CN107287106A

CN107287106A - The novel method and system of detection of nucleic acids are carried out using quantitative PCR and digital pcr

Info

Publication number: CN107287106A
Application number: CN201710681242.5A
Authority: CN
Inventors: 陈海肃; 格雷戈里·金茨; 李耀辉; 李晨
Original assignee: Cajudi Biotechnology Yixing Co Ltd
Current assignee: Coyote Bioscience Co Ltd
Priority date: 2017-08-10
Filing date: 2017-08-10
Publication date: 2017-10-24

Abstract

The invention provides the novel method and system that detection of nucleic acids is carried out for quantitative PCR and digital pcr.

Description

The novel method and system of detection of nucleic acids are carried out using quantitative PCR and digital pcr

Background technology

Nucleic acid amplification method allows selectively to expand and identify core interested from complex mixture such as biological sample Acid.In order to detect the nucleic acid in biological sample, generally biological sample is handled, with from the other components of biological sample and its He may interfere with other materials of nucleic acid and/or amplification and isolates nucleic acid.Core interested is being isolated from biological sample , can be for example, by amplification method, the method (for example, polymerase chain reaction (PCR)) such as based on thermal cycle, to interested after acid Nucleic acid expanded.After nucleic acid interested is expanded, amplified production can be detected, and detection knot is understood by end user Really.However, when needing to carry out multiple or multiple amplified reactions, methods described is probably cumbersome, tediously long and inefficiency.

Have been reported and deliver droplets on as the chemistry and biochemical reaction in container progress confined volume (for example, nucleic acid expands Increase), and developed a variety of methods to generate such droplet.However, these technologies generally differ with droplet size, group Into different, relatively low flux, and/or the problems such as monodispersity droplet can not be generated.

The content of the invention

This area is present for analyzing quick, the accurate and high-throughout preparation of the nucleic acid from complex sample type, method With the demand of device.This method and apparatus can be used for, for example, realizing rapid sample-response detection (sample to Answer detection) and reply can be by the disease of its detection of nucleic acids.

The invention provides for effectively amplification of nucleic acid, such as RNA and DNA molecular method and system are particularly used for With the method and system of a large amount of different nucleic acid molecules of high flux and/or parallel amplification and analysis.The nucleic acid product of amplification can be rapid Ground is detected with high sensitive.

In one aspect, the invention provides a kind of target nucleic acid for being used to identify the biological sample for obtain since subject The presence or absence of molecule, or relative quantity method.Methods described may include：(a) generate emulsion, the emulsion comprising fluorinated oil, Electric liquid and the surfactant based on bay ether, wherein the emulsion includes multiple droplets, the droplet includes (i) Multiple nucleic acid molecules of the biological sample from the subject, and (ii) carry out DNA (DNA) amplification and Reagent needed for optional reverse transcription amplification；(b) the multiple nucleic acid molecules in the multiple droplet are being enough to produce The DNA cloning is carried out under conditions of the amplified production of the multiple nucleic acid molecules；And (c) detection comes from the multiple small liquid The signal of one or more of drop droplet, wherein the target nucleic acid point in the multiple nucleic acid molecules of the signal designation The presence or absence of son or relative quantity.

In some embodiments, the DNA cloning is polymerase chain reaction (PCR).

In some embodiments, the biological sample is directly obtained from the subject, without preculture, non-selective Enrichment, selective enrichment, it is plated on differentiation culture medium, and/or the biomedicine identification of expected property.In some embodiments, institute State tissue or fluid of the biological sample from the subject.

In some embodiments, the reagent in (a) includes the report agent for generating the signal.In some embodiment party In case, the intensity of the signal is proportional to the amount of the amplified production of the multiple nucleic acid molecules.In some embodiments In, the report agent is dyestuff.In some embodiments, the dyestuff is fluorescent dye.

In some embodiments, the droplet is encapsulated in the aqueous phase of oil phase, wherein the aqueous phase is comprising described Multiple nucleic acid molecules and the reagent.

In some embodiments, the droplet is stable at a temperature of higher than 95 DEG C, does not deform or closes And.

In some embodiments, be contained in the multiple droplet the multiple nucleic acid molecules it is un-extracted and/or Purifying.

In some embodiments, the emulsion by by aqueous first fluid and non-aqueous second fluid mix come Generation.In some embodiments, the emulsion is by the way that aqueous first fluid and non-aqueous second fluid are mixed to produce Raw mixture, then is vortexed to generate to the mixture.

In some embodiments, the electric liquid is electrically insulating liquid.In some embodiments, the electronics liquid Body is the liquid based on fluorohydrocarbon.In some embodiments, the electric liquid includes perfluor triamylamine.

In some embodiments, the surfactant based on bay ether is glycolic polyoxyethanyl bay ether. In some embodiments, the emulsion also includes another surfactant.In some embodiments, another surface Activating agent is polyoxyethylene sorbitan monolaurate.

In some embodiments, wherein (b) includes carry out reverse transcription amplification parallel with the DNA cloning.In some realities Apply in scheme, the multiple nucleic acid molecules are carried out with reverse transcription amplification to produce complementary DNA (cDNA) point wherein (b) includes (i) Son, and (ii) carry out the DNA cloning parallel with the reverse transcription amplification to produce at least one subset of the cDNA molecules The amplified production.

In some embodiments, methods described also includes exporting the information on the amount of the amplified production to reception Person.In some embodiments, wherein described information is used as report output.

In one aspect, the invention provides a kind of target nucleic acid for being used to identify the biological sample for obtain since subject The presence or absence of molecule, or relative quantity system.The system may include：Droplet maker, it generates emulsion, the emulsion Surfactant comprising fluorinated oil, electric liquid and based on bay ether, wherein the emulsion includes multiple droplets, it is described small Drop includes multiple nucleic acid molecules of the described biological sample of (i) from the subject, and (ii) carries out DNA (DNA) amplification and the reagent needed for optional reverse transcription amplification；Controller, it operationally connects with the droplet maker Connect, the multiple nucleic acid molecules in the multiple droplet are being enough to produce institute wherein the controller is programmed to (i) State and carry out the DNA cloning under conditions of the amplified production of multiple nucleic acid molecules；And (ii) detection comes from the multiple small liquid The signal of one or more of drop droplet, wherein the target nucleic acid point in the multiple nucleic acid molecules of the signal designation The presence or absence of son or relative quantity.

In some embodiments, the system further comprises detector, and the detector is used to detect the signal, Wherein described controller is operably coupled to the detector, and manipulates the detector detection signal.

In some embodiments, the controller is programmed to (i) to the multiple core in the multiple droplet Acid molecule carries out the DNA cloning under conditions of being enough to produce the amplified production of the multiple nucleic acid molecules, wherein with it is described The parallel carry out reverse transcription amplification of DNA cloning；And (ii) detection is from one or more of the multiple droplet droplet Signal, wherein presence or absence or the relative quantity of the target nucleic acid molecule in the multiple nucleic acid molecules of the signal designation.

In some embodiments, the controller is programmed to (i) and carries out reverse transcription expansion to the multiple nucleic acid molecules Increase to produce complementary DNA (cDNA) molecule, and at least one subset of the cDNA molecules is carried out and the reverse transcription amplification Parallel DNA cloning is to produce the amplified production；And (ii) detection is from one or more of the multiple droplet The signal of droplet, wherein presence or absence or the phase of the target nucleic acid molecule in the multiple nucleic acid molecules of the signal designation To amount.

In some embodiments, the controller also includes output module, and the output module is used for will be on described The information of the amount of amplified production is exported to recipient.In some embodiments, described information is used as report output.

In one aspect, it is used for the system of droplet or biology sample detection the invention provides a kind of.The system can Including：Sample holder, it is arranged to detecting that one or more of the subset or the subset of multiple droplets are raw During thing sample, the multiple droplet is kept along single plane；Detection unit, including：(i) at least one excitaton source, its quilt It is configured to guide exciting light in multiple detection zones in the single plane into given detection zone, and the inspection of (ii) optics Survey device, its be arranged in the given detection zone along optical window detect the multiple droplet the subset or One or more of biological samples；And at least one actuator, it is arranged to make the optical window along described many Individual detection zone relative movement.

In some embodiments, at least one described actuator is a part for the detection unit.In some implementations In scheme, at least one described actuator is multiple actuators.In some embodiments, at least one actuator coupling To the detection unit.In some embodiments, at least one described actuator is coupled to the sample holder.

In some embodiments, the excitaton source of energy is arranged to guide into the exciting light described to regular inspection Survey region.

In some embodiments, the system may also include one or more computer processors, its operationally coupling The detection unit and at least one described actuator are bonded to, wherein one or more of computer processors are individually or always It is programmed for body：(i) at least one described actuator is guided, so as to when the multiple droplet is along the single plane phase During adjacent to the sample holder so that the optical window is relatively moved along the multiple detection zone, wherein described relative The optical window is positioned in the given detection zone by movement, and at least one described excitaton source of (ii) guiding swashs described It is luminous to guide the given detection zone, and (iii) guiding fluorescence detector into detect in the given detection zone The subset or one or more of biological samples of the multiple droplet in the optical window.

In some embodiments, one or more of computer processors individually or are generally programmed for drawing At least one described actuator is led, so that each detection along the multiple detection zone of the optical window in order Region is relatively moved.

In some embodiments, one or more of computer processors individually or are generally programmed for： (i) at least one illumination correction chart picture is collected from the multiple detection zone using the fluorescence detector, and (ii) uses institute At least one illumination correction chart picture is stated to compensate or correct the multiple droplet come from the multiple detection zone Illuminate heterogeneity.

In some embodiments, at least one described excitaton source is multiple excitaton sources.In some embodiments, it is described Multiple excitaton sources are multiple light emitting diodes (LED).In some embodiments, the multiple LED each LED transmittings are different The light of frequency.In some embodiments, the multiple LED produces white light.In some embodiments, the multiple excitaton source Produce the light of multiple color.In some embodiments, the multiple color includes at least three kinds colors.In some embodiments In, the multiple color includes at least four colors.In some embodiments, the multiple color includes at least five kinds face Color.

In some embodiments, at least one described excitaton source includes light emitting diode (LED).In some embodiments In, the LED transmitting boundaries are at 390 nanometers (nm) to the light in 700 nanometer ranges.

In some embodiments, at least one described excitaton source includes radiator.In some embodiments, it is described to dissipate Hot device is fin.

In some embodiments, at least one described excitaton source includes one or more optical units, for by described in Exciting light is guided into and/or focused in the given detection zone.In some embodiments, one or more of optics lists Member includes condenser lens.In some embodiments, one or more of optical units include directional mirror.In some realities Apply in scheme, the detection unit does not include spectroscope.

In some embodiments, the detection unit further comprises one or more filters, its be used for select by Given frequency or the light of frequency range that the fluorescence detector is detected.In some embodiments, it is one or many Individual filter is multiple filters.In some embodiments, the multiple filter includes each filter rotatably selected Light device.In some embodiments, the fluorescence detector includes charge coupling device (CCD) camera.

In some embodiments, the detection unit is arranged to move along three degree of freedom.In some embodiment party In case, the detection unit is arranged to along the planar movement parallel to the single plane.In some embodiments, institute Detection unit is stated to be arranged to along the planar movement for being orthogonal to the single plane.

In some embodiments, the system can further comprise fluid stream unit, and it is arranged to adjacent to institute Sample holder is stated to provide the multiple droplet.

In some embodiments, at least one described excitaton source, which is oriented to, guides the exciting light into the optical window Mouthful.In some embodiments, at least one described excitaton source is oriented to is introduced only into the optical window by the exciting light. In some embodiments, at least one described excitaton source is oriented to guides the exciting light along first axle into, wherein described Fluorescence detector is oriented to along the second not parallel with first axle axle to detect the subset of the multiple droplet Or one or more of biological samples.

In some embodiments, the detection unit is arranged to so that the exciting light and the optical window pair Relatively moved simultaneously in the multiple detection zone.

In one aspect, it is used for the method for droplet or biology sample detection the invention provides a kind of.Methods described can Including：(a) detecting system is activated, the detecting system includes a. sample holders, it is arranged to detecting multiple small liquid During one or more of the subset of drop or the subset biological sample, the multiple droplet is kept along single plane；b. Detection unit, including：(i) at least one excitaton source, it is arranged to guide into exciting light multiple in the single plane Given detection zone in detection zone, and (ii) fluorescence detector, it is arranged to the edge in the given detection zone Optical window detects the subset or one or more of biological samples of the multiple droplet；C. at least one is activated Device, it is arranged to make the optical window relatively move along the multiple detection zone；And (b) is at least one described in Individual actuator, so as to when the multiple droplet along the single plane adjacent to the sample holder when so that the light Learn window to relatively move along the multiple detection zone, wherein the optical window is positioned at described give by the relative movement In detection zone, (c) guides the exciting light into the given detection zone using at least one described excitaton source；And (d) The multiple droplet in the optical window in the given detection zone is detected using the fluorescence detector The subset or one or more of biological samples.

In some embodiments, methods described can further comprise that there is provided adjacent to the sample holder before (b) The multiple droplet.

In some embodiments, one or more of biological samples include DNA (DNA) molecule.One In a little embodiments, one or more of biological samples are included in the core of the nucleic acid amplification reaction carried out on template nucleic acid molecule Sour amplified production.

In some embodiments, methods described can further comprise before (b) so that the template nucleic acid molecule experience It is sufficient for the condition of the nucleic acid amplification reaction.In some embodiments, when the multiple droplet is adjacent to the sample During product supporter, the nucleic acid amplification reaction is carried out.In some embodiments, the condition is included the multiple droplet Temperature circulated between at least two temperature of multiple temperature levels.In some embodiments, the condition is included by described in The temperature of multiple droplets is maintained within given temperature or given temperature range.

In some embodiments, methods described further comprises at least one described actuator of guiding, so that institute Each detection zone along the multiple detection zone of optical window in order is stated to relatively move.

In some embodiments, methods described further comprises that (i) uses the fluorescence detector from the multiple inspection Survey region and collect at least one illumination correction chart picture, and (ii) is compensated or corrected using at least one described illumination correction chart picture Come from the illumination heterogeneity of the multiple droplet in the multiple detection zone.

In some embodiments, methods described further comprises so that the exciting light and the optical window are for institute Multiple detection zones are stated to relatively move simultaneously.

In some embodiments, methods described further comprises the subset or described for detecting the multiple droplet Intensity difference in one or more biological samples.

In some embodiments, methods described further comprises so that the optical window is relative according to priority along track Mobile, the track includes multiple translation vectors along the multiple detection zone.In some embodiments, it is the multiple flat Moving vector includes second group of vector that first group of vector is orthogonal to first group of vector.

According to following detailed description, other aspects of the present invention and advantage will become to those skilled in the art It is clear that the illustrative embodiment of the present invention wherein only has shown and described.It will recognize that, present disclosure can be wrapped Other and different embodiments are included, and its some details can be modified at multiple obvious aspects, it is all these All without departing from present disclosure.Correspondingly, accompanying drawing and description will be considered to be illustrative and not restrictive naturally.

Quote and be incorporated to

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

Brief description of the drawings

The novel feature of the present invention is specifically explained in the appended claims.By reference to below to wherein using this The detailed description and the accompanying drawings (also referred to as " scheming " herein) that the illustrated embodiment of inventive principle is illustrated by, it will obtain The features and advantages of the present invention are better understood from, in the accompanying drawings：

Fig. 1 is the schematic diagram for depicting example system.

Fig. 2 is the photo of the droplet prepared according to the method for the present invention.

Fig. 3 is the photo that HBV viruses are detected according to the method for the present invention.

Fig. 4 is the top view for describing the detection unit in detecting system of the invention.

Fig. 5 is the schematic diagram for describing the detecting system of the present invention.

Fig. 6 is the bottom view for the detection unit for describing Fig. 4 of the present invention.

Fig. 7 is the schematic diagram for the lightray propagation for describing the detecting system of the present invention.

Fig. 8 is the schematic diagram of the motion track for the optical window for describing the detecting system of the present invention.

Detailed description of the invention

It is aobvious for those skilled in the art although each embodiment of the present invention has been illustrated and described herein And be clear to, these embodiments are only provided in an illustrative manner.Those skilled in the art are in the situation without departing substantially from the present invention Down it is contemplated that many changes, change and replacement.It should be appreciated that can be replaced using the various of invention as described herein embodiment For scheme.

As used in the present specification and claims, singulative " one ", " one kind " and "the" include multiple Several reference things, unless the context.For example, term " cell " includes multiple cells, including its mixing Thing.

As used in the present specification and claims, when term " about " is before numerical value is placed in, unless up and down Text is expressly stated otherwise, and referring to the numerical value can fluctuate no more than 10%.It is no more than for example, the numerical value can fluctuate 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% etc..

As it is used herein, term " amplification (amplifying) " and " expanding (amplification) " is interchangeable makes With, and it is often referred to generate the one or more copies or " amplified production " of nucleic acid.Term " DNA cloning " is often referred to generate DNA One or more copies of molecule or " DNA product of amplification ".Term " reverse transcription amplification " is often referred to the effect through reverse transcriptase From ribonucleic acid (RNA) template generation DNA (DNA).

As it is used herein, term " cycle threshold " or " Ct " are often referred to the circulation in Thermal Cycling, in the circulation In due to caused by amplified production detectable signal increase up to the statistically significant level higher than background signal.

As it is used herein, term " denaturation (denaturing) " and " being denatured (denaturation) " is interchangeable makes With, and be often referred to the helical structure of double-strandednucleic acid and completely or partially untwist, and refer to single-chain nucleic acid in some cases Secondary structure is untwisted.Denaturation may include the inactivation of pathogen cells wall or virus coat, and inhibitor protein matter inactivation. The condition that can be denatured includes " denaturation temperature " and " denaturation duration ", and " denaturation temperature " is often referred to allow what is be denatured Temperature, " denaturation duration " is often referred to occur the time quantum that denaturation is distributed.

As it is used herein, term " incubation " is often referred under conditions of progress or without vibration and stirring so that Sample, mixture or solution maintain certain constant temperature section time." incubation temperature " is often referred to allow to be incubated the temperature occurred. " being incubated the duration " is often referred to distribute to the length for being incubated the time occurred.

As it is used herein, term " extension (elongation) " is often referred to mix nucleotides in the way of template-directed Enter nucleic acid.Extension can be occurred by means of such as enzyme of polymerase or reverse transcriptase.The condition that can extend includes " prolonging Stretch temperature " and " extension duration ", " elongating temperature " is often referred to the temperature for allowing to extend, and " extension duration " is usual Refer to occur the time quantum that extension is distributed.

As it is used herein, term " nucleic acid " is often referred to nucleotides (deoxyribonucleotide or the ribose of any length The polymerized form of nucleotides or its analog.Nucleic acid can have any three-dimensional structure, and executable any known or unknown Function.The non-limiting examples of nucleic acid include DNA, RNA, gene or the code area of genetic fragment or noncoding region, by chain point Analyse the one or more locus determined, extron, introne, mRNA (mRNA), transfer RNA, rRNA, short interference RNA (siRNA), short hairpin RNA (shRNA), Microrna (miRNA), ribozyme, cDNA, recombinant nucleic acid, branching nucleic acid, plasmid, Carrier, the DNA of the arbitrary sequence of separation, RNA, nucleic acid probe and the primer of the arbitrary sequence of separation.Nucleic acid can comprising a kind of or The nucleotides of a variety of modifications, such as methylated nucleotide and nucleotide analog.If it exists, the modification to nucleotide structure It can be carried out before or after nucleic acid assembling.The nucleotide sequence of nucleic acid can be interrupted by non-nucleotide component.Nucleic acid can be poly- Further modified after conjunction, such as by being coupled or combining with report agent.

As it is used herein, term " primer extension reaction " is often referred to double-strandednucleic acid denaturation, primer and the nucleic acid of denaturation One or two chain combinations, then extension primer.In some cases, template nucleic acid can be single in the case of without denaturation (for example, part is single-stranded) of chain, and primer can be combined in the single-chain nucleic acid, then extension primer.

As it is used herein, term " reactant mixture " is often referred to include for completing nucleic acid amplification (for example, DNA expands Increase, RNA amplification) necessary to reagent composition, the non-limiting examples of these reagents include having target RNA or target DNA Specific primer sets, the DNA produced by RNA reverse transcription, archaeal dna polymerase, reverse transcriptase are (for example, the reverse for RNA Record), suitable buffer solution (including zwitterionic buffer), co-factor (for example, divalence and monovalent cation), dNTP and other Enzyme (for example, uracil-DNA glycosylase (UNG) etc.).In some cases, reactant mixture can also include one or more Report agent.

As it is used herein, " report agent " is often referred to produce the composition of detectable signal, the presence of the signal or not It whether there is in the presence of available for detection amplified production.

As it is used herein, term " target nucleic acid " be often referred to it is in the starter population of nucleic acid molecules, with certain seed nucleus The nucleic acid molecules of nucleotide sequence, its presence, amount and/or sequence or one of which or multinomial change need to be measured.Target Nucleic acid can be any kind of nucleic acid, including DNA, RNA and their analog.As it is used herein, " target nucleus ribosomal ribonucleic acid (RNA) " it is often referred to the target nucleic acid as RNA.As it is used herein, " target DNA (DNA) " is often referred to as DNA Target nucleic acid.

As it is used herein, term " subject " be often referred to can test or detectable hereditary information entity or Medium.Subject can be people or individual.Subject can be vertebrate, such as mammal.Mammal it is unrestricted Property example include mouse, ape and monkey, people, domestic animal, sport animals (sport animal) and pet.Other examples of subject include food Thing, plant, soil and water.

As it is used herein, term " fluid " is often referred to liquid or gas.Fluid is unable to maintain that the shape of restriction, and It can be flowed in the time range of observable, so as to fill in the container that it is placed in.Therefore, the fluid can have any allow Its appropriate viscosity flowed.If there is two or more fluids, every kind of fluid can be selected independently by those skilled in the art From substantially any fluid (liquid, gas etc.).

