises the magnetic beads 100 that still is combined with DNA once more.Reduce arm 10, thus sheath 54 is reduced in the second damping fluid chamber 66.Once more magnet 14 is reduced entering sheath, and once more pearl 100 is attracted on the sheath 54.Promote sheath 54 and magnet 14 simultaneously, rotation platform is located immediately under the sheath 54 reaction vessel 68.Reduce arm 10, so that sheath 54 is reduced in the top part 92 of reaction vessel 68.As preceding, do not reduce magnet 14 so that pearl 100 no longer attracts on sheath 54.Pearl 100 is released in the upper area 92 of reaction vessel 68.As preceding, slightly promote and reduce arm 10 and sheath 54, guarantee that all pearls all discharge from sheath 54.By warm water 96 DNA elution from the pearl 100 is got off then.Promoting arm 10 takes out sheath 54 from reaction vessel 68.

Fig. 9 show have the analyte 100 that is used for combination discharge into reaction vessel 68 magnet 14 be the viewgraph of cross-section of the operation of the functional part of sheath 54 herein.Magnetic beads 100 is released in the top part 92 of reaction vessel 68, and Jia Re water 96 comes out DNA elution from magnetic beads 100 herein.

Rotation platform once more makes to add the reagent chamber 62 that is used for the necessary reagent of nucleic acid amplification reaction in advance therein and be located immediately under the sheath 54.Reduce arm 10, thus sheath 54 is reduced in the reagent chamber 62.The reagent (not shown) is made according to prescription in advance, makes them be also coupled on the magnetic bead (not shown).In case sheath 54 is in place in reagent chamber 62, magnet 14 is reduced in the sheath 54, the magnetic beads that is attached with reagent attracted on the sheath 54.Sheath 54 and magnet 14 are promoted jointly, so that the reagent (not shown) is taken out from reagent chamber 62.Rotation platform 2 then, and reaction vessel 68 is located immediately under the sheath 54.Reduce arm 10 then, thus sheath 54 is reduced to the top part of reaction chamber 92.Do not reduce magnet 14 once more, the magnetic beads that is combined with reagent is discharged into the upper area 92 of reaction vessel 68 from sheath 54.With warm water 96 reagent elution on the magnetic beads is got off.After finishing elution, reduce arm 10 once more, make sheath 54 in place.Magnet 14 is reduced in the sheath 54, all magnetic beads in the top part 92 of reaction vessel 68 (promptly from analyte and be used for those of reagent) are attracted to sheath 54.Mention sheath 54 and magnet 14 simultaneously, to take out pearl, rotation platform 2.Then with pearl as waste deposits to in the damping fluid chamber of crossing.After pearl is discharged from sheath 54, pass through the rotation of the mobile and platform 2 of arm 10 once more, sheath is turned back to its original position on platform 2.

The top part 92 of reaction vessel 68 comprises the nucleic acid samples of purifying and is used for all required reagent of amplified reaction now.This arm 10 is used to extract cutter 50 now.Rotation platform 2 is located immediately under the cutter 50 reaction vessel 68 once more.Reduce this arm 10, thus cutter 50 is reduced in the reaction vessel 68.Cutter 50 pierces through film 94, and the water 96 that will comprise DNA and reagent splashes in the bottom part 90 of reaction vessel 68.Do not use arm to take out cutter 50, but cutter is retained in the reaction vessel 68, be used for the stopper of sealed reaction vessel 68 in this its conduct.

Fig. 9 shows the viewgraph of cross-section of reaction vessel 68, has the functional part with sealed reaction vessel 68 in place herein, is cutter 50 at this.Cutter 50 also is used to pierce through the top part 92 of separating reaction vessel 68 middle and upper part chambers and the film 94 of bottom part 90, makes the water 96 that comprises the DNA analysis thing and be used for examining the reagent of amplified reaction can enter kapillary 680.Cutter 50 is held in place with sealed reaction vessel 68, makes that solvent can not evaporate from chamber during amplified reaction.Selectively, can on reaction vessel 68, use lid or other closing member.

