CN109642197A

CN109642197A - system and method for thermal cycling

Info

Publication number: CN109642197A
Application number: CN201680088726.2A
Authority: CN
Inventors: 李响; 于飞
Original assignee: Coyote Bioscience Co Ltd
Current assignee: Coyote Bioscience Co Ltd
Priority date: 2016-06-24
Filing date: 2016-06-24
Publication date: 2019-04-16
Also published as: WO2017219350A1

Abstract

The present disclosure provides nucleic acid amplification systems and methods of nucleic acid amplification using such systems.

Description

System and method for thermal cycle

Background technique

Nucleic acid amplification method permission selectively expands and identifies interested core from complex mixture such as biological sample Acid.Can be via amplification method known in the art, the method based on thermal cycle such as including polymerase chain reaction (PCR), To expand interested nucleic acid.During or after expanding interested nucleic acid, amplified production can detecte, and by end user Interpret testing result.Traditional nucleic acid amplification and detection method is usually directed to the heat circulating equipment for needing high-voltage power to input.It is real When round pcr be related to using can real-time detection from experience nucleic acid amplification sample signal detector.Combined thermal cycle A degree of power input is needed with detection, which limits the use of thermal cycler.

Point-of-care (POC) detection has under the conditions of laboratory infrastructure poor resource-constrained or real receiving It tests the delay of room result and the potentiality from far-off regions for promoting infectious disease detection and disposition that follow-up may be more complex is carried out to patient.So And nucleic acid amplification is carried out in POC setting and faces many challenges.For example, being inputted if necessary to high-voltage power to carry out in real time PCR, then thermal cycler will have limited portability.For example, battery may exhaust quickly using such legacy system. Similarly, can be used for being limited for the power supply of such system power supply, thus prevent in the case that varying environment and sufficiently Use thermal cycler.

Summary of the invention

To the improved system and method for low-power thermal cycle, there are demands.Such low-power thermal cycle can make heat Recycle unit is portable and operable in varied situations.For example, this kind of heat circulating equipment can be brought to scene or take to not Easily obtain some areas of the country of normal power supplies.This disclosure provides be for quickly and easily carry out thermal cycle System and method, to make to adapt to different point-of-care (POC) to be set to possibility.

The one aspect of present disclosure provides a kind of system for nucleic acid amplification, which includes pedestal, reaction Container, shell, fluid flow components (or Fluid flow unit) and controller.

In some embodiments, the pedestal may include heating element (or heating unit).In some embodiments, The occupied area of the pedestal may be less than or equal to about 5000mm²。

In some embodiments, the reaction vessel may include reaction chamber.In some embodiments, the reacting part Part can be adjacent and in thermal communication with the heating element.In some embodiments, during use, the heating element Thermal energy can be provided to the reaction mixture in the reaction chamber.In some embodiments, the reaction mixture may include core Reagent necessary to sour sample and nucleic acid amplification.

In some embodiments, the shell can be releasably attached on the pedestal.In some embodiments In, the shell can encapsulate the reaction vessel when being installed on the pedestal.In some embodiments, the shell It may include at least one outlet, described in the excessively described reaction chamber of the source stream which allows convective fluid from the convective fluid reaches At least one outlet.

In some embodiments, the fluid flow components can make the convective fluid from the source stream of the convective fluid It crosses the reaction chamber and reaches at least one described outlet.

In some embodiments, the controller can be operably coupled to the heating element and the fluid stream Dynamic component.In some embodiments, the controller can be programmed to that the reaction mixture is made to be subjected to one or more add Hot and cold but recycles, to promote the nucleic acid amplification reaction to the reaction mixture.It in some embodiments, can be by (i) The heating element is guided to provide thermal energy to the reaction chamber, and (ii) guides the fluid flow components so that the convection current The excessively described reaction chamber of the source stream of body from the convective fluid reaches at least one described outlet, be subjected to the reaction mixture One or more heating and cooling cycle, to promote the nucleic acid amplification reaction to the reaction mixture.

In some embodiments, the occupied area of the reaction chamber may be less than or equal to about 1000mm²。

In some embodiments, the occupied area may be less than or equal to about 500mm²。

In some embodiments, the occupied area may be less than or equal to about 300mm²。

In some embodiments, the occupied area of the pedestal may be less than or equal to about 2000mm²。

In some embodiments, the occupied area for the shell being installed on the pedestal may be less than or equal to about 5000mm²。

In some embodiments, the cross section of the reaction chamber is smaller than the cross section of the heating element.

In some embodiments, the ratio between the surface area of the reaction chamber and volume can be at least 100mm^-1。

In some embodiments, the system can further comprise the power supply powered to the heating element.

In some embodiments, the power supply can power to the fluid flow components.

In some embodiments, the system can further comprise being operably connected in the pedestal or the shell To the switch of the power supply.In some embodiments, the switch is adjustable from the power supply to the electricity of the heating element Power supply.

In some embodiments, when the shell is installed on the pedestal, the switch is bootable from the electricity Power supply of the source to the heating element.

In some embodiments, the shell may include room, which encapsulates described anti-when being installed on the pedestal Answer container.

In some embodiments, the system can further comprise that the sample being installed on the heating element is supported Device.In some embodiments, the sample holder can receive and fix the reaction vessel.

In some embodiments, when the reaction vessel is fixed on the sample holder, the reaction chamber can It is adjacent and in thermal communication with the heating element.

In some embodiments, the pedestal can further include the excitation for being operably coupled to the reaction vessel Energy source.In some embodiments, the excitation energy source can provide excitation energy to the reaction vessel, to induce instruction The present or absent signal of the amplified production of the nucleic acid amplification reaction.

In some embodiments, the system can further comprise the first sensing with the reaction vessel sensing communication Device.In some embodiments, the present or absent signal of the detectable instruction amplified production of the first sensor.

In some embodiments, the first sensor can be optical sensor, and the signal can be light Learn signal.

In some embodiments, the system can further comprise the second sensing with the reaction vessel sensing communication Device.In some embodiments, the second sensor can detect the reaction indoor one or more of the reaction vessel Temperature at a position.

In some embodiments, the system can further comprise being operatively coupled in the pedestal or the shell To the display of the first sensor.In some embodiments, the display can be configured to display and indicate the expansion Increase production the present or absent signal of object.

In some embodiments, the system can further comprise being operatively coupled in the pedestal or the shell To the first sensor and/or the display of the second sensor.In some embodiments, the display can be matched 1) display is set to indicate the present or absent signal of the amplified production and/or 2) show the described of the reaction vessel React the temperature at indoor one or more of positions.

In some embodiments, the excitation energy source can be light source.

In some embodiments, the convective fluid can be convection gas.

In some embodiments, the convection gas can be air.

In some embodiments, the fluid flow components can be fan.

In some embodiments, the fluid flow components can be installed on the shell or the pedestal.

In some embodiments, the source of the convective fluid can be refrigeration unit.

In some embodiments, the heating element may include infrared heating unit.

In some embodiments, the heating element may include peltier (Peltier) heating unit.

In some embodiments, the heating element may include heating unit containing aluminium.

In some embodiments, the heating element may include electrical resistor heating element.

In some embodiments, the controller can be programmed to that the heating element is guided to provide to the reaction chamber Thermal energy until the reaction mixture reaches the first temperature, and guides the fluid flow components so that the convective fluid The reaction chamber is flowed through, until the reaction mixture reaches the second temperature lower than first temperature.

In some embodiments, the controller can be programmed to reach the second temperature when the reaction mixture When guide the fluid flow components to reduce or terminate the flowing of the convective fluid.

In some embodiments, the controller may include in the susceptor.

In some embodiments, the reaction mixture may include one or more primers and polymerase.

In some embodiments, the reaction mixture may include buffer.

In some embodiments, the reaction mixture may include the active cation for adjusting the polymerase.

In some embodiments, the cation may include Mg²⁺Or Mn²⁺。

In some embodiments, one or more primers can have for HBV, HCV, FluA, FluB, CA16, EV71, enterovirus, EBOV, EBV, measles virus, salmonella, HPV and/or the nucleic acid sequence of HIV selection.

In some embodiments, the nucleic acid amplification reaction can be polymerase chain reaction (PCR).

In some embodiments, the mean temperature of the convective fluid can be below about 15 DEG C.

In some embodiments, the mean temperature of the convective fluid can be below about 10 DEG C.

In some embodiments, the controller can be by using the thermal energy provided by the heating element to control The rate of heat addition of reaction chamber is stated, and flows through the flowing of the reaction chamber using the convective fluid to control the reaction chamber Cooling rate, to be heated and/or cooled to reaction mixture offer.

In some embodiments, when the rate of heat addition is greater than the cooling rate, the reaction mixture can be made It is subjected to heating.

In some embodiments, when the rate of heat addition is less than the cooling rate, the reaction mixture can be made It is subjected to cool to.

In some embodiments, the rate of heat addition can be at least 5 DEG C/s.

In some embodiments, the cooling rate can be at least 5 DEG C/s.

In some embodiments, in the case where the switch is not opened, the controller can not make described anti- Mixture is answered to be subjected to one or more of heating and cooling cycle.

In some embodiments, after the switch is opened after postponing after a period of time, the controller can The reaction mixture is set to be subjected to one or more of heating and cooling cycle.

In some embodiments, the reaction vessel can further include sampling unit.In some embodiments, institute Stating sampling unit may include the sampler chamber being in fluid communication with the reaction chamber.

In some embodiments, the sampling unit can further include the collecting part for collecting nucleic acid samples.

In some embodiments, the sampling unit can further include the sealing for sealing the opening of the sampler chamber Part.

In some embodiments, during use, the collecting part can pierce the seal member with by the core Sour sample is discharged into the sampler chamber.

In some embodiments, the thickness of the side of the reaction chamber adjacent with the heating element is smaller than about 1mm。

The one aspect of present disclosure provides a kind of for nucleic acid amplification method comprising:

Activation system, the system include (i) pedestal, (ii) reaction vessel, (iii) shell and (iv) fluid flow components；And

The reaction mixture is set to be subjected to one or more heating and cooling cycle, to promote the nucleic acid to the reaction mixture Amplified reaction.

In some embodiments, the pedestal may include heating element.In some embodiments, the pedestal accounts for Ground area may be less than or equal to about 5000mm²。

In some embodiments, the reaction vessel may include reaction chamber.In some embodiments, the reacting part Part can be adjacent and in thermal communication with the heating element.In some embodiments, the reaction chamber may include that reaction is mixed Object is closed, which includes reagent necessary to nucleic acid samples and nucleic acid amplification.

In some embodiments, the shell can be releasably attached on the pedestal.In some embodiments In, the shell can encapsulate the reaction vessel.In some embodiments, the shell may include at least one outlet, should Outlet allows convective fluid to reach at least one described outlet from the excessively described reaction chamber of the source stream of the convective fluid.

In some embodiments, the heating element can be guided to provide thermal energy, and (ii) to the reaction chamber by (i) The fluid flow components are guided so that described in the excessively described reaction chamber arrival of the source stream of the convective fluid from the convective fluid At least one outlet, to make the reaction mixture be subjected to one or more heating and cooling cycle, to promote to the reaction The nucleic acid amplification reaction of mixture.

In some embodiments, may be mounted to the occupied area of the shell on the pedestal less than or equal to about 5000mm²。

In some embodiments, the activation in (a) can be carried out by being powered with power supply to the heating element.

In some embodiments, the power supply can power to the fluid flow components.

In some embodiments, the power supply can be operably coupled to by opening in the pedestal or the shell Switch supply electric power.

In some embodiments, the switch can be opened by the way that the shell to be installed on the pedestal.

In some embodiments, the method can further comprise that the reaction vessel is fixed to peace before (a) It is attached on the sample holder on the heating element.

In some embodiments, the method can further comprise providing excitation energy to the reaction vessel, to lure Lead the present or absent signal for indicating the amplified production of the nucleic acid amplification reaction.

In some embodiments, the method can further comprise first provided with the reaction vessel sensing communication Sensor, to detect the present or absent signal for indicating the amplified production.

In some embodiments, the method can further comprise second provided with the reaction vessel sensing communication Sensor, to detect the temperature of the indoor one or more positions of the reaction of the reaction vessel.

In some embodiments, the method can further comprise providing in the pedestal or the shell operationally It is coupled to the display of first sensor and/or second sensor.In some embodiments, the display can be configured Are as follows: (1) display indicates that the present or absent signal of the amplified production, and/or (2) show the described of the reaction vessel React the temperature at indoor one or more of positions.

In some embodiments, the method can further comprise providing in the pedestal or the shell operationally It is coupled to the display of first sensor.In some embodiments, the display can be configured to display and indicate the expansion Increase production the present or absent signal of object.

In some embodiments, the excitation energy source can be light source.

In some embodiments, the convective fluid can be convection gas.

In some embodiments, the convection gas can be air.

In some embodiments, the fluid flow components can be fan.

In some embodiments, the method can further comprise that will be equipped with the shell thereon before (a) The pedestal is placed in refrigeration unit.

In some embodiments, the heating element may include infrared heating unit.

In some embodiments, the heating element may include peltier heating unit.

In some embodiments, thermal energy can be provided to the reaction chamber, until the reaction mixture reaches the first temperature Degree.In some embodiments, the convective fluid can be made to flow through the reaction chamber, be lower than until the reaction mixture reaches The second temperature of first temperature.

In some embodiments, the method can further comprise when the reaction mixture reaches the second temperature When reduce or terminate the flowing of the convective fluid.

In some embodiments, the reaction mixture may include buffer.

In some embodiments, the cation may include Mg²⁺Or Mn²⁺。

In some embodiments, the method can further comprise being controlled using the thermal energy provided by the heating element The rate of heat addition of the reaction chamber is made, and flows through the flowing of the reaction chamber using the convective fluid to control the reaction The cooling rate of room.

In some embodiments, the rate of heat addition can be greater than the cooling rate, to make the reaction mixture It is subjected to heating.

In some embodiments, the rate of heat addition is smaller than the cooling rate, to make the reaction mixture It is subjected to cool to.

In some embodiments, the rate of heat addition can be at least 5 DEG C/s.

In some embodiments, the cooling rate can be at least 5 DEG C/s.

In some embodiments, in (b), in the case where the switch is not opened, the reaction can not be made Mixture is subjected to one or more of heating and cooling cycle.

In some embodiments, in (b), after the switch is opened after postponing after a period of time, can make The reaction mixture is subjected to one or more of heating and cooling cycle.

In some embodiments, the method can further comprise deactivating the system after (b).

In some embodiments, the method can further comprise that the nucleic acid samples are placed on institute before (a) It states in reaction vessel.

In some embodiments, the reaction vessel can further include sampling unit, and the sampling unit packet Containing collecting part and the sampler chamber being in fluid communication with the reaction chamber.In some embodiments, can by on it or The collecting part for wherein having collected the nucleic acid samples pierces through the seal member for sealing the opening of the sampler chamber, by the nucleic acid Sample is placed in the reaction vessel.

The one aspect of present disclosure provides a kind of system for nucleic acid amplification, which includes pedestal, reaction Container, shell and controller.

In some embodiments, the shell can be releasably attached on the pedestal.In some embodiments In, the shell can encapsulate the reaction vessel.In some embodiments, the shell may include cooling-part, the cooling Component when the shell is installed on the pedestal with the reaction vessel thermal communication.

In some embodiments, the controller can be coupled to the heating element.In some embodiments, described Controller can be programmed to that the reaction mixture is made to be subjected to one or more heating and cooling cycle, to promote to the reaction The nucleic acid amplification reaction of mixture.In some embodiments, the reaction can be adjusted using the heating element by (i) to hold The rate of heat addition of device, and (ii) adjust the cooling rate of the reaction vessel using the cooling-part, to keep the reaction mixed It closes object and is subjected to one or more heating and cooling cycle, to promote the nucleic acid amplification reaction to the reaction mixture.