As it is used herein, term " aqueous fluids " is often referred to water, by water or from the fluid of water preparation, or comprising The fluid of water.For example, aqueous fluids can be with the aqueous solution of the water as solvent.The aqueous fluids of the present invention, which can be included, to be used to carry out The reagent of required chemical reaction (such as polymerase chain reaction (PCR)).The non-limiting example of aqueous fluids includes but not limited In water and other aqueous solution for including water, such as cell or Biomedia, ethanol, salting liquid.

As it is used herein, term " non-aqueous fluid " be often referred to the liquid except water, by the liquid except water or From the fluid of the liquid preparation except water.The non-limiting example of non-aqueous fluid includes but is not limited to oil, such as hydro carbons, silicone oil, Fluorocarbon oil, organic solvent etc..

As it is used herein, term " crossover location " is often referred to a passage through another passage, or and another Point or region that passage converges.

As it is used herein, term " subregion " is often referred to be split into or the part being distributed as or share.The example of subregion Including droplet and hole.

As it is used herein, term " droplet " is often referred to the part of the separation of first fluid (such as aqueous fluids), It is surrounded by second fluid (such as non-aqueous fluid).Emulsion may include first fluid (such as liquid) in second fluid Dispersion.The first fluid is probably immiscible in the second fluid.In some embodiments, described first Fluid and the second fluid substantially immiscible.The fluid of the present invention can be spherical, or can use any shape, such as example Shape with oval cross section.For the droplet of aspherical, its a diameter of and described aspherical droplet has same volume Perfect mathematics spheroid diameter.The droplet of the present invention can be single emulsion, double emulsion or triple emulsion etc..

Droplet can include skin zone (skin).The skin zone can be formed when droplet is heated.The skin zone it is comparable in There is higher viscosity inside droplet.In some cases, the skin zone can prevent the droplet from melting with other droplets Close.

As it is used herein, term " microfluidic device " be often referred to comprising at least one having less than about 10mm, 1mm, Chip, region, product or the system of the fluid passage of 0.5mm or 0.1mm cross sectional dimensions.

As it is used herein, " cross sectional dimensions " of passage can be substantially square relative to the flow of fluid in the passage To vertically being measured.

As it is used herein, term " passage " is often referred in device or matrix (such as chip) or the feature in it, its Flow of fluid is guided at least in part.Passage can have any shape of cross section (circle, ring-type, ellipse, triangle, not advise Then shape, square or rectangle etc.), and can be covering or unlapped.When passage is completely covered, the passage is extremely A few part can have the cross section encapsulated completely, or whole passage is encapsulated over the whole length, only its import or export Or opening exception.The passage of the present invention can be any suitable length.The passage can be it is straight, it is substantially straight, or it can Including one or more bendings or bending etc..For example, the passage can have snakelike or helical configuration.In some embodiments In, the passage includes one or more branches, and it is partly or entirely connected with one or more of the other passage.When passage exists When corner or turning point bending or bending, the corner or turning point can have circular contour, so that fluid or subregion It will not be trapped at the corner or turning point.

Passage can also have at least about 2:1st, at least about 3:1st, at least about 4:1st, at least about 5:1st, at least about 6:1st, at least about 8:1st, at least about 10:1st, at least about 15:1st, at least about 20:1st, at least about 30:1st, at least about 40:1st, at least about 50:1st, at least about 60:1st, at least about 70:1st, at least about 80:1st, at least about 90:1st, at least about 100:(length is to average horizontal stroke for 1 or higher length-width ratio The ratio between sectional dimension).Open channel would generally have the feature for being easy to control fluid delivery, such as architectural feature (contracting of extension Enter), and/or physically or chemically feature (hydrophobicity is to hydrophily), or other can be to the spy of fluid applying power (such as confining force) Levy.The non-limiting example of the power actuator of suitable power, which can be produced, includes piezo-activator, pressure valve, the electricity for applying AC electric fields Pole etc..Fluid in passage can partially or even wholly be full of the passage., can be for example using surface when using open channel Tension force (i.e. protrusion or recessed new lunar surface) is kept fluids in passage.

As it is used herein, term " excitaton source " is often referred to produce exciting with multiple different wave lengths or frequency Device, equipment or the device of signal, light emitting diode used in embodiment of the invention (" LED ").Here, wavelength Can be discrete wavelength, and excitation signal can be exciting light.In some embodiments, excitaton source can additionally or can Selection of land includes one or more optical units, for guiding and/or focusing on exciting light in given detection zone into.

As it is used herein, term " fluorescence detector " is often referred to be applicable the unit or device of detection optical signal, It is for example including one or more optical units or light propagation path.The unit or device generally include one or many Individual lens, one or more prisms, IMAQ or acquisition equipment, imaging sensor.IMAQ or acquisition equipment are, for example, All kinds of cameras (such as charge coupling device (" CCD ") camera), it is alternatively supported adjustment resolution ratio, shoots static or move Motion video, change burnt poly-, camera lens zoom in or out, changes imaging pattern, the adjustment depth of field, change the time for exposure, adjust visual angle or The functions such as the visual field.In one embodiment of the invention, fluorescence detector be adapted to detect for coming from sample (for example droplet or Biological sample) excite produced optical signal through excitaton source, such as various fluorescence or other detectable light, so as to utilize All kinds of optical detecting methods realize the detection to droplet or biological sample, and optical detecting method here includes but is not limited to Fluorimetry and UV-Visible absorption.

As it is used herein, term " actuator " is often referred to drive one or more destination objects along one or many Device, equipment or the mechanism of individual direction movement or rotation.Usual actuator drives mesh by the movement (such as rotating) of itself Mark the movement of object.Actuator can be driven by various forms of energy in itself, for example electric energy, luminous energy, magnetic energy, heat energy, machine Tool energy, hydraulic pressure or air pressure.In terms of concrete application, actuator can be all kinds of motors or motor.For example, motor can be certainly Rectification or outside commutating machine.Motor can also be exchange (AC) or direct current (DC) motor.Motor can include direct-drive motor, Stepper motor, electrodeless motor or servomotor.Motor may be configured to rotate along single direction, or can be to phase Anti- direction rotates.In some embodiments of the present invention, actuator can be stepper motor, and can be at least partly Ground is located in detection unit, or is integrated into detection unit.In some embodiments of the present invention, actuator can be with one Or multiple computer processor couplings, so as to receive the instruction for coming from computer processor, so that optical window Relatively move in the detection area.

In one embodiment, the surface of emulsion of the invention comprising fluorinated oil, electric liquid and based on bay ether is lived Property agent.The emulsion can include multiple droplets.The droplet can be comprising (i) biological sample from the subject Multiple nucleic acid molecules, and (ii) carry out the reagent needed for DNA (DNA) amplification and optional reverse transcription amplification.

For example, the emulsion can include fluorinated oil.Any suitable fluorinated oil can be used in the present invention.Suitable fluorinated oil Non-limiting example includes HFE 7100, HFE 7500, FC-40, FC-43, FC-70, FC-3208, or its combination.

For example, the emulsion can include electric liquid.Any suitable electric liquid can be used in the present invention.Suitable electronics Liquid can be any electric liquid suitable for electronic application.The electric liquid can have the property of surfactant.It is described Electric liquid can be electrical conduction liquid, or, the electric liquid can be electrically insulating liquid.The electric liquid may be based on fluorine For the liquid of hydrocarbon.For example, the electric liquid can include perfluor triamylamine.Or, the electric liquid can include other fluoro Hydrocarbon, including perfluoro-hydrocarbon.In some embodiments, the electric liquid is Fluorinert electric liquids.For example, the electricity Sub- liquid may be selected from Fluorinert FC-87, FC-72, FC-84, FC-77, FC3255, FC-3283, FC-40, FC-43, FC- 70、FC-5312.For example, the electric liquid can be Fluorinert FC-70.

For example, the emulsion can include surfactant.Any suitable surfactant can be used in the present invention.For example, Surfactant of the present invention can be hydrophilic surfactant active or lipophilic surfactant.

Hydrophilic surfactant active can be ionic or non-ionic.Suitable ionic surfactant include but It is not limited to alkylammonium salt；Fusidic Acid salt；The derivative of fatty acid of amino acid, oligopeptides and polypeptide；Amino acid, oligopeptides and polypeptide Glyceride ester derivatives；Lecithin and hydrolecithin；Lysolecithin and hydrogenated lyso lecithin；Phosphatide and its derivative；It is molten Blood phospholipid and its derivative；Carnitine fatty acid ester salt；Alkyl sulfate；Soap；Docusate sodium；Acyl-lactate；Glycerine The monoacylated and diacetylation tartrate of monoesters and diglyceride；The monoglyceride and diglyceride of succinylation；Glycerine The citrate of monoesters and diglyceride；And its mixture.

In foregoing group, some ionic surfactants include, for example：Lecithin, lysolecithin, phosphatide, haemolysis Phosphatide and its derivative；Carnitine fatty acid ester salt；Alkyl sulfate；Soap；Docusate sodium；Acyl-lactate；Glycerine list The monoacylated and diacetylation tartrate of ester and diglyceride；The monoglyceride and diglyceride of succinylation；Glycerine list The citrate of ester and diglyceride；And its mixture.

Ionic surface active agent can be lecithin, lysolecithin, phosphatidyl choline, phosphatidyl-ethanolamine, phosphatidyl Glycerine, phosphatidic acid, phosphatidylserine, lysophosphatidyl choline, lysophosphatidyl ethanolamine, lysophosphatidyl glycerol, haemolysis Phosphatidic acid, hemolytic phosphatidylserine, PEG- phosphatidyl-ethanolamines, PVP- phosphatidyl-ethanolamines, the lactate of aliphatic acid, tristearin Acyl -2- lactates, stearoyl lactylates, succinylated monoglycerides, the mono bis acetylation winestone of monoglyceride/diglyceride Acid esters, the citrate of monoglyceride/diglyceride, cholylsarcosine, capronate, caprylate, decylate, laurate, meat Myristate, palmitate, oleate, ricinoleate, linoleate, linolenate, stearate, lauryl sulfate ester, Myristyl sulfate ester (teracecyl sulfate), many storehouse esters, Laurylcarnitine, palmitoyl carnitine, C14 and The ionized form of its salt and mixture.

Hydrophilic non-ionic surfactant may include but be not limited to APG；Alkylmaltosides；Alkylthio Glucoside；Lauryl LABRAFIL M 1944CS；Polyoxyalkylene alkyl such as polyethylene glycol alkyl ether；Polyoxyalkylene alkyl phenol is as gathered Ethylene glycol alkyl phenol；Polyoxyalkylene alkyl phenol fatty acid ester such as polyethylene glycol fatty acid monoesters and polyethylene glycol fatty acid diester； Polyethylene glycol glycerol fatty acid ester；Polyglyceryl fatty acid ester；Such as polyethylene glycol dehydration of polyoxyalkylene sorbitan fatty acid ester Span；In polyalcohol and the group being made up of glyceride, vegetable oil, hydrogenated vegetable oil, aliphatic acid and sterol at least The hydrophily product of transesterification reaction of one member；Polyoxyethylene sterol, its derivative and analog；Oxyethylated vitamin And its derivative；Polyox-yethylene-polyoxypropylene block copolymer；And its mixture；Polyethylene glycol sorbitan fatty acid esters And polyalcohol is exchanged with the hydrophilic ester of at least one member in the group being made up of triglycerides, vegetable oil and hydrogenated vegetable oil Reaction product.The polyalcohol can be glycerine, ethylene glycol, polyethylene glycol, sorbierite, propane diols, pentaerythrite or sugar.

Other hydrophilic non-ionic surfactants include but is not limited to PEG-10 laurates, PEG-12 laurates, PEG-20 laurates, PEG-32 laurates, PEG-32 dilaurates, PEG-12 oleates, PEG-15 oleates, PEG- 20 oleates, PEG-20 dioleates, PEG-32 oleates, PEG-200 oleates, PEG-400 oleates, PEG-15 stearic acid Ester, PEG-32 distearates, PEG-40 stearates, PEG-100 stearates, PEG-20 dilaurates, PEG-25 are sweet Oily trioleate, PEG-32 dioleates, PEG-20 glyceryl laurate esters, PEG-30 glyceryl laurate esters, PEG-20 stearic acid Glyceride, PEG-20 oleins, PEG-30 oleins, PEG-30 glyceryl laurate esters, PEG-40 lauric acids Ester, PEG-40 palm-kernel oils, PEG-50 rilanit specials, PEG-40 castor oil, Cremophor ELP, PEG-60 castor oil, Cremophor RH40 PEG-60 rilanit specials, PEG-60 corn oils, PEG-6 capric acid/glycerol caprylate, PEG-8 capric acid/ Glycerol caprylate, Natrulon H-10-laurate, PEG-30 cholesterol, PEG-25 phytosterols, PEG-30 sojasterols, PEG- 20 trioleates, PEG-40 sorbitan monooleates, PEG-80 anhydrous sorbitols laurate, polysorbate 20, poly- sorb Ester 80, POE-9 lauryl ethers, POE-20 lauryl ethers, POE-10 oleyl ethers, POE-20 oleyl ethers, POE-20 stearyl ethers, life Educate phenol PEG-100 succinates, PEG-24 cholesterol, Natrulon H-10-oleate, polysorbate40, polysorbate60, sucrose monostearate Ester, sucrose monolaurate, sucrose palmitic acid ester, PEG 10-100 nonylphenol series, PEG15-100 octyl phenols series And poloxamer.

Only for example, suitable lipophilic surfactant includes：Fatty alcohol；Fatty acid glyceride；Acetylated glycerol Fatty acid ester；Lower alcohol fatty acid esters；Methyl glycol fatty acid ester；Sorbitan fatty acid ester；Polyethylene glycol anhydrous sorbitol Fatty acid ester；Sterol and sterol derivative；Oxyethylated sterol and sterol derivative；Polyethylene glycol alkyl ether；Sugar ester；Sugar Ether；The lactic acid derivative of monoglyceride and diglyceride；Polyalcohol with by glyceride, vegetable oil, hydrogenated vegetable oil, aliphatic acid and The hydrophobicity product of transesterification reaction of at least one member in the group of sterol composition；Fat soluble vitamin/vitamin derivative；And Its mixture.In the group, some lipophilic surfactants include fatty acid glyceride, methyl glycol fatty acid ester and its mixing The hydrophobicity of at least one member in thing, or polyalcohol and the group that is made up of vegetable oil, hydrogenated vegetable oil and triglycerides Product of transesterification reaction.

Conventional use of surfactant also includes such as oleic acid, lecithin, sorbitan trioleate, chlorination 16 Alkyl pyridine, benzalkonium chloride, polyoxyethylene (20) sorbitan mono-laurate, polyoxyethylene (20) anhydrous sorbitol list are hard Resin acid ester, polyoxyethylene (20) sorbitan monooleate, polyoxypropylene/polyoxyethylene block copolymer, polyoxypropylene/poly- Oxygen ethene/ethylenediamine block copolymer, ethoxylated castor oil etc..Other suitable surfactants/emulsifiers are for ability Field technique personnel are known, and in CTFA International Cosmetic Ingredient Dictionary Listed in and Handbook, Vol.2, the 7th edition (1997).

In some embodiments, the surfactant is the surfactant based on bay ether.For example, the surface Activating agent is glycolic polyoxyethanyl bay ether.

In some embodiments, the emulsion also includes another surfactant.For example, another surface is lived Property agent is any another surfactant specifically described herein.In one embodiment, another surfactant It is polyoxyethylene sorbitan monolaurate (Tween 20).

In some embodiments, the surfactant is fluoro surfactants.For example, the surfactant can Include hydrophobicity tail groups and hydrophilic head group, tail groups and hydrophilic head group based on polymer, based on polymer Tail groups and the head group based on polymer, tail groups of fluorination and be hydrophilic head group, or be based on fluorinated polymer Group and the head group based on hydrophilic polymer.In some embodiments, the surfactant belongs to diblock copolymer Or triblock copolymers type.For example, the surfactant can be block copolymer, such as by two PFPE blocks and The triblock copolymer of one polyethylene glycol block composition.In some embodiments, the surfactant is selected from the group： PFPE-PEG-PFPE (PFPE-polyethylene glycol-PFPE), triblock copolymer EA- surfactants (RainDance ) and DMP (dimorpholino phosphoric acid)-surfactant (Baret, Kleinschmidt etc., 2009) Technologies.Polymer The length of PEG in species (including polymeric surfactant), can be any suitable length, and can according to it is used not Change with type of polymer.

In some embodiments, be contained in the multiple droplet the multiple nucleic acid molecules it is un-extracted and/or Purifying.For example, the multiple nucleic acid molecules are not yet purified when being contained in the multiple droplet.In some embodiments, The multiple nucleic acid molecules are when being contained in the multiple droplet, and the nucleic acid molecules are un-extracted.For example, the multiple core Acid molecule can be a part for biological sample, and the multiple nucleic acid molecules are the multiple when being contained in the multiple droplet Nucleic acid molecules are extracted not from the biological sample.In addition, in some embodiments, the multiple nucleic acid molecules comprising When the multiple droplet, the target nucleic acid (for example, target RNA or target DNA) being present in the multiple nucleic acid molecules may be not Concentrated or enrichment.

For example, the emulsion can include emulsifying agent.Any suitable emulsifying agent can be used in the present invention.Described suitable breast Agent can cause emulsion-stabilizing of the present invention.The emulsifying agent can have the property of surfactant.The emulsifying agent Can be oil-water emulsifiers or water-in-oil emulsifier.The non-limiting example of emulsifying agent includes fatty glyceride, sucrose ester, mountain Pears sugar alcohol ester, soybean lecithin, glyceryl laurate ester, propylene glycol ester etc..In some embodiments, the emulsifying agent is sorbose Alcohol ester.In some embodiments, the emulsifying agent is the emulsifying agent based on monostearate.It is for example described to be based on monostearate The emulsifying agent of ester can be the serial emulsifying agents of Tween, such as polyoxyethylene sorbitan monostearate (Tween 60).

After the emulsion of the present invention is generated, multiple nucleic acid molecules in multiple droplets in emulsion can be enough Produce and carry out DNA cloning under conditions of the amplified production of the multiple nucleic acid molecules.The side of any suitable DNA cloning can be used Method carries out the DNA cloning, DNA cloning method including but not limited to described herein.

In some embodiments, the DNA cloning also includes carry out reverse transcription amplification parallel with the DNA cloning.Can The reverse transcription amplification, reverse transcription amplification including but not limited to described herein are carried out using the method for any reverse transcription amplification Method.The parallel DNA cloning of any method progress and reverse transcription amplification can be used, it is including but not limited to described herein parallel DNA cloning and reverse transcription amplification method.

For example, the DNA cloning includes first the multiple nucleic acid molecules being carried out reverse transcription amplification to produce complementary DNA (cDNA) molecule, and the DNA cloning parallel with least one subset progress of the reverse transcription amplification to the cDNA molecules with Produce the amplified production.

In some embodiments, the droplet is stable at a temperature of higher than 95 DEG C, does not deform or closes And.The droplet can be herein stable in described amplification process, do not deform or merge.For example, institute Stating droplet can be stable under any temperature herein in described amplification process or in any Thermal Cycling, no Deform or merge.

In another aspect, the present invention relates to a kind of target nucleic acid for being used to identify the biological sample for obtain since subject The presence or absence of molecule, or relative quantity system.The system may include：Droplet maker, and controller, its with it is described Droplet maker is operably connected.

The droplet maker can be any suitable droplet maker, as long as it can the method according to the invention life Into emulsion.For example, the droplet maker can generate emulsion of the present invention.For example, the emulsion includes fluorinated oil, electricity Sub- liquid and the surfactant based on bay ether, wherein the emulsion includes multiple droplets, the droplet comes comprising (i) Carry out DNA (DNA) amplification from multiple nucleic acid molecules of the biological sample of the subject, and (ii) and appoint Reagent needed for the reverse transcription amplification of choosing.

The controller can be any suitable controller, include but is not limited to the present invention described in controller and can be real Any device and instrument of existing control function.The controller is programmed to execute the side of the present invention in some embodiments Method.Such as described controller can be programmed to (i) and the multiple nucleic acid molecules in the multiple droplet are being enough to produce The DNA cloning is carried out under conditions of the amplified production of the multiple nucleic acid molecules；And (ii) detection is from the multiple small The signal of one or more of drop droplet, wherein the target nucleic acid in the multiple nucleic acid molecules of the signal designation The presence or absence of molecule or relative quantity.

In some embodiments, the system further comprises detector, and the detector is used to detect the signal. The detector can detect the signal, method including but not limited to described herein with any suitable method.Detection The non-limiting examples of method include optical detection, spectral detection, electrostatic detection, Electrochemical Detection etc..Optical detecting method bag Include but be not limited to fluorimetry and UV-Visible absorption.Spectral method of detection includes but is not limited to mass spectrography, nuclear magnetic resonance (NMR) spectral method and infra-red sepectrometry.Electrostatic detection methods include but is not limited to the technology based on gel, for example, gel electrophoresis. The electrochemistry that electrochemical detection method includes but is not limited to amplified production after the high performance liquid chromatography separation of amplified production is examined Survey.The detector may include real-time detector device.The non-limiting examples of real-time detector device are followed including various real-time PCR heat Ring instrument, real-time PCR system, sequence detection system, intelligent circulation system etc..

In some embodiments, the controller is operably coupled to the detector, and manipulates the detector Detect signal.

In one aspect, the present invention relates to the method for forming subregion.Methods described includes the water that guiding includes nucleic acid samples Property fluid by first passage, and guiding non-aqueous fluid is by second channel, both is flowed to multiple intersections position in chip Put, so that multiple subregions are formed in the multiple crossover location when the aqueous fluids and the non-aqueous fluid are in contact, Each subregion of wherein the multiple subregion is required for nucleic acid amplification including (i) described nucleic acid samples or part thereof, and (ii) Reagent.In the second channel, the non-aqueous fluid can be substantially free of the sample and the reagent.