Enter in the kapillary 680 of reaction vessel 68 in order to drive the water 96 that comprises DNA and this reagent, with platform 2 high speed rotating.Centrifugal force drive fluid 96 enters in the kapillary 680.Owing in rotary course, use the axle 72 that is installed in the socket 74, reaction vessel 68 can be that axle rotates with the platform, so can assist it.In order to prevent that the liquid that is included in chamber 56,58,60,64 and 66 from overflowing in this high speed rotating process, the design of these chambers has oval cross section, and has annular notch in the bottom, as shown in Figure 3 in addition.This indoor design prevents any overflowing.

After the water 96 that comprises DNA and nuclear amplification entered kapillary 680, sample was ready to take place nucleic acid amplification reaction.Yet, one or more other reagent are added in the part 90 of reaction vessel 68 in this stage.This can promote cutter 50 by at first using arm 10, introduces these one or more other reagent then and realizes.It can further comprise and carries out the required PCR reagent of nested PCR reaction, or in signal system required reagent.

Here, can reaction vessel 68 be covered, perhaps as mentioned above by introducing sealing cutter 50 with the lid that is used in particular for this purpose.

In this stage, can manually take out reaction vessel 68 1 from installing, be used for carrying out another device of nucleic acid amplification reaction.Yet in this case, single device is applicable to and carries out nucleic acid amplification and optical detection thereof in addition.These operations are to carry out in the latter half (not shown) of device 1.

For making this process full automation, reaction vessel 68 has protuberance 98, makes it can use the mode the same with other functional part 50 and 54 by 10 operations of device arm.

Reduce arm 10, rotation platform 2 makes the protuberance 98 of reaction vessel 68 be meshed with the fork spare 12 of arm 10.Promote arm 10 then, promote reaction vessel 68 thus.The reaction vessel 68 that promotes has been shown in Fig. 1 and 2.Rotation platform then is aligned in below the reaction vessel 68 of lifting notch portion 78.Reduce arm then, reduce the bottom of reaction vessel thus by these notch portion 78 accesss to plant 1.

When being positioned at the bottom of device 1, be used for the thermocirculator and the fluorescence detector that is used to detect final product of the reaction mixture of heating and cooling kapillary 680, carry out nucleic acid amplification reaction.By apply kapillary 680 auxiliary these reactions with the electric conductive polymer 681 that can make kapillary 680 Fast Heating and cooling.Especially, this reaction vessel 68 can be put into the socket of controllable electric power, make contact 682 and 683 and polymkeric substance 681 form circuit.Electric power control to circuit can make polymkeric substance 681 heating or cooling, realizes rapid thermal cycles.

In case this reaction is finished, reaction vessel 68 can be turned back on the platform 2, to carry out second centrifugation step, preferably use arm 10.This can drive described one or more other reagent and enter kapillary 680.Can make reaction vessel through further handling, with the specific other reagent that is fit to add then.For example, it can be by repeating aforementioned process through another thermal cycle, perhaps makes signal system be shown its processing and/or detect.

In order to detect, sealing lower surface 684 capillaceous is preferably transparent, makes to use suitable checkout equipment (for example spectrofluorimeter) to observe visible signal by it.This can realize by various measures, for example comprise and by arm 10 reaction vessel 68 being transferred in the spectrofluorimeter equipment.Yet most preferably, when reaction vessel 68 is arranged in the socket of the controllable electric power that is used for the thermal cycle process, arrange this spectrofluorimeter from reaction vessel 68, to read signal.

After finishing this operation, will comprise cutter 50, sheath 54 and chamber 56,58,60,62,64 and 66 and the platform 2 of reaction vessel 68 all from device, take out and dispose.To comprise then in the new platform introducing device of required element, and make it can be used for another sample preparation once more.