In some embodiments, the cooling-part can be that at least about material of 0.2J/g*K is formed by thermal capacity.

In some embodiments, the cooling-part can be that at least about material of 0.3J/g*K is formed by thermal capacity.

In some embodiments, the cooling-part can be that at least about material of 0.4J/g*K is formed by thermal capacity.

In some embodiments, the cooling-part can be that at least about material of 0.5J/g*K is formed by thermal capacity.

In some embodiments, the cooling-part can be that at least about material of 1.0J/g*K is formed by thermal capacity.

In some embodiments, the cooling-part is the solid comprising copper.

The one aspect of present disclosure provides one kind for nucleic acid amplification method, this method comprises:

Activation system, the system include (i) pedestal, (ii) reaction vessel and (iii) shell；And

In some embodiments, the rate of heat addition of the reaction vessel can be adjusted using the heating element by (i), (ii) adjusts the cooling rate of the reaction vessel using the cooling-part, come make the reaction mixture be subjected to one or Multiple heating and cooling cycle, to promote the nucleic acid amplification reaction to the reaction mixture.

In some embodiments, the method can further comprise providing in the pedestal or the shell operationally It is coupled to the display of first sensor and/or second sensor.In some embodiments, the display is configured as: (1) display indicates that the present or absent signal of the amplified production, and/or (2) show the described anti-of the reaction vessel Answer the temperature at indoor one or more of positions.

In some embodiments, the method can further comprise providing in the pedestal or the shell operationally It is coupled to the display of first sensor.In some embodiments, the display is configured as display and indicates the amplification The present or absent signal of product.

According to the detailed description of the following illustrative embodiment that present disclosure only has shown and described, present disclosure Other aspect and advantage will become obvious to those skilled in the art.It will recognize that present disclosure energy Enough includes other and different embodiments, and its several details can be modified at multiple apparent aspects, is owned These are all without departing from present disclosure.Correspondingly, attached drawing and description will be considered to be illustrative and not restrictive naturally.

It quotes and is incorporated to

The all publications, patents and patent applications referred in this specification are both incorporated herein by reference, and degree is such as It is same particularly and individually to point out that each individual publication, patent or patent application are incorporated by reference into.

Detailed description of the invention

Novel feature of the invention is specifically explained in the appended claims.By reference to below to wherein utilize this hair The detailed description and the accompanying drawings that the illustrative embodiment of bright principle is illustrated, it will obtain to the features and advantages of the present invention It better understands, in the drawings:

Figure 1A and Figure 1B illustrates the example of the system of present disclosure.

Fig. 2 illustrates the example of the system of present disclosure.

Fig. 3 illustrates the internal structure of the system of present disclosure.

Fig. 4 illustrates the internal structure of the system of present disclosure.

It may include the display in the system of present disclosure that Fig. 5, which is illustrated,.

Fig. 6 illustrates the reaction vessel of present disclosure.

Fig. 7 shows the viewgraph of cross-section of the reaction vessel of present disclosure.

Fig. 8 shows the bottom view of the reaction vessel of present disclosure.

Fig. 9 shows the computer control system for being programmed or being otherwise configured to realize the method for present disclosure System.

Specific embodiment

It is aobvious for those skilled in the art although the preferred embodiments of the invention have been illustrated and described herein And be clear to, these embodiments only provide in an illustrative manner.Those skilled in the art are not departing from situation of the invention Down now it will be appreciated that a variety of variations, change and replacement.It should be appreciated that the various substitutions of embodiment of the present invention described herein Scheme can be used for implementing the present invention.

As used herein, term " sample " typically refers to containing or suspects any sample containing nucleic acid molecules.For example, Samples subjects can be the biological sample containing one or more nucleic acid molecules.The biological sample can be from the body sample of subject Product obtain (for example, extract or separation), the optional autoblood of the body sample (for example, whole blood), blood plasma, serum, urine, saliva, Mucosal secretion, sputum, excrement and tear.The fluid or tissue sample that the body sample can be subject are (for example, skin-like Product).In some instances, which is obtained from the cell-free body fluid of subject, such as whole blood.In this case, which can Including Cell-free DNA and/or cell-free RNA.In some other examples, the sample be environmental sample (for example, soil, waste, Surrounding air etc.), production piece (for example, sample from any industrial process) and foodstuff samples are (for example, dairy produce, plant Product and meat products).

In some embodiments, sample directly is obtained without being further processed from subject.In some embodiments In, in the pre-treatment sample of biological or chemical reaction (for example, nucleic acid amplification).For example, can be in addition biological sample and nucleic acid Decomposition agent is added in sample holder before reagent necessary to expanding.The example of decomposition agent include Tris-HCl, EDTA, Detergent (for example, Triton X-100, SDS), lysozyme, glucolase, protease E, viral endolysin, outer lysin, enzymatic hydrolysis Enzyme, lyticase, Proteinase K, the endolysin from bacteriophage and outer lysin, come from withered grass at the endolysin from bacteriophage PM2 The endolysin of bacillus (B.subtilis) bacteriophage PBSX comes from lactobacillus prophage Lj928, Lj965, bacteriophage The endolysin of 15Phiadh, the endolysin from streptococcus pneumonia bacteriophage Cp-I, Streptococcusagalactiae bacteriophage B30 it is difunctional Peptide glycan lysin, the endolysin from prophage bacterium and outer lysin come from the interior of Listeria (Listeria) bacteriophage Lysin, cave albumen-endolysin, 20 lysis genes of cell, holWMY Staphylococcus warneri (Staphylococcus wameri) M Bacteriophage varphiWMY, the Iy5WMY of Staphylococcus warneri M bacteriophage varphiWMY, polysorbas20, PEG, KOH, NaCl and its Combination.In some embodiments, decomposition agent is sodium hydroxide (NaOH).In some embodiments, biological sample is not spent Dirty agent processing.

In some embodiments, sample is purified (for example, pure by filtering, centrifugation, column purification and/or magnetism Change, for example, by using magnetic bead (for example, super-paramagnetic bead)) to obtain the nucleic acid purified.

Sample can have any suitable size or volume.In some instances, small size includes no more than about 5mL；No More than about 4mL；No more than about 3mL；No more than about 2mL；No more than about 1mL；No more than about 500 μ L；No more than about 250 μ L；No More than about 100 μ L；No more than about 90 μ L；No more than about 80 μ L；No more than about 70 μ L；No more than about 60 μ L；No more than about 50 μ L； No more than about 40 μ L；No more than about 30 μ L；No more than about 25 μ L；No more than about 20 μ L；No more than about 15 μ L；No more than about 10 μ L；No more than about 8 μ L；No more than about 6 μ L；No more than about 5 μ L；No more than about 4 μ L；No more than about 3 μ L；No more than about 2 μ L；No More than about 1 μ L；No more than about 0.8 μ L；No more than about 0.5 μ L；No more than about 0.3 μ L；No more than about 0.2 μ L；No more than about 0.1 μL；No more than about 0.05 μ L；Or no more than about 0.01 μ L.

As used herein, term " body fluid " typically refers to any fluid that can be obtained from subject.Body fluid may include but It is not limited to, for example, blood, urine, saliva, tear, sweat, body exudates, body excretions or being originated from subject or can be from Any other fluid that subject obtains.Particularly, body fluid includes but is not limited to blood, serum, blood plasma, marrow, saliva, urine Liquid, spinal fluid, tear, excrement, mucus, sweat, earwax, oil, glandular secretion object, celiolymph, sperm, vaginal secretion, comes gastric juice Derived from the interstitial fluid of tumor tissues, ocular fluid, placental fluids, amniotic fluid, Cord blood, lymph, chamber liquid, sputum, fester, meconium, breast milk And/or other secretion or excreta.

As used herein, term " nucleic acid " typically refers to the molecule comprising one or more nucleic acid subunits.Nucleic acid can Including one or more selected from adenosine (A), cytimidine (C), guanine (G), thymidine (T) and uracil (U) or its variant A subunit.Nucleotide may include A, C, G, T or U or its variant, which includes but is not limited to peptide nucleic acid (PNA).Nucleotide It may include any subunit that can be mixed in the nucleic acid chains of growth.Such subunit can be A, C, G, T or U, or to one A or multiple complementation A, C, G, T or U have specificity or with purine (that is, A or G or its variant) or pyrimidine (that is, C, T or U, Or its variant) complementary any other subunit.Subunit can make each nucleic acid base or base group (for example, AA, TA, AT, GC, CG, CT, TC, GT, TG, AC, CA or its uracil counterpart) it can be differentiated.In some instances, nucleic acid is deoxidation Ribonucleic acid (DNA) or ribonucleic acid (RNA) or derivatives thereof.Nucleic acid can be single-stranded or double-strand.Nucleic acid may include one Kind or the nucleotide of a variety of modifications, for example, methylated nucleotide and nucleotide analog.

As used herein, term " polymerase " typically refers to any enzyme for capableing of catalytic polymerization.The reality of polymerase Example includes such as nucleic acid polymerase, transcriptase or ligase.Polymerase can be polymerase (polymerization enzyme) Or polymerism enzyme (polymerizing enzyme).

As used herein, term " subject " typically refer to have can test or the entity of detectable hereditary information or Medium.Subject can be people or individual.Subject can be vertebrate, such as mammal.The example of subject includes Muroid, apes, the mankind, farm-animals, sport animals, pet, birds, dog, cat, horse, ox, sheep, pig, dolphin, rodent (for example, mouse, rat) or insect.Other examples of subject include, for example, food, plant, soil and water.Subject can To be living body or dead volume.The subject can be human or animal.

As used herein, term " about " or " close " typically refer to reasonably change, for example, specified amount +/- 10%, +/- 9%, within +/- 8%, +/- 7%, +/- 6%, +/- 5%, +/- 4%, +/- 3%, +/- 2% or +/- 1%.

As used herein, term " reaction mixture " is typically referred to comprising for completing nucleic acid amplification (for example, DNA expands Increase, RNA amplification) necessary to reagent composition, the non-limiting example of this kind of reagent includes having to target RNA or target DNA The primer sets of specificity, the DNA generated by the reverse transcription of RNA, archaeal dna polymerase, reverse transcriptase are (for example, be used for the reverse of RNA Record), suitable buffer (including zwitterionic buffer), co-factor (for example, divalent and monovalent cation), dNTP and other Enzyme (for example, uracil-DNA glycosylase (UNG) etc.).In some cases, reaction mixture also may include one or more Report agent.

As used herein, " report agent " typically refers to generate the composition of detectable signal, the presence of the signal or not It whether there is in the presence of can be used for detecting amplified production.

As used herein, term " target nucleic acid " typically refers to have certain nucleosides in the starter population of nucleic acid molecules The nucleic acid molecules of acid sequence, presence, amount and/or sequence or one of them or multinomial variation needs are measured.Target nucleus Acid can be any kind of nucleic acid, including DNA, RNA and the like.As used herein, " target nucleus ribosomal ribonucleic acid (RNA) " is logical Refer to the target nucleic acid as RNA.As used herein, " target DNA (DNA) " typically refers to the target nucleus as DNA Acid.

In one aspect, this disclosure provides the systems for nucleic acid amplification.The system may include pedestal, the pedestal It may include heating element, and the occupied area of the pedestal may be less than or equal to about 9000mm².For example, the land occupation face of the pedestal Product may be less than or equal to about 8000mm², about 7000mm², about 6000mm², about 5000mm², about 4500mm², about 4000mm², about 3500mm², about 3000mm², about 2500mm², about 2000mm², about 1500mm², about 1000mm², about 900mm², about 800mm², about 700mm², about 600mm², about 500mm², about 400mm², about 300mm², about 200mm², about 100mm²Deng.

The system can further comprise the reaction vessel comprising reaction chamber.The reaction chamber can it is adjacent with heating element and with Thermal communication.During use, which can provide thermal energy to the reaction mixture in reaction chamber.The reaction mixture can Include reagent necessary to nucleic acid samples and nucleic acid amplification.The occupied area of the reaction chamber may be less than or equal to about 2000mm².Example Such as, the occupied area of the reaction chamber may be less than or equal to about 1500mm², about 1000mm², about 900mm², about 800mm², about 700mm², about 600mm², about 500mm², about 400mm², about 300mm², about 200mm², about 100mm²Deng.In some embodiments In, the cross section of reaction chamber is less than the cross section of heating element.

On the other hand, this disclosure provides the methods for nucleic acid amplification.This method may include activation system, should System includes the pedestal that (i) includes heating element, and wherein the occupied area of the pedestal is less than or equal to about 5000mm², (ii) packet Reaction vessel containing reaction chamber, wherein the reaction chamber is adjacent and in thermal communication with heating element.The reaction chamber may include reaction Mixture, the reaction mixture include reagent necessary to nucleic acid samples and nucleic acid amplification.The system can further comprise (iii) The shell being releasably attached on pedestal.The shell can encapsulate reaction vessel, and the shell may include at least one outlet, The outlet allows convective fluid to cross at least one described outlet of reaction chamber arrival from the source stream of convective fluid.The system may also include (iv) so that convective fluid is crossed reaction chamber from the source stream of convective fluid and reach at least one fluid flow components exported.This method can It further comprise that reaction mixture is made to be subjected to one or more heating and cooling cycle, to promote the nucleic acid to reaction mixture to expand Increase reaction, this can guide heating element to provide thermal energy, and (ii) guidance fluid flow components so that convection current to reaction chamber by (i) Fluid, which from the source stream of convective fluid crosses reaction chamber and reaches at least one outlet, to carry out.

In some embodiments, the occupied area of the pedestal may be less than or equal to about 9000mm².For example, the pedestal Occupied area may be less than or equal to about 8000mm², about 7000mm², about 6000mm², about 5000mm², about 4500mm², about 4000mm², about 3500mm², about 3000mm², about 2500mm², about 2000mm², about 1500mm², about 1000mm², about 900mm², about 800mm², about 700mm², about 600mm², about 500mm², about 400mm², about 300mm², about 200mm², about 100mm²Deng.

At any aspect of the multiple aspect, the shell and/or pedestal can have more than one outlet, for example, institute 2,3,4,5,6,7,8,9 or more outlets can be had by stating shell and/or pedestal, these outlets allow convective fluid from convection current The source stream of fluid crosses reaction chamber and reaches at least one outlet.

The reaction vessel can be any container (for example, PCR pipe) that can receive the solution comprising nucleic acid samples.It should Reaction vessel may include reaction chamber, and chemistry and/or biological respinse can occur in the reaction chamber.The reaction vessel can have Different size, shape, weight and configurations.In some embodiments, which is round or oval tubulose.One In a little embodiments, which is rectangle, square, diamond shape, circle, ellipse or triangle.The reaction vessel can be with It is regular shape or irregular shape.For example, reaction vessel can be pipe, hole, capillary, cassette, cuvette, centrifuge tube or shifting Liquid pipe tip.In some embodiments, the ratio between the surface area of the reaction vessel and volume are at least 100mm^-1、200mm^-1、 300mm^-1、350mm^-1、400mm^-1、450mm^-1、500mm^-1Or it is bigger.The thickness of the side of the reaction chamber adjacent with heating element It is smaller than about 3mm.For example, the thickness of the side of the reaction chamber adjacent with heating element be smaller than about 3mm, 2.5mm, 2.0mm, 1.5mm, 1.0mm, 0.9mm, 0.8mm, 0.7mm, 0.6mm, 0.5mm, 0.4mm, 0.3mm, 0.2mm, 0.1mm, 0.05mm etc..