Methods described can further comprise the condition of (b) in the amplified production for being enough to produce the nucleic acid samples or part thereof Under to described nucleic acid samples in each subregion in the multiple subregion or part thereof carry out nucleic acid amplification reaction.In some realities Apply in scheme, (b) can be carried out in collecting zone.In some embodiments, (b) can be carried out on chip.

In some embodiments, (b) can follow any nucleic acid amplification reaction scheme and carry out, such as described in this specification Those.

Step (b) may include to carry out thermal cycle to each in the multiple subregion.The thermal cycle may include to make institute The temperature for stating each subregion in multiple subregions is circulated between the first temperature and the second temperature higher than the first temperature.One In the case of a little, the thermal cycle may include to make the temperature of each subregion in the multiple subregion more than two different temperatures it Between circulate.

The aqueous fluids can be comprising nucleic acid samples and for reagent necessary to nucleic acid amplification.

In one aspect, the present invention relates to the method for forming subregion.Methods described, which includes working as, includes the aqueous of nucleic acid samples When fluid is in contact with non-aqueous fluid, multiple subregions are formed, wherein each subregion of the multiple subregion includes (i) described core Sour sample or part thereof, and (ii) is for reagent necessary to nucleic acid amplification.

Methods described can further comprise the condition of (b) in the amplified production for being enough to produce the nucleic acid samples or part thereof Under to described nucleic acid samples in each subregion in the multiple subregion or part thereof carry out nucleic acid amplification reaction.

Methods described can be further after (b), including (c) is when the multiple zone configuration is in substantially planar collection After region, the signal for indicating the amplified production presence or absence is detected simultaneously in the multiple subregion.

In some embodiments, methods described further comprises guiding the multiple subregion to the collecting zone.

In some embodiments, (b) is carried out in the collecting zone.

The collecting zone may be included in chip.

In some embodiments, the collecting zone includes multiple intervals, and wherein in (c), in the multiple area Between in given interval detect signal simultaneously.

May be sized to of the collecting zone accommodates the multiple subregion in one layer.In some cases, it is described The size of collecting zone may be alternatively provided as accommodating the multiple subregion in multilayer.

In some embodiments, (b) is carried out on chip.

In one aspect, the invention provides a kind of method for forming subregion.Methods described, which includes working as, includes nucleic acid samples Aqueous fluids when being in contact with non-aqueous fluid, multiple subregions are formed, wherein each subregion of the multiple subregion includes (i) Described nucleic acid samples or part thereof, and (ii) is for reagent necessary to nucleic acid amplification.

Methods described can further after (b), including (c) when the multiple subregion by hole be fixed on collecting zone it Afterwards, the signal for indicating the amplified production presence or absence is detected simultaneously in the multiple subregion, wherein the chi that each hole has Very little (such as length, width, depth) is less than the average diameter of the given subregion in the multiple subregion.

In some embodiments, (b) is carried out in the collecting zone.

The collecting zone may be included in chip.

In some embodiments, (b) is carried out on chip.

In some embodiments, the hole is sized to accommodate the single subregion in the multiple subregion.

The non-aqueous fluid can include hydrophobic liquid.The non-limiting example of hydrophobic liquid include oil, such as hydro carbons, Silicone oil, fluorocarbon oil, fluorinated oil, organic solvent etc..In some embodiments, the oil is fluorinated oil, such as HFE 7100, HFE 7500, FC-40, FC-43, FC-70, FC-3208, or its combination.In some embodiments, the oil is mineral oil, Such as atoleine, light mineral oil, white oil, refined mineral oil, naphthenic oil, aromatic oil, or its combination.The oil can for it is any can Oil for preparing droplet.

The non-aqueous fluid can include surfactant.The surfactant can include hydrophobicity tail groups and hydrophilic Property head group, tail groups and hydrophilic head group based on polymer, the tail groups based on polymer and the head based on polymer Group, tail groups of fluorination and be hydrophilic head group, or the tail group based on fluorinated polymer and based on hydrophilic polymer Head group.In some embodiments, the surfactant belongs to diblock copolymer or triblock copolymers type.Example Such as, the surfactant can be block copolymer, for example, be made up of two PFPE blocks and a polyethylene glycol block Triblock copolymer.In some embodiments, the surfactant is selected from the group：(perfluor gathers PFPE-PEG-PFPE Ether-polyethylene glycol-PFPE), triblock copolymer EA- surfactants (RainDance Technologies) and DMP (dimorpholino phosphoric acid)-surfactant (Baret, Kleinschmidt etc., 2009).Type of polymer (including polymer table Face activating agent) in PEG length, can be any suitable length, and can be become according to used different type of polymer Change.The surfactant can be with of 0.0001% to 5% (w/w), such as 0.001% to 4% (w/w), 0.01% to 3% (w/w), 0.1% to 2% (w/w), 0.1% to 1% (w/w) concentration is present in non-aqueous fluid.In some embodiment party In case, the surfactant is with least about, at most about or about about 0.1% (w/w), 0.2% (w/w), 0.3% (w/ W), 0.4% (w/w), 0.5% (w/w), 0.6% (w/w), 0.7% (w/w), 0.8% (w/w), 0.9% (w/w), 1.0% (w/ W), 1.2% (w/w), 1.4% (w/w), 1.6% (w/w), 1.8% (w/w), 2.0% (w/w), 2.5% (w/w), 3.0% (w/ W), 3.5% (w/w), 4.0% (w/w), 4.5% (w/w), 5.0% (w/w), 7.0% (w/w), 10.0% (w/w), 15.0% (w/w), 20.0% (w/w) or more or less concentration are present in non-aqueous fluid.

The first passage can be any suitable length.The first passage can be substantially straight.Or, it is described First passage can include one or more bendings or bending etc..The first passage can have snakelike or spiral shape to set.It is described First passage can have one or more branches.Part or all of the branch can include secondary channel so that first leads to The main channel in road is connected with one or more second channels.The first passage is also connected to fluid source as described herein.

The first passage can have at least about 1mm, at least about at least about 2mm, at least about 3mm, at least about 5mm, 7mm, At least about 1cm, at least about 1.5cm, at least about 2cm, at least 2.5cm, at least about 3cm, at least about 5cm, at least about 7cm, at least About 10cm length.The first passage can have no more than about 10cm, no more than about no more than about 7cm, 5cm, no more than about 3cm, no more than about 2.5cm, no more than about 2cm, no more than about 1.5cm, no more than about 1cm, no more than about 7mm, no more than about 5mm, no more than about 3mm, or no more than about 2mm length.When referring to the length of the first passage, the length also includes Its all branch and the length of secondary channel.

The cross-sectional area of the first passage can be substantial constant, it is possibility to have be changed.In some implementations In scheme, the cross-sectional area of the first passage is varied from position of the fluid stream in the first passage.Institute The cross-sectional area for stating first passage can be average at least about 1,000 μm², at least about 2,000 μm², at least about 3,000 μm², extremely It is few about 5,000 μm², at least about 10,000 μm², at least about 20,000 μm², at least about 30,000 μm², at least about 50,000 μm², extremely It is few about 100,000 μm², at least about 200,000 μm², at least about 300,000 μm², at least about 500,000 μm², at least about 1,000, 000μm²Or it is higher.The cross-sectional area of the first passage can be averagely no more than about 1,000,000 μm², no more than about 500, 000μm², no more than about 300,000 μm², no more than about 200,000 μm², no more than about 100,000 μm², no more than about 50,000 μm², no more than about 30,000 μm², no more than about 20,000 μm², no more than about 10,000 μm², no more than about 5,000 μm², do not surpass Cross about 3,000 μm², or is no more than about 2,000 μm².These cross-sectional area numerical value also can independent assortment.

The cross-sectional area of the first passage can be varied from along the length direction of passage.Such as described first passage Cross-sectional area can be in average cross sectional area about 75% to about 125%, about 80% to about 120%, about 90% to about 110%, about 95% to about 105%, change in the range of about 97% to about 103% or about 99% to about 101%.Described first leads to Road can have any suitable shape of cross section, such as circle, annular, ellipse, triangle, irregular shape, square or square Shape etc..

The first passage can have any suitable cross-sectional dimension.Cross-sectional dimension is often referred to accommodate Full-size in the cross section of the first passage, wherein the cross section refers to the mean square with fluid stream in first passage To orthogonal face.For example, the cross-sectional dimension can be no more than 1mm, and no more than about 800 μm, no more than about 600 μm, No more than about 500 μm, no more than about 400 μm, no more than about 300 μm, no more than about 250 μm, no more than about 200 μm, it is no more than About 100 μm, no more than about 75 μm, no more than about 50 μm, no more than about no more than about 25 μm, 10 μm etc..For example, described maximum horizontal Sectional dimension can be at least about 5 μm, at least about 10 μm, at least about 25 μm, at least about 50 μm, at least about at least about 75 μm, 100 μ M, at least about 200 μm, at least about 250 μm, at least about 300 μm, at least about 400 μm, at least about 500 μm, at least about 600 μm, extremely It is few about 800 μm etc..

The first passage can be with one or more second channel fluid communications.The second channel can be microfluid dress Put.However, in some embodiments, the first passage and/or second channel are not microfluidic devices.

The second channel can with first passage at a distance of relative constancy distance.And/or, the first passage and Second channel can be substantially parallel to each other.In some embodiments, the first passage and second channel are average at a distance of both About the 75% to about 125% of distance, e.g., from about 80% to about 120%, about 90% to about 110%, about 95% to about 105%, about 97% to about 103% or about 99% to about 101%.

In some embodiments, more than one second channel may be present.Each second channel can lead to described first Road fluid communication.If more than a second channel, each second channel can with the first passage at a distance of identical or Different distances.In addition, multiple second channels can have identical or different length, cross-sectional area or other properties.It is described Second channel also fluid communication or each other not fluid communication each other.

The second channel can have any suitable shape.The second channel can be substantially straight.Described second Passage can have one or more bendings or bending.The second channel can have the shape substantially the same with the first passage Shape allows for example described second channel and the first passage at a distance of the distance of substantial constant.The second channel also may be used There are different shapes from the first passage.

The second channel can have any suitable length.For example, the length of the second channel can be at least about 1mm, at least about 2mm, at least about 3mm, at least about 5mm, at least about 7mm, at least about 1cm, at least about 1.5cm, at least about 2cm, At least 2.5cm, at least about 3cm, at least about 5cm, at least about 7cm, at least about 10cm etc..For example, the length of the second channel It can be no more than about 10cm, no more than about no more than about 7cm, no more than about 5cm, no more than about 3cm, 2.5cm, be no more than about 2cm, no more than about 1.5cm, no more than about 1cm, no more than about 7mm, no more than about 5mm, no more than about 3mm, or no more than about 2mm.The length of the second channel may include the length of its any branch or secondary channel.

The cross-sectional area of the second channel can be substantial constant, it is possibility to have be changed.In some implementations In scheme, the cross-sectional area of the second channel is varied from position of the fluid stream in the second channel.Institute The cross-sectional area for stating second channel can be average at least about 1,000 μm², at least about 2,000 μm², at least about 3,000 μm², extremely It is few about 5,000 μm², at least about 10,000 μm², at least about 20,000 μm², at least about 30,000 μm², at least about 50,000 μm², extremely It is few about 100,000 μm², at least about 200,000 μm², at least about 300,000 μm², at least about 500,000 μm², at least about 1,000, 000μm²Or it is higher.The cross-sectional area of the second channel can be averagely no more than about 1,000,000 μm², no more than about 500, 000μm², no more than about 300,000 μm², no more than about 200,000 μm², no more than about 100,000 μm², no more than about 50,000 μm², no more than about 30,000 μm², no more than about 20,000 μm², no more than about 10,000 μm², no more than about 5,000 μm², do not surpass Cross about 3,000 μm², or no more than about 2,000 μm².These cross-sectional area numerical value also can independent assortment.

In some embodiments, the cross-sectional area of the second channel can be varied from.For example, the second channel Cross-sectional area can be varied from along the length direction of passage.The cross-sectional area of such as described second channel can be average Cross-sectional area about 75% to about 125%, about 80% to about 120%, about 90% to about 110%, about 95% to about 105%, about Change in the range of 97% to about 103% or about 99% to about 101%.The cross-sectional area of the second channel can with it is described The cross-sectional area of first passage is identical or different.In addition, the second channel can have any suitable shape of cross section, example Such as circle, annular, ellipse, triangle, irregular shape, square or rectangle.The shape of cross section of the second channel can With identical or different with the shape of cross section of the first passage.

The second channel can have any suitable cross-sectional dimension.Cross-sectional dimension is often referred to accommodate Full-size in the cross section of the second channel, wherein the cross section refers to the mean square with fluid stream in second channel To orthogonal face.For example, the cross-sectional dimension can be no more than 1mm, and no more than about 800 μm, no more than about 600 μm, No more than about 500 μm, no more than about 400 μm, no more than about 300 μm, no more than about 250 μm, no more than about 200 μm, it is no more than About 100 μm, no more than about 75 μm, no more than about 50 μm, no more than about no more than about 25 μm, 10 μm etc..For example, described maximum horizontal Sectional dimension can be at least about 5 μm, at least about 10 μm, at least about 25 μm, at least about 50 μm, at least about at least about 75 μm, 100 μ M, at least about 200 μm, at least about 250 μm, at least about 300 μm, at least about 400 μm, at least about 500 μm, at least about 600 μm, extremely It is few about 800 μm etc..The cross-sectional dimension of the second channel can be with the first passage cross-sectional dimension phase It is same or different.

The first passage can include main channel and multiple secondary channels.The secondary channel can multiple crossover locations with The second channel intersects.Flow the aqueous fluids from the main channel of the first passage and may pass through one or more secondary channels, To enter non-aqueous fluid contained in the second channel.The aqueous fluids can make substantially with the non-aqueous fluid It is immiscible, and therefore can form the droplet for the aqueous fluids being contained in non-aqueous fluid.In some embodiments, The secondary channel is substantially of similar shape or size, and/or its cross-sectional area is substantially less than main channel or The cross-sectional area of two passages so that mainly influenceed on the resistance (resistance) of fluid stream by the size of secondary channel, This may be such that the droplet to be formed is substantially monodispersed.

In some embodiments, during mean resistance of the secondary channel to fluid stream is first passage and/or second channel Resistance it is at least about 3 times high, it is at least about 10 times high for example, at least about 5 times are high, it is at least about 20 times high, it is at least about 30 times high, It is at least about 50 times high, it is at least about 75 times high, it is at least about 100 times high, it is at least about 200 times high, it is at least about 300 times high, at least about 500 It is high again, or at least about 1, it is 000 times high.In some embodiments, mean resistance of the secondary channel to fluid stream is no more than first Resistance in passage and/or second channel it is about 1,000 times high or 500 times high.The secondary channel can have substantially the same Mean resistance.For example, mean resistance of the secondary channel to fluid stream can be averaged in all secondary channels to fluid stream About the 75% to about 125% of resistance, about 80% to about 120%, about 90% to about 110%, about 95% to about 105%, about 97% Change in the range of to about 103% or about 99% to about 101%.

In some embodiments, using with relatively small cross-sectional area or relatively small minimum or maximum transversal The secondary channel of face size produces the high-drag to fluid stream.Or, it is in some embodiments, described transversal except controlling Outside face area or cross sectional dimensions, using other technologies produce high-drag, for example the secondary channel is coated and/or Build relatively sinuous secondary channel.Correspondingly, the secondary channel can be to be substantially straight, or the secondary channel can be wrapped Containing one or more bendings or bending etc..If there is multiple secondary channels, these secondary channels can each have identical or not Same shape.For example, part or all of secondary channel can make it is substantially straight.Appoint in addition, the secondary channel can have What suitable shape of cross section, such as circle, annular, ellipse, triangle, irregular shape, square or rectangle.Described time Level passage can independently have identical or different shape of cross section.The shape of cross section of the secondary channel can be with the master The shape of cross section of passage and/or second channel is identical or different.

The secondary channel can have any suitable cross-sectional dimension.Cross-sectional dimension is often referred to accommodate Full-size in the cross section of the secondary channel, wherein the cross section refers to the mean square with fluid stream in secondary channel To orthogonal face.For example, the cross-sectional dimension can be no more than 1mm, and no more than about 800 μm, no more than about 600 μm, No more than about 500 μm, no more than about 400 μm, no more than about 300 μm, no more than about 250 μm, no more than about 200 μm, it is no more than About 100 μm, no more than about 75 μm, no more than about 50 μm, no more than about no more than about 25 μm, 10 μm etc..For example, described maximum horizontal Sectional dimension can be at least about 5 μm, at least about 10 μm, at least about 25 μm, at least about 50 μm, at least about at least about 75 μm, 100 μ M, at least about 200 μm, at least about 250 μm, at least about 300 μm, at least about 400 μm, at least about 500 μm, at least about 600 μm, extremely It is few about 800 μm etc..The height of the secondary channel can be identical or different with the height of described first or second channel.

In some embodiments, cross sectional dimensions minimum in secondary channel is to the ratio of maximum cross sectional dimensions At least about 1:1.1, at least about 1:1.5, at least about 1:2, at least about 1:3, at least about 1:5, at least about 1:7, at least about 1:10, extremely Few about 1:15, at least about 1:20, at least about 1:25, at least about 1:30, at least about 1:35, at least about 1:40, at least about 1:50, extremely Few about 1:60, at least about 1:70, at least about 1:80, at least about 1:90, at least about 1:100 etc..For example, the ratio is can be for not More than about 1:100, no more than about 1:90, no more than about 1:80, no more than about 1:70, no more than about 1:60, no more than about 1:50, No more than about 1:40, no more than about 1:35, no more than about 1:30, no more than about 1:25, no more than about 1:20, no more than about 1: 15, no more than about 1:10, no more than about 1:7, no more than about 1:5, no more than about 1:3, no more than about 1:2, no more than about 1:1.5 Deng.

The secondary channel can have any suitable length.In some embodiments, the length of the secondary channel Determined by the distance between main channel and second channel.For example, the average length of the secondary channel can be at least about 10 μm, extremely It is few about 20 μm, at least about 30 μm, at least about 50 μm, at least about 100 μm, at least about 200 μm, at least about at least about 300 μm, 500 μ M, at least about 1,000 μm, or at least about 2,000 μm etc..For example, the length of the secondary channel can be no more than about 2,000 μm, No more than about 1,000 μm, no more than about 500 μm, no more than about 300 μm, no more than about 200 μm, no more than about 100 μm, do not surpass Cross about 50 μm, no more than about 30 μm, no more than about 20 μm, or no more than about 10 μm.The length of the secondary channel can be basic Upper identical, or the length can all secondary channels average length (or the spacing of the first passage and second channel From) about 75% to about 125%, about 80% to about 120%, about 90% to about 110%, about 95% to about 105%, about 97% to Change in the range of about 103% or about 99% to about 101%.

The cross-sectional area of the secondary channel can be average at least about 20 μm², at least about 30 μm², at least about 50 μm², extremely It is few about 75 μm², at least about 100 μm², at least about 300 μm², at least about 400 μm², at least about 500 μm², at least about 750 μm², at least About 1,000 μm², at least about 1,600 μm², at least about 2,000 μm², at least about 3,000 μm², at least about 4,000 μm², at least about 5, 000μm², at least about 6,000 μm², at least about 6,400 μm², at least about 7,000 μm², at least about 8,000 μm², at least about 9,000 μ m², at least about 10,000 μm²Deng.The cross-sectional area of the secondary channel can be averagely no more than about 10,000 μm², it is no more than About 9,000 μm², no more than about 8,000 μm², no more than about 7,000 μm², no more than about 6,400 μm², no more than about 6,000 μm², No more than about 6,000 μm², no more than about 5,000 μm², no more than about 4,000 μm², no more than about 3,000 μm², no more than about 2, 000μm², no more than about 1,600 μm², no more than about 1,000 μm², no more than about 750 μm², no more than about 500 μm², no more than about 400μm², no more than about 300 μm², no more than about 100 μm², no more than about 75 μm², no more than about 50 μm², no more than about 30 μm², No more than about 20 μm²Deng.

In some embodiments, the cross-sectional area of the secondary channel can all secondary channels average cross-section About the 75% to about 125% of area, about 80% to about 120%, about 90% to about 110%, about 95% to about 105%, about 97% Change in the range of to about 103% or about 99% to about 101%.The cross-sectional area of the secondary channel can be substantial constant Or be varied from.For example, the cross-sectional area of the secondary channel can be varied from along the length direction of passage.Such as institute The cross-sectional area for stating secondary channel can be in average cross sectional area about 75% to about 125%, about 80% to about 120%, about 90% to about 110%, about 95% to about 105%, change in the range of about 97% to about 103% or about 99% to about 101%.

In some embodiments, the volume of the secondary channel can be in the volume of all secondary channels about 75% to about 125%, about 80% to about 120%, about 90% to about 110%, about 95% to about 105%, about 97% to about 103% or about 99% Change in the range of to about 101%.

In some embodiments, the cross-sectional area of the main channel and/or the second channel is described secondary logical It is at least about 10 times of minimum cross-sectional area high in the cross-sectional area in road, it is for example, at least about 15 times high, it is at least about 20 times high, It is at least about 30 times high, it is at least about 40 times high, it is at least about 50 times high, it is at least about 75 times high, it is at least about 100 times high, at least about 200 times Height, at least about 300 times high, at least about 500 times high, at least about 1,000 times high, at least about 2,000 times high, at least about 3,000 times Height, or at least about 5, it is 000 times high.In some embodiments, the cross-sectional area of the main channel and/or the second channel Be the secondary channel cross-sectional area in minimum cross-sectional area it is no more than about 5,000 times high, no more than about 3,000 It is high again, no more than about 2, it is 000 times high, no more than about 1, it is 000 times high, it is no more than about 500 times high, it is no more than about 300 times high, do not surpass Cross about 200 times high, it is no more than about 100 times high, it is no more than about 75 times high, it is no more than about 50 times high, it is no more than about 40 times high, do not surpass Cross about 30 times high, or it is no more than about 20 times high.