Figure 11 A has described the setting that reaction vessel (150) is placed the upper chamber (685) of reaction vessel (68).This container (150) surface 1 54 thereon has plastic closure, has the paper tinsel diaphragm at its lower surface (152).The sidewall of container is compressible.This container is separated into each compartment (153) (Figure 11 B) that ring-type is provided with.Each compartment comprises different reagent.

Lid (154) (Figure 11 C) has a plurality of puncture needles (156), and it is provided for piercing through the paper tinsel surface, bottom (152) of each compartment of container (150) (153).When exerting pressure on lid 154, for example in the step of method (3) as mentioned above, all pins 156 pierce through lower surface (152), and reagent is discharged in the upper chamber (685).

Yet if desired, can be separately or pierce through each compartment in order, for example by move link to each other with this device pierce through rod or cutter, make its required compartment of independent aligning in needs.

The variant form of this container has been shown among Figure 12.In this case, container (160) has the single chamber that is limited by last paper tinsel film and following paper tinsel film (151,152), comprises liquid reagent (161).Its also at an upper portion thereof the zone have a pair of annular flange (162,163), between it, define the profiled member (164) that is used for the crawl arm (not shown).

Therefore this container can be introduced automatically in the reaction vessel (68) perhaps actual the introducing in any other required reaction vessel.Thread a needle or the independent rod that pierces through by the thorn on the lid, pierce through paper tinsel 151,152, the content of chamber 161 can be discharged.

Therefore this container can be used for method as mentioned above, perhaps is used for release reagent, for example it is discharged in the upper chamber (685), then it is removed from neighbouring.Selectively, with comprising under the situation that the thorn lid of threading a needle covers it, its original position can be retained in the upper chamber (685) for example, this its as sealing element.

Alternative multi-compartment container has been shown among Figure 13.In this case, container (170) comprises three kinds of reagent (171,172,173), and wherein the third comprises magnetic beads (174), and these three kinds of reagent all are arranged in the independent compartment that is limited by last paper tinsel film (151) and following paper tinsel film (152).

Use rod (175) pierces through film (151) and (152) can make reagent 171,172 and 174 orders mix.The interpolation of magnetic beads makes can carry out the magnetic immuno separation method.

Figure 14 shows the rod that can be used for for example reagent (for example PCR instant pearl) being transferred to (for example in preliminary step) in the container 686.In case be arranged in container, this pearl can dissolve, and is undertaken by for example adding sample.Selectively, this pearl can be transferred in the different containers, is used for preliminarily solubilised, and for example using then, pipette system shifts final solution.

But rod shown in Figure 14 has the stock (1 ') of the material (for example polystyrene or latex) of static electrification or static electrification.It has head construction (2 '), and described head construction makes it can be connected the equipment that is used for automatically it being moved to from a position another position.

The lower surface (3 ') of bar (1) has groove (4 ') (Figure 15), and reagent bead can be contained in wherein.

In use (Figure 16) should be placed on the automatic sensing equipment (not shown) by head (2 ') by rod.At least the bottom static electrification of bar (1 ') is for example by rubbing with insulating material.Place it in first container (the 5 ') top (Fig. 1 6A) that holds reagent bead (6 ') then.This rod is reduced in the container (5 '), till pearl (6 ') attracted on the powered surfaces (3 '), is placed in groove (4 ') (Figure 16 B) at this.

Can will should from first container (5 '), promote (Figure 16 C) together with the pearl (6 ') that adheres to by rod.Then second container (7 ') is placed on below this rod.Second container comprises solvent or solution (8 ').Pearl reduces this rod surface (3 ') is immersed in the solution, so can be assigned to (Figure 16 D) in the solution.