The reaction vessel may include sampling unit.The sampling unit may include the sampler chamber being in fluid communication with reaction chamber. The sampling unit may include the collecting part for collecting nucleic acid samples.In some embodiments, which includes that sealing is adopted The seal member of the opening of specimen chamber.During use, which can pierce seal member and is adopted with being discharged into nucleic acid samples In specimen chamber.

The seal member can be any conjunction for separating at least two volumes or separating volume and external environment Suitable structure.Seal member can be synthesis film, such as by solid-state material (for example, semiconductor, metal, semimetal or nonmetallic) Or the film that polymeric material (for example, polymer film) is formed.For example, seal member can be by sealing sampler chamber and by itself and external environment Opaque, the transparent or semitransparent material separated is formed.In some embodiments, which is made of parafilm Polymer film.

The system may include the shell being releasably attached on pedestal.The shell can the encapsulating when being installed on pedestal Reaction vessel, and the shell may include at least one outlet, and which allows the source stream of convective fluid from convective fluid excessively anti- Room is answered to reach at least one described outlet.In some embodiments, which comprises more than one outlet, for example, 2,3,4, 5,6,7 or more outlets.The occupied area of the shell may be less than or equal to about 9000mm².For example, the land occupation face of the shell Product may be less than or equal to about 8000mm², about 7000mm², about 6000mm², about 5000mm², about 4500mm², about 4000mm², about 3500mm², about 3000mm², about 2500mm², about 2000mm², about 1500mm², about 1000mm², about 900mm², about 800mm², about 700mm², about 600mm², about 500mm², about 400mm², about 300mm², about 200mm², about 100mm²Deng.The shell may include pacifying The room of reaction vessel is encapsulated when being attached on pedestal.In some embodiments, the shell may include on being installed to pedestal up to The room of small part encapsulating reaction vessel.

In addition, the system may include making convective fluid cross reaction chamber from the source stream of convective fluid to reach at least one outlet Fluid flow components.The convective fluid can be convection gas, such as air or the mixture of one or more gases.The convection current The source of fluid can be refrigeration unit, such as refrigerator.The fluid flow components can be fan.In some embodiments, by this Fluid flow components are installed on shell or pedestal.

The system can further comprise the controller for being operably coupled to heating element and fluid flow components.The control Device processed can be programmed to that reaction mixture is made to be subjected to one or more heating and cooling cycle, to promote the core to reaction mixture Sour amplified reaction.This can guide heating element to provide thermal energy to reaction chamber for example, by (i), and (ii) guides fluid flow components So that convective fluid from the source stream of convective fluid crosses reaction chamber and reaches at least one outlet and carries out.The controller may include in base In seat.

The system can further comprise power supply.The power supply can be configured to heating element and/or fluid flow components Power supply.The activation of the system can be realized by being powered with power supply to heating element and/or fluid flow components.

The system can include switch in pedestal and/or shell.The switch can be operably coupled to power supply, and It is adjustable from power supply to heating element and/or the power supply of fluid flow components.For example, when shell is installed on pedestal, The switch may be guided from power supply to heating element and/or the power supply of fluid flow components.It can be by opening pedestal or shell In be operably coupled to the switch of power supply to supply electric power.The switch can be opened by the way that shell to be installed on pedestal.

The system may include sample holder.The sample holder can receive and fix reaction vessel.Work as reaction vessel When fixed on sample holder, the reaction chamber in the reaction vessel can be adjacent and in thermal communication with heating element.Some In embodiment, which is installed on heating element；This can ensure that reaction vessel or in which sample in fixation When into sample holder or on sample holder with heating element thermal communication.

In the system of present disclosure, the pedestal can further include the excitation for being operably coupled to reaction vessel Energy source.The excitation energy source can provide excitation energy to reaction vessel, to induce the amplified production of instruction nucleic acid amplification reaction Present or absent signal.The excitation energy source can be light source or other inductive energy sources, such as light emitting diode, swash Light device or other energy sources.The excitation energy source can be activated guiding excitation energy to comprising sample in the reaction chamber, from And generate the generation of chemical or biological reactionss (for example, nucleic acid amplification reaction) and/or the transmitting signal (example of result on instruction sample Such as, optical signalling, fluorescence signal and/or electrostatic signal).

The system may include one or more sensors, for example, 2,3,4,5,6,7 or more sensors.For example, The system may include the first sensor with reaction vessel sensing communication.The presence of the detectable instruction amplified production of first sensor Or the signal being not present.First sensor can be optical sensor, inductance sensor, electrochemical sensor, electrostatic transducer And/or impedance transducer.The signal can be optical signalling, fluorescence signal and/or electrostatic signal.

The system can further comprise the second sensor with reaction vessel sensing communication.Second sensor is detectable anti- Answer the temperature of the indoor one or more positions of reaction of container.

The system can further comprise that first sensor and/or the second sensing are operably coupled in pedestal or shell One or more (for example, 1,2,3 or more) display of device.The display can be configured to display instruction amplified production Present or absent signal and/or reaction vessel the indoor one or more positions of reaction temperature.

The heating element may include that infrared heating unit, peltier heating unit, heating unit containing aluminium and/or resistance add Hot cell.

The controller can be programmed to guidance heating element and provide thermal energy to reaction chamber, until reaction mixture reaches the One temperature, and fluid flow components are guided so that convective fluid flows through reaction chamber, until reaction mixture reaches lower than the first temperature The second temperature of degree.The controller can be programmed to guide fluid flow components to subtract when reaction mixture reaches second temperature Less or terminate convective fluid flowing.

First temperature can be about 80 DEG C to about 100 DEG C.For example, the first temperature can be about 87 DEG C to about 95 DEG C or about 90 DEG C to about 95 DEG C.First temperature can be about 92 DEG C to about 95 DEG C.First temperature can be greater than or equal to about 90 DEG C, 91 DEG C, 92 DEG C, 93 DEG C, 94 DEG C, 95 DEG C, 96 DEG C, 97 DEG C, 98 DEG C, 99 DEG C or 100 DEG C.Second temperature can be about 40 DEG C to about 70 DEG C or about 50 DEG C to about 60 DEG C.Second temperature can less than or equal to about 40 DEG C, 45 DEG C, 50 DEG C, 51 DEG C, 52 DEG C, 53 DEG C, 54 DEG C, 55 DEG C, 56 DEG C, 57 DEG C, 58 DEG C, 59 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C, 80 DEG C or 85 DEG C.

The reaction mixture may include one or more primers and polymerase.In some embodiments, reaction mixing Object may include buffer.The reaction mixture may include the active cation for adjusting polymerase.The cation may include Mg²⁺ And/or Mn²⁺.One or more primers can have the sequence complementary with target nucleic acid sequence.

At any aspect of the multiple aspect, nucleic acid amplification reaction can be carried out using the primer sets for target nucleic acid. Primer sets generally comprise one or more primers.For example, primer sets can include about 1,2,3,4,5,6,7,8,9,10,20,30, 40,50 kinds or more primers.In some embodiments, primer sets include to be directed to different amplified productions or different nucleic acid The primer of amplified reaction.For example, primer sets may include the first primer and second primer complementary with nucleic acid chains product, the first primer It is necessary to generating the first chain of the nucleic acid product complementary at least part of target nucleic acid, the second primer is generation and nucleic acid Necessary to second chain of the nucleic acid product of at least part complementation of the first chain of product.

For example, primer sets can be directed to target RNA.The primer sets may include can be used for generate it is mutual at least part of target RNA The first primer of the first chain of nucleic acid product of benefit.In the case where reverse transcription reaction, the first chain of nucleic acid product can be DNA. The primer sets also may include that can be used for generating nucleic acid product second chain complementary at least part of the first chain of nucleic acid product Second primer.In the case where the reverse transcription reaction carried out parallel with DNA cloning, the second chain of nucleic acid product be can be and oneself One chain of nucleic acid (for example, DNA) product of the DNA chain complementation of RNA template generation.

If it is desired, any suitable number of primer sets can be used.It is, for example, possible to use about 1,2,3,4,5,6,7,8, 9,10 or more primer sets.When using multiple primer sets, one or more primer sets can respectively correspond to specific core Sour amplified reaction or amplified production.

In some embodiments, using archaeal dna polymerase.Any suitable archaeal dna polymerase can be used, including commercially available Archaeal dna polymerase.Archaeal dna polymerase typically refers to the enzyme that can be incorporated into nucleotide in such a way that template combines in DNA chain. The non-limiting example of archaeal dna polymerase includes Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, VENT polymerization Enzyme, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Expand polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, Pho polymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerization Enzyme, Tih polymerase, Tfi polymerase, Platinum Taq polymerase, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow piece Section and their variant, modified outcome and derivative.For certain thermal starting polymerase, it may be necessary at about 92 DEG C to 95 For about 2 minutes to the denaturing step of 10 minutes sections at a temperature of DEG C (for example, 94 DEG C to 95 DEG C), this is according to different poly- Synthase may change heat distribution.

In some embodiments, using reverse transcriptase.Any suitable reverse transcriptase can be used.Reverse transcriptase is usually Refer to the enzyme that nucleotide can be incorporated into DNA chain when in conjunction with RNA template.The non-limiting example of reverse transcriptase includes HIV-1 reverse transcriptase, M-MLV reverse transcriptase, AMV reverse transcriptase, reverse transcriptase of telomere and their variant, modification Product and derivative.

Target nucleic acid sequence can be related to disease.The disease can be related to virus such as RNA virus or DNA virus.In some realities It applies in scheme, which can be selected from human immunodeficiency virus I (HIV I), human immunodeficiency virus II (HIV II), just gluing Virus, Ebola virus, dengue virus, influenza virus, hepatitis virus, hepatitis A virus, hepatitis type B virus, hepatitis C Virus, Hepatitis D virus, Hepatitis E virus, HGV RNA, Epstein-Barr virus, monocytosis,mononucleosis virus, giant cell Virus, SARS virus, west nile fever virus, poliovirus, measles virus, herpes simplex virus, variola virus, adenopathy Poison and varicella virus.In some embodiments, which is selected from H1N1 virus, H3N2 virus, H7N9 virus and H5N1 Virus.In some embodiments, which is 55 type adenovirus (ADV55) or 7 type adenovirus (ADV7).In some implementations In scheme, which is to have the RNA- Hepatitis C Virus (RNA-HCV) of first.In some embodiments, the disease Disease can be with pathogenic bacteria (for example, mycobacterium tuberculosis (Mycobacterium tuberculosis)) or pathogenic protozoa (for example, plasmodium (Plasmodium)) is related.

In some embodiments, the disease is cancer.The non-limiting example of cancer includes colorectal cancer, bladder Cancer, oophoroma, carcinoma of testis, breast cancer, cutaneum carcinoma, lung cancer, cancer of pancreas, gastric cancer, the cancer of the esophagus, the cancer of the brain, leukaemia, liver cancer, uterus Endometrial carcinomas, prostate cancer and head and neck cancer.

In some embodiments, one or more primers have for HBV, HCV, FluA, FluB, CA16, EV71, enterovirus, EBOV, EBV, measles virus, salmonella, HPV and/or the nucleic acid sequence of HIV selection.

Target nucleic acid that multiple nucleic acids amplified reaction can be used in amplification of nucleic acid sample simultaneously generates amplified production.In addition, nucleic acid Amplification can be it is linear, exponential or combinations thereof.The non-limiting example of nucleic acid amplification method includes reverse transcription, primer Extension, ligase chain reaction, relies on the amplification of unwindase (for example, after contacting nucleic acid with unwindase at polymerase chain reaction Amplification), non-symmetric amplification, rolling circle amplification and multiple displacement amplification (MDA).In some embodiments, which can be with It is DNA.In the case where expanding to target RNA, DNA can be obtained by the reverse transcription of RNA, and can be used then DNA cloning generates the DNA product of amplification.The DNA product of amplification can indicate there is target RNA in the biological sample.To DNA In the case where being expanded, using any DNA cloning method known in the art.The non-limiting example of DNA cloning method Modification including polymerase chain reaction (PCR), PCR is (for example, PCR, ApoE gene, assembly PCR, asymmetric in real time PCR, digital pcr, emulsion-based PCR, transfer to PCR (dial-out PCR), rely on the PCR of unwindase, nest-type PRC, heat start PCR, Inverse PCR, methylation status of PTEN promoter, micro- primer PCR, multiplex PCR, nest-type PRC, overlapping-extension PCR, hot asymmetric interlaced PCR, fall progressively PCR) and ligase chain reaction (LCR).In some embodiments, DNA cloning is linear.In some implementations In scheme, DNA cloning is exponential.In some embodiments, DNA cloning is realized using nest-type PRC, this can improve inspection Survey the sensitivity of the DNA product of amplification.

At any aspect of the multiple aspect, nucleic acid amplification reaction as described herein can carry out in parallel.In general, parallel Amplified reaction is the amplified reaction occurred in same reaction chamber simultaneously.Parallel nucleic acid amplification reaction can carry out as follows: example It such as, in the reaction chamber include and keeping this anti-for reagent necessary to each nucleic acid amplification reaction to obtain reaction mixture Mixture is answered to be subjected to for condition necessary to each nucleic acid amplification reaction.For example, reverse transcription amplification and DNA cloning can be put down as follows It carries out capablely: is provided in the reaction chamber for reagent necessary to both amplification methods to obtain reaction mixture, and make this Reaction mixture is subjected to being adapted for the condition of the two amplified reactions.By RNA reverse transcription generate DNA can in parallel into Row is expanded to generate the DNA product of amplification.Any suitable number of nucleic acid amplification reaction can be carried out in parallel.In some implementations In scheme, carry out at least 1 in parallel, 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,30, 40,50,100,200,300,400,500,1000,10000 or more nucleic acid amplification reactions.

The mean temperature of the convective fluid can be below about 25 DEG C.For example, the mean temperature of the convective fluid can be below about 24℃、23℃、22℃、21℃、20℃、19℃、18℃、17℃、16℃、15℃、14℃、13℃、12℃、11℃、10℃、9 ℃、8℃、7℃、6℃、5℃、4℃、3℃、2℃、1℃、0℃。

The controller can control the rate of heat addition of reaction chamber by using the thermal energy provided by heating element, and make Flow through the flowing of reaction chamber with convective fluid to control the cooling rate of reaction chamber, thus to reaction mixture provide heating and/ Or it is cooling.When the rate of heat addition is greater than cooling rate, reaction mixture can be made to be subjected to heating.When the rate of heat addition is less than cooling rate When, reaction mixture can be made to be subjected to cool to.The rate of heat addition can be at least 0.1 DEG C/s.For example, the rate of heat addition can be at least 0.5 DEG C/ s、1℃/s、2℃/s、3℃/s、4℃/s、5℃/s、6℃/s、7℃/s、8℃/s、9℃/s、10℃/s、11℃/s、12℃/s、 13 DEG C/s, 14 DEG C/s, 15 DEG C/s, 16 DEG C/s, 17 DEG C/s, 18 DEG C/s, 19 DEG C/s, 20 DEG C/s, 21 DEG C/s etc..Cooling velocity can be At least 0.1 DEG C/s.For example, cooling rate can be at least 0.5 DEG C/s, 1 DEG C/s, 2 DEG C/s, 3 DEG C/s, 4 DEG C/s, 5 DEG C/s, 6 DEG C/s, 7℃/s、8℃/s、9℃/s、10℃/s、11℃/s、12℃/s、13℃/s、14℃/s、15℃/s、16℃/s、17℃/s、18 DEG C/s, 19 DEG C/s, 20 DEG C/s, 21 DEG C/s etc..