Any appropriate number of secondary channel may be present.In some embodiments, substantial amounts of secondary channel can be used for Higher rate produces droplet.If in addition, secondary channel to the resistance of fluid stream relative to first and/or second channel convection current The resistance of body stream is relatively large, then greater number of secondary channel may not substantially influence droplet produce speed and/or The monodispersity of droplet.Therefore, in some embodiments, there is connection main channel and the second channel of of a relatively high quantity Secondary channel.In some embodiments, have at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 800, at least 1,000, At least 1,200, at least 1,500, at least 2,000, at least 2,500 connection main channels and second channel secondary channel.

The multiple secondary channel can be into any suitable angle relative to main channel and/or second channel.For example, secondary Level passage can be into about 90 ° of intersecting angle relative to main channel and/or second channel.Each secondary channel can be with substantially Identical angle intersects with the main channel and/or second channel, or the intersecting angle can be independently identical or different 's.In addition, the intersecting angle between main channel and second channel can also be identical or different.In some embodiments, The secondary channel it is each with the main channel and/or second channel into about 45 ° to about 135 °, about 45 ° to about 100 °, about 70 ° to about 100 °, about 80 ° to about 100 °, about 85 ° to about 95 °, about 88 ° to about 92 ° of angle.In some embodiments, it is secondary Level passage and main channel and/or second channel are at least about 10 °, about 15 °, about 20 °, about 25 °, about 30 °, about 35 °, about 40 °, about 45 °, about 50 °, about 55 °, about 60 °, about 65 °, about 70 °, about 75 °, about 80 °, about 85 °, about 90 °, about 95 °, about 100 °, about 105 °, about 110 °, about 115 °, about 120 °, about 125 °, about 130 °, about 135 °, about 140 °, about 145 °, about 150 °, about 155 °, about 160 °, about 165 °, the angle (e.g., from about 90 ° to about 170 °) of the scope between about 170 ° of angle, or these any numerical value.

The secondary channel can be arranged between main channel and second channel with any suitable configuration.For example, secondary Passage can be equidistant arrangement so that the distance between neighboring secondary passage is substantially the same, or in all adjacency channels About the 75% to about 125% of average distance, about 80% to about 120%, about 90% to about 110%, about 95% to about 105%, about Change in the range of 97% to about 103% or about 99% to about 101%.When the cross-sectional area of secondary channel is substantial constant When, the spacing between secondary channel can be used for the size for determining droplet.

Chip can include multigroup first passage, second channel and multiple crossover locations.For example, the chip can include 2,3, 4,5,6,7,8,9,10 or more first passages and/or second channel.Each first passage can comprising main channel and one or Multiple secondary channels.

The quantity of crossover location can be 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more.Crossover location can be logical first Between the main channel in road and second channel, between the secondary channel and second channel of first passage, and/or first passage secondary Formed between passage and main channel.Multiple crossover locations can be arranged linearly or nonlinearly.For example, multiple crossover locations can be arranged Configured for two-dimensional array.

The subregion of the present invention can be the separate section of first fluid (such as aqueous fluids), and it has second fluid (example completely Such as non-aqueous fluid) surround.In some embodiments, subregion is droplet.Subregion (such as droplet) can be any shape Shape, and be not necessarily spherical.In aspherical subregion, its a diameter of and described aspherical droplet has same volume The diameter of perfect mathematics spheroid.

The present invention multiple subregions can first fluid (such as aqueous fluids) a part it is (such as non-by second fluid Aqueous fluids) around when formed.As it is used herein, enclosed when closure can be drawn around first fluid, and the closure circle only leads to When crossing second fluid, it is believed that a part for first fluid " is surrounded " by second fluid.It is first-class when that can surround in any direction Body draw closure circle, and the closure circle only by second fluid when, it is believed that a part for first fluid " is enclosed completely by second fluid Around ".When can be dependent on direction around first fluid draw closure circle, and the closure circle only by second fluid when, it is believed that first A part for fluid is by second fluid " substantially around ".

As described herein, the multiple subregion can be in the main channel of first passage, second channel and/or first passage Formed at the one or more crossover locations formed between secondary channel.It is multiple that the multiple subregion can be formed from them Expel or deflect from by one or more power at crossover location, for example, putting on the one or more imports and/or outlet of passage Power.The power such as can apply as pump, gravity, capillarity, surface tension, electro-osmosis, centrifugal force, by subregion from it Expel and/or deflect from multiple crossover locations for being formed.Vacuum be it is also possible to use (such as by vavuum pump or other are suitable Vacuum source).The non-limiting example of pump includes syringe pump, peristaltic pump, pressure stream pump etc..In some embodiments, it is the multiple Subregion can be formed between the main channel by first passage and multiple secondary channels of first passage.In some embodiments, The multiple passage is located at the same side of the first passage.

The average-size of subregion (such as droplet) may depend on one or more properties (such as flow velocity, viscosity) of fluid And/or the size of chip, (such as spacing, passage between the length and width of passage, adjacency channel exist for configuration or geometry Pore size of crossover location etc.).

The chip of the present invention can include passage and multiple crossover locations.One or more passages are in some embodiments Microfluidic channel.In some embodiments, at least part passage is not microfluidic device.

The chip can include any amount of passage, including microfluidic channel.The passage can be arranged as any suitable Configuration.All passages can homogeneously connect, or the network of more than one passage may be present.Passage can be independently straight , bending, bending etc..In some embodiments, in chip passage (when being added together) is micro- with least about 100 Rice, at least about 300 microns, at least about 500 microns, at least about 1mm, at least about 3mm, at least about 5mm, at least about 10mm, at least About 30mm, at least 50mm, at least about 100mm, at least about 300mm, at least about 500mm, at least about 1m, at least about 2m, or at least About 3m total length.In some embodiments, chip of the invention has at least two passage, at least three passage, at least four Passage, at least five passage, at least ten passage, at least 20 passages, at least 30 passages, at least 40 passages, at least 50 Passage, at least 60 passages, at least 70 passages, at least 80 passages, at least 90 passages, at least 100 passages etc..

In some embodiments, at least some of the passage in the chip are microfluidic channels.For example, the chip In fluid in all or part of cross-sectional dimension be less than about 2mm, in some cases, less than about 1mm.One In the case of a little, all fluid passages in chip are microfluidic channels.In some embodiments, passage in the chip Cross-sectional dimension is less than about 500 microns, less than about 200 microns, less than about 100 microns, less than about 50 microns, or is less than About 25 microns.

Passage in chip can be arranged with any suitable configuration.Different passages can be used to arrange manipulate fluid, Other key elements in subregion (such as droplet) and/or passage.For example, the passage in device can be configured to generation subregion (for example Discrete droplet, single emulsion, double emulsion, multiple emulsion etc.), fluid and/or subregion or other key elements are mixed, screening Or sort fluid and/or subregion or other key elements, fluid and/or subregion are separated, cause reaction occur (such as two fluids it Between, or between the material and second fluid of first fluid carrying, or the reaction between two kinds of materials of two fluids carryings) etc. Deng.

Fluid can be delivered to the passage in chip via one or more fluid sources.Any suitable fluid can be used Source, and in some embodiments, more than one fluid source can be used.Pump, gravity, capillarity, surface tension, electricity can be used The fluid of the fluid source in future such as infiltration, centrifugal force is delivered into one or more of chip passage.It it is also possible to use vacuum (for example By vavuum pump or other suitable vacuum sources).The non-limiting example of pump includes syringe pump, peristaltic pump, pressure stream pump etc..Fluid Source can be the bank comprising corresponding fluids, and the bank can be with one or more of chip passage fluid communication.Bank can Comprising one or more outlets, and the passage in chip can include one or more entrances.The outlet and entrance can flow each other Body UNICOM.In some cases, hydrodynamic resistance device (hydrodynamic can be set at bank outlet and/or feeder connection Resistor), fluid stream is controlled such as valve, filter, sieve, coiled pipe.The hydrodynamic resistance device can be comprising total length 0.1mm, 0.5mm, 1mm, 1.5mm, 2mm, 2.5mm snakelike tube passage.The snakelike tube passage can turn back repeatedly, for example, 2, 3,4,5,6,7,8 times or more times.

The chip can have associated any amount of fluid source, such as 1,2,3,4,5,6,7,8,9,10, Or more fluid source.The fluid source is without fluid is delivered into identical passage.Different fluid sources can deliver fluid Enter different passages.For example, can deliver aqueous fluids into first passage, and non-aqueous fluid is delivered into second channel.

The invention further relates to after the multiple zone configuration is in collecting zone, detected simultaneously in the multiple subregion Indicate the signal of the amplified production presence or absence.The collecting zone can be in the downstream of multiple crossover locations.In some implementations In scheme, detected simultaneously in all subregions in the multiple subregion or partial-partition indicate the amplified production exist with No signal.In some cases, the collecting zone includes multiple intervals, therefore can be in the multiple interval given area part Detect the signal simultaneously.

The method of the present invention also includes guiding the multiple subregion to the collecting zone.Third channel for example can be used The multiple subregion is guided to the collecting zone from multiple crossover locations.The diameter of the third channel can be more than described many The cross sectional dimensions of each in individual subregion.

One or more first passages and/or second channel can be connected by the third channel with collecting zone.Herein In description and restriction to first passage and/or second channel, unless clearly contradicted, be otherwise applied to the third channel.

The third channel can join with one or more first passages, second channel, and/or the collecting zone fluid It is logical.

The collecting zone may be included in the chip.Or, the collecting zone can be separated with the chip.Work as receipts When collection region is not included in the chip, it can be with the chip fluid UNICOM.Can by multiple crossover locations formed it is multiple One or more passages that subregion is included by the chip and/or the collecting zone are guided to the collecting zone.

The collecting zone can have suitable shape and/or configuration.For example, the collecting zone can be substantially planar 's.In one embodiment, the collecting zone tool curved surface.In some embodiments, the collecting zone can be annular 's.In some embodiments, the collecting zone is inclined.The collecting zone can be separated with chip.The collecting region Domain can be rotatable.

In some embodiments, it can be included in the side of rotatable supporting construction (such as rotatable symmetrical disk) Multiple chips.Each chip in the multiple chip can be comprising passage (such as first passage and second channel) for generation institute State multiple subregions and collecting zone.In some embodiments, the rotatable supporting construction includes multiple collecting zone (examples The collecting zone such as separated with chip).The rotation of the supporting construction can be driven by the motor that can adjust the speed of rotation.It is described many Individual chip or multiple collecting zones can be placed relative to the Central Symmetry of the supporting construction.In some embodiments, it is described Multiple chips or the insertion of the multiple collecting zone are integrated into the rotatable support structure.

May be sized to of the collecting zone accommodates the multiple subregion in simple layer.For example, the collecting region The mode of the stacking that may be sized to avoid or do not have or hardly have the multiple subregion in domain.In some embodiment party In case, the collecting zone is surrounded by two layers of parallel plane, and two layers of parallel planes bound collecting zone is flat Equal distance.In some embodiments, the height of the collecting zone is the average diameter of the subregion about or less than about generation.Example Such as, the height of the collecting zone may be less than about 2000 μm, less than about 1000 μm, small less than about 500 μm less than about 750 μm In about 400 μm, less than about 300 μm, less than about 200 μm, less than about 100 μm, less than about 90 μm, less than about 80 μm, less than about 70 μ M, less than about 60 μm, less than about 50 μm, less than about 45 μm, less than about 40 μm, less than about 35 μm, less than about 30 μm, less than about 25 μ M, less than about 20 μm, less than about 15 μm, less than about 10 μm, less than about 5 μm, less than about 1 μm, less than about 0.1 μm, less than about 0.01 μm or it is smaller.The diameter of the collecting zone (or when applied, the plane that the collecting zone is included) can be at least or about 0.01 μm, 0.1 μm, 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 150 μ M, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1mm, 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, 9mm, 10mm, or it is bigger.However, in some cases, the collecting region The size in domain also may be configured as accommodating the multiple subregion in multiple layers.

The collecting zone can include hole, and being dimensioned so as to of the hole accommodates the single subregion in the multiple subregion. The size (such as width, length, depth) in each hole can be the average diameter less than the given subregion in the multiple subregion.Example Such as, the size in each hole is smaller than about 500 μm, less than about 400 μm, less than about 300 μm, less than about 200 μm, less than about 100 μm, Less than about 90 μm, less than about 80 μm, less than about 70 μm, less than about 60 μm, less than about 50 μm, less than about 45 μm, less than about 40 μm, Less than about 35 μm, less than about 30 μm, less than about 25 μm, less than about 20 μm, less than about 15 μm, or less than about 10 μm, or it is smaller.

Hole can accommodate whole droplet, or single droplet a part.Or, hole can accommodate multiple droplets, such as extremely Few 2,3,4,5 or 10 droplets, or more.

In some embodiments, in collecting zone, each region in the multiple subregion is in addressable alone Position.For example, each subregion in the multiple subregion may correspond to structure or the space limited, these structures and space are with energy Enough identifications are present in the structure of any such restriction or the mode of the subregion in space is encoded, arranges or arranged.In some realities Apply in scheme, the structure of restriction or space can be the hole for being sized to accommodate single subregion.

Can be used for the chip is that outside source of thermal power is followed to each subregion progress heat in the multiple subregion Ring.For example, the source of thermal power can be infrared power source.In some embodiments, it can be used and be incorporated into the chip Source of thermal power carries out thermal cycle to each subregion in the multiple subregion.For example, the source of thermal power be thermoelectric element (for example Peltier elements) or stratie.Or, the source of thermal power can be inductive heating element.

In some embodiments, thermoelectric element (such as Peltier elements) attaches to the side (example of the collecting zone Such as downside).The thermoelectric element can be in close contact with the collecting zone, and its area can be as big as at least whole collecting region of covering Domain.

In some embodiments, the side (such as bottom side) of the collecting zone can be prepared by heat absorption material, described Material can convert light energy into heat.For example, the collecting zone with can be by by the relative side (such as top side) of heat absorption material The material (such as transparent material) for being transmissive to light is made.Therefore, light is (such as from the infrared lamp above the collecting zone The light of transmitting) side is may pass through, and available for the temperature (for example, changing the temperature in subregion) for improving collecting zone.

In some embodiments, in collecting zone can comprising one or more temperature sensors with monitoring temperature (for example Real-time monitoring temperature).The temperature sensor can provide anti-to the control system of the control power source (such as described IR lamps) Feedforward information.After temperature sensor receive information, the control system can be compiled then by one or more independent or entirety Journey is controlled by the computer processor of the adjustment power source.

When multiple chips or control area are contained in or are integrated in the rotatable supporting construction, the power source (such as one or more infrared lamps) can be arranged such that the multiple chip or collecting zone with the rotation of the supporting construction Turn to be evenly exposed to the power source (such as light source), so that it is guaranteed that different chips or collecting zone in the supporting construction It is heated properly.

The chip can include one or more entrances and one or more outlets.Each entrance can with it is one or more Bank fluid communication.Bank can be filled with fluid (aqueous fluids or non-aqueous fluid), and the fluid is to be supplied in one or many The passage included in individual chip.Thick mouth can be located at an end for including passage in chip and/or collecting zone.One can be driven Or multiple subregions, fluid or waste flow through outlet and flow to and the bank for exporting fluid communication.In some embodiments, institute State entrance and/or outlet can be comprising elastic ring such as rubber ring or connector such as rubber tube with the inlet/outlet and institute State formation sealedly connection between bank.In some cases, first passage and/or second channel and the outlet can be used Between pressure drop the aqueous fluids and non-aqueous fluid are flowed.The pressure drop can be at least about 0.1psi, extremely Few about 0.5psi, at least about at least about 1psi, at least about 5psi, at least about 10psi, at least about 15psi, 20psi, at least about 30psi, at least about 40psi, at least about 50psi, at least about 60psi, at least about 70psi, at least about 80psi, at least about 90psi, at least about 100psi, at least about 150psi, at least about 200psi, at least about 250psi, at least about 300psi, at least about 350psi, at least about 400psi, at least about 450psi, at least about 500psi, at least about 750psi or bigger.The pressure drop can At most about 750psi, at most about 500psi, at most about 450psi, at most about 400psi, at most about 350psi, at most about 300psi, at most about 250psi, at most about 200psi, at most about 150psi, at most about 100psi, at most about 90psi, at most about 80psi, at most about 70psi, at most about 60psi, at most about 50psi, at most about 40psi, at most about 30psi, at most about 20psi, at most about 15psi, at most about 10psi, at most about 5psi, at most about 1psi, at most about 0.5psi, at most about 0.1psi Or it is smaller.In some embodiments, by controlling the import, the first passage and/or second channel and the outlet Between pressure drop substantial constant, generate it is multiple be substantially monodispersed subregions.

The chip and/or one or more banks can include filter or enriching apparatus and come from nucleic acid sample to remove Desired composition in the undesirable material of product, and/or the enrichment nucleic acid samples.Filter or enriching apparatus it is non-limiting Property example include filter membrane, such as nitrocellulose, cellulose acetate, makrolon, polypropylene and polyvinylidene fluoride micropore Film, and milipore filter (for example, those prepared by polysulfones, polyvinylidene fluoride, cellulose).In some embodiments, drive Dynamic stream is from the aqueous fluids and/or non-aqueous fluid of one or more of banks by one or more filters to enter State the entrance of chip.In some embodiments, permeate is collected in single bank.

In order to detect the signal for indicating the amplified production presence or absence, inspection of the present invention simultaneously in multiple subregions Survey can further comprise guiding excitation energy to the multiple subregion, and detect the signal launched from the multiple subregion.It is described The detector integrated with the chip can be used to detect for signal.In some cases, the chip exterior can be used in the signal Detector detection.For example, the detector can be charge coupling device (CCD) camera.

The excitation energy can be by providing with the excitation energy source that the chip is integrated.In some cases, it is described to excite Energy can be provided by the excitation energy source of the chip exterior.For example, the excitation energy source can be by light emitting diode or laser There is provided.The signal can be optical signalling (such as fluorescence signal), photochemistry signal, and/or electrostatic signal.In some embodiment party In case, provided in the side (such as collecting zone above) of the chip optical image capturing apparatus (such as CCD camera) and Adjoint fluorescence excitation light source.

In detection of the present invention, the flow velocity that the multiple subregion flows through the collecting zone is smaller than about 10ml/ H, for example, less than about 9ml/h, less than about 8ml/h, less than about 7ml/h, less than about 6ml/h, less than about 5ml/h, less than about 4ml/ H, less than about 3ml/h, less than about 2ml/h, less than about 1ml/h, less than about 0.5ml/h, 0.1ml/h, 0.01ml/h or smaller. In some embodiments, in detection of the present invention, the multiple subregion is substantially static.

The method of the present invention can further comprise after the detection has been carried out (for example, indicating described expand have detected After the signal for the presence or absence for increasing production thing), guide the multiple subregion to leave the collecting zone, flow direction outlet.It is described go out Mouth can be under negative pressure.In some cases, the negative pressure may be less than or about -5 bars, -4 bars, -3 bars, -2 bars, -1 bar, -0.9 Bar, -0.8 bar, -0.7 bar, -0.6 bar, -0.5 bar, -0.4 bar, -0.3 bar, -0.2 bar, -0.1 bar, -0.05 bar, -0.04 bar, - 0.03 bar, -0.02 bar, -0.01 bar, -0.005 bar, -0.001 bar, -0.0001 bar, or it is smaller.The first passage and/or Two passages can be in direct draught relative to the outlet.In some cases, first passage and/or second channel and institute can be used The pressure drop between outlet is stated so that the aqueous fluids, non-aqueous fluid and/or the subregion (such as droplet) flow It is dynamic.The pressure drop can be at least about 0.1psi, at least about at least about 0.5psi, at least about 1psi, at least about 5psi, 10psi, At least about 15psi, at least about 20psi, at least about 30psi, at least about 40psi, at least about 50psi, at least about 60psi, at least About 70psi, at least about 80psi, at least about 90psi, at least about 100psi, at least about 150psi, at least about 200psi, at least about 250psi, at least about 300psi, at least about 350psi, at least about 400psi, at least about 450psi, at least about 500psi, at least About 750psi or bigger.The pressure drop can be up to about 750psi, at most about at most about 500psi, 450psi, at most about 400psi, at most about 350psi, at most about 300psi, at most about 250psi, at most about 200psi, at most about 150psi, at most About 100psi, at most about 90psi, at most about 80psi, at most about 70psi, at most about 60psi, at most about 50psi, at most about 40psi, at most about 30psi, at most about 20psi, at most about 15psi, at most about 10psi, at most about 5psi, at most about 1psi, At most about 0.5psi, at most about 0.1psi or smaller.

The amplified production can be detected with least about 90% susceptibility.For example, the amplified production can be down to Few 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher susceptibility is detected.As it is used herein, susceptibility is often referred to correctly be accredited as the positive Signal accounts for measuring for positive signal ratio.

The amplified production can be gone out with least about 90% specific detection.For example, the amplified production can be down to Few 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher specific detection goes out.As it is used herein, specificity is often referred to correctly be accredited as feminine gender Signal accounts for measuring for negative signal ratio.

The chip or its component (for example, the passage, described collecting zone etc.), can use multiple material and method system It is standby.For example, the chip or its component can be formed from solid material, wherein the passage can pass through micro manufacturing, thin film deposition work Skill such as spin coating and chemical vapors deposition, physical vapor deposition, laser manufacture, photoetching technique, engraving method (including wet-chemical or are waited Ion processes), electro-deposition etc. formed.A variety of manufacturing process (such as soft lithographic, hot moulding, injection molding and laser ablation) can For producing chip and its component.