Claims (46)

1. method of carrying out multistep reaction, described method comprises:

1) add one or more first reagent in reaction vessel, described reaction vessel comprises the upper chamber that can hold reagent, and this upper chamber is communicated with the lower chamber that restriction reagent flows,

2) make described reaction vessel bear centrifugal force, enter in the lower chamber to drive described one or more first reagent;

3) in this first chamber, add other reagent, and close described chamber;

4) one of make in upper chamber or the lower chamber at least the condition that makes described one or more first reagent or described other reagent participate in first reaction respectively or reach required reaction conditions of standing;

5) make described reaction vessel bear centrifugal force, enter in the lower chamber, and it can be interacted with the content in the lower chamber to drive described other reagent;

Wherein carry out automatically step (2)～(5) at least.

2. according to the process of claim 1 wherein that the described lower chamber of described reaction vessel is a kapillary.

3. according to the method for claim 1 or 2, wherein in claim 1, make lower chamber stand the condition that makes described one or more first reagent participate in first reaction or reach required reaction conditions.

4. according to each method in the claim 1～3, the cover closing reaction vessel by suitable shape in step (3) wherein.

5. according to each method in the aforementioned claim, wherein said one or more first reagent comprise the PCR reaction mixture, and in step (4), make described reagent stand thermal cycle.

6. according to the method for claim 4, wherein thermal cycle realizes by conducting polymer by making electric current, and described conducting polymer constitutes upper chamber or lower chamber or adjacent with upper chamber or lower chamber.

7. according to the method for claim 5 or 6, wherein said other reagent comprises that carrying out the 2nd PCR reacts one or more required other reagent.

8. according to the method for claim 5 or 6, wherein said other reagent comprises the signal system that is used to detect amplification of nucleic acid.

9. according to the method for claim 8, wherein this signal system is included in the following reagent that produces fluorescence, chemiluminescence or bioluminescence signal of existence of DNA amplification.

10. according to the method for claim 9, wherein this signal system can detect under the situation of not opening reaction vessel.

11. method according to claim 9 or 10, wherein this signal system comprises the combination of fluorescence labeling probe and DNA bond, the sequence-specific that can find in this DNA if described fluorescence labeling probe has been amplified in the PCR course of reaction with DNA combines, and described DNA bond can be by absorbing the fluorescent energy that sent by this probe or by provide fluorescent energy and this fluorescence labeling probe to interact for probe.

12. according to the method for claim 11, wherein this DNA bond is the bond that can not discharge fluorescence when combining with DNA.

13. according to each method in the claim 1～4, wherein this one or more first reagent or in addition reagent comprise and carry out the required reagent of subject matter reverse transcriptase method, described one or more first reagent or in addition another in the reagent then comprise the required PCR reagent of cDNA amplification that can obtain by described subject matter reverse transcriptase method.

14. according to the method for claim 5, wherein in addition reagent is the reagent of this amplified reaction product of can degrading.

15. according to each method in the claim 1～4, wherein said one or more first reagent comprise and are used to carry out some required reagent of amplified reaction, and wherein said other reagent comprise described amplified reaction essential, be not included in the reagent in described one or more first reagent, and wherein, in step (4), described one or more first reagent are reached the temperature conditions of being convenient to take place suitable amplification.

16., wherein before adding, this one or more first reagent and/or this other reagent are provided at the cylindrical shell or the frangible container of the upper chamber inside that is arranged in out on the opening of lower chamber according to each method in the aforementioned claim.

17.,, this one or more first reagent and/or this other reagent are added in the reaction vessel wherein by breaking described cylindrical shell or frangible container according to the method for claim 16.

18., wherein use goad or cutter to break described cylindrical shell or frangible container according to the method for claim 17.

19. according to method arbitrary in the aforementioned claim, wherein, in step 1), these one or more first reagent is solid reagent, uses to comprise that following method transfers to it in reaction vessel:

(i) rod is placed near the described solid reagent in first container or the primary importance, described rod comprises the material of static electrification, and described rod can electrostatic attraction and kept described solid reagent in its surface, extracting a certain amount of described solid reagent,

(ii) move this rod and/or this first container or reaction vessel, make this rod be positioned at reaction vessel near;

(iii) remove this solid reagent, make it put into second container or the second place from described rod.