In some embodiments, in the case where switch is not opened, the controller passes through reaction mixture By one or more heating and cooling cycle.For example, the controller only can be such that reaction mixture is subjected to when switch is opened It is heated or cooled.In some embodiments, after switch is opened after postponing after a period of time, which makes to react Mixture is subjected to one or more heating and cooling cycle.

In some embodiments, before activation system, the method can further comprise being fixed to reaction vessel It is installed on the sample holder of heating element.When reaction vessel is fixed on sample holder, the reaction of the reaction vessel It room can be adjacent and in thermal communication with heating element.In some embodiments, sample holder is installed on heating element； This can ensure that reaction vessel or in which sample be fixed to sample holder in or sample holder on when with heating element heat Connection.

The method of present disclosure can further comprise providing excitation energy to reaction vessel, to induce instruction nucleic acid amplification The present or absent signal of the amplified production of reaction.Excitation energy can be by light source or other inductive energy sources, such as shine Diode, laser or other energy sources provide.Excitation energy can be guided to comprising sample in the reaction chamber, to generate Indicate that the generation of chemical or biological reactionss (for example, nucleic acid amplification reaction) and/or the transmitting signal of result are (for example, light on sample Learn signal, fluorescence signal and/or electrostatic signal).

In the method for present disclosure, it is possible to provide one or more sensors are (for example, 2,3,4,5,6,7 or more A sensor).For example, this method may include the first sensor provided with reaction vessel sensing communication, with detection instruction amplification The present or absent signal of product.First sensor can be optical sensor, inductance sensor, electrochemical sensor, Electrostatic transducer and/or impedance transducer.The signal can be optical signalling, fluorescence signal and/or electrostatic signal.

In some embodiments, the method includes providing the second sensor with reaction vessel sensing communication, with inspection Survey the temperature of the indoor one or more positions of reaction of reaction vessel.The reaction chamber of second sensor detectable response container The temperature of interior one or more positions.

The method may include providing in pedestal or shell to be operably coupled to first sensor and/or second sensor One or more (for example, 1,2,3 or more) display.The display can be configured to display instruction amplified production The temperature of the indoor one or more positions of the reaction of present or absent signal and/or reaction vessel.

Before activation system, the pedestal for being equipped with shell thereon can be placed on to refrigeration unit (for example, refrigerator, refrigeration Room etc.) in.

In the method for present disclosure, thermal energy can be provided to reaction chamber, until reaction mixture reaches the first temperature, and And convective fluid can be made to flow through reaction chamber, until reaction mixture reaches the second temperature lower than the first temperature.This method can be into One step includes reducing or terminating the flowing of convective fluid when reaction mixture reaches second temperature.

The reaction vessel may include sampling unit；The sampling unit may include collecting part and be in fluid communication with reaction chamber Sampler chamber.It can be by piercing through the opening of sealing sampler chamber with the collecting part for having collected nucleic acid samples on or in which Seal member places nucleic acid samples in the reaction vessel.

On the other hand, this disclosure provides the systems for nucleic acid amplification.The system may include comprising heating part The pedestal of part, and the occupied area of the pedestal may be less than or equal to about 9000mm².For example, the occupied area of the pedestal can be small In or equal to about 8000mm², about 7000mm², about 6000mm², about 5000mm², about 4500mm², about 4000mm², about 3500mm²、 About 3000mm², about 2500mm², about 2000mm², about 1500mm², about 1000mm², about 900mm², about 800mm², about 700mm², about 600mm², about 500mm², about 400mm², about 300mm², about 200mm², about 100mm²Deng.

The system may include the reaction vessel comprising reaction chamber.The reaction chamber can be adjacent with heating element and heat connects therewith It is logical.During use, heating element can provide thermal energy to the reaction mixture in reaction chamber；The reaction mixture may include nucleic acid Reagent necessary to sample and nucleic acid amplification.The occupied area of the reaction chamber may be less than or equal to about 2000mm².For example, this is anti- The occupied area of room is answered to may be less than or equal to about 1500mm², about 1000mm², about 900mm², about 800mm², about 700mm², about 600mm², about 500mm², about 400mm², about 300mm², about 200mm², about 100mm²Deng.In some embodiments, reaction chamber Cross section is less than the cross section of heating element.In some embodiments, the occupied area of reaction chamber is less than accounting for for heating element Ground area.

The system may include the shell being releasably attached on pedestal.The shell can be when being installed on pedestal at least Reaction vessel is partially or even wholly encapsulated, and the shell may include cooling-part, which is installed to base in shell When on seat with reaction vessel thermal communication.

The system may include the controller for being operably coupled to heating element.The controller can be programmed to make to react Mixture is subjected to one or more heating and cooling cycle, to promote the nucleic acid amplification reaction to reaction mixture.This can pass through (i) the cooling speed of reaction vessel is adjusted using cooling-part using the rate of heat addition of heating element adjusting reaction vessel and (ii) Rate carries out.

On the other hand, this disclosure provides the methods for nucleic acid amplification.This method may include activation system, should System includes the pedestal that (i) includes heating element, and the occupied area of the pedestal may be less than or equal to about 5000mm², (ii) includes The reaction vessel of reaction chamber, the reaction chamber can be adjacent and in thermal communication with heating element, and the reaction chamber may include reaction Mixture, the reaction mixture include reagent necessary to nucleic acid samples and nucleic acid amplification, and (iii) is releasably attached to Shell on pedestal, which can encapsulate reaction vessel, and the shell may include cooling-part, and the cooling-part is in the shell When being installed on pedestal with reaction vessel thermal communication.This method can further comprise that reaction mixture is made to be subjected to one or more add Hot and cold but recycles, and to promote the nucleic acid amplification reaction to reaction mixture, this can adjust reaction using heating element by (i) The rate of heat addition of container and (ii) are realized using the cooling rate of cooling-part adjusting reaction vessel.In some embodiments In, the occupied area of the pedestal is less than or equal to about 9000mm²、8000mm², about 7000mm², about 6000mm², about 5000mm², about 4500mm², about 4000mm², about 3500mm², about 3000mm², about 2500mm², about 2000mm², about 1500mm², about 1000mm², about 900mm², about 800mm², about 700mm², about 600mm², about 500mm², about 400mm², about 300mm², about 200mm², about 100mm² Deng.

The cooling-part can be at least about 0.1J/g*K, for example, at least about 0.2J/g*K, at least about 0.3J/ by thermal capacity G*K, at least about 0.4J/g*K, at least about 0.5J/g*K, at least about 1.0J/g*K, at least about 1.5J/g*K, at least about 2.0J/g* K, at least about 2.5J/g*K or bigger material are formed.In some embodiments, which is the solid comprising copper.

The nucleic acid samples can be biological sample, the biological sample such as obtained from subject.In the presence of for from subject Obtain a variety of methods of biological sample.It include: directly into cyclic system from the non-limiting example that subject obtains biological sample System (for example, via syringe or other needles are intravenous or intra-arterial entrance) collects the biological sample of secretion (for example, excrement, urine Liquid, sputum, saliva etc.), surgical operation (for example, biopsy), wiping (for example, cheek swab, oropharynx swab), liquid relief and breathing.This Outside, biological sample can be obtained from any region of anatomy of subject locating for desired biological sample.

The biological sample directly obtained from subject can usually refer in addition to collecting biological sample from subject for into one It walks except processing, the biological sample not being further processed after being obtained from subject.For example, can by following steps directly from Subject obtains blood: into the circulatory system of subject, taking out blood (for example, passing through needle) from subject, and make to take out Blood enter in storage.The storage may include reagent (for example, anti-coagulants), so that blood sample can be used for further dividing Analysis.In another example, swab can be used to obtain the epithelial cell on the oralpharyngeal surfaces of subject.Biology is being obtained from subject After sample, the swab containing biological sample can be made to contact with fluid (for example, buffer), to collect biology stream from the swab Body.Alternatively, can be pre-processed before biological sample is supplied to system to it.

In some embodiments, when providing biological sample in the reaction vessel, the biological sample is not yet purified.One In a little embodiments, when biological sample is supplied to reaction vessel, the nucleic acid of the biological sample is not yet extracted.It will for example, working as It, can not be from the RNA or DNA extracted in biological sample in the biological sample when biological sample is supplied to reaction vessel.In addition, In some embodiments, before biological sample is supplied to reaction vessel, it can not be concentrated and be present in the biological sample Target nucleic acid (for example, target RNA or target DNA).Alternatively, the dilution or dense of the sample can be carried out before sample is supplied to system Contracting.

The reaction mixture also may include the reagent of the target nucleic acid of detection amplification.The reagent, which can be, can generate and can examine The report agent of signal is surveyed, the existence or non-existence instruction amplified production of the detectable signal whether there is.Detectable signal it is strong Degree can be proportional to the amount of amplified production.For example, detectable signal can with the amount of amplified production directly linearly, index ratio Example, inverse proportion, or the proportionate relationship with any other type.In some cases, when amplified production with initial by expanding The different types of nucleic acid of target nucleic acid it is generated when, the intensity of detectable signal is proportional to the amount of the target nucleic acid initially expanded. For example, in the case where the DNA obtained by parallel reverse transcription and amplification from reverse transcription is to expand target RNA, for the two Reagent necessary to reacting may also include the report agent that can produce detectable signal, and the DNA of detectable signal instruction amplification is produced The presence of object and/or the target RNA of amplification.The intensity of detectable signal can be with the DNA product of amplification and/or the initial target of amplification The amount of RNA is proportional.Using for report agent also makes it possible real-time amplification method, including for the real-time of DNA cloning PCR。

Report agent can be connected by covalently or non-covalently key with the nucleic acid including amplified production.Non-covalent bond it is non- Limitative examples include ionic interaction, Van der Waals force, hydrophobic interaction, hydrogen bonding and combinations thereof.In some implementations In scheme, report agent reports that the variation of agent level can be used for detecting amplified production in conjunction with initial reactant.In some realities It applies in scheme, report agent is only detectable (or undetectable) when nucleic acid amplification carries out.In some embodiments, light Reactive dye (for example, fluorescent dye) is learned with agent of giving a report.Reagent for detecting the target nucleic acid of amplification can be nucleic acid and combine Dyestuff.The dyestuff can be DNA intercalative dye.The non-limiting example of dyestuff includes Eva green, and SYBR is green, and SYBR is blue, DAPI, Propidium iodide (propidium iodine), Hoeste, SYBR gold, ethidium bromide, acridine, proflavin, acridine orange, acridine yellow, Fluorescent coumarin (fluorcoumanin), ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, Homidium Bromide (homidium), mithramycin, more pyridine rutheniums (ruthenium polypyridyl), Anthramycin (anthramycin) are luxuriant and rich with fragrance Pyridine and acridine, ethidium bromide, propidium iodide, the own ingot of iodate (hexidium iodide), dihydro second ingot, second ingot homodimer- 1 and second ingot homodimer -2, single Azide second ingot (ethidium monoazide) and ACMA, Hoechst33258, Hoechst 33342, Hoechst 34580, DAPI, acridine orange, 7-AAD, actinomycin D, LDS751, hydroxystilbamidine (hydroxystilbamidine), SYTOX is blue, and SYTOX is green, SYTOX orange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO- 3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen, SYBR gold, SYBR green I, SYBR green II, SYBR DX, SYTO-40, -41, -42, -43, -44, - 45 (indigo plants), SYTO-13, -16, -24, -21, -23, -12, -11, -20, -22, -15, -14, -25 (green), SYTO-81, -80, - 82, -83, -84, -85 (orange), SYTO-64, -17, -59, -61, -62, -60, -63 (red), fluorescein, fluorescein isothiocynate (FITC), tetramethylrhodamine isothiocyanates (TRITC), rhodamine, tetramethylrhodamine, R-PE, Cy-2, Cy- 3, Cy-3.5, Cy-5, Cy5.5, Cy-7, texas Red (Texas Red), Phar-Red, allophycocyanin (APC), Sybr Green I, Sybr green II, Sybr gold, CellTracker is green, 7-AAD, second ingot homodimer I, second ingot homodimer II, second ingot Homodimer III, ethidium bromide, umbelliferone, eosin, green fluorescent protein, erythrosine, cumarin, methylcoumarin, pyrene, Peacock green, Stilbene, fluorescein, cascade blue (cascade blue), dichlorotriazine amine fluorescein, dansyl Cl, fluorescence lanthanide series metal Complex compound (such as those include the complex compound of europium and terbium), carboxyl tetrachlorofluorescein, 5 and/or 6- Fluoresceincarboxylic acid (FAM), 5- (or 6-) iodacetyl amido fluorescein, 5- { [2 (and 3) -5- (acetyl group sulfydryl)-succinyl group] amino } fluorescein (SAMSA- fluorescence Element), Sulforhodamine B sulfonic acid chloride, 5- and/or 6- carboxyrhodamine (ROX), 7- amino-methyl-cumarin, 7- amino -4- Methylcoumarin -3- acetic acid (AMCA), BODIPY fluorogen, 8- methoxyl group pyrene -1,3,6- trisulfonic acid trisodium salt, 3,6- bis- sulphurs Acid -4- amino-naphthalimide, phycobniliprotein, AlexaFluor 350,405,430,488,532,546,555,568, 594,610,633,635,647,660,680,700,750 and 790 dyestuff, DyLight 350,405,488,550,594,633, 650,680,755 and 800 dyestuffs or other fluorogens.

In some embodiments, report agent is sequence specific oligonucleotide probes, can be miscellaneous with amplified production There is optical activity when friendship.Since probe is in conjunction with the sequence-specific of amplified production, the use of oligonucleotide probe be can be improved The specificity and sensitivity of detection.Probe can be connected to any optical activity report agent (for example, dyestuff) as described herein, and It may also include the optically active quencher that can block associated dyestuff.The non-limiting reality of the probe for agent of giving a report can be used Example includes TaqMan probe, TaqMan Tamara probe, TaqMan MGB probe or Lion probe.

Report agent can be RNA oligonucleotide probe, may include the optical activity dyestuff (example being adjacently located on probe Such as, fluorescent dye) and quencher.Dyestuff and quencher in close proximity to can blocked dye optical activity.Probe can with wait expand The target sequence of increasing combines.Once the exonuclease activity of archaeal dna polymerase is broken probe during amplification, then quencher and dye Material separation, and free dyestuff regains its optical activity, which can then be detected.

Report agent can be molecular beacon (molecular beacon).Molecular beacon may include, for example, in hairpin conformation Oligonucleotides one end on the quencher that connects.It is optical activity dyestuff in the other end of the oligonucleotides, for example, fluorescence contaminates Material.In hairpin structure, optical activity dyestuff and quencher are tightly enough approached, and enable the light of quencher blocked dye Learn activity.However, once hybridize with amplified production, the oligonucleotides, that is, linear conformation and with the target sequence on the amplified production Hybridization.The linearisation of oligonucleotides leads to the separation of optical activity dyestuff and quencher, so that optical activity is restored, and It can be detected.Molecular beacon can improve the specific and sensitive of detection to the sequence-specific of the target sequence on amplified production Degree.

In some embodiments, report agent is radioactive species.The non-limiting example of radioactive species includes¹⁴C、¹²³I、¹²⁴I、¹²⁵I、¹³¹I、Tc99m、³⁵S or³H。

In some embodiments, report agent is the enzyme that can generate detectable signal.Detectable signal can pass through enzyme pair Its substrate, or the activity of specific substrates is generated in the case where enzyme has multiple substrates.It can be non-with the enzyme for agent of giving a report Limitative examples include alkaline phosphatase, horseradish peroxidase, I2- galactosidase, alkaline phosphatase, beta galactose glycosides Enzyme, acetylcholinesterase and luciferase.

Reagent necessary to reaction vessel nucleic acid expands can be provided to nucleic acid samples.In some embodiments, it tries Agent includes one or more of: (i) reverse transcriptase, (ii) archaeal dna polymerase and (iii) drawing for target nucleic acid (for example, RNA) Object group.It includes reverse transcriptase (for example, Sensiscript and Omniscript turns that some examples of reagent, which may include commercially available, Record enzyme), the premix of archaeal dna polymerase (for example, HotStarTaq archaeal dna polymerase) and dNTPs is (for example, Qiagen One- Step RT-PCR or One-Step RT-qPCR kit).

In some embodiments, sample is provided in sample container such as reaction vessel.It can be provided in reaction vessel Any component of sample, including target nucleic acid, the reagent of the target nucleic acid of detection amplification and/or for the reagent of nucleic acid amplification, to obtain Obtain reaction mixture.Any suitable reaction vessel can be used.In some embodiments, reaction vessel includes main body, the master Body may include inner surface, outer surface, open end and opposite closed end.In some embodiments, reaction vessel includes cap.It should Cap can be configured in its end in contact main body that is open, so that closing the open end of reaction vessel when being contacted.Some In the case of, the cap and reaction vessel are permanently connected, so that it remains attached to reaction vessel in the case where opening and closing configuration.One In a little situations, which is dismountable, so that cap is separated with reaction vessel when reaction vessel is opened.In some embodiments In, reaction vessel can be sealed, it is optionally gas-tight seal.The reaction vessel can be close (fluid-tight) of liquid.

Reaction vessel may include main body and cap；The cap can be removably attached in main body or permanently connected with main body. The main body may include the one or more walls to form reaction chamber and sampler chamber, and sampler chamber can be in fluid communication with reaction chamber.Some In embodiment, reaction chamber and sampler chamber are spatially at least partially separated from each other.For example, between reaction chamber and sampler chamber There may be sealings, at least partly prevent fluid from flowing between reaction chamber and sampler chamber.In some embodiments, instead Answer the sealing between room and sampler chamber that can be pierced (for example, puncture) to form opening, so that fluid can be flowed to from reaction chamber Sampler chamber, or vice versa.Sampler chamber may include opening, and seal member can be located at opening or the inside of sampler chamber, from And indoor content (for example, reaction mixture) and outdoor environment are separated.The cap may include being configured as obtaining simultaneously Retain the collecting part (for example, needle or recess) of sample.The size of collecting part and the opening of sampler chamber configure in the following manner: When opening cap closure, collecting part can be adapted in the opening, pierce through seal member and by be retained thereon or in which sample Product are discharged into sampling room (for example, be discharged into reaction mixture or include in other solution in sampler chamber).

Any size can be provided for reaction vessel.The reaction vessel, which can be configured to have, contains no more than about 100 μ L Reaction mixture volume.For example, the reaction vessel can be configured to have containing no more than about 0.01 μ L, 0.03 μ L, 0.05μL、0.07μL、0.1μL、0.5μL、1μL、2μL、3μL、4μL、5μL、6μL、7μL、8μL、9μL、10μL、15μL、20μL、 25 μ L, 30 μ L, 35 μ L, 40 μ L, 45 μ L, 50 μ L, 55 μ L, 60 μ L, 65 μ L, 70 μ L, 80 μ L, 90 μ L, 100 μ L, 150 μ L or 200 μ The volume of L reaction mixture.The reaction vessel can have be configured as containing be no more than fall into any two value described herein it Between in the range of volume volume.

The height of reaction vessel may be less than or equal to about 0.1mm, 0.2mm, 0.3mm, 0.4mm, 0.5mm, 1mm, 1.5mm, 2mm、2.5mm、3mm、4mm、5mm、6mm、7mm、8mm、9mm、10mm、15mm、20mm、25mm、30mm、35mm、40mm、 50mm, 60mm or 70mm.The height of reaction vessel can be fallen between any two value as described herein.

The cross-sectional area of reaction vessel can be at least about 1mm²、5mm²、10mm²、20mm²、30mm²、40mm²、50mm²、 60mm²、70mm²、80mm²、90mm²、100mm²、150mm²、200mm²、250mm²、300mm²、350mm²、400mm²、450mm²、 500mm²、550mm²、600mm²、650mm²、700mm²、750mm²、800mm²、850mm²、900mm²、950mm²、1000mm²、 1100mm²、1200mm²、1300mm²、1400mm²Or 1500mm².The cross-sectional area of reaction vessel can fall into as described herein any Between two values.

The thickness of the wall of reaction vessel can no more than about 0.01mm, about 0.05mm, about 0.1mm, about 0.15mm, about 0.2mm, About 0.25mm, about 0.3mm, about 0.35mm, about 0.4mm, about 0.5mm, about 0.6mm, about 0.7mm, about 0.8mm, about 0.9mm, about 1.0mm, about 2.0mm, about 3.0mm, about 4.0mm, about 5.0mm, about 6.0mm, about 7.0mm, about 8.0mm, about 9.0mm, about 10mm, About 15mm, about 20mm etc..

Reaction vessel can be made of any suitable material, and the non-limiting example of such material includes glass, metal, modeling Material and combinations thereof.Reaction vessel can by allow optical signalling in reaction vessel leave reaction vessel optical clear or Trnaslucent materials is made.Reaction vessel can be left the material of the optical signalling of reaction vessel and be made by may filter that or do not filter.? In some embodiments, reaction vessel is by allowing the transparent material inside detector observing response container to be formed.In some realities It applies in scheme, the inside of reaction vessel can be imaged.Alternatively, can detect and measure the optical signalling for leaving reaction vessel Amount.

Sample holder can receive reaction vessel.Reaction vessel can be removably supplied to sample holder. Reaction vessel can be inserted into sample holder or take out it from sample holder.Reaction vessel can be placed on the disclosure It is removed from support component in the support component of the system of content or by it.

It may be through after a period of time when nucleic acid amplification reaction occurs.The system may include can be anti-in nucleic acid amplification The detector of signal is detected during should occurring.The detector can be able to detect signal without removing sample from system.

At any aspect of the multiple aspect, detector can detect amplified production (for example, the DNA product of amplification, amplification RNA product).Any suitable detection method known in the art can be used in the detection of amplified production (DNA including amplification) To realize.The concrete type of used detection method may depend on, for example, specific amplified production, the reaction for amplification Whether the type of container, other reagents in reaction mixture, report agent include in the reactive mixture, and using reporting The concrete type of used report agent when agent.The non-limiting example of detection method includes optical detection, spectral detection, electrostatic Detection, Electrochemical Detection etc..Optical detecting method includes but is not limited to fluorimetry and UV-Visible absorption.Spectrum inspection Survey method includes but is not limited to mass spectrography, nuclear magnetic resonance (NMR) spectral method and infra-red sepectrometry.Electrostatic detection methods include but not It is limited to the technology based on gel, for example, gel electrophoresis.Electrochemical detection method includes but is not limited to the efficient liquid in amplified production To the Electrochemical Detection of amplified production after phase chromatographic isolation.

The detector can be able to detect the optical signalling from sample.The optical signalling can be from the glimmering of sample Light or other luminous signals.Optical signalling can be generated in the stimulation light for being supplied to the sample by sample response.Stimulate light can It is provided by light source.The light source can be in system.In some embodiments, light is absorbed by the sample, and electromagnetic radiation light.Hair The light penetrated can be in identical or different wavelength with the light of absorption.In some embodiments, optical signalling is from light source Light reflection.Alternatively, light can be irradiated across sample, and detector can be able to detect the light across sample.

It in some embodiments, will be about the presence of amplified production (for example, DNA product of amplification) and/or the letter of amount Breath output is to recipient.The information about amplified production can be exported there are many method.It, can be real when nucleic acid amplification is carrying out When such information is provided.In other cases, once nucleic acid amplification is completed, so that it may provide the information.In some embodiment party In case, some data can be provided in real time, and once expand completion, so that it may other data be presented.

In some embodiments, this type of information is visually (for example, over the display) or oral (for example, by medical treatment Practitioner) it is supplied to recipient.In some embodiments, this type of information provides in report.Report may include any number Desired element, the non-limiting example of the element includes about subject (for example, gender, age, race, healthy shape Condition etc.) initial data, processed data (for example, graphical display (for example, figure, chart, tables of data, data summarization), determine Cycle threshold, target polynucleotide initial amount calculated value) information, about whether there are the conclusion of target nucleic acid, diagnostic message, Prognosis information, disease information, etc., and combinations thereof.The report (for example, hard copy) that this report can be used as printing provides, or Electronic report offer is provided.In some embodiments (including the case where wherein providing electronic report), this type of information via Such as monitor or television, the screen operationally being connect with the unit for obtaining amplified production, tablet computer screen, mobile system The electronic consoles output such as system screen.The report of printing and electronic report can be stored respectively in file or database, so that it Can be accessed for being compared with later report.

Any suitable communication media (including for example, network connection, wireless connection, cloud or internet connection) can be used Report is sent to the recipient of Local or Remote position.In some embodiments, the system for report being sent to recipient, Such as personal computer, phone, plate or other systems.This report can check online, be stored in the system of recipient or beat It prints off and.In the presence of other suitable methods for transmitting report, the non-limiting example of this method includes mailing hard copy report It accuses so that reception and/or recipient are checked.

The information for including in report or report can be exported to various types of recipient.The non-limit of such recipient Property example processed includes therefrom obtaining the subject, doctor, the doctor for treating subject, the clinic for clinical test of biological sample Monitor, nurse, researcher, Laboratory Technician, the representative of drugmaker, health care companies, biotech company, doctor Institute, human assistance's tissue, health care management person, electronic system (for example, one of medical records of storage such as subject or Multiple stage computers and/or one or more computer server), Public Health Practice person, other medical workers and other medical treatment Facility.

Shell can partially or even wholly surround the component (for example, reaction vessel) of the system.Shell can laterally and/ Or the component (for example, reaction vessel) of the system is surrounded in top and bottom.Shell can be flexible or rigid structure.Detection Device may include in shell.In some embodiments, detector is located at the outside of the shell of the system.Detector, which can be, is The component part of system.Alternatively, detector can be dismountable or can separate with system.

Optical path can be provided between sample and detector.Signal from sample can reach detector via the optical path.Come It may pass through optical path from the optical signalling of sample and reach detector.The optical path may include the direct sight between sample and detector. In some embodiments, one or more optical elements can be provided between sample and detector.The example of optical element can Optics member including lens, reflecting mirror, prism, diffusing globe, condenser, optical filter, dichroscope, optical fiber or any other type Part.

Optical path can be disposed entirely in the shell of system.Optically optical path can be isolated with ambient enviroment for the shell.Example Such as, which can be lighttight, so that the interference optics that may interfere with optical path can be provided seldom in shell or not provided Signal.Light from hull outside may cannot be introduced into enclosure interior.The optics detected by detector can be advantageously reduced in this The inaccuracy of signal.

Optical path can be retained while nucleic acid amplification occurs.When nucleic acid amplification occurs via optical path, detector can be with The signal from sample can constantly or periodically be detected.

In some embodiments, use battery pack as the power supply to system power supply.The battery pack can be used for heating Component, fluid flow components and/or detector power supply.

The power supply can provide low-voltage for the system of present disclosure.In some embodiments, which is less than Or be equal to about 60V, 50V, 48V, 40V, 30V, 24V, 20V, 18V, 16V, 15V, 14V, 13V, 12V, 11V, 10V, 9V, 8V, 7V, 6V, 5V, 4V, 3V, 2V or 1V.In some embodiments, using less than or equal to about 50V, 40V, 30V, 24V, 20V, 18V, The low-voltage of 16V, 15V, 14V, 13V, 12V, 11V, 10V, 9V, 8V, 7V, 6V, 5V, 4V, 3V, 2V or 1V to heating element, stream Body mobile parts, excitation energy source and/or detector power supply.

In some embodiments, the method that low-power can be used for operating system or execute present disclosure.For example, can make With about 84W come operating system or the method for executing present disclosure.In some embodiments, low-power less than or equal to about 250W、200W、150W、130W、120W、110W、100W、90W、85W、84W、83W、80W、75W、70W、65W、60W、55W、 50W, 45W, 40W, 35W, 30W, 25W, 20W, 15W, 10W, 5W, 1W, 500mW, 100mW, 50mW, 10mW, 5mW or 1mW.For The amount of the power of operating system or execution method can be fallen between any two value as described herein.

In many aspects, amplified production is generated using primer extension reaction.Primer extension reaction is typically included in Reaction mixture was incubated into for the first duration and under the second temperature lower than the first temperature by reaction mixture at a temperature of one Incubate the circulation of the second duration.First temperature can be denaturation temperature.First duration can be the denaturation duration. Second temperature can be elongating temperature.Second duration, which can be, extends the duration.

Denaturation temperature can according to the specific nucleic acid sample for example analyzed, in nucleic acid samples target nucleic acid particular source (example Such as, virion, bacterium), used reagent and/or desired reaction condition and change.For example, denaturation temperature can be about 80 DEG C to about 110 DEG C.In some instances, denaturation temperature can be about 90 DEG C to about 100 DEG C.In some instances, denaturation temperature It can be about 90 DEG C to about 97 DEG C.In some instances, denaturation temperature can be about 92 DEG C to about 95 DEG C.In other other examples In, denaturation temperature can be at least or be equal to about 80 DEG C, 81 DEG C, 82 DEG C, 83 DEG C, 84 DEG C, 85 DEG C, 86 DEG C, 87 DEG C, 88 DEG C, 89 DEG C, 90 DEG C, 91 DEG C, 92 DEG C, 93 DEG C, 94 DEG C, 95 DEG C, 96 DEG C, 97 DEG C, 98 DEG C, 99 DEG C or 100 DEG C.

The denaturation duration can according to the specific nucleic acid sample for example analyzed, in nucleic acid samples target nucleic acid particular source (for example, virion, bacterium), used reagent and/or desired reaction condition and change.For example, when denaturation continues Between can less equal than about 300 seconds, 240 seconds, 180 seconds, 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds or 1 second.For example, denaturation the duration can be no more than 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds or 1 second.

Elongating temperature can according to the specific nucleic acid sample for example analyzed, in nucleic acid samples target nucleic acid particular source (example Such as, virion, bacterium), used reagent and/or desired reaction condition and change.For example, elongating temperature can be about 30 DEG C to about 80 DEG C.In some instances, elongating temperature can be about 35 DEG C to about 72 DEG C.In some instances, elongating temperature can It is about 45 DEG C to about 65 DEG C.In some instances, elongating temperature can be about 35 DEG C to about 65 DEG C.In some instances, extend temperature Degree can be about 40 DEG C to about 60 DEG C.In some instances, elongating temperature can be about 50 DEG C to about 60 DEG C.In other other examples In, elongating temperature can be to be no more than or be equal to about 35 DEG C, 36 DEG C, 37 DEG C, 38 DEG C, 39 DEG C, 40 DEG C, 41 DEG C, 42 DEG C, 43 DEG C, 44 ℃、45℃、46℃、47℃、48℃、49℃、50℃、51℃、52℃、53℃、54℃、55℃、56℃、57℃、58℃、59 ℃、60℃、61℃、62℃、63℃、64℃、65℃、66℃、67℃、68℃、69℃、70℃、71℃、72℃、73℃、74 DEG C, 75 DEG C, 76 DEG C, 77 DEG C, 78 DEG C, 79 DEG C or 80 DEG C.

Extend the duration can according to the specific nucleic acid sample for example analyzed, in nucleic acid samples target nucleic acid particular source (for example, virion, bacterium), used reagent and/or the desired reaction condition needed and change.Continue for example, extending Time can be less equal than about 300 seconds, 240 seconds, 180 seconds, 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 Second, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds or 1 second.For example, 120 seconds, 90 can be no more than by extending the duration Second, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds or 1 second.

At any aspect of the multiple aspect, the primer extension reaction of multiple circulations can be carried out.It is any appropriate to carry out The circulation of number.For example, the recurring number carried out can be followed with less than about 100,90,80,70,60,50,40,30,20,10 or 5 Ring.The recurring number of progress may depend on, for example, obtaining detectable amplified production (for example, there are targets in nucleic acid samples for instruction The DNA amplification product of the detectable amount of RNA) necessary to recurring number (for example, cycle threshold (Ct)).For example, can detect Amplified production (for example, instruction there are the DNA products of the detectable amount of target RNA in nucleic acid samples) necessary to recurring number can It is followed with less equal than about 100 circulations, 75 circulations, 70 circulations, 65 circulations, 60 circulations, 55 circulations, 50 Ring, 40 circulations, 35 circulations, 30 circulations, 25 circulations, 20 circulations, 15 circulations, 10 circulations or 5 circulations.This Outside, in some embodiments, it is being less than about 100,75,70,65,60,55,50,45,40,35,30,25,20,15,10 or 5 Cycle threshold (Ct) under obtain the amplifiable product of detectable amount (there are the detectable of target RNA for example, in instruction biological sample The DNA product of amount)

Amplification generate instruction exist expanded target nucleic acid detectable amount amplified production needed for the time can according to from The particular cycle of the middle nucleic acid samples for obtaining target nucleic acid, the specific nucleic acid amplified reaction and desired amplified reaction that will carry out It counts and changes.For example, the amplification of target nucleic acid can be 120 minutes or shorter, 90 minutes or shorter, 60 minutes or shorter, 50 minutes Or shorter, 45 minutes or shorter, 40 minutes or shorter, 35 minutes or shorter, 30 minutes or shorter, 25 minutes or shorter, 20 points Clock or it is shorter, 15 minutes or shorter, periods of 10 minutes or shorter or 5 minutes or shorter generate instruction there are target nucleic acid can The amplified production of detection limit.

In some embodiments, the amplification of target RNA is at 120 minutes or shorter, 90 minutes or shorter, 60 minutes or more It is short, 50 minutes or shorter, 45 minutes or shorter, 40 minutes or shorter, 35 minutes or shorter, 30 minutes or shorter, 25 minutes or It is shorter, 20 minutes or shorter, 15 minutes or shorter, periods of 10 minutes or shorter or 5 minutes or shorter generates instruction and exists The DNA amplification product of the detectable amount of target RNA.

In some embodiments, reaction mixture can be made to undergo the primer extension reaction of multiple series.The multiple system Single series in column may include the specific primer extension of multiple circulations, which is characterized in that, for example, as herein its Condition is specifically denaturalized and extended described in his place.In general, for example, for Denaturing and/or extension condition, Mei Gedan A series is different from other single series of at least one of the multiple series.For example, when continuing with regard to denaturation temperature, denaturation Between, elongating temperature and extend in the duration any one, two, three or all for four, single series can be different Another single series in the multiple series.In addition, multiple series may include any number of single series, for example, At least about or about 2,3,4,5,6,7,8,9,10 or more single serial.

For example, the primer extension reaction of multiple series may include First Series and second series.First Series, for example, can Including more than ten recycle primer extension reaction, wherein each circulation of First Series include (i) by reaction mixture about It incubates and is no more than 30 seconds at 92 DEG C to about 95 DEG C, subsequent (ii) incubates the reaction mixture at about 35 DEG C to about 65 DEG C not to be surpassed Spend about one minute.Second series, such as, it may include the primer extension reaction recycled more than ten, wherein each of second series Circulation includes that (i) incubated reaction mixture no more than 30 seconds at about 92 DEG C to about 95 DEG C, and subsequent (ii) mixes the reaction Object incubates no more than about 1 minute at about 40 DEG C to about 60 DEG C.In this specific example, first and second series they It is different in elongating temperature condition.However, the example is not intended to limit, because times of different extensions and Denaturing can be used Meaning combination.

In some embodiments, gradual time the time spent in (that is, system from temperature transition to an another temperature) And/or ramp rate can be vital factor in amplification.For example, amplification generates instruction, there are the detectable amounts of target nucleic acid Amplified production needed for temperature and time can be changed according to ramp rate and/or gradual time.Ramp rate can influence to use In the temperature and time of amplification.

Gradual time and/or ramp rate can be different between cycles.However in some embodiments, it recycles Between gradual time and/or ramp rate be identical.Gradual time and/or ramp rate can be based on the samples handled It is adjusted.

In some embodiments, such as according to the property of sample and reaction condition it determines gradual between different temperatures Time.Accurate temperature and incubative time can also be determined according to the property of sample and reaction condition.In some embodiments, Single sample is handled into (for example, being subjected to amplification condition) repeatedly using multiple thermal cycles, each thermal cycle is for example gradual It is different on time, temperature and/or incubative time.It then can the best or optimal thermal cycle for specific sample selection.This is provided For tested specific sample or the steady and efficient method of sample combination discretion thermal cycle.

In some embodiments, target nucleic acid is subjected to Denaturing before primer extension reaction starting.In multiple series Primer extension reaction in the case where, target nucleic acid can be subjected to Denaturing before executing the multiple series, or can be in institute It states and is subjected to Denaturing between multiple series.For example, target nucleic acid can be between the First Series and second series in multiple series It is subjected to Denaturing.The non-limiting example of such Denaturing includes denaturation temperature distribution (for example, one or more denaturation temperature Degree) and denaturant.

In some embodiments, nucleic acid samples are preheated before carrying out primer extension reaction.Preheat the temperature of nucleic acid samples It spends (for example, preheating temperature) and the duration (for example, pre- thermal endurance) can be according to the specific nucleic acid samples for example analyzed And change.In some instances, by nucleic acid samples preheat no more than about 60 minutes, 50 minutes, 40 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, 45 Second, 30 seconds, 20 seconds, 15 seconds, 10 seconds or 5 seconds.In some instances, by nucleic acid samples about 80 DEG C to about 110 DEG C at a temperature of Preheating.In some instances, by nucleic acid samples about 90 DEG C to about 100 DEG C at a temperature of preheat.In some instances, by nucleic acid Sample about 90 DEG C to about 97 DEG C at a temperature of preheat.In some instances, the temperature by nucleic acid samples at about 92 DEG C to about 95 DEG C The lower preheating of degree.In some instances, nucleic acid samples are being no more than or are being equal to about 80 DEG C, 81 DEG C, 82 DEG C, 83 DEG C, 84 DEG C, 85 DEG C, 86 DEG C, 87 DEG C, 88 DEG C, 89 DEG C, 90 DEG C, 91 DEG C, 92 DEG C, 93 DEG C, 94 DEG C, 95 DEG C, 96 DEG C, 97 DEG C, 98 DEG C, 99 DEG C or 100 It is preheated at a temperature of DEG C.

The time needed for any aspect of the multiple aspect, the element of Method Of Accomplishment can be according to the specific step of this method Suddenly change.For example, the time quantum of the element of Method Of Accomplishment can be about 5 minutes to about 120 minutes.In other instances, it completes The time quantum of the element of method can be about 5 minutes to about 60 minutes.In other instances, the time quantum of the element of Method Of Accomplishment can It is about 5 minutes to about 30 minutes.In other instances, the time quantum of the element of Method Of Accomplishment can shorter than or be equal to 120 minutes, it is short In or equal to 90 minutes, shorter than or equal to 75 minutes, shorter than or equal to 60 minutes, shorter than or equal to 45 minutes, shorter than or be equal to 40 minutes, shorter than or be equal to 35 minutes, shorter than or be equal to 30 minutes, shorter than or be equal to 25 minutes, shorter than or be equal to 20 minutes, it is short In or be equal to 15 minutes, shorter than or be equal to 10 minutes or shorter than or be equal to 5 minutes.

The system can accurately control the temperature of sample to obtain desired temperature curve.The system can incite somebody to action Temperature control about ± 5 DEG C, ± 4 DEG C, ± 3 DEG C, ± 2 DEG C, ± 1.2 DEG C, ± 1 DEG C, ± 0.7 DEG C, ± 0.5 DEG C, ± 0.3 DEG C, ± In 0.1 DEG C, ± 0.05 DEG C, ± 0.01 DEG C, ± 0.005 DEG C or ± 0.001 DEG C.The system can be advantageously able in low-voltage And/or the temperature control of high quality is provided when operating under low-power.The system can provide while with small size The temperature of high quality controls.

The signal for detecting the sample from experience amplification can carry out in the whole process.The detection can be in amplification procedure It is carried out continuously or is carried out in one or more points.Sample can emit optical signalling in the whole process.The optical signalling can be with The amount of the target nucleic acid expanded in sample is related.

It can be with real-time display data related with the signal detected.For example, can be shown while expanding with The process of nucleic acid amplification and/or the related data of the result of nucleic acid amplification.In some embodiments, one or more displays It can be built in system.For example, display may be provided on the shell and/or pedestal of system/in.Any description of display It is applicable to any kind of output module.Display may include the audio or tactile output of visual displays and information.It is aobvious Show that device can show information on screen or other kinds of user interface (UI).For example, screen can be built in system.

In some embodiments, data are shown in individually display equipment.The individual display equipment can be System communication.In some embodiments, communication can occur via connection.The connection can be rigid line connection or be wirelessly connected.System Direct communication can occur between system and display equipment.For example, can occur bluetooth, infrared communication, radio, WiFi or other directly Communication.In other cases, indirect communication can occur between system and display equipment.For example, network (such as local area network can be passed through (LAN)) or wide area network (WAN) such as internet) communicate.In some embodiments, using telecommunication network (for example, honeycomb Telephone network, data network).In some instances, it is communicated using 3G, 4G or 5G network.One can be used in the communications A or multiple intermediate systems, such as relay system (for example, tower) or router.Alternatively, not using intermediate system.

System can have input module, which receives amplification and be present in from the nucleic acid samples that subject obtains The user of target nucleic acid (for example, target RNA, target DNA) requests.Any suitable mould that can receive such user's request can be used Block.The input module may include, for example, the device comprising one or more processors.The input module is built-in in system. The input module can be integrated into the shell or pedestal of system and/or can enter from hull outside.

Alternatively, the input module can be individually or can separate with system.The input module can pass through connection (those of description connection elsewhere in such as present disclosure) and system communication.The non-limiting reality of system comprising processor Example include: desktop computer, laptop computer, tablet computer (for example, iPad、 Galaxy Tab), cellular phone, smart phone (for example, iPhone、The phone of support), it is personal Digital assistants (PDA), video game console, TV, music playing system (for example,IPod), video playing system System, pager and calculator.Processor can be with other lists of one or more controllers, computing unit and/or computer system Member is associated, or is implanted into firmware when needed.If implemented in software, routine (or program) can be stored in any meter In calculation machine readable memory such as RAM, ROM, flash memory, disk, laser disk or other storage mediums.Similarly, the software It can be transported to system via any of transfer approach, this method includes, for example, through communication channel, such as telephone wire, Yin Te Net, local Intranet, wireless connection etc., or via portable medium, such as computer readable disk, flash disc drives.Respectively A step can be used as various district's groups, operation, tool, module or technology to implement, the latter again can hardware, firmware, software or Implement in any combination thereof.When implementing within hardware, some or all of these district's groups, operation, technology etc. can be for example Customize integrated circuit (IC), specific integrated circuit (ASIC), Field Programmable Logic Array (FPGA), programmable logic array (PLA) etc. implement in.

In some embodiments, the input module, which is configured as receiving, carries out target nucleic acid amplification in amplification module User's request.The amplification module may include for completing one or more heating and cooling cycle and/or one or more primers The component of extension.The input module can be directly (for example, by input system, such as by the keyboard of user's operation, mouse Or touch screen) or indirectly (for example, by wired or wireless connection, including via internet) receive user's request.Input mould Block can provide the request of user to amplification module via output electronic device.In some embodiments, input module may include User interface (UI), such as graphic user interface (GUI), which is configured to that user is made to provide amplification target nucleic acid Request.GUI may include text, figure and/or audio frequency component.GUI can be provided on an electronic display, which includes The display of system containing computer processor.Such display may include resistance-type or capacitive touch screen.

In some embodiments, the output module includes as being with processor for described in input module System.The output module may include input equipment as described herein, and/or may include defeated for communicating with amplification module Enter electronic device.In some embodiments, which is electronic console, as on nucleic acid amplification system display or Individually display equipment.In some embodiments, which includes UI.In some embodiments, the output mould Block is operably coupled to the communication interface of computer network such as internet.In some embodiments, which makes Information is transmitted with any suitable telecommunication media (including computer network, wireless network, cloud, local Intranet or internet) To the recipient for being in Local or Remote position.In some embodiments, which can analyze connects from amplification module The data received.The output module can analyze information while expanding and occurring in real time.Post analysis one can be completed in amplification A little data.In some cases, which includes the report generation that can be generated report and report is sent to recipient Device, this report may include the amount as described elsewhere herein about amplified production and/or existing any information.Some In embodiment, which may be in response to automatically transmit information from the information that amplification module receives, such as with original Data or by include expand module in software carry out data analysis form.Alternatively, the output module can be used receiving Information is transmitted after the instruction of family.It can electronically be checked by the information that output module transmits or be printed by printer Come.

Input module, amplification one of module and output module or it is a variety of may include that in the same system, or can wrap Containing one or more identical components.For example, amplification module also may include that input module, output module or both all include.? In other examples, the system comprising processor both may include that can also reside in output module in input module.User can make It is requested to expand target nucleic acid with the system, and the system also can be used and be sent to the information about amplified production Recipient.In some cases, the system comprising processor is comprised in all three module, so that should include processor It includes the component expanded in module or any other module that system, which can also be used for control, provides instruction to the component, and receive The information returned from the instrument.

Figure 1A and Figure 1B provides the example of the system of present disclosure.System 100 may include the pedestal 107 of bottom.? In pedestal 107, there may be excitation energy source (for example, light sources) 106 guiding energy to including in reaction vessel 104 Sample, to generate from the signal for being present in any amplified production in reaction vessel 104.Pedestal 107 also may include power supply (example Such as, battery) and/or detector.The system also may include be located at pedestal 107 on and the heating part with 104 thermal communication of reaction vessel Part (for example, heat block) 105.It can be by including that the power supply in pedestal 107 is powered to heating element 105.The system can also wrap Containing the shell 102 being releasably attached on pedestal 107.Shell 102 can encapsulate reaction vessel when being installed on pedestal 107 104.Shell 102 may include at least one outlet 103, the outlet 103 allow convective fluid from the source of convective fluid (for example, by Such as fan of fluid flow components 101 generates) flow through at least one outlet 103 of the arrival of reaction vessel 104.Shell 102 also may include It can be used as the touch point 108 of switch.For example, touch point 108 and pedestal can be made when shell 102 to be installed on pedestal 107 107 contacts, to power on.Then, power supply will start heating element 105 (for example, passing through controller), be included in improving With the temperature of the reaction mixture in the reaction vessel 104 of 105 thermal communication of heating element.When the temperature in reaction vessel 104 reaches When to the first temperature levels, heating (for example, by deactivating heating element 105) can be stopped, and can star fluid flowing Component 101 is to generate the flowing of convective fluid, so that the convective fluid from its source stream crosses reaction vessel 104 and reaches at least one and Mouth 103, to reduce the temperature in reaction vessel 104.It, can when the temperature in reaction vessel 104 reaches second temperature level To deactivate fluid flow components 101, and a heating-cooling circulation can be completed.The circulation can be repeated as many times as desired.

Fig. 2 provides the example of the system of present disclosure.Show the external appearance of system 200.System 200 may include Pedestal 202 and the shell 205 being installed on pedestal 202.Shell 205 may include at least one outlet 203,204 and of switch button Display screen 201.When needed, switch button 204 and/or display screen 201 can include by pedestal 202.

Fig. 3 shows the internal structure of the system 300 of present disclosure.System 300 may include pedestal 305.Fluid flowing Component 301 may be mounted on pedestal 305, and heating element 303 can be installed in parallel pedestal with fluid flow components 301 On 305.Sample holder 302 can be kept by the support member 307 being mounted on pedestal 305 near heating element 303, so that Sample holder 302 and any reaction vessel fixed by the sample holder will be with 303 thermal communications of heating element.The system can The controller for being operably coupled to heating element 303 and/or fluid flow components 301 is further included (for example, printed circuit Plate) 304.Controller 304 can by comprising in systems and be operably coupled to controller 304 switch 306 start and/or stop With.

Fig. 4 provides the bottom view of the internal structure of the system 400 of present disclosure.The system may include front cover 401, preceding Lid 401 may include aperture array 402 to allow fluid (for example, air stream) to flow in and out the system.The system can be further Comprising power supply 403 (for example, battery), to power to heating element 404 and/or fluid flow components 405.

It may include the display 501 in the system of present disclosure that Fig. 5 A, which is illustrated,.Fig. 5 B to Fig. 5 D is provided can be The example at the interface shown on display 501.Fig. 5 B show can displays temperature (for example, comprising reaction in the reaction vessel The real time temperature of mixture) mode example.Fig. 5 C and Fig. 5 D, which are shown, can show that negative (Fig. 5 C) or positive (Fig. 5 D) expands Increase the example of the mode of result.

Fig. 6 provides the example of the reaction vessel 600 of present disclosure.Reaction vessel 600 may include cap 605 and main body 606.Main body 606 may include reaction chamber 604 and the sampler chamber 603 with the fluid communication of reaction chamber 604.Sampler chamber 603 may include out Mouth 602, and when cap closure, the collecting part 601 for including by cap 605 can be fitted in opening 602.

Fig. 7 shows the viewgraph of cross-section of the reaction vessel 700 of present disclosure.Reaction vessel 700 may include reaction chamber 705 and sampler chamber 703, the sealing element 704 for being partially or wholly separated reaction vessel 705 and sampling container 703 may be present.It can deposit Sealing sampler chamber opening and prevent wherein content to be externally exposed the seal member 702 of environment.Reaction vessel 700 may be used also Comprising the cap with collecting part 701, collecting part 701 can be fitted in the opening of sampler chamber and penetrate seal member 702.

Fig. 8 shows the bottom view of the reaction vessel of present disclosure.The reaction vessel may include main body and cap 801, and And the main body may include reaction chamber 803 and sampler chamber 802.The bottom side 804 of the reaction vessel can be thickness no more than about 0.3mm Layer.

Computer control system

This disclosure provides the computer control systems for the method for being programmed to realize present disclosure.Fig. 9 is shown Computer system 901, is programmed or is otherwise configured for sample treatment and analysis, such as droplet generate with And nucleic acid amplification and detection.The various aspects of the method and system of present disclosure are adjusted in computer system 901.

Computer system 901 includes central processing unit (CPU, also referred herein as " processor " and " computer disposal Device ") 905, it can be single or multiple core processor, or multiple processors for parallel processing.Computer system 901 is also Including memory or storage location 910 (for example, random access memory, read-only memory, flash memory), Electronic saving list First 915 (for example, hard disks), the communication interface 920 (for example, network adapter) for being communicated with one or more other systems with And peripheral equipment 925, such as cache memory, other memories, data storage and/or electronical display adapter.Storage Device 910, storage unit 915, interface 920 and peripheral equipment 925 pass through communication bus (solid line) and 905 phases of CPU such as mainboard Communication.Storage unit 915 can be data storage cell (or data repository) for storing data.Computer system 901 can Computer network (" network ") 930 is operably coupled to by means of communication interface 920.Network 930 can be internet, internet And/or extranet, or the Intranet and/or extranet that are communicated with internet.In some cases, network 930 be telecommunications and/ Or data network.Network 930 may include one or more computer servers, which can support distributed meter It calculates, such as cloud computing.In some cases, network 930 can realize peer-to-peer network by means of computer system 901, this can make with The equipment that computer system 901 couples can play the role of client or server.

A series of machine readable instructions can be performed in CPU 905, which may be embodied in program or software. The instruction is storable in the storage locations such as memory 910.The instruction can be directed to CPU 905, and CPU 905 is then programmable Or otherwise configure method of the CPU 905 to realize present disclosure.Example by the operation executed of CPU 905 may include Extraction, decoding, execution and write-back.

CPU 905 can be a part of the circuits such as integrated circuit.One or more other assemblies of system 901 can Including in circuit.In some cases, which is specific integrated circuit (ASIC).

Storage unit 915 can storage file, such as program of driver, library and preservation.Storage unit 915 can store use User data, for example, user preference and user program.In some cases, computer system 901 may include one or more additional Data storage cell, the additional-data storage unit are located at outside computer system 901, such as positioned at by Intranet or because of spy On the remote server that net and computer system 901 communicate.

Computer system 901 can be communicated by network 930 and one or more remote computer systems.For example, calculating Machine system 901 can be communicated with the remote computer system of user.The example of remote computer system includes personal computer (example Such as, portable PC), plate or plate PC (for example, iPad、Galaxy Tab), phone, Smart phone (for example,IPhone, support Android equipment,) or individual digital help Reason.User can access computer system 901 via network 930.

Method can be realized by way of machine (for example, computer processor) executable code as described herein, The machine executable code is stored on the Electronic saving position of computer system 901, such as in memory 910 or Electronic saving On unit 915.Machine executable code or machine readable code can provide in the form of software.In use, the generation Code can be executed by processor 905.In some cases, the code can be retrieved from storage unit 915 and is stored in memory In case being obtained by processor 905 on 910.In some cases, electronic memory module 915 can be excluded, and machine can be performed and refer to Order is stored on memory 910.

The code can by precompile and be configured to have be adapted for carrying out the code processor machine together with make With, or can be compiled during operation.The code can be provided with programming language, and programming language may be selected so that the code can It is executed in a manner of precompile or Just-In-Time (as-compiled).

On the other hand, this disclosure provides the non-transitory computer readable mediums comprising machine executable code Matter, the machine executable code are realized when being executed by one or more computer processors for carrying out chemistry to nucleic acid samples Or the method for biological respinse.This method may include activation system, which includes the pedestal that (i) includes heating element, wherein should The occupied area of pedestal is less than or equal to about 5000mm², (ii) includes the reaction vessel of reaction chamber, the wherein reaction chamber and heating Component is adjacent and in thermal communication.The reaction chamber may include reaction mixture, which includes nucleic acid samples and nucleic acid Reagent necessary to expanding.The system can further comprise the shell that (iii) is releasably attached on pedestal.The shell can wrap Reaction vessel is sealed, and the shell may include at least one outlet, which allows source stream mistake of the convective fluid from convective fluid Reaction chamber reaches at least one described outlet.The system may include that (iv) makes convective fluid cross reaction chamber from the source stream of convective fluid Reach the fluid flow components of at least one outlet.This method can further comprise guiding heating element to anti-by (i) Room is answered to provide thermal energy, and (ii) guidance fluid flow components so that convective fluid crosses reaction chamber arrival from the source stream of convective fluid At least one described outlet, makes reaction mixture be subjected to one or more heating and cooling cycle, to promote to reaction mixture Nucleic acid amplification reaction.

On the other hand, this disclosure provides the non-transitory computer readable mediums comprising machine executable code Matter, the machine executable code realize the side for nucleic acid samples amplification when being executed by one or more computer processors Method.This method may include activation system, which includes the pedestal that (i) includes heating element, and the occupied area of the pedestal can be small In or equal to about 5000mm², (ii) includes the reaction vessel of reaction chamber, which can be adjacent with heating element and heat connects therewith It is logical, and the reaction chamber may include reaction mixture, and which includes examination necessary to nucleic acid samples and nucleic acid amplification Agent, and (iii) are releasably attached to the shell on pedestal, which can encapsulate reaction vessel, and the shell may include Cooling-part, the cooling-part when the shell is installed on pedestal with reaction vessel thermal communication.This method can further comprise Reaction mixture is set to be subjected to one or more heating and cooling cycle, to promote the nucleic acid amplification reaction to the reaction mixture, This can adjust the rate of heat addition of reaction vessel using heating element by (i) and (ii) uses cooling-part adjusting reaction vessel Cooling rate realize.

The various aspects of system and method provided herein, such as computer system 901, may be embodied in programming.This technology Many aspects be considered " product " or " product ", generally on a type of machine readable media carry or Machine (or processor) executable code of embodiment and/or the form of associated data.Machine executable code is storable in such as On the electronic memory modules such as memory (for example, read-only memory, random access memory, flash memory) or hard disk.It " deposits Storage " type medium may include any or all of tangible memory of computer, processor etc. or its relating module, such as various half Conductor memory, tape drive, disc driver etc. can provide non-transitory storage at any time for software programming. The all or part of the software can be communicated sometimes by internet or various other telecommunication networks.Such communication, example Such as, it can enable software to be loaded into another computer or processor from a computer or processor, for example, from management service Device or host are loaded into the computer platform of application server.Therefore, the another type of medium packet of software elements can be carried Include light wave, electric wave and electromagnetic wave, such as across between local device physical interface, pass through wired and optics land line network and logical It crosses various airlinks and uses.The physical component of this kind of wave, wired or wireless link, optical link etc. are carried, It is considered the medium of carrying software.As used herein, except tangible " storage " medium of non-transitory is not limited to, otherwise The terms such as computer or machine " readable medium ", which refer to, participates in providing instruction to processor for any medium of execution.

Therefore, machine readable media, such as computer-executable code can take many forms, including but not limited to: having Shape storage medium, carrier media or physical transmission medium.Non-volatile memory medium includes such as CD or disk, such as any Any storage equipment in computer etc., such as can be used for realizing database etc. as shown in the drawings.Volatile storage medium Including dynamic memory, the main memory of such as such computer platform.Tangible transmission media includes coaxial cable；Copper wire and Optical fiber, including conducting wire, the conducting wire include the bus in computer system.Carrier wave transmission media can take electric signal or electromagnetic signal Or the form of sound wave or light wave, such as in radio frequency (RF) and infrared (IR) data communication process generate those of electric signal or Electromagnetic signal or sound wave or light wave.Therefore, the common form of computer-readable medium includes, for example: floppy disk, flexible disk (flexible disk), hard disk, tape, any other magnetic medium, CD-ROM, DVD or DVD-ROM, any other optics are situated between Matter, card punch paper tape, any other physical storage medium, RAM, ROM, PROM and EPROM, FLASH- with hole patterns The cable or link of carrier wave as EPROM, any other storage chip or cassette, transmission data or the carrier wave of instruction, transmission (link) or computer can therefrom read any other media of programming code and/or data.These computer-readable medium shapes Many in formula, which may participate in, is carried to processor for one or more sequences of one or more instruction for executing.

Computer system 901 may include electronic console 935 or communicate that electronic console 935 includes at any time Between the user interface (UI) 940 of such as nucleic acid sequence information is provided.The example of UI includes but is not limited to graphic user interface (GUI) With network-based user interface.

The method and system of present disclosure can be realized by one or more algorithms.Algorithm can be by central processing list Member 905 passes through software realization when executing.Algorithm can such as regulator control system or realization method provided herein.

The devices, systems, and methods of present disclosure can be with other devices, system or method, such as PCT/CN14/ Those devices, system or method described in 094914 and PCT/CN14/078022 are combined, and each of these applications is all It is incorporated herein by reference in their entirety.

Although the preferred embodiments of the invention have been shown and described herein, those skilled in the art are come It says it is evident that these embodiments only provide in an illustrative manner.It is not intended to the specific example by providing in specification To limit the present invention.Although describing the present invention by reference to aforementioned specification, the description of this paper embodiment and diagram are not It should be explained with restrictive meaning.Those skilled in the art are without departing from the present invention now it will be appreciated that a variety of changes Change, change and replaces.In addition, it should be understood that all aspects of the invention be not limited to it is set forth herein it is specific describe, configuration or Relative scale, but depend on a variety of conditions and variable.It should be appreciated that the various of embodiment of the present invention described herein are replaced It can be used for implementing the present invention for scheme.It is therefore contemplated that the present invention should also cover any such substitution, modification, variation Or equivalent.Following following claims is intended to limit the scope of the invention, thus the method being included in the scope of these claims and Structure and its equivalent.

Claims

1. a kind of system for nucleic acid amplification comprising:

Pedestal comprising heating element, wherein the occupied area of the pedestal is less than or equal to about 5000mm²；

Reaction vessel comprising reaction chamber, wherein the reaction chamber is adjacent with the heating element and connects with the heating element heat It is logical, wherein during use, reaction mixture of the heating element into the reaction chamber provides thermal energy, the reaction mixing Object includes reagent necessary to nucleic acid samples and nucleic acid amplification；

The shell being releasably attached on the pedestal, wherein the shell encapsulated when being installed on the pedestal it is described anti- Container is answered, and wherein the shell includes at least one outlet, the outlet allows convective fluid from the convective fluid The excessively described reaction chamber of source stream reaches at least one described outlet；

Fluid flow components make described in the convective fluid reaches from the excessively described reaction chamber of the source stream of the convective fluid At least one outlet；And

It is operably coupled to the controller of the heating element and the fluid flow components, wherein the controller is programmed To guide the heating element to provide thermal energy to the reaction chamber by (i), and (ii) guidance fluid flow components so that The excessively described reaction chamber of the source stream for obtaining the convective fluid from the convective fluid reaches at least one described outlet, makes described Reaction mixture is subjected to one or more heating and cooling cycle, to promote the nucleic acid amplification reaction to the reaction mixture.

2. system according to claim 1, wherein the occupied area of the reaction chamber is less than or equal to about 1000mm²。

3. system according to claim 2, wherein the occupied area is less than or equal to about 500mm²。

4. system according to claim 3, wherein the occupied area is less than or equal to about 300mm²。

5. system according to claim 1, wherein the occupied area of the pedestal is less than or equal to about 2000mm²。

6. system according to claim 1, wherein the occupied area for the shell being installed on the pedestal be less than or Equal to about 5000mm²。

7. system according to claim 1, wherein the cross section of the reaction chamber is less than the cross section of the heating element.

8. system according to claim 1, wherein the ratio between the surface area of the reaction chamber and volume are at least 100mm^-1。

9. system according to claim 1 further comprises the power supply powered to the heating element.

10. system according to claim 9, wherein the power supply is powered to the fluid flow components.

11. system according to claim 9 further comprises being operably coupled to institute in the pedestal or the shell The switch of power supply is stated, wherein the switch-mode regulation is from the power supply to the power supply of the heating element.

12. system according to claim 11, wherein when the shell is installed on the pedestal, the switch guidance From the power supply to the power supply of the heating element.

13. system according to claim 1, wherein the shell includes room, the room is when being installed on the pedestal Encapsulate the reaction vessel.

14. system according to claim 1 further comprises the sample holder being installed on the heating element, institute Sample holder is stated to receive and fix the reaction vessel.

15. system according to claim 14, wherein when the reaction vessel is fixed on the sample holder, institute It is adjacent and in thermal communication with the heating element to state reaction chamber.

16. system according to claim 1, wherein the pedestal, which further includes, is operably coupled to the reaction appearance The excitation energy source of device indicates the core wherein the excitation energy source provides excitation energy to the reaction vessel with induction The present or absent signal of the amplified production of sour amplified reaction.

17. system according to claim 16 further comprises the first sensor with the reaction vessel sensing communication, Wherein the first sensor detection indicates the present or absent signal of the amplified production.

18. system according to claim 17, wherein the first sensor is optical sensor, and the wherein letter It number is optical signalling.

19. according to claim 1 or system described in 17, further comprising the second sensing with the reaction vessel sensing communication Device, wherein the second sensor detects the temperature of the indoor one or more positions of the reaction of the reaction vessel.

20. system according to claim 17 further comprises being operably coupled in the pedestal or the shell The display of the first sensor, wherein the display be configured as display indicate the amplified production the presence or The signal being not present.

21. system according to claim 19 further comprises being operably coupled in the pedestal or the shell The display of the first sensor and/or the second sensor, wherein the display is configured as:

1) display indicates the present or absent signal of the amplified production, and/or

2) temperature at the indoor one or more of positions of the reaction of the reaction vessel is shown.

22. system according to claim 16, wherein the excitation energy source is light source.

23. system according to claim 1, wherein the convective fluid is convection gas.

24. system according to claim 23, wherein the convection gas is air.

25. system according to claim 1, wherein the fluid flow components are fans.

26. system according to claim 1, wherein the fluid flow components are installed to the shell or the pedestal On.

27. system according to claim 1, wherein the source of the convective fluid is refrigeration unit.

28. system according to claim 1, wherein the heating element includes infrared heating unit.

29. system according to claim 1, wherein the heating element includes peltier heating unit.

30. system according to claim 1, wherein the heating element includes heating unit containing aluminium.

31. system according to claim 1, wherein the heating element includes electrical resistor heating element.

32. system according to claim 1, wherein the controller is programmed to guide the heating element to described anti- It answers room to provide thermal energy until the reaction mixture reaches the first temperature, and guides the fluid flow components so that described right Stream fluid flows through the reaction chamber until the reaction mixture reaches the second temperature lower than first temperature.

33. system according to claim 32, wherein the controller is programmed to reach institute when the reaction mixture The fluid flow components are guided when stating second temperature to reduce or terminate the flowing of the convective fluid.

34. system according to claim 1, wherein the controller is included in the pedestal.

35. system according to claim 1, wherein the reaction mixture includes one or more primers and polymerase.

36. system according to claim 35, wherein the reaction mixture includes buffer.

37. system according to claim 35, wherein the reaction mixture includes the active of the adjusting polymerase Cation.

38. the system according to claim 37, wherein the cation includes Mg²⁺Or Mn²⁺。

39. system according to claim 35, wherein one or more primers have for HBV, HCV, FluA, FluB, CA16, EV71, enterovirus, EBOV, EBV, measles virus, salmonella, HPV and/or the nucleic acid sequence of HIV selection.

40. system according to claim 1, wherein the nucleic acid amplification reaction is polymerase chain reaction (PCR).

41. system according to claim 1, wherein the mean temperature of the convective fluid is below about 15 DEG C.

42. system according to claim 41, wherein the mean temperature of the convective fluid is below about 10 DEG C.

43. system according to claim 1, wherein the controller as the heating element by using described in being provided Thermal energy is come to control the rate of heat addition of the reaction chamber, and using the flowing that the convective fluid flows through the reaction chamber The cooling rate of the reaction chamber is controlled, to heat and/or cool to reaction mixture offer.

44. system according to claim 43, wherein when the rate of heat addition is greater than the cooling rate, the reaction Mixture is subjected to heating.

45. system according to claim 43, wherein when the rate of heat addition is less than the cooling rate, the reaction Mixture is subjected to cool to.

46. system according to claim 43, wherein the rate of heat addition is at least 5 DEG C/s.

47. system according to claim 43, wherein the cooling rate is at least 5 DEG C/s.

48. system according to claim 1, wherein the controller is not in the case where the switch is not opened The reaction mixture is set to be subjected to one or more of heating and cooling cycle.

49. system according to claim 1, wherein after the switch is opened after postponing after a period of time, institute Stating controller makes the reaction mixture be subjected to one or more of heating and cooling cycle.

50. system according to claim 1, wherein the reaction vessel further includes sampling unit, and wherein institute Stating sampling unit includes the sampler chamber being in fluid communication with the reaction chamber.

51. system according to claim 50, wherein the sampling unit further includes the collection for collecting nucleic acid samples Component.

52. system according to claim 51, wherein the sampling unit, which further includes, seals opening for the sampler chamber The seal member of mouth.

53. system according to claim 52, wherein during use, the collecting part pierce through the seal member with The nucleic acid samples are discharged into the sampler chamber.

54. system according to claim 1, wherein the thickness of the side of the reaction chamber adjacent with the heating element Less than about 1mm.

55. a kind of method for nucleic acid amplification comprising:

(a) system is activated, the system comprises the pedestals that (i) includes heating element, wherein the occupied area of the pedestal is small In or equal to about 5000mm², (ii) includes the reaction vessel of reaction chamber, wherein the reaction chamber is adjacent with the heating element simultaneously With the heating element thermal communication, wherein the reaction chamber includes reaction mixture, the reaction mixture includes nucleic acid samples With reagent necessary to nucleic acid amplification, (iii) is releasably attached to the shell on the pedestal, wherein shell encapsulated institute Reaction vessel is stated, wherein the shell includes at least one outlet, the outlet allows convective fluid from the convective fluid The source stream reaction chamber excessively reaches at least one described outlet, and (iv) fluid flow components, make the convective fluid from The excessively described reaction chamber of the source stream of the convective fluid reaches at least one described outlet；And

(b) heating element is guided to provide thermal energy to the reaction chamber by (i), and (ii) guides the fluid flow components So that the excessively described reaction chamber of the source stream of the convective fluid from the convective fluid reaches at least one described outlet, make The reaction mixture is subjected to one or more heating and cooling cycle, anti-to the nucleic acid amplification of the reaction mixture to promote It answers.

56. method according to claim 55, wherein the occupied area of the reaction chamber is less than or equal to about 1000mm²。

57. method according to claim 55, wherein the occupied area of the pedestal is less than or equal to about 2000mm²。

58. method according to claim 55, wherein the occupied area for the shell being installed on the pedestal is less than Or it is equal to about 5000mm²。

59. method according to claim 55, wherein the cross section of the reaction chamber is less than the transversal of the heating element Face.

60. method according to claim 55, wherein the ratio between the surface area of the reaction chamber and volume are at least 100mm^-1。

61. method according to claim 55, wherein the activation in (a) be by with power supply to the heating element Power supply and carry out.

62. method according to claim 61, wherein the power supply is powered to the fluid flow components.

63. method according to claim 61, wherein being operably connected by opening in the pedestal or the shell The electric power is supplied to the switch of the power supply.

64. method according to claim 63, wherein by the shell is installed on the pedestal open it is described Switch.

65. method according to claim 55, wherein the shell includes room, the room is when being installed on the pedestal Encapsulate the reaction vessel.

66. method according to claim 55 further comprises that the reaction vessel is fixed to installation before (a) On sample holder on to the heating element.

67. method according to claim 66, wherein when the reaction vessel is fixed on the sample holder, institute It is adjacent and in thermal communication with the heating element to state reaction chamber.

68. method according to claim 55 is further comprised providing excitation energy to the reaction vessel, is referred to induction Show the present or absent signal of the amplified production of the nucleic acid amplification reaction.

69. method according to claim 68 further comprises providing to pass with the first of the reaction vessel sensing communication Sensor, to detect the present or absent signal for indicating the amplified production.

70. method according to claim 69, wherein the first sensor is optical sensor, and the wherein letter It number is optical signalling.

71. method according to claim 69 further comprises providing to pass with the second of the reaction vessel sensing communication Sensor, to detect the temperature of the indoor one or more positions of the reaction of the reaction vessel.

It further comprise that operationally coupling is provided in the pedestal or the shell 72. method according to claim 71 It is bonded to the display of the first sensor and/or the second sensor, wherein the display is configured as: (1) showing Indicate that the present or absent signal of the amplified production, and/or (2) show the described anti-of the reaction vessel Answer the temperature at indoor one or more of positions.

It further comprise that operationally coupling is provided in the pedestal or the shell 73. method according to claim 69 It is bonded to the display of the first sensor, wherein the display, which is configured as display, indicates that the described of the amplified production is deposited Or the signal that is not present.

74. method according to claim 68, wherein the excitation energy source is light source.

75. method according to claim 55, wherein the convective fluid is convection gas.

76. the method according to claim 75, wherein the convection gas is air.

77. method according to claim 55, wherein the fluid flow components are fans.

78. method according to claim 55, wherein the fluid flow components are installed to the shell or the base On seat.

79. method according to claim 55, wherein the source of the convective fluid is refrigeration unit.

80. method according to claim 55 further comprises that will be equipped with the institute of the shell thereon before (a) Pedestal is stated to be placed in refrigeration unit.

81. method according to claim 55, wherein the heating element includes infrared heating unit.

82. method according to claim 55, wherein the heating element includes peltier heating unit.

83. method according to claim 55, wherein the heating element includes heating unit containing aluminium.

84. method according to claim 55, wherein the heating element includes electrical resistor heating element.

85. method according to claim 55, wherein thermal energy is provided to the reaction chamber, until the reaction mixture reaches To the first temperature, and the convective fluid is wherein made to flow through the reaction chamber, until the reaction mixture reaches lower than institute State the second temperature of the first temperature.

86. the method according to claim 85, further comprise when the reaction mixture reaches the second temperature, Reduce or terminate the flowing of the convective fluid.

87. method according to claim 55, wherein the reaction mixture includes one or more primers and polymerase.

88. the method according to claim 87, wherein the reaction mixture includes buffer.

89. the method according to claim 87, wherein the reaction mixture includes the active of the adjusting polymerase Cation.

90. the method according to claim 89, wherein the cation includes Mg²⁺Or Mn²⁺。

91. the method according to claim 87, wherein one or more primers have for HBV, HCV, FluA, FluB, CA16, EV71, enterovirus, EBOV, EBV, measles virus, salmonella, HPV and/or the nucleic acid sequence of HIV selection.

92. method according to claim 55, wherein the nucleic acid amplification reaction is polymerase chain reaction (PCR).

93. method according to claim 55, further comprise using the thermal energy provided by the heating element Lai The rate of heat addition of the reaction chamber is controlled, and flows through the flowing of the reaction chamber using the convective fluid to control State the cooling rate of reaction chamber.

94. the method according to claim 93, wherein the rate of heat addition is greater than the cooling rate, to make described anti- Mixture is answered to be subjected to heating.

95. the method according to claim 93, wherein the rate of heat addition is less than the cooling rate, to make described anti- Mixture is answered to be subjected to cool to.

96. the method according to claim 93, wherein the rate of heat addition is at least 5 DEG C/s.

97. the method according to claim 93, wherein the cooling rate is at least 5 DEG C/s.

98. method according to claim 55, wherein in the case where the switch is not opened, not making in (b) The reaction mixture is subjected to one or more of heating and cooling cycle.

99. method according to claim 55, wherein being prolonged after a period of time after the switch is opened in (b) After late, the reaction mixture is made to be subjected to one or more of heating and cooling cycle.

100. method according to claim 55 further comprises deactivating the system after (b).

101. method according to claim 55 further comprises being placed on the nucleic acid samples described before (a) In reaction vessel.

102. method described in 01 according to claim 1, wherein the reaction vessel further includes sampling unit, and wherein The sampling unit include collecting part and with the reaction chamber be in fluid communication sampler chamber, wherein by on it or its In have collected the collecting parts of the nucleic acid samples and pierce through the seal member for sealing the opening of the sampler chamber, by the core Sour sample is placed in the reaction vessel.

103. a kind of system for nucleic acid amplification comprising:

Reaction vessel comprising reaction chamber, wherein the reaction chamber is adjacent and in thermal communication with the heating element, wherein During use, reaction mixture of the heating element into the reaction chamber provides thermal energy, and the reaction mixture includes core Reagent necessary to sour sample and nucleic acid amplification；

The shell being releasably attached on the pedestal, wherein the shell encapsulated when being installed on the pedestal it is described anti- Container is answered, and wherein the shell includes cooling-part, the cooling-part is when the shell is installed on the pedestal With the reaction vessel thermal communication；

It is operably coupled to the controller of the heating element, wherein the controller is programmed to by described in (i) use Heating element adjusts the rate of heat addition of the reaction vessel, and (ii) adjusts the cold of the reaction vessel using the cooling-part But rate makes the reaction mixture be subjected to one or more heating and cooling cycle, to promote to the reaction mixture Nucleic acid amplification reaction.

104. system described in 03 according to claim 1, wherein the cooling-part is at least about 0.2J/g*K's by thermal capacity Material is formed.

105. system described in 04 according to claim 1, wherein the cooling-part is at least about 0.3J/g*K's by thermal capacity Material is formed.

106. system described in 05 according to claim 1, wherein the cooling-part is at least about 0.4J/g*K's by thermal capacity Material is formed.

107. system described in 06 according to claim 1, wherein the cooling-part is at least about 0.5J/g*K's by thermal capacity Material is formed.

108. system described in 07 according to claim 1, wherein the cooling-part is at least about 1.0J/g*K's by thermal capacity Material is formed.

109. system described in 03 according to claim 1, wherein the cooling-part is the solid comprising copper.

110. a kind of method for nucleic acid amplification comprising:

(a) system is activated, the system comprises the pedestals that (i) includes heating element, wherein the occupied area of the pedestal is small In or equal to about 5000mm², (ii) includes the reaction vessel of reaction chamber, wherein the reaction chamber is adjacent with the heating element simultaneously In thermal communication, wherein the reaction chamber includes reaction mixture, the reaction mixture includes nucleic acid samples and nucleic acid amplification Necessary reagent, and (iii) are releasably attached to the shell on the pedestal, wherein reacting described in the shell encapsulated Container, wherein the shell includes cooling-part, the cooling-part when the shell is installed on the pedestal with it is described Reaction vessel thermal communication；And

(b) rate of heat addition of the reaction vessel is adjusted using the heating element by (i), and (ii) uses the cooling end Part adjusts the cooling rate of the reaction vessel, and the reaction mixture is made to be subjected to one or more heating and cooling cycle, with Promote the nucleic acid amplification reaction to the reaction mixture.

111. method described in 10 according to claim 1, wherein the occupied area of the reaction chamber is less than or equal to about 1000mm²。

112. method described in 10 according to claim 1, wherein the occupied area of the pedestal is less than or equal to about 2000mm²。

113. method described in 10 according to claim 1, wherein the occupied area for the shell being installed on the pedestal is small In or equal to about 5000mm²。

114. method described in 10 according to claim 1 further comprises providing excitation energy to the reaction vessel, with induction Indicate the present or absent signal of the amplified production of the nucleic acid amplification reaction.

115. method described in 14 according to claim 1 further comprises first provided with the reaction vessel sensing communication Sensor, to detect the present or absent signal for indicating the amplified production.

116. method described in 15 according to claim 1, wherein the first sensor is optical sensor, and wherein described Signal is optical signalling.

117. method described in 15 according to claim 1 further comprises second provided with the reaction vessel sensing communication Sensor, to detect the temperature of the indoor one or more positions of the reaction of the reaction vessel.

118. method described in 17 according to claim 1 further comprises providing in the pedestal or the shell operationally It is coupled to the display of the first sensor and/or the second sensor, wherein the display is configured as: (1) showing Show that the present or absent signal for indicating the amplified production, and/or (2) show the described of the reaction vessel React the temperature at indoor one or more of positions.

119. method described in 15 according to claim 1 further comprises providing in the pedestal or the shell operationally It is coupled to the display of the first sensor, wherein the display, which is configured as display, indicates the described of the amplified production The present or absent signal.