In some embodiments, the chip or its component are prepared by polymer.The polymer can be flexible polymer Thing such as dimethyl silicone polymer (" PDMS "), polytetrafluoroethylene (PTFE) (" PTFE " or) etc..For example, passage (such as miniflow Body passage) it can be realized by using the separately fabricated microfluid system of PDMS or other soft lithographies.Other potential conjunctions The example of suitable polymer includes but is not limited to PET (PET), polyacrylate, polymethylacrylic acid Ester, makrolon, polystyrene, polyethylene, polypropylene, polyvinyl chloride, cyclenes copolymer (COC), polytetrafluoroethylene (PTFE), fluoro gather Compound, siloxanes (such as dimethyl silicone polymer), polyvinylidene chloride, dibenzo cyclobutane (BCB), polyimides, polyamides are sub- Fluoro derivatives of amine etc..In addition, combination, copolymer or the blend of above-mentioned polymer be may also include.

In some embodiments, the chip or its component are by polymeric material and/or flexible material and/or elastomeric material Prepare, and can be manufactured in order to isomorphism molding easily by hardenable fluid preparation (for example duplicating molded, injection molding, Cast molding etc.).The hardenable fluid substantially can be induced solidification to be any, or spontaneous solid turns to solid Fluid, the solid can accommodate or transport the fluid for fluid network.In one embodiment, the hardenable stream Body includes polymeric liquid or liquid polymer precursor (i.e. prepolymer).Suitable polymeric liquid includes, such as thermoplasticity Polymer, thermosetting polymer, wax, metal, or its mixture or compound, it is heated to more than fusing point.In some implementations In scheme, suitable polymeric liquid includes the solution of one or more polymer in a suitable solvent, and the solution is being gone Solid polymer is formed during except solvent (such as by evaporation).Such polymeric material can evaporate from molten condition or by solvent Solidification.There are a variety of suitable polymeric materials is used to form molding.The non-limiting example of such a polymeric material includes Siloxanes, epoxy polymer and polyacrylate.

The invention further relates to the method for amplification of nucleic acid.

In one aspect, it is present in directly from the target in the biological sample that subject obtains the invention provides a kind of amplification The method of ribonucleic acid (RNA).This method includes：(a) provide comprising biological sample and for expanding with DNA (DNA) Increase the reaction vessel of reagent necessary to abreast carrying out reverse transcription amplification, to obtain reactant mixture, the reagent is inverse comprising (i) Transcriptase, (ii) archaeal dna polymerase, and (iii) are directed to target RNA primer sets；And (b) passes through the reactant mixture in reaction vessel The primer extension reaction of multiple circulations is gone through, to generate the DNA amplification product for indicating there is target RNA, each circulation includes：(i) will Reactant mixture is incubated the denaturation duration less equal than 60 seconds under denaturation temperature, and subsequent (ii) exists reactant mixture The extension duration less equal than 60 seconds is incubated under elongating temperature, so as to expand target RNA.

On the other hand, it is present in directly from the target in the biological sample that subject obtains the invention provides a kind of amplification The method of ribonucleic acid (RNA).This method includes：(a) biological sample obtained from subject is received；(b) provide comprising life Thing sample and the reaction for expanding necessary reagent for progress reverse transcription amplification and optional DNA (DNA) are held Device, to obtain reactant mixture, the reagent is directed to target RNA primer sets comprising (i) reverse transcriptase and (ii)；(c) mix reaction Compound undergoes the primer extension reaction of multiple circulations, to produce the expansion for the detectable amount for indicating to exist in biological sample target RNA Increase DNA product；(d) amount of the DNA amplification product of detection (c)；And (e) by the information on the amount of DNA amplification product export to Recipient, wherein for completing (a)-(e) time quantum less than or equal to about 30 minutes.

In one aspect, it is present in the invention provides a kind of amplification from the target nucleic acid in the biological sample that subject obtains Method.This method includes：(a) provide comprising biological sample and the reaction of reagent necessary to for carrying out nucleic acid amplification is held Device, to obtain reactant mixture, the reagent includes (i) DNA (DNA) polymerase and optional reverse transcriptase, and (ii) primer sets of target nucleic acid are directed to；And (b) makes the multiple serial primer extends of reactant mixture experience in reaction vessel anti- It should indicate there is the amplified production of target nucleic acid in biological sample to generate, each series following is followed including two or more Ring：(i) the incubation reaction mixture under with denaturation temperature and the Denaturing that is characterized of denaturation duration, subsequent (ii) with Incubation reaction mixture under the conditions of the extension that elongating temperature and extension duration are characterized, wherein with regard to Denaturing and/or prolonging Stretch for condition, single series is different from least one other single series in the multiple series.

In one aspect, the invention provides a kind of method for being used to detect the target nucleic acid molecule in biological sample.It is described Method may include：A) biological sample is mixed to obtain mixture with lysis buffer；B) by the mixture higher than The incubation duration of no more than about 15 minutes is incubated under 15 DEG C of incubation temperature, or without being incubated；C) will be from b) it is mixed Compound is added to reaction vessel, and the reaction vessel includes the reagent needed for carrying out nucleic acid amplification, and the reagent includes：(i) take off Oxygen ribonucleic acid (DNA) polymerase, and (ii) are directed to the primer sets of the target nucleic acid molecule, to obtain reactant mixture；And D) reactant mixture in the reaction vessel is undergone the primer extension reaction of multiple circulations, indicated with generating described There is the amplified production of the target nucleic acid molecule in sample, each circulation includes：(i) reaction is incubated under denaturation temperature mixed Compound is less equal than denaturation duration of 60 seconds, then (ii) be incubated under elongating temperature the reactant mixture be less than or The extension duration equal to 60 seconds, so as to expand the target nucleic acid molecule.The amplified production can be DNA product.

In one aspect, the invention provides a kind of method for being used to detect the target nucleic acid molecule in biological sample.It is described Method may include：A) biological sample is mixed to obtain mixture with lysis buffer；B) by the mixture higher than The incubation duration of no more than about 15 minutes is incubated under 15 DEG C of incubation temperature, or without being incubated；C) will be from b) it is mixed Compound is added to reaction vessel, and the reaction vessel includes the reagent needed for carrying out nucleic acid amplification, and the reagent includes：(i) take off Oxygen ribonucleic acid (DNA) polymerase, and optionally, reverse transcriptase, and (ii) are directed to the primer sets of the target nucleic acid, it is anti-to obtain Answer mixture；And the reactant mixture in the reaction vessel is undergone multiple serial primer extension reactions, with Generation indicates there is the amplified production of the target nucleic acid in the sample, and each series following is followed including two or more Ring：(i) reactant mixture is incubated under the Denaturing being characterized with denaturation temperature and denaturation duration, subsequent (ii) The reactant mixture is incubated under the conditions of the extension being characterized with elongating temperature and extension duration, wherein the just denaturation For condition and/or the extension condition, single series is different from least one other single series in the multiple series. The biological sample can include fecal specimens.The biological sample can include milk sample.The amplified production can be DNA productions Thing.

Any aspect in many aspects of the present invention, " incubation temperature " can be greater than about 15 DEG C, for example, in height It is high in about 20 DEG C, greater than about 25 DEG C, greater than about 30 DEG C, greater than about 35 DEG C, greater than about 40 DEG C, greater than about 45 DEG C, greater than about 50 DEG C It is high in about 55 DEG C, greater than about 60 DEG C, greater than about 65 DEG C, greater than about 70 DEG C, greater than about 75 DEG C, greater than about 80 DEG C, greater than about 85 DEG C It is high in about 90 DEG C, greater than about 91 DEG C, greater than about 92 DEG C, greater than about 93 DEG C, greater than about 94 DEG C, greater than about 95 DEG C, greater than about 96 DEG C It is incubated at a temperature of about 97 DEG C, greater than about 98 DEG C, greater than about 99 DEG C, greater than about 100 DEG C, or, at about 20 DEG C to about Be incubated at a temperature of 90 DEG C, for example, about 25 DEG C to about 85 DEG C, about 30 DEG C to about 80 DEG C, about 40 DEG C to about 70 DEG C, about 40 DEG C to about 95 DEG C, about 45 DEG C to about 90 DEG C, about 50 DEG C to about 85 DEG C, about 55 DEG C to about 80 DEG C, about 60 DEG C to about 75 DEG C, about 65 DEG C To being incubated at a temperature of about 75 DEG C or about 65 DEG C to about 70 DEG C.

Any aspect in many aspects of the present invention, described " being incubated the duration " can be not more than 20 minutes.Example Such as, described " be incubated duration " can be not more than 19 minutes, not more than 18 minutes, not more than 17 minutes, not more than 16 minutes, Not more than 15 minutes, not more than 14 minutes, not more than 13 minutes, not more than 12 minutes, not more than not more than 11 minutes, 10 points Clock, not more than 9 minutes, not more than 8 minutes, not more than 7 minutes, not more than 6 minutes, not more than 5 minutes, not more than 4 minutes, no More than 3 minutes, not more than 2 minutes, not more than 1 minute, not more than 50 seconds, not more than not more than 40 seconds, 30 seconds, not more than 20 Second, not more than 15 seconds, or not more than 10 seconds.In some embodiments, the incubation duration can be zero, i.e., without incubating Educate.

Any aspect in many aspects of the present invention, the lysis buffer can comprising NaCl, PBS and/or HEPES.Conventional buffer solution may include but be not limited to about 0.9% sodium chloride solution, phosphate buffer, Lactated Lin Ge Family name's solution, acetylizad Ringer's solution, phosphate buffered saline (PBS), citrate buffer, sodium carbonate buffer, sodium acid carbonate Buffer solution, borate buffer solution, Tris buffer solutions, histidine buffering liquid, HEPES buffer solution, MOPOS buffer solutions, glycine delay Fliud flushing, L-Glycylglycine buffer solution etc..

Any aspect in many aspects of the present invention, it is being mixed by the biological sample with the lysis buffer Before, the biological sample can in the solution be suspended, to obtain the homogeneous prepared product for including the biological sample.The solution can For buffer suspension liquid.The buffer suspension liquid can include NaCl, PBS and/or HEPES.The buffer solution according to workable for the present invention May include but be not limited to about 0.9% sodium chloride solution, phosphate buffer, Lactated Ringer's solution, acetylizad woods Grignard solution, phosphate buffered saline (PBS), citrate buffer, sodium carbonate buffer, sodium bicarbonate buffer liquid, boric acid salt buffer Liquid, Tris buffer solutions, histidine buffering liquid, HEPES buffer solution, MOPOS buffer solutions, glycine buffer, the sweet ammonia of N- glycyl Acid buffer etc..

Any aspect in many aspects of the present invention, the homogeneous prepared product can be at a temperature of greater than about 40 DEG C It is incubated, for example, can be at greater than about 45 DEG C, greater than about 50 DEG C, greater than about 55 DEG C, greater than about 60 DEG C, greater than about 65 DEG C, greater than about 70 DEG C, greater than about 75 DEG C, greater than about 80 DEG C, greater than about 85 DEG C, greater than about 90 DEG C, greater than about 91 DEG C, greater than about 92 DEG C, greater than about 93 DEG C, greater than about 94 DEG C, greater than about 95 DEG C, greater than about 96 DEG C, greater than about 97 DEG C, greater than about 98 DEG C, greater than about 99 DEG C, greater than about It is incubated at a temperature of 100 DEG C.

Any aspect in many aspects of the present invention, the homogeneous prepared product can be incubated not more than 20 minutes, example Such as, not more than 19 minutes, not more than 18 minutes, not more than 17 minutes, not more than not more than 16 minutes, 15 minutes, not more than 14 Minute, not more than 13 minutes, not more than 12 minutes, not more than 11 minutes, not more than not more than 10 minutes, 9 minutes, not more than 8 Minute, not more than 7 minutes, not more than 6 minutes, not more than 5 minutes, not more than 4 minutes, not more than 3 minutes, not more than 2 minutes, Not more than 1 minute, not more than 50 seconds, not more than 40 seconds, not more than 30 seconds, not more than 20 seconds, not more than 15 seconds, or not more than 10 Second.

In some embodiments, can be to the life before the biological sample is mixed with the lysis buffer Thing sample is centrifuged, to obtain the supernatant and precipitation comprising biological sample.In some embodiments, in the biological sample Before product are mixed with the lysis buffer, the biological sample can be centrifuged, to obtain supernatant and comprising biological sample The precipitation of product.

In some embodiments, can be to institute after the mixture of the biological sample and lysis buffer is incubated Mixture is stated to be centrifuged to obtain supernatant.The supernatant can include biological sample.Then, the supernatant can be used as Mixture in subsequent reactions.For example, the supernatant can be added to comprising for carrying out the reagent needed for nucleic acid amplification In reaction vessel.

In some cases, before the primer extension reaction is carried out, can make the target nucleic acid molecule undergo one or Multiple Denaturings.One or more of Denaturings may be selected from denaturation temperature distribution and denaturant.

In some cases, can be by the biological sample at 90 DEG C to 100 DEG C before the primer extension reaction is carried out Preheating temperature under preheating be not more than the pre- thermal endurance of 10 minutes.In some embodiments, the pre- thermal endurance Not more than 1 minute.

Any aspect in many aspects of the present invention, can be in the case where not carrying out DNA or RNA extractions by the life The mixture of thing sample and lysis buffer is added to reaction vessel, and the reaction vessel includes the examination needed for carrying out nucleic acid amplification Agent.In some cases, can in the case where not purified by the mixture be added to the reaction vessel.In some feelings Under condition, the mixture is added to the reaction vessel under conditions of can concentrating or be enriched with not carrying out DNA or RNA.

Any aspect in many aspects of the present invention, has expanded the nucleic acid from the biological sample derived from subject. In some embodiments, the biological sample can be obtained directly from source.For example, the biological sample can be directly from it Source is obtained, and without preculture, non-selective enrichment, selective enrichment, be plated on differentiation culture medium, and/or the life of expected property Thing medical verification." preculture " is often referred to before the method for the present invention is carried out, to one or more target kind (examples in sample Such as microorganism) expanded, or increase the method for its quantity." non-selective enrichment " is often referred to non-selectively in population mixture The method of the middle all or most of species (such as microorganism) of increase." selective enrichment " is often referred in population mixture increase by one The ratio and/or amount of kind or a variety of particular species (such as microorganism), and suppress the method for other species.This suppression can be Due to the selective toxicity of medium component such as compound, and organism by the use of medium component when be used as microbial metabolism End-product and the selective toxicity of material produced." differentiation culture medium " is often referred to include the indicant of one or more additions, Allow to distinguish the culture medium of the specified chemical reaction occurred in growth course." it is expected that the identification of property biomedicine " is often referred to Microorganism is made based on the observation of growth, Gram's staining result etc. to colony characteristicses, on primary isolation medium The Preliminary Identification gone out.

In some embodiments, the biological sample increases bacterium through culture.For example, before being mixed with lysis buffer, Biological sample can be made to undergo the culture duration under the conditions of enrichment culture.The enrichment culture condition may include suitably training Support base (such as Tryptic Soy nutrient solution (TBS), modified Tryptic Soy nutrient solution, tryptone, nutrient medium, bacteriolyze culture Liquid (LB), Gram-negative nutrient solution, peptone, Tryptic Soy nutrient solution or salmonella culture medium containing yeast) in close At suitable temperature (for example, about 23 DEG C to about 40 DEG C, such as about 25 DEG C, about 30 DEG C, about 35 DEG C or about 37 DEG C etc.) vibration or not Biological sample is cultivated under conditions of vibration.In some embodiments, the culture medium is salmonella culture medium, its relative to Other bacteriums, are more beneficial for salmonella propagation.Exemplary salmonella culture medium includes bismuth sulfite agar (BS), xylose Lysine deoxycholic acid agar (XLD), selenium bright green sulfanilamide (SN) nutrient solution (SBG) etc., but not limited to this.When the culture continues Between can be about 0.5 hour to 10 hours, for example, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, about 6 hours, about 6.5 hours, about 7 hours, it is about 7.5 small When, about 8 hours, about 8.5 hours, about 9 hours, about 9.5 hours or about 10 hours.In some embodiments, the culture is held The continuous time is no more than about 7 hours, for example, no more than about 6.5 hours, no more than about 6 hours, no more than about 5.5 hours, do not surpass Cross about 5 hours, be no more than about 4.5 hours, be no more than about 4 hours, be no more than about 3.5 hours, be no more than about 3 hours, be no more than About 2.5 hours, no more than about 2 hours, no more than about 1.5 hours, no more than about 1 hour or no more than about 0.5 hour.

In some embodiments, before the biological sample experienced the culture duration under the conditions of enrichment culture And/or afterwards, the biological sample can be centrifuged, to obtain the supernatant and precipitation comprising biological sample.In some realities Apply in scheme, the biological sample was experienced under the conditions of enrichment culture before or after the culture duration, can be to described Biological sample is centrifuged, to obtain supernatant and precipitation comprising biological sample.

In some embodiments, the biological sample experienced under the conditions of enrichment culture culture the duration it Afterwards, the biological sample can be mixed with lysis buffer, and do not suffer from selective enrichment, be plated on differentiation culture medium, and/or It is expected that the biomedical identification of property.

In some embodiments, the biological sample experienced under the conditions of enrichment culture culture the duration it Afterwards, lysis buffer can be added to the mixture.The lysis buffer can be alkaline.For example, the cracking buffering Liquid can include NaOH.The pH of the lysis buffer can be about 7 to about 14, such as about 8 to about 13, about 9 to about 12, about 10 to about 11.For example, the pH of the lysis buffer can be about 7,7.5,8,8.5,9,9.5,10,10.5,11,11.5,12,12.5,13, 13.5 or 14.

Any aspect in the multiple aspect, the present invention relates to obtain biological sample from subject.In certain situation Under, the biological sample is directly obtained from subject.The biological sample directly obtained from subject is often referred to such biological sample： It does not enter after subject obtains in addition to any means further handled for gathering biological sample from subject Row further processing.For example, directly obtaining blood from subject by following steps：Into the circulatory system of subject, from by Blood (for example, by pin) is taken out in examination person, and the blood of taking-up is entered in storage.The storage can be comprising reagent (for example, anti- Coagulant), to cause blood sample to can be used for further analysis.In another example, swab can be used to obtain subject's Epithelial cell on oralpharyngeal surfaces.In another example, swab can be used to obtain the fecal specimens of subject.Obtained from subject Obtain after biological sample, the swab containing biological sample can be made to be contacted with fluid (for example, buffer solution), to collect biological from swab Fluid.

In some embodiments, the biological sample is fecal specimens.In some embodiments, the fecal specimens For solid manure sample.In some embodiments, the fecal specimens are liquid manure sample.In some embodiments, The solid-like fecal specimens can be suspended in appropriate buffer solution, as suspension fecal specimens.In some embodiments, institute It can be watery diarrhea thing to state liquid fecal specimens.

In some embodiments, the biological sample is obtained by swab.For example, the available swab scraping solid excrement Just sample surfaces, to obtain fecal specimens.Or, the swab can be immersed liquid manure sample or suspension fecal specimens, with Obtain fecal specimens.The swab can be sterile swab.The swab can be sterile flocking swab.

Also fecal specimens can be obtained with other methods, for example, pipette, microsyringe, syringe, pipette can be used Pipe, pipettor etc. obtain liquid manure sample or suspension fecal specimens.For example, small spoon, liquid relief point, tweezers etc. can be used to obtain Solid manure sample.

Any aspect in many aspects of the present invention, the weight of the biological sample can be about 50mg to about 5g, example Such as, the weight of the biological sample can be about 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg、500mg、550mg、600mg、650mg、700mg、750mg、800mg、850mg、900mg、950mg、1.0g、1.1g、 1.2g, 1.3g, 1.4g, 1.5g, 1.6g, 1.7g, 1.8g, 1.9g, 2.0g, 2.5g, 3.0g, 3.5g, 4.0g, 4.5g or 5.0g, Or can be any value or scope between any of the above-described numerical value.

Any aspect in many aspects of the present invention, the volume of liquid biological sample or the suspension biological sample can It is about 50 μ l to about 5ml, for example, the volume of the biological sample can be about 50 μ l, 100 μ l, 150 μ l, 200 μ l, 250 μ l, 300 μl、350μl、400μl、450μl、500μl、550μl、600μl、650μl、700μl、750μl、800μl、850μl、900μl、 950μl、1.0ml、1.1ml、1.2ml、1.3ml、1.4ml、1.5ml、1.6ml、1.7ml、1.8ml、1.9ml、2.0ml、 2.5ml, 3.0ml, 3.5ml, 4.0ml, 4.5ml or 5.0ml, or can be any value or scope between any of the above-described numerical value.

In some embodiments, biological sample is not yet purified when being provided in reaction vessel.In some embodiments In, when biological sample is provided into reaction vessel, the nucleic acid of biological sample is not yet extracted.Biological sample is provided for example, working as During into reaction vessel, RNA or DNA in biological sample may be extracted not from biological sample.In addition, in some embodiments In, before biological sample is provided into reaction vessel, it is present in the target nucleic acid in biological sample (for example, target RNA or target DNA) may be not concentrated.

The present invention many aspects any one in, the mixture of the biological sample and the lysis buffer can be Do not undergo under conditions of DNA or ribonucleic acid (RNA) are extracted and be added in reaction vessel.In some cases, the mixture It can be added under conditions of not undergoing purifying in reaction vessel.In some cases, the mixture can not undergo DNA or It is added under conditions of RNA concentrations in reaction vessel.The reaction vessel can be to include the examination needed for for carrying out nucleic acid amplification The reaction vessel of agent.

For example, the mixture of the biological sample and the lysis buffer is incubated as described in other parts herein Afterwards, can be not purified added to reaction vessel by the mixture.For example, can be by the mixture without DNA or ribose core Sour (RNA) is extracted and is added to reaction vessel.For example, the mixture can be concentrated without DNA or ribonucleic acid (RNA) and added Add to reaction vessel.The reaction vessel can be to include the reaction vessel of the reagent needed for for carrying out nucleic acid amplification.

For example, after supernatant can be produced mixture specifically described herein is centrifuged, by the supernatant not It is purified to be added to reaction vessel.For example, after supernatant can be produced mixture specifically described herein is centrifuged, will The supernatant extracts without DNA or ribonucleic acid (RNA) and is added to reaction vessel.For example, can be will be specifically described herein Mixture is centrifuged after producing supernatant, the supernatant to be concentrated without DNA or ribonucleic acid (RNA) and is added to Reaction vessel.The reaction vessel can be to include the reaction vessel of the reagent needed for for carrying out nucleic acid amplification.

Any suitable biological sample comprising nucleic acid can be obtained from subject.Biological sample can be solid matter (example Such as, biological tissue), or can be fluid (for example, biofluid).Generally, biofluid may include related to live organism Any fluid.The non-limiting examples of biological sample include any anatomical location from subject (for example, tissue, circulation System, marrow) obtain blood (or the composition of blood-for example, leucocyte, red blood cell, blood platelet), from any of subject Cell that anatomical location is obtained, skin, heart, lung, kidney, expiratory air, marrow, excrement, seminal fluid, vaginal secretion, from swollen It is the tissue fluid of tumor tissue, mammary gland, pancreas, cerebrospinal fluid, tissue, brush,throat, biopsy article, placental fluids, amniotic fluid, liver, muscle, flat Sliding flesh, bladder, gall-bladder, colon, intestines, brain, chamber liquid, phlegm, purulence, micropopulation (microbiota), meconium, milk, prostate, food Road, thyroid gland, serum, saliva, urine, gastric juice and digestive juice, tear, ocular fluids, sweat, mucus, earwax, oil, body of gland point Secretion, spinal fluid, hair, nail, Skin Cell, blood plasma, nose swab or nasopharynx washing lotion, spinal fluid, Cord blood, lymph (lymphatic fluid) and/or other excretas or bodily tissue.

In other cases, the biological sample may be from soil or food.For example, the foodstuff samples can be dairy products Sample.In some cases, the dairy products sample may include milk.

From subject biological sample can be obtained by a variety of methods.Means for directly obtaining biological sample from subject Non-limiting examples include：Into the circulatory system (for example, through syringe or other pins be intravenous or intra-arterial entrance), collect The biological sample (for example, excrement, urine, phlegm, saliva etc.) of secretion, surgical operation (for example, biopsy), wipe (for example, oral cavity is wiped Son, oropharynx swab, procto swab), liquid relief and breathing.In addition, any solution that can be from subject residing for desired biological sample Cut open position and obtain biological sample.In some embodiments, the biological sample can be obtained from its packing material, for example, including food The packing material of thing (such as milk) or soil.Soil can be life under mineral matter, organic substance, gas, liquid and certain situation The mixture of object.

Any aspect in the multiple aspect, is expanded to generate amplified production to target nucleic acid.Target nucleic acid can be with It is target RNA or target DNA.Target nucleic acid be target RNA in the case of, target RNA can be any kind of RNA, including herein other RNA types described in place.In some embodiments, target RNA is viral RNA.In some embodiments, viral RNA can Can be pathogenic for subject.Causative virus RNA non-limiting examples include human immunodeficiency virus I (HIV I), people HIV II (HIV II), orthomyxovirus, Ebola virus, dengue virus, influenza virus (for example, H1N1, H3N2, H7N9 or H5N1), hepatitis viruse, hepatitis A virus, hepatitis type B virus, HCV (for example, tool first RNA- HCV virus), Hepatitis D virus, HEV, HGV RNA, Epstein-Barr virus, monocytosis,mononucleosis it is viral, huge Cell virus, SARS virus, west nile fever virus, poliovirus, measles virus, enterovirus (such as Coxsackie virus, Such as coxsackie virus A 16) and norovirus (such as norovirus GI or norovirus GII).

In the case where target nucleic acid is target DNA, target DNA can be any kind of DNA, including described in elsewhere herein DNA types.In some embodiments, target DNA is viral DNA.In some embodiments, viral DNA may for by Examination person is pathogenic.The non-limiting examples of DNA virus include herpes simplex virus, variola virus, adenovirus (for example, 55 Type adenovirus, 7 type adenovirus) and varicella virus (for example, fowl pox).

In some cases, target DNA can be DNA of bacteria.DNA of bacteria may be from being pathogenic bacterium to subject, For example, the pathogenic bacteria can be Gram-positive or Gram-negative pathogenic bacteria.For example, the pathogenic bacteria can Selected from staphylococcus aureus (Staphylococcus aureus), Listeria monocytogenes (Listeria Monocytogenes), Escherichia coli (Escherichia coli), Enterobacter sakazakii (Enterobacter sakazakii), Vibrio parahaemolytious (Vibrio Parahemolyticus), Shigella (Shigella spp.) and mycobacterium tuberculosis --- One kind, which has been notified, causes phthisical bacterium.In some cases, target DNA can be (for example, a kind of from pathogenic protozoa Or the protozoan of a variety of plasmodium types for causing malaria) DNA.

Any aspect in many aspects of the present invention, when the biological sample is mixed to obtain with the buffer suspension liquid When obtaining homogeneous prepared product, the biological sample can be about 5 to the ratio of the buffer suspension liquid:1 (wt/vol) to about 1:500 (wt/vol), for example, about 1:1 (wt/vol) to about 1:100(wt/vol).For example, the biological sample is to the buffer suspension The ratio of liquid can be about 5:1 (wt/vol), about 4:1 (wt/vol), about 3:1 (wt/vol), about 2:1 (wt/vol), about 1:1(wt/ Vol), about 1:2 (wt/vol), about 1:3 (wt/vol), about 1:4 (wt/vol), about 1:5 (wt/vol), about 1:6 (wt/vol), about 1:7 (wt/vol), about 1:8 (wt/vol), about 1:9 (wt/vol), about 1:10 (wt/vol), about 1:20 (wt/vol), about 1:30 (wt/vol), about 1:40 (wt/vol), about 1:50 (wt/vol), about 1:60 (wt/vol), about 1:70 (wt/vol), about 1:80 (wt/vol), about 1:90 (wt/vol), about 1:100 (wt/vol), about 1:110 (wt/vol), about 1:120 (wt/vol), about 1: 130 (wt/vol), about 1:140 (wt/vol), about 1:150 (wt/vol), about 1:160 (wt/vol), about 1:170 (wt/vol), About 1:180 (wt/vol), about 1:190 (wt/vol), about 1:200 (wt/vol), about 1:250 (wt/vol), about 1:300(wt/ Vol), about 1:350 (wt/vol), about 1:400 (wt/vol), about 1:450 (wt/vol), or about 1:500(wt/vol).

Any aspect in many aspects of the present invention, when lysis buffer is added into the homogeneous prepared product, The lysis buffer can be about 50 to the ratio of the homogeneous prepared product:1 (vol/vol) to about 1:50 (vol/vol), for example About 5:1 (vol/vol) to about 1:5(vol/vol).For example, the lysis buffer can be to the ratio of the homogeneous prepared product About 50:1 (vol/vol), 40:1 (vol/vol), 30:1 (vol/vol), 20:1 (vol/vol), 10:1 (vol/vol), 9:1 (vol/vol), 8:1 (vol/vol), 7:1 (vol/vol), 6:1 (vol/vol), 5:1 (vol/vol), about 4:1 (vol/vol), About 3:1 (vol/vol), about 2:1 (vol/vol), about 1:1 (vol/vol), about 1:2 (vol/vol), about 1:3 (vol/vol), about 1:4 (vol/vol), about 1:5 (vol/vol), about 1:6 (vol/vol), about 1:7 (vol/vol), about 1:8 (vol/vol), about 1: 9 (vol/vol), about 1:10 (vol/vol), about 1:20 (vol/vol), about 1:30 (vol/vol), about 1:40 (vol/vol), or About 1:50(vol/vol).

Any aspect in many aspects of the present invention, the biological sample obtained from subject or from described herein Mixture, supernatant, suspension or homogeneous prepared product derived from biological sample with for carrying out nucleic acid amplification institute in reaction vessel Required reagent provides to obtain reactant mixture together.Besides or furthermore, any mixture obtained from biological sample, supernatant Liquid, suspension or homogeneous prepared product can with for carrying out providing together with reagent to obtain necessary to nucleic acid amplification in reaction vessel Reactant mixture.Any suitable reaction vessel can be used.In some embodiments, reaction vessel includes main body, the main body It may include inner surface, outer surface, openend and relative blind end.In some embodiments, reaction vessel may include lid.Institute Stating lid can be configured as in its openend and body contact so that the openend closing of the reaction vessel when being contacted. Under certain situation, the lid is for good and all associated with reaction vessel so that it remains attached to anti-under opening and closing configuration Answer container.In some cases, the lid is removable, so that when reaction vessel is opened, lid is separated with reaction vessel. In some embodiments, reaction vessel can be sealed, in some cases to be gas-tight seal.

Reaction vessel can have different sizes, shape, weight and configuration.In some instances, reaction vessel can be Circular or ellipse tubulose.In some embodiments, reaction vessel can be rectangle, square, rhombus, circle, ellipse Shape or triangle.Reaction vessel can be regular shape or irregular shape.In some embodiments, the closing of reaction vessel End can have taper, circular or flat surface.The non-limiting examples of type of reaction vessel include pipe, hole, capillary, cylinder, ware, Centrifuge tube or head of pipette.Reaction vessel can be constructed by any suitable material, and the non-limiting examples of these materials include glass Glass, metal, plastics and combinations thereof.

In some embodiments, reaction vessel is a part for reaction vessel array.Reaction vessel array is especially useful Multiple samples are handled in automatic mode and/or simultaneously.For example, the microwell plate that reaction vessel can be made up of many holes Hole.In another example, reaction vessel may be housed in the hole of hot block of thermal cycler, and wherein thermal cycle block includes each can Receive multiple holes of shuttle.The array being made up of reaction vessel may include any an appropriate number of reaction vessel.For example, battle array Row may include at least 2,4,6,8,10,12,14,16,18,20,25,35,48,96,144,384 an or more reaction vessels. The reaction vessel part of reaction vessel array can also individually be addressed by fluid treating device so that the fluid treating device can be just Really identification reaction vessel, and appropriate fluent material is assigned in reaction vessel.Fluid treating device can be used for fluid material Expect the addition automation into reaction vessel.

In some embodiments, reaction vessel can include multiple hot-zones.Hot-zone in reaction vessel can be by making reaction The different zones of container are realized exposed to different Thermal cycling conditions.For example, reaction vessel may include top hot-zone with Portion hot-zone.Top hot-zone can receive biological sample and examination necessary to for obtaining the reactant mixture for nucleic acid amplification Agent.The reactant mixture can then undergo the first thennocycling protocols.After the circulation of desired number, for example, reactant mixture Slowly but continuously lower hotspot can be leaked into from top hot-zone.In lower hotspot, reactant mixture is then subjected to and top The circulation of the desired number of the second different thennocycling protocols of the scheme of hot-zone.These strategies are when using nested PCR amplification DNA May be particularly useful.In some embodiments, in reaction under the auxiliary for the temperature-sensitive layered material that hot-zone can be in reaction vessel Produced in container.In tiiese cases, reactant mixture is discharged into from a hot-zone using the heating of temperature-sensitive layered material In next hot-zone.In some embodiments, reaction vessel include 2,3,4,5,6,7,8,9,10,11,12,13,14,15 Or more hot-zone.

In some embodiments, the reaction vessel comprising hot-zone can be used for handling biological sample before nucleic acid amplification.Example Such as, decomposition agent can be added to the first of reaction vessel before addition biological sample and for reagent necessary to nucleic acid amplification Hot-zone.When in the reaction vessel that biological sample and reagent are added to comprising decomposition agent, acquisition can crack the biological sample The reactant mixture of interior species (for example, cell or virion).Or, can be by decomposition agent and biological sample and reagent simultaneously It is added in the first hot-zone of reactant mixture.The temperature conditionss for making the first hot-zone be subjected to being suitable for decomposition agent effect can be used to The cell and virion in biological sample are cracked in first hot-zone so that the nucleic acid in the biological sample is discharged into reaction mixing In thing.After cracking, reactant mixture can be then set to enter the second hot-zone of reaction vessel, for using amplification as described herein Method is expanded to the nucleic acid of release.

The lysis buffer can include any suitable decomposition agent, including commercially available decomposition agent.The non-limit of decomposition agent Property example processed includes Tris-HCl, EDTA, detergent (for example, Triton X-100, SDS), lysozyme, glucolase (glucolase), protease E, viral endolysin, outer lysin (exolysin), zymolase (zymolose), lyticase (Iyticase), Proteinase K, endolysin and outer lysin from bacteriophage, the endolysin from bacteriophage PM2, from withered grass Bacillus (B.subtilis) bacteriophage PBSX endolysin, from lactobacillus prophage Lj928, Lj965, bacteriophage 15Phiadh endolysin, the endolysin from streptococcus pneumonia bacteriophage Cp-I, Streptococcusagalactiae bacteriophage B30's is difunctional Peptide glycan lysin, endolysin and outer lysin from prophage bacterium, from the interior of Listeria (Listeria) bacteriophage Lysin, cave albumen (holin)-endolysin, the lysis genes of cell 20, holWMY walsh staphylococcuses (Staphylococcus Wameri) M bacteriophages varphiWMY, walsh staphylococcus M bacteriophages varphiWMY Iy5WMY, Tween 20, PEG, KOH, NaCl, and combinations thereof.In some cases, buffer solution can include decomposition agent (for example, lysis buffer).Lysis buffer An example be sodium hydroxide (NaOH).In some embodiments, the biological sample is without detergent-treatment.For example, The lysis buffer can be free of any detergent.

The pH of the lysis buffer is about 7 to about 14, such as about 8 to about 13, about 9 to about 12, about 10 to about 11.Example Such as, the pH of the lysis buffer can be about 7,7.5,8,8.5,9,9.5,10,10.5,11,11.5,12,12.5,13,13.5, Or 14.

Any aspect in many aspects of the present invention, when the biological sample is mixed with the lysis buffer to obtain When obtaining mixture, the biological sample can be about 5 to the ratio of the lysis buffer:1 (wt/vol) to about 1:10(wt/ Vol), for example, about 1:1 (wt/vol) to about 1:10(wt/vol).For example, ratio of the biological sample to the buffer suspension liquid Example can be about 5:1 (wt/vol), about 4:1 (wt/vol), about 3:1 (wt/vol), about 2:1 (wt/vol), about 1:1 (wt/vol), About 1:2 (wt/vol), about 1:3 (wt/vol), about 1:4 (wt/vol), about 1:5 (wt/vol), about 1:6 (wt/vol), about 1:7 (wt/vol), about 1:8 (wt/vol), about 1:9 (wt/vol), or about 1:10(wt/vol).

In some embodiments, the reaction vessel includes the reagent needed for carrying out nucleic acid amplification.For example, the reagent It may include to carry out the reagent needed for reverse transcription amplification and/or DNA (DNA) amplification.

Any kind of nucleic acid amplification reaction is used equally for amplification target nucleic acid and generates amplified production.In addition, the expansion of nucleic acid Increasing can be linear, exponential form or its combination.Amplification can be emulsion-based or can be with right and wrong emulsion-based.Nucleic acid The non-limiting examples of amplification method include reverse transcription, primer extend, polymerase chain reaction, ligase chain reaction, unwindase according to Bad amplification, non-symmetric amplification, rolling circle amplification and multiple displacement amplification (MDA).In some embodiments, amplified production can be with It is DNA.In the case where being expanded to target RNA, DNA can be obtained by RNA reverse transcription and using subsequent DNA Expand to generate the DNA product of amplification.The DNA product of amplification can indicate there is target RNA in biological sample.Enter to DNA In the case of row amplification, a variety of DNA cloning methods can be used.The non-limiting examples of DNA cloning method include polymerase chain React (PCR), PCR modification (for example, in real time PCR, ApoE gene, assembling PCR, asymmetric pcr, digital pcr, Emulsion-based PCR, PCR, nest-type PRC, heat start PCR, inverse PCR, the methyl for transfering to PCR (dial-out PCR), unwindase to rely on Change the asymmetric friendship of specific PCR, micro- primer PCR (miniprimer PCR), multiplex PCR, nest-type PRC, overlapping-extension PCR, heat Wrong PCR (thermal asymmetric interlaced PCR), fall progressively PCR) and ligase chain reaction (LCR).At some In the case of, DNA cloning is linear.In some cases, DNA cloning is exponential.In some cases, DNA cloning is adopted Realized with nest-type PRC, it can improve the sensitivity of the DNA product of detection amplification.In some embodiments, it is specifically described herein Amplification can refer to primer extension reaction.

In many aspects, these nucleic acid amplification reactions as described herein parallel can be carried out.Generally, parallel amplified reaction is The amplified reaction occurred simultaneously in same reaction vessel.Parallel nucleic acid amplification reaction can be carried out as follows：For example, in reaction Container is included for reagent necessary to each nucleic acid amplification reaction to obtain reactant mixture, and makes the reactant mixture It is subjected to for condition necessary to each nucleic acid amplification reaction.For example, reverse transcription amplification and DNA cloning can abreast be entered as follows OK：There is provided for reagent necessary to both amplification methods to form and obtain reactant mixture, and make in reaction vessel The reactant mixture is subjected to being adapted for the condition of the two amplified reactions.The DNA produced by RNA reverse transcription can be abreast Expanded to produce the DNA product of amplification.Any suitable number of nucleic acid amplification reaction can be carried out abreast.In some feelings Under condition, abreast carry out at least 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or more Individual nucleic acid amplification reaction.

The parallel advantage for carrying out nucleic acid amplification reaction may include the rapid translating between the nucleic acid amplification reaction of coupling.Example Such as, target nucleic acid (for example, target RNA, target DNA) can be extracted or discharged from biological sample in the heating period of parallel nucleic acid amplification. In the case of target RNA, for example, the biological sample comprising target RNA can be heated and target RNA is discharged from biological sample.Released The target RNA put can immediately begin to reverse transcription (via reverse transcription amplification) to produce complementary DNA.Then the complementation can be expanded immediately DNA, generally in the magnitude of several seconds.Target RNA discharged from biological sample and target RNA reverse transcriptions be complementary DNA between in short-term Between interval can help to make to may interfere with reverse transcription in biological sample and/or the influence of the inhibitor of DNA cloning is minimized.

Any aspect in these many aspects, can be used the primer sets for target nucleic acid anti-to carry out nucleic acid amplification Should.For example, the reagent needed for the progress nucleic acid amplification may include one or more primer sets.Primer sets generally comprise it is a kind of or A variety of primers.For example, primer sets can include about 1,2,3,4,5,6,7,8,9,10 kind or more kind primer.In some cases, Primer sets can include the primer for different amplified production or different nucleic acid amplification reactions.For example, primer sets can be comprising the One primer and second primer complementary with nucleic acid chains product, the first primer are the generation core complementary with least a portion of target nucleic acid Necessary to first chain of acid product, the second primer is that generation is produced with the complementary nucleic acid of at least a portion of the chain of nucleic acid product first Necessary to second chain of thing.

For example, primer sets can be directed to target RNA.Primer sets, which can be included, can be used for generation and target RNA at least a portion complementary The chain of nucleic acid product first the first primer.In the case of reverse transcription reaction, the first chain of nucleic acid product can be DNA.Draw Thing group can also include the second of the chain of nucleic acid product second that can be used for generation complementary with least a portion of the chain of nucleic acid product first Primer.In the case of the reverse transcription reaction of progress parallel with DNA cloning, the second chain of nucleic acid product can be with from RNA moulds One chain of nucleic acid (for example, DNA) product for the DNA complementation that plate is produced.

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, 9th, 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 any aspect of many aspects, the reagent can include forward primer.The reagent can be included and is adapted for The forward primer of any amount of nucleic acid amplification.For example, the reagent can include about less than 0.01 μM, 0.01 μM, 0.05 μM, 0.06 μM、0.07μM、0.08μM、0.09μM、0.1μM、0.2μM、0.3μM、0.4μM、0.5μM、0.6μM、0.7μM、0.8μM、0.9μ M、1.0μM、1.1μM、1.2μM、1.3μM、1.4μM、1.5μM、1.6μM、1.7μM、1.8μM、1.9μM、2.0μM、2.5μM、3.0 μM、3.5μM、4.0μM、4.5μM、5.0μM、6.0μM、7.0μM、8.0μM、9.0μM、10.0μM、11.0μM、12.0μM、13.0μ M, 14.0 μM, 15.0 μM, more than 15.0 μM dNTP, or between any of the above-described numerical value range of concentrations forward primer, for example, about 0.01 to 10.0 μM, 0.05 to 5.0 μM, 0.05 to 4.0 μM, 0.05 to 3.0 μM, 0.05 to 2.0 μM, 0.05 to 1.0 μM, 0.05 To 0.5 μM, 0.1 to 5.0 μM, 0.1 to 4.0 μM, 0.1 to 3.0 μM, 0.1 to 2.0 μM, 0.1 to 1.0 μM, 0.1 to 0.9 μM, 0.1 To 0.8 μM, 0.1 to 0.7 μM, 0.1 to 0.6 μM, 0.1 to 0.5 μM of forward primer.In one aspect, the reagent can include about 0.1 to 1.0 μM of forward primer.

In any aspect of many aspects, the reagent can include reverse primer.The reagent can be included and is adapted for The reverse primer of any amount of nucleic acid amplification.For example, the reagent can include about less than 0.01 μM, 0.01 μM, 0.05 μM, 0.06 μM、0.07μM、0.08μM、0.09μM、0.1μM、0.2μM、0.3μM、0.4μM、0.5μM、0.6μM、0.7μM、0.8μM、0.9μ M、1.0μM、1.1μM、1.2μM、1.3μM、1.4μM、1.5μM、1.6μM、1.7μM、1.8μM、1.9μM、2.0μM、2.5μM、3.0 μM、3.5μM、4.0μM、4.5μM、5.0μM、6.0μM、7.0μM、8.0μM、9.0μM、10.0μM、11.0μM、12.0μM、13.0μ M, 14.0 μM, 15.0 μM, more than 15.0 μM dNTP, or between any of the above-described numerical value range of concentrations reverse primer, for example, about 0.01 to 10.0 μM, 0.05 to 5.0 μM, 0.05 to 4.0 μM, 0.05 to 3.0 μM, 0.05 to 2.0 μM, 0.05 to 1.0 μM, 0.05 To 0.5 μM, 0.1 to 5.0 μM, 0.1 to 4.0 μM, 0.1 to 3.0 μM, 0.1 to 2.0 μM, 0.1 to 1.0 μM, 0.1 to 0.9 μM, 0.1 To 0.8 μM, 0.1 to 0.7 μM, 0.1 to 0.6 μM, 0.1 to 0.5 μM of reverse primer.In one aspect, the reagent can include about 0.1 to 1.0 μM of reverse primer.

In some embodiments, using archaeal dna polymerase.Any suitable archaeal dna polymerase can be used, including it is commercially available Archaeal dna polymerase.Archaeal dna polymerase is often referred to the enzyme that can be incorporated into nucleotides in the way of template is combined in DNA.DNA The non-limiting examples of polymerase include Taq polymerase, Tth polymerases, Tli polymerases, Pfu polymerase, VENT polymerases, DEEPVENT polymerases, EX-Taq polymerases, LA-Taq polymerases, Expand polymerases, Sso polymerases, Poc polymerases, Pab Polymerase, Mth polymerases, Pho polymerases, ES4 polymerases, Tru polymerases, Tac polymerases, Tne polymerases, Tma polymerases, Tih polymerases, Tfi polymerases, Platinum Taq polymerases, Hi-Fi polymerases, Tbr polymerases, Tfl polymerases, Pfutubo polymerases, Pyrobest polymerases, Pwo polymerases, KOD polymerases, Bst polymerases, Sac polymerases, Klenow pieces Section, and their variant, the product and derivative of modification.For certain thermal starting polymerase, it may be necessary at 94 DEG C -95 DEG C The denaturing step of lower 2 minutes to 10 minutes, this may change heat distribution according to different polymerases.

In some embodiments, the reagent needed for the progress nucleic acid amplification may include reverse transcriptase.In some implementations In scheme, reagent described in the present invention can include reverse transcriptase.For example, any suitable reverse transcriptase can be used.Reverse Record enzyme is often referred to that nucleotides can be incorporated into the enzyme in DNA when being combined with RNA templates.The non-limiting reality of reverse transcriptase Example include HIV1-RT, M-MLV reverse transcriptases, AMV reverse transcriptases, reverse transcriptase of telomere, and they variant, The product and derivative of modification.DNA and RNA polymerase many specifically described herein or known in the art can draw in template Nucleotide analog is utilized in the primer extension reaction led.Broad category of nucleotide analog known in the art.Ucleotides Include ribonucleotide and deoxyribonucleotide analogs like the non-limiting examples of thing.Generally, the analog include adenosine, Thymidine, uridine, cytidine, the analog of uridine and these bases.The analog may include ribonucleoside triphosphote, or it may also include Extra phosphate group, such as four phosphoric acid, five phosphoric acid, six phosphoric acid, seven phosphoric acid or more.The example of some in these analogs is retouched It is set forth in, for example U.S. Patent Application Publication 2003-0124576 and 2007-0072196, and U.S. Patent number 7,223,541 With 7,052,839, its full text is incorporated herein with regard to all purposes by quoting.

In many aspects, amplified production is generated using primer extension reaction.Primer extension reaction generally includes following Circulation：Reactant mixture is incubated to one section of denaturation duration under denaturation temperature, and by reactant mixture in elongating temperature It is lower to be incubated one section of extension duration.

In some cases, before the primer extension reaction is carried out, by the biological sample at about 90 DEG C to about 100 DEG C pre-heating temperature under the preheating no more than about pre-add thermal endurance of 10 minutes.In some embodiments, it is described pre- The duration of heat is no more than 1 minute.

Denaturation temperature can be according to the particular organisms sample, the particular source (example of biological sample target nucleic acid for example analyzed 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 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.

Being denatured the duration can be according to the particular organisms sample, the particular source of biological sample target nucleic acid for example analyzed (for example, virion, bacterium), used reagent and/or desired reaction condition and change.For example, when denaturation continues Between can less equal than 300 seconds, 240 seconds, 180 seconds, 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 Second, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds or 1 second.For example, denaturation the duration can no more than 120 seconds, 90 seconds, 60 Second, 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 be according to the particular organisms sample, the particular source (example of biological sample target nucleic acid for example analyzed 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, temperature is extended 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 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 DEG C, 45 DEG C, 46 DEG C, 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℃、75℃、76℃、77 DEG C, 78 DEG C, 79 DEG C or 80 DEG C.

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

Any aspect in the multiple aspect, can carry out the primer extension reaction of multiple circulations.It can carry out any suitable When the circulation of number.For example, the period carried out can with less than about 100,90,80,70,60,50,40,30,20,10 or 5 follow Ring.The period of progress may depend on, for example, obtaining detectable amplified production (for example, indicating there is target in biological sample The DNA amplification product of RNA detectable amount) necessary to period (for example, cycle threshold (Ct)).For example, can detect Amplified production (for example, indicate exist in biological sample target RNA detectable amount DNA product) necessary to period can With less than about or be about 100 circulation, 75 circulation, 70 circulation, 65 circulation, 60 circulation, 55 circulation, 50 follow Ring, 40 circulations, 35 circulations, 30 circulations, 25 circulations, 20 circulations, 15 circulations, 10 circulations or 5 circulations.This Outside, in some embodiments, the amplified production of detectable amount is (for example, indicate there is the detectable of target RNA in biological sample The DNA product of amount) can be with the circulation less than 100,75,70,65,60,55,50,45,40,35,30,25,20,15,10 or 5 Threshold value (Ct) is obtained.

Amplification produce indicate exist expanded target nucleic acid detectable amount amplified production needed for time can according to from The particular cycle of the middle biological sample for obtaining target nucleic acid, the specific nucleic acid amplified reaction that will be carried out and desired amplified reaction Count and change.For example, the amplification of target nucleic acid can be at 120 minutes or shorter, 90 minutes or shorter, 60 minutes or shorter, 50 minutes Or it is 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, the period of 10 minutes or shorter or 5 minutes or shorter produces and indicates there is target nucleic acid The amplified production of detectable amount.

In some embodiments, target RNA amplification can 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, the period of 10 minutes or shorter or 5 minutes or shorter produces and indicates exist The DNA amplification product of target RNA detectable amount.

In some embodiments, reactant mixture can be made to undergo multiple serial primer extension reactions.The multiple system Single series in row may include the specific primer extension of multiple circulations, and the reaction is characterised by, for example, as herein its Specific denaturation and extension condition described in his place.Generally, for example, for Denaturing and/or extension condition, Mei Gedan Individual series is different from least one other single series in the multiple series.For example, with regard to oblique variable Rate, denaturation temperature, change Property duration, elongating temperature and in the extension duration any one, two, three, four or all for five, it is single Individual series may differ from another single series in the multiple series.In addition, multiple series may include any number of list Individual series, for example, at least about or about 2,3,4,5,6,7,8,9,10 or more a single series.

For example, multiple serial primer extension reactions may include First Series and second series.First Series, for example, can Primer extension reaction including more than ten circulations, wherein each circulation of First Series include (i) by reactant mixture about It is incubated and is no more than 30 seconds at 92 DEG C to about 95 DEG C, reactant mixture is incubated by subsequent (ii) at about 35 DEG C to about 65 DEG C is no more than About one minute.Second series, for example, it may include more than the primer extension reaction of ten circulations, wherein each of second series follows Ring includes (i) and is incubated reactant mixture no more than 30 seconds at about 92 DEG C to about 95 DEG C, and subsequent (ii) exists reactant mixture It is incubated at about 40 DEG C to about 60 DEG C no more than about 1 minute.In this instantiation, the first and second series are in their extension It is different in temperature conditionss.However, the example is not intended to limit, because any group of different extensions and Denaturing can be used Close.

In each series in multiple serial primer extension reactions, any an appropriate number of circulation can be carried out.For example, In each series in multiple serial primer extension reactions the period that carries out can with less than about 100,90,80,70,60, 50th, 40,30,20,10 or 5 circulations.

The advantage for carrying out multiple serial primer extension reactions may is that, and under the conditions of similar denaturation and extension Single a series of primer extension reaction is compared, and multiple serial methods are produced with relatively low cycle threshold and indicated in biological sample There is the amplified production of the detectable amount of target nucleic acid.Compared with the single series under the conditions of similar denaturation and extension, use Multiple serial primer extension reactions can by these cycle thresholds reduce at least about or about 1%, 5%, 10%, 15%, 20%, 25%th, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

In some embodiments, tiltedly become the time (that is, thermal cycler from a temperature transition to another temperature spent when Between) and/or tiltedly variable Rate is the key factor in amplification.For example, amplification produces the expansion for the detectable amount for indicating to exist target nucleic acid Temperature and time needed for volume increase thing can change according to oblique variable Rate and/or oblique change time.Oblique variable Rate can influence to be used to expand The temperature and time of increasing.

In some cases, oblique change time and/or oblique variable Rate can be different between cycles.But in some feelings Under condition, oblique change time and/or oblique variable Rate between circulation can be identicals.Oblique change time and/or oblique variable Rate can be based on The sample handled is adjusted.

In some cases, the oblique change between different temperatures can be for example determined according to the property and reaction condition of sample Time.Also accurate temperature and incubation time can be determined according to the property and reaction condition of sample.In some embodiments, It can be used multiple thermal cycles that single sample is handled into (for example, being subjected to amplification condition) repeatedly, each thermal cycle is for example oblique It is different on change time, temperature and/or incubation time.Best or optimal thermal cycle can be then selected for the specific sample.This is carried The efficient method for tested specific sample or sample combination tailoring thermal cycle sane is supplied.

In some embodiments, target nucleic acid can be subjected to Denaturing before primer extension reaction startup.In multiple systems In the case of the primer extension reaction of row, target nucleic acid can be subjected to Denaturing before the multiple series is performed, or can be Denaturing is subjected between the multiple series.For example, target nucleic acid can First Series and the second series in multiple series it Between be subjected to Denaturing.The non-limiting examples of these Denaturings include denaturation temperature and are distributed (for example, one or more denaturation Temperature) and denaturant.

In some embodiments, biological sample can be preheated before primer extension reaction is carried out.The biological sample of preheating The temperature (for example, preheating temperature) of product and duration (for example, pre- thermal endurance) can be according to the specific lifes for example analyzed Thing sample and change.In some instances, can be no more than about 60 minutes, 50 minutes, 40 minutes, 30 points by biological sample preheating Clock, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 points Clock, 1 minute, 45 seconds, 30 seconds, 20 seconds, 15 seconds, 10 seconds or 5 seconds.In some instances, can be in about 80 DEG C to about 110 DEG C of temperature Lower preheating biological sample.In some instances, biological sample can be preheated at a temperature of about 90 DEG C to about 100 DEG C.One In a little examples, biological sample can be preheated at a temperature of about 90 DEG C to about 97 DEG C.In some instances, can be at about 92 DEG C to about Biological sample is preheated at a temperature of 95 DEG C.In other other examples, can 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 Biological sample is preheated at a temperature of 100 DEG C.

In some embodiments, it is described carry out nucleic acid amplification needed for reagent (including for carry out parallel nucleic acid amplification Required reagent) it may also include report agent.The report agent can produce detectable signal.The detectable signal may indicate that amplification Product whether there is.For example, the existence or non-existence of detectable signal may indicate that amplified production whether there is.Detectable signal Intensity can be proportional to the amount of amplified production.In some cases, when amplified production is by the target nucleic acid inhomogeneity with initially expanding When the nucleic acid of type is generated, the intensity of detectable signal can be proportional to the amount of the target nucleic acid initially expanded.For example, by flat Row ground reverse transcription and the DNA that is obtained from reverse transcription of amplification are come in the case of expanding target RNA, for being tried necessary to the two reactions Agent, which may also include, can produce the report agent of detectable signal, and the detectable signal indicates the DNA product of amplification and/or the target of amplification RNA presence.The intensity of detectable signal can be proportional to the amount of the DNA product of amplification and/or the initial target RNA of amplification.Report Accuse using for agent and also make it possible real-time amplification method, including the real-time PCR for DNA cloning.

Report agent can be connected by covalently or non-covalently interacting with the nucleic acid including amplified production.It is non-covalent The non-limiting examples of interaction include ionic interaction, Van der Waals force, hydrophobic interaction, hydrogen bonding and its group Close.In some embodiments, report agent can be combined with initial reactant, and report that the change of agent level can be used for detection to expand Increase production thing.In some embodiments, report agent can only be detectable (or undetectable) when nucleic acid amplification is carried out. In some embodiments, the available agent of giving a report of optical activity dyestuff (for example, fluorescent dye).The non-limiting examples bag of dyestuff Include SYBR green, 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 are held mould partially Plain D, chromomycin, Homidium Bromide (homidium), mithramycin, many pyridine rutheniums (ruthenium polypyridyl) pacify aspergillus Plain (anthramycin), phenanthridines and acridine, ethidium bromide, propidium iodide, the own pyridine of iodate (hexidium iodide), dihydro second Ingot, second ingot homodimer -1 and second ingot homodimer -2, single Azide second ingot (ethidium monoazide) and ACMA, Hoechst 33258, Hoechst 33342, Hoechst 34580, DAPI, acridine orange, 7-AAD, actinomycin D, LDS751, Hydroxystilbamidine (hydroxystilbamidine), SYTOX is blue, and SYTOX is green, SYTOX oranges, 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, - 42nd, -43, -44, -45 (indigo plant), 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 are different Thiocyanic acid fluorescein (FITC), tetramethylrhodamine isothiocyanates (TRITC), rhodamine, tetramethylrhodamine, R- algae red eggs In vain, VIC, NED, PET, 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 homodimeric Body I, second ingot homodimer II, second ingot homodimer III, ethidium bromide, umbelliferone, eosin, green fluorescent protein, red moss It is red, cumarin, methylcoumarin, pyrene, peacock green, Stilbene, fluorescein, cascade blue (cascade blue), dichlorotriazine amine fluorescence Element, dansyl Cl, fluorescence Lanthanide Complexes (such as those include complex compounds of europium and terbium), carboxyl tetrachlorofluorescein (TET), 5 and/ Or 6- Fluoresceincarboxylic acids (FAM), 5- (or 6-) iodacetyl amido fluorescein, 5- { [2 (and 3) -5- (acetyl group sulfydryl)-succinyls Base] amino fluorescein (SAMSA- fluoresceins), Sulforhodamine B sulfonic acid chloride, 5 and/or 6 carboxyrhodamines (ROX), 5- and/ Or 6- carboxyls tetramethylrhodamine (TAMRA), 6- carboxyls -4 ', 5 '-two chloro- 2 ', 7 '-dimethoxyfluoresceins (JOE), 6- carboxylics Base -2', 4,4', 5', 7,7'- chlordene fluoresceins (HEX), 7- amino-methyls-cumarin, 7- amino -4- methylcoumarins -3- Acetic acid (AMCA), BODIPY fluorogens, 8- methoxyl group pyrenes -1,3,6- trisulfonic acid trisodium salts, 3,6- disulfonic acid -4- amino-naphthalene two Carboximide, phycobniliprotein, AlexaFluor 350,405,430,488,532,546,555,568,594,610,633,635, 647th, 660,680,700,750 and 790 dyestuff, DyLight 350,405,488,550,594,633,650,680,755 and 800 Dyestuff, or other fluorogens.

In some embodiments, report agent can have optically active sequence specific when hybridizing with amplified production Property oligonucleotide probe.Because probe is combined with the sequence-specific of amplified production, the use of oligonucleotide probe can improve inspection The specificity of survey and sensitivity.Probe may be connected to any optical activity report agent (for example, dyestuff) as described herein, and also It may include the optically active quencher that can block associated dyestuff.The non-limiting examples of the probe for agent of giving a report can be used Including TaqMan probe, TaqMan Tamara probes, TaqMan MGB probes or Lion probes.

In some embodiments, report agent can be optically active sequence with blocking when hybridizing with amplified production Row specific oligonucleotide probe.In some embodiments, the oligonucleotide probe shows optical activity in fracture.Example Such as, the report agent can be oligonucleotide probe, and it is comprising optical activity dyestuff (for example, fluorescent dye) and is adjacently located on Quencher on probe.Dyestuff and quencher in close proximity to can blocked dye optical activity.Probe can be with target to be amplified Sequence is combined.Once the exonuclease activity of archaeal dna polymerase is broken probe during expanding, then quencher and dyestuff point From, and free dyestuff regains its optical activity, the activity can be then detected.The oligonucleotide probe can be RNA oligonucleotide probe or DNA oligonucleotide probe.

In some embodiments, report agent can be molecular beacon (molecular beacon).Molecular beacon includes, For example, the quencher connected on one end of the oligonucleotides of hairpin conformation.It is optical activity in the other end of the oligonucleotides Dyestuff, for example, fluorescent dye.In hairpin structure, optical activity dyestuff and quencher are tightly enough approached so that quencher It is capable of the optical activity of blocked dye.However, once with amplified production hybridize, the oligonucleotides be linear conformation and with the expansion Increase production the target sequence hybridization on thing.The linearisation of oligonucleotides causes the separation of optical activity dyestuff and quencher, so that Optical activity is recovered, and can be detected.Molecular beacon can improve inspection to the sequence-specific of the target sequence on amplified production The specificity of survey and sensitivity.

In any aspect of many aspects, the reagent can include one or more oligonucleotide probes or molecule letter Mark.The reagent can include the one or more oligonucleotide probes or molecular beacon of any amount for being adapted for nucleic acid amplification. For example, the reagent can include about less than 0.01 μM, 0.01 μM, 0.05 μM, 0.06 μM, 0.07 μM, 0.08 μM, 0.09 μM, 0.1 μM、0.2μM、0.3μM、0.4μM、0.5μM、0.6μM、0.7μM、0.8μM、0.9μM、1.0μM、1.1μM、1.2μM、1.3μM、 1.4μM、1.5μM、1.6μM、1.7μM、1.8μM、1.9μM、2.0μM、2.5μM、3.0μM、3.5μM、4.0μM、4.5μM、5.0μ M, 6.0 μM, 7.0 μM, 8.0 μM, 9.0 μM, 10.0 μM, 11.0 μM, 12.0 μM, 13.0 μM, 14.0 μM, 15.0 μM, more than 15.0 μ The one or more oligonucleotide probes or molecular beacon of range of concentrations between M dNTP, or any of the above-described numerical value, for example, about 0.01 to 10.0 μM, 0.05 to 5.0 μM, 0.05 to 4.0 μM, 0.05 to 3.0 μM, 0.05 to 2.0 μM, 0.05 to 1.0 μM, 0.05 To 0.5 μM, 0.1 to 5.0 μM, 0.1 to 4.0 μM, 0.1 to 3.0 μM, 0.1 to 2.0 μM, 0.1 to 1.0 μM, 0.1 to 0.9 μM, 0.1 To 0.8 μM, 0.1 to 0.7 μM, 0.1 to 0.6 μM, 0.1 to 0.5 μM of one or more oligonucleotide probes or molecular beacon. On one side, the reagent can include about 0.1 to 0.5 μM of one or more oligonucleotide probes or molecular beacon.

In some embodiments, report agent can be radioactive species.The non-limiting examples of radioactive species include¹⁴C、¹²³I、¹²⁴I、¹²⁵I、¹³¹I、Tc99m、³⁵S or³H。

In some embodiments, report agent can produce the enzyme of detectable signal.Detectable signal can pass through Enzyme is produced to its substrate, or in the case where enzyme has multiple substrates to the activity of specific substrates.The enzyme of agent of giving a report can be used Non-limiting examples include alkaline phosphatase, horseradish peroxidase, I2- galactosidases, alkaline phosphatase, beta galactose Glycosides enzyme, acetylcholinesterase and luciferase.

In the amplified reaction of parallel progress, a variety of report agent can be used to detect a variety of amplified productions.A variety of reports Each accused in agent can produce detectable signal, and the detectable signal that a variety of report agent are produced is different from each other.It is described Each in a variety of detectable signals may indicate that its corresponding amplified production whether there is.In a variety of detectable signals The intensity of each can corresponding amplified production amount it is proportional.For example, a kind of corresponding detectable signal of amplified production can For FAM fluorescence, and the corresponding detectable signal of another amplified production can be ROX fluorescence.The different detectable letter of Parallel testing Number it may be such that the presence or absence that can compare different amplified productions and amount.Or or the moreover, different detectable letter of Parallel testing Number may be such that can determine the amount of amplified production for internal reference thing.

In any aspect of many aspects, the reagent can further include MgCl₂.The reagent can comprising be suitable into The MgCl of any amount of row nucleic acid amplification₂.For example, the reagent can include about less than 0.01mM, 0.01mM, 0.05mM, 0.06mM、0.07mM、0.08mM、0.09mM、0.1mM、0.2mM、0.3mM、0.4mM、0.5mM、0.6mM、0.7mM、0.8mM、 0.9mM、1.0mM、1.1mM、1.2mM、1.3mM、1.4mM、1.5mM、1.6mM、1.7mM、1.8mM、1.9mM、2.0mM、 2.5mM、3.0mM、3.5mM、4.0mM、4.5mM、5.0mM、6.0mM、7.0mM、8.0mM、9.0mM、10.0mM、11.0mM、 12.0mM, 13.0mM, 14.0mM, 15.0mM, more than 15.0mM MgCl₂, or range of concentrations between any of the above-described numerical value MgCl₂, for example, about 0.01 to 15.0mM, 0.05 to 14.0mM, 0.1 to 13.0mM, 0.2 to 12.0mM, 0.3 to 11.0mM, 0.4 to 10.0mM, 0.5 to 9.0mM, 0.6 to 8.0mM, 0.7 to 7.0mM, 0.8 to 6.0mM, 0.9 to 5.0mM, 1.0 to 4.0mM, 1.1 to 3.0mM, 1.2 to 2.5mM, 1.3 to 2.0mM, 1.4 are to 1.6mM MgCl₂.In one aspect, the reagent can Include about 1.5mM MgCl₂。

In any aspect of many aspects, the reagent can include dNTP.The reagent can include and be adapted for nucleic acid The dNTP of any amount of amplification.For example, the reagent can include about less than 0.01mM, 0.01mM, 0.05mM, 0.06mM, 0.07mM、0.08mM、0.09mM、0.1mM、0.2mM、0.3mM、0.4mM、0.5mM、0.6mM、0.7mM、0.8mM、0.9mM、 1.0mM、1.1mM、1.2mM、1.3mM、1.4mM、1.5mM、1.6mM、1.7mM、1.8mM、1.9mM、2.0mM、2.5mM、 3.0mM、3.5mM、4.0mM、4.5mM、5.0mM、6.0mM、7.0mM、8.0mM、9.0mM、10.0mM、11.0mM、12.0mM、 13.0mM, 14.0mM, 15.0mM, the dNTP more than range of concentrations between 15.0mM dNTP, or any of the above-described numerical value, for example, about 0.01 to 10.0mM, 0.05 to 5.0mM, 0.05 to 4.0mM, 0.05 to 3.0mM, 0.05 to 2.0mM, 0.05 to 1.0mM, 0.05 To 0.5mM, 0.1 to 5.0mM, 0.1 to 4.0mM, 0.1 to 3.0mM, 0.1 to 2.0mM, 0.1 to 1.0mM, 0.1 to 0.9mM, 0.1 To 0.8mM, 0.1 to 0.7mM, 0.1 to 0.6mM, 0.1 to 0.5mM dNTP.In one aspect, the reagent can include about 0.1 To 0.5mM dNTP.

In many aspects, amplified production (for example, the DNA product of amplification, RNA products of amplification) can detect.Amplified production The detection of (DNA for including amplification) can be realized using any suitable detection method.The concrete kind of used detection method Type be may depend on, for example, specific amplified production, for the type of the reaction vessel of amplification, other in reactant mixture are tried Whether agent, report agent includes in the reactive mixture, and the used particular type for reporting agent when using report agent.Inspection The non-limiting examples of survey method include optical detection, spectral detection, electrostatic detection, Electrochemical Detection etc..Optical detecting method Including but not limited to fluorimetry and UV-Visible absorption.It is common that spectral method of detection includes but is not limited to mass spectrography, nuclear-magnetism Shake (NMR) spectral method and infra-red sepectrometry.Electrostatic detection methods include but is not limited to the technology based on gel, for example, gel is electric Swimming.Electrochemical detection method includes but is not limited to the electrochemistry after the high performance liquid chromatography separation of amplified production to amplified production Detection.

In some embodiments, amplified production can be detected in the primer extension reaction.In some embodiments, Amplified production can be detected in multiple serial primer extension reactions.In some embodiments, can be in multiple serial primers Amplified production is detected in each single series in extension.In some embodiments, it can prolong in multiple serial primers Some single series in reaction are stretched, but do not detect amplified production in other single series.For example, the multiple serial draws Thing extension may include First Series and second series.Amplified production can not be detected in First Series, and in second series Middle detection amplified production.Or, amplified production can be detected in First Series, and amplified production is not detected in second series. Or, amplified production can be detected in First Series and second series, or do not detected in First Series and second series Amplified production.

In some embodiments, the detectable signal produced by report agent can be detected in the primer extension reaction. In some embodiments, the detectable signal produced by report agent can be detected in multiple serial primer extension reactions. In some embodiments, detect what is produced by report agent in each single series that can be in multiple serial primer extension reactions Detectable signal.In some embodiments, some single series that can be in multiple serial primer extension reactions, but do not exist The detectable signal produced by report agent is detected in other single series.For example, the multiple serial primer extension reaction can Including First Series and second series.The detectable signal produced by report agent can not be detected in First Series, and second The detectable signal produced by report agent is detected in series.Or, it can be detected in First Series by examining that report agent is produced Signal is surveyed, and does not detect the detectable signal produced by report agent in second series.Or, can be in First Series and second The detectable signal produced by report agent is detected in row, or does not detect and is produced by report agent in First Series and second series Raw detectable signal.

Any aspect in the multiple aspect, the time needed for the key element of Method Of Accomplishment can be according to the specific of this method Step and change.For example, the time quantum for the key element of Method Of Accomplishment can be about 5 minutes to about 120 minutes.In other examples In, the time quantum for the key element of Method Of Accomplishment can be about 5 minutes to about 60 minutes.In other instances, for Method Of Accomplishment The time quantum of key element can be about 5 minutes to about 30 minutes.In other instances, the time quantum for the key element of Method Of Accomplishment can With less than or equal to 120 minutes, less than or equal to 90 minutes, less than or equal to 75 minutes, less than or equal to 60 minutes, be less than or Equal to 45 minutes, less than or equal to 40 minutes, less than or equal to 35 minutes, less than or equal to 30 minutes, less than or equal to 25 points Clock, less than or equal to 20 minutes, less than or equal to 15 minutes, less than or equal to 10 minutes, or less than or equal to 5 minutes.

In some embodiments, can be by the presence of the amplified production DNA product of amplification (for example) and/or amount Information is exported to recipient.Information on amplified production can be exported via a variety of methods.In some embodiments, these are believed Breath can be verbally provided to recipient.In some embodiments, these information can be provided in report.Report may include any number The desired element of purpose, the non-limiting examples of the element are included on subject (for example, sex, age, race, health Situation etc.) initial data, processed data (for example, figure shows (for example, figure, chart, tables of data, data summarization), really Fixed cycle threshold, the calculated value of target polynucleotide initial amount) information, about whether the conclusion that there is target nucleic acid, diagnosis letter Breath, prognosis information, disease information, etc., and combinations thereof.This report can be provided as the report (for example, hard copy) of printing, or Person can provide as electronic report.In some embodiments (including wherein providing the situation of electronic report), these information can Via such as monitor or television, operationally with for obtaining screen, tablet PC screen that the unit of amplified production is connected, moving Electronic console (for example, electronic display) output such as dynamic device screen.The report of printing and electronic report can be stored respectively in In file or database so that they can be accessed for being compared with later report.

In addition, can be used any suitable communication media (including, for example, network connection, wireless connection or internet connect Connect) transmission will be reported to the recipient of Local or Remote position.In some embodiments, report can be sent to recipient's Device, such as personal computer, phone, flat board or other devices.This report can online be watched, is stored on the device of recipient Or printing.Can also be by transmitting report for transmitting any other means of information, the non-limiting examples of the means include postal Hard copy report is posted to check for reception and/or recipient.

In addition, these information can be exported to various types of recipient.The non-limiting examples of these recipients Including therefrom obtain the subject of biological sample, doctor, the doctor for treating subject, the clinical monitor for clinical test, Nurse, researcher, Laboratory Technician, the representative of drugmaker, health care companies, biotech company, hospital, the mankind Aid organization, health care management person, electronic system are (for example, one or more meter of the medical records of storage such as subject Calculation machine and/or one or more computer server), Public Health Practice person, other medical workers and other medical facilities.

In one aspect, the invention provides a kind of system for implementing the method according to any method disclosed herein. In another aspect, it is present in directly from the target nucleus in the biological sample that subject obtains for expanding the invention provides a kind of The system of ribosomal ribonucleic acid (RNA).The system includes：(a) input module, its user for receiving the target RNA in amplification biological sample please Ask；(b) module is expanded, it is asked in response to the user：Reactant mixture is received in reaction vessel, the reactant mixture is included Biological sample and reagent, the reagent necessary to for abreast carrying out reverse transcription amplification with DNA (DNA) amplification Comprising (i) reverse transcriptase, (ii) archaeal dna polymerase, and (iii) are directed to target RNA primer sets；And, make the reaction in reaction vessel Mixture undergoes the primer extension reaction of multiple circulations, to generate the DNA amplification product for indicating there is target RNA, each circulation bag Include：(i) reactant mixture is incubated to the denaturation duration less equal than 60 seconds under denaturation temperature, subsequent (II) will react Mixture is incubated the extension duration less equal than 60 seconds under elongating temperature, so as to expand target RNA；And (c) is operable Ground is coupled to the output module of the amplification module, and the wherein output module exports the information on target RNA or DNA product to connecing Receipts person.

In another aspect, the invention provides a kind of biological sample for being used to expand and being present in and directly being obtained from subject In target nucleus ribosomal ribonucleic acid (RNA) system.The system includes：(a) input module, it receives the target RNA in amplification biological sample User request；(b) module is expanded, it is asked in response to the user：(i) reactant mixture is received in reaction vessel, this is anti- Mixture is answered comprising the biological sample obtained from subject and for carrying out reverse transcription amplification and optional deoxyribose core Reagent necessary to sour (DNA) amplification, the reagent includes (1) reverse transcriptase, and (2) are directed to target RNA primer sets；And (ii) makes Reactant mixture undergoes the primer extension reaction of multiple circulations, indicates there is the detectable of target RNA in biological sample to produce The DNA amplification product of amount；(iii) amount of the DNA amplification product of detection (iii)；And (iv) is by the amount of DNA amplification product Information is exported to recipient, wherein for completing (i)-(iv) time quantum less than or equal to about 30 minutes；And (c) is operationally Coupled to the output module of the amplification module, described information is sent to recipient by the wherein output module.

In another aspect, it is present in the invention provides a kind of for expanding from the biological sample that subject obtains The system of target nucleic acid.The system includes：(a) input module, it receives user's request of the target RNA in amplification biological sample；(b) Module is expanded, it is asked in response to the user：Reactant mixture is received in reaction vessel, the reactant mixture includes biological sample Product and reagent necessary to for carrying out nucleic acid amplification, the reagent include (i) archaeal dna polymerase and optional reverse transcriptase, and (ii) primer sets of target nucleic acid are directed to；And, the reactant mixture in reaction vessel is undergone multiple serial primer extension reactions, Indicate there is the amplified production of target nucleic acid in biological sample to generate, each series following is followed including two or more Ring：(i) reactant mixture is incubated under the Denaturing being characterized with denaturation temperature and denaturation duration, subsequent (ii) exists To be incubated the reactant mixture under the conditions of elongating temperature and the extension that is characterized of extension duration, wherein with regard to Denaturing and/ Or for extension condition, single series is different from least one other single series in the multiple series；And (c) is operable Ground is coupled to the output module of the amplification module, and the wherein output module exports the information on target RNA or DNA product to connecing Receipts person.

In another aspect, it is used to expand from the target nucleic acid in the biological sample that subject obtains the invention provides a kind of System.The system may include the electronic display of the user interface with display graphic element, and the graphic element can be by user Access, to perform to expand the amplification scheme of the target nucleic acid in biological sample.The system may also include computer processor (bag Include any suitable device with computer processor as described elsewhere herein), it is coupled to the electronic display And be programmed to perform the amplification scheme when user selects the graphic element.The amplification scheme may include：Make comprising biology The multiple serial primer extension reactions of reactant mixture experience of sample and reagent necessary to for carrying out nucleic acid amplification, with life Into amplified production.The amplified production may indicate that the presence of biological sample target nucleic acid.In addition, the primer extension reaction of each series It may include two or more following circulations：It is incubated under the Denaturing being characterized with denaturation temperature and denaturation duration Reactant mixture, then the incubation reaction mixture under the conditions of with elongating temperature and the extension that is characterized of extension duration.Just For Denaturing and/or extension condition, single series may differ from least one other single system in the multiple series Row.

In addition, any restriction and description of the method to the present invention, unless substantially runed counter to, are otherwise also applied for the present invention's System.

In one aspect, the present invention relates to a kind of system for being used to detect the target nucleic acid molecule in biological sample.The system System may include：Input module, biological sample described in its reception processing is asked with the user for detecting the target nucleic acid molecule；And one Individual or multiple computer processors being operatively connected with the input block.One or more of computer processors can be single Solely or generally it is programmed for carrying out any method described herein.In one aspect, at one or more of computers Reason device can individually or generally be programmed for a) mixing to obtain mixture with lysis buffer by the biological sample；B) by institute The incubation duration that mixture is incubated no more than about 15 minutes under the incubation temperature higher than 15 DEG C is stated, or without being incubated； C) reaction vessel will be added to from mixture b), the reaction vessel includes the reagent needed for carrying out nucleic acid amplification, described Reagent includes：(i) DNA (DNA) polymerase, and (ii) are directed to the primer sets of the target nucleic acid molecule, anti-to obtain Answer mixture；And d) make the primer extension reaction of the multiple circulations of reactant mixture experience in the reaction vessel, with Generation indicates the amplified production that there is the target nucleic acid molecule in the sample, and each circulation includes：(i) under denaturation temperature The denaturation duration that the reactant mixture is less equal than 60 seconds is incubated, then (ii) is incubated described anti-under elongating temperature The extension duration for answering mixture to be less equal than 60 seconds, so as to expand the target nucleic acid molecule.The amplified production can be DNA product.

In one aspect, the present invention relates to a kind of system for being used to detect the target nucleic acid molecule in biological sample.The system System may include：Input module, biological sample described in its reception processing is asked with the user for detecting the target nucleic acid molecule；And one Individual or multiple computer processors being operatively connected with the input block.One or more of computer processors can be single Solely or generally it is programmed for carrying out any method described herein.In one aspect, at one or more of computers Reason device can individually or generally be programmed for a) mixing to obtain mixture with lysis buffer by the biological sample；B) by institute The incubation duration that mixture is incubated no more than about 15 minutes under the incubation temperature higher than 15 DEG C is stated, or without being incubated； C) reaction vessel will be added to from mixture b), the reaction vessel includes the reagent needed for carrying out nucleic acid amplification, described Reagent includes：(i) DNA (DNA) polymerase, and optionally, reverse transcriptase, and (ii) is for the target nucleic acid Primer sets, to obtain reactant mixture；And the reactant mixture in the reaction vessel is undergone multiple serial Primer extension reaction, to generate the amplified production for indicating there is the target nucleic acid in the sample, each series includes two Or more following circulation：(i) it is incubated under the Denaturing being characterized with denaturation temperature and denaturation duration described anti- Mixture is answered, subsequent (ii) is incubated the reaction under the conditions of the extension being characterized with elongating temperature and extension duration and mixed Thing, wherein for the Denaturing and/or the extension condition, single series is different from the multiple series at least One other single series.The biological sample can include fecal specimens.The biological sample can include milk sample.It is described to expand It can be DNA product to increase production thing.

The system of the present invention may also include biological sample processing module, and it causes the biological sample to be buffered with the cracking Liquid mixes to obtain the mixture.The biological sample processing module can include the mixture.The biological sample processing Module can be incubated the mixture.For example, the biological sample processing module may include heat block or incubator, the heat block Or incubator can be by the mixture of the biological sample and the lysis buffer at greater than about 15 DEG C, for example, greater than about 20 DEG C, greater than about 25 DEG C, greater than about 30 DEG C, greater than about 35 DEG C, greater than about 40 DEG C, greater than about 45 DEG C, greater than about 50 DEG C, greater than about 55 DEG C, greater than about 60 DEG C, greater than about 65 DEG C, greater than about 70 DEG C, greater than about 75 DEG C, greater than about 80 DEG C, greater than about 85 DEG C, greater than about 90 DEG C, greater than about 91 DEG C, greater than about 92 DEG C, greater than about 93 DEG C, greater than about 94 DEG C, greater than about 95 DEG C, greater than about 96 DEG C, greater than about It is incubated at a temperature of 97 DEG C, greater than about 98 DEG C, greater than about 99 DEG C, greater than about 100 DEG C, or, at about 20 DEG C to about 90 DEG C At a temperature of be incubated, for example, about 25 DEG C to about 85 DEG C, about 30 DEG C to about 80 DEG C, about 40 DEG C to about 70 DEG C, about 40 DEG C extremely About 95 DEG C, about 45 DEG C to about 90 DEG C, about 50 DEG C to about 85 DEG C, about 55 DEG C to about 80 DEG C, about 60 DEG C to about 75 DEG C, about 65 DEG C to about It is incubated at a temperature of 75 DEG C or about 65 DEG C to about 70 DEG C.

The mixture of the biological sample and the lysis buffer can be incubated few by the biological sample processing module In the incubation time of 20 minutes.For example, the incubation duration can be not more than 19 minutes, not more than 18 minutes, it is not more than 17 minutes, not more than 16 minutes, not more than 15 minutes, not more than 14 minutes, not more than 13 minutes, not more than 12 minutes, seldom In 11 minutes, not more than 10 minutes, not more than 9 minutes, not more than 8 minutes, not more than not more than 7 minutes, 6 minutes, not more than 5 Minute, not more than 4 minutes, not more than 3 minutes, not more than 2 minutes, not more than 1 minute, not more than 50 seconds, not more than 40 seconds, no More than 30 seconds, not more than 20 seconds, not more than 15 seconds, or not more than 10 seconds.

The homogeneous prepared product can be incubated by the biological sample processing module at a temperature of greater than about 40 DEG C, for example, Greater than about 65 DEG C, greater than about 70 DEG C, it can be higher than at greater than about 45 DEG C, greater than about 50 DEG C, greater than about 55 DEG C, greater than about 60 DEG C About 75 DEG C, greater than about 80 DEG C, greater than about 85 DEG C, greater than about 90 DEG C, greater than about 91 DEG C, greater than about 92 DEG C, greater than about 93 DEG C, it is higher than About 94 DEG C, greater than about 95 DEG C, greater than about 96 DEG C, greater than about 97 DEG C, greater than about 98 DEG C, greater than about 99 DEG C, greater than about 100 DEG C of temperature Degree is lower to be incubated.

The homogeneous prepared product can be incubated the incubation time of not more than 20 minutes by the biological sample processing module.Example Such as, it is described incubation the duration can be not more than 19 minutes, not more than 18 minutes, not more than 17 minutes, not more than 16 minutes, no More than 15 minutes, not more than 14 minutes, not more than 13 minutes, not more than 12 minutes, not more than 11 minutes, not more than 10 minutes, Not more than 9 minutes, not more than 8 minutes, not more than 7 minutes, not more than 6 minutes, not more than 5 minutes, not more than 4 minutes, seldom In 3 minutes, not more than 2 minutes, not more than 1 minute, not more than 50 seconds, not more than 40 seconds, not more than 30 seconds, not more than 20 seconds, Not more than 15 seconds, or not more than 10 seconds.

The system of the present invention may also include the amplification module being operably connected with the biological sample processing module, wherein The amplification module (i) adds a certain amount of mixture to reaction vessel from biological sample processing module, and (ii) makes reaction Reactant mixture experience primer extension reaction in container is to generate amplified production, and the amplified production indicates the target nucleic acid point The presence of son.

In some embodiments, the mixture of amplification module biological sample in the future processing module is added to reaction Container, the reaction vessel includes the reagent needed for carrying out nucleic acid amplification, and the reagent includes：(i) DNA (DNA) Polymerase, and optionally, reverse transcriptase, and (ii) one or more primer sets, each primer sets can specifically bind target nucleus Acid sequence or its variant, to obtain reactant mixture；And the reactant mixture in the reaction vessel is undergone one Or multiple serial primer extension reactions, to generate the amplified production for indicating there is the target nucleic acid sequence in the sample, Each series includes two or more following circulations：(i) in the denaturation being characterized with denaturation temperature and denaturation duration Under the conditions of be incubated the reactant mixture, subsequent (ii) is under the conditions of the extension being characterized with elongating temperature and extension duration The reactant mixture is incubated, wherein when carrying out multiple serial primer extension reactions, with regard to the Denaturing and/or described For extension condition, single series is different from least one other single series in the multiple series.

Or, the mixture can be added to the reaction vessel manually, and the amplification module can hold the reaction The reactant mixture in device undergoes one or more serial primer extension reactions, there is the target nucleic acid to generate instruction The amplified production of sequence.

In some embodiments, once identify included in the kit together applied with the system on for The information of one or more relevant parameters of primer extension reaction is carried out, the amplification module can make the reaction vessel automatically In the reactant mixture undergo one or more serial primer extension reactions, indicate exist in the sample to generate The amplified production of the target nucleic acid sequence.Described information can be recognized by the identification module communicated with the amplification module, such as this In text described in other parts.

The system of the present invention may also include the output module being operably connected with one or more computer processors, its Described in output module the information on the target nucleic acid molecule or the amplified production is exported to recipient.

In the system of the present invention, it is one or more of to calculate before biological sample is mixed with lysis buffer Machine processor can individually or be generally programmed in the solution suspend the biological sample, and the biology is included to obtain The homogeneous prepared product of sample.In some embodiments, one or more of computer processors can individually or generally It is programmed to centrifuge the biological sample, to obtain the supernatant and precipitation comprising biological sample, or obtains supernatant With the precipitation comprising biological sample.

In some embodiments, after the mixture of the biological sample and lysis buffer is incubated, described one Individual or multiple computer processors can individually or generally be programmed to that the mixture is centrifuged to obtain supernatant. The supernatant can include biological sample.Then, the supernatant can be used as to the mixture in subsequent reactions.For example, can be by The supernatant is added to comprising in the reaction vessel for carrying out the reagent needed for nucleic acid amplification.

In some embodiments, one or more of computer processors can individually or generally be programmed to make The biological sample increases bacterium through culture.For example, before being mixed with lysis buffer, one or more of computer processors It can individually or generally be programmed to make biological sample undergo the culture duration under the conditions of enrichment culture.The enrichment training Foster condition may include in suitable culture medium (such as Tryptic Soy nutrient solution (TBS), modified Tryptic Soy nutrient solution, tryptose Peptone, nutrient medium, bacteriolyze nutrient solution (LB), Gram-negative nutrient solution, peptone, the Tryptic Soy nutrient solution containing yeast, Or salmonella culture medium) at a suitable temperature (for example, about 23 DEG C to about 40 DEG C, such as about 25 DEG C, about 30 DEG C, about 35 DEG C or about 37 DEG C etc.) vibration or it is non-oscillating under the conditions of cultivate biological sample.In some embodiments, the culture medium It is salmonella special