20. be used to store the frangible container of reagent, described container has reagent chamber therein, this reagent chamber has the wall that at least one can pierce through at its lower surface, wherein upper surface comprises the wall that another can pierce through or comprises the lid that pierces through utensil, and it can cause reagent to discharge from this chamber through being arranged so that piercing through of the described wall that pierces through.

21. according to the container of claim 20, wherein this wall that can pierce through is metal or lamination metallic film surface.

22. according to the container of claim 20 or 21, wherein this reagent chamber has more than a compartment.

23. according to the container of claim 22, wherein this compartment setting adjacent one another are.

24. according to the container of claim 22, wherein this compartment is arranged on the top of each other.

25. according to each container in the claim 20～24, it further comprises and is used to make this container to be automatically moved to the utensil of the position in the reaction vessel.

26. according to the container of claim 25, wherein said utensil comprises one or more annular flanges.

27. one kind solid reagent transferred to the method for second container or the second place from first container or primary importance, described method comprises

(i) rod is placed near the described solid reagent in first container or the primary importance, described rod comprises the material of static electrification, and described rod can electrostatic attraction and kept described solid reagent in its surface, extracting a certain amount of described solid reagent,

(ii) move this rod and/or this first container or second container, this rod is positioned near second container or the position;

(iii) remove this solid reagent, make it put into second container or the second place from described rod.

28. according to the method for claim 27, it carries out automatically.

29. according to the method for claim 27 or 28, wherein this solid reagent is the aggregation through freezing or spray-dired reagent.

30. according to the method for claim 29, wherein this solid reagent is to comprise the pearl that is used to carry out one or more required reagent of nucleic acid amplification reaction.

31. according to the method for claim 30, wherein this amplified reaction is a polymerase chain reaction.

32. according to each method in the claim 27～31, static electrification material that wherein should rod is polystyrene or latex.

33., wherein in preliminary step,, on this rod, produce or increase electric charge by this rod that rubs with insulator according to each method in the claim 27～32.

34. according to the method for claim 33, wherein this insulator is a synthetic textiles.

35. according to the method for claim 33 or 34, wherein this friction step is carried out automatically.

36. according to each method in the claim 27～35, wherein this rod is a hollow.

37. according to the method for claim 36, wherein provide magnet, it can be contained in this rod.

38. according to each method in the claim 27～37, the profile of at least a portion outside surface that wherein should rod can make reagent be contained in this profile.

39. according to the method for claim 38, wherein this profile comprises one or more lacunas or groove.

40. according to the method for claim 38 or 39, lower surface that wherein should rod is a contoured.

41. according to each method in the claim 27～40, wherein step (iii) in, this rod is immersed in the liquid, to remove any solid reagent from this rod.

42. according to the method for claim 42, wherein this solid reagent is to comprise the pearl that is applicable to the reagent that carries out PCR, this liquid is again buffer suspension liquid or DNA/RNA extract.

43., wherein should after use, can handle arbitrarily by rod according to each method in the claim 27～42.

44. a device, but comprise the rod of the dielectric material that comprises static electrification, and the utensil that is used for described rod is transferred to from first container second container.

45. according to the device of claim 44, it comprises:

(iv) platform, it comprises:

(c) be applicable to the chamber that receives sample; With

(d) functional part;

(the v) arm that can raise and reduce, it comprises and is used for the utensil that is connected with this functional part removeability, makes described parts to raise and reduce with this arm; And

(vi) be used for moving the utensil of this platform, make any chamber or functional part to aim at respect to this arm,

Wherein said functional part is this rod.

46. a rod, but it comprises the dielectric material of static electrification, its through contoured with the groove or lacuna solid reagent received by static capacity on its outer surface in.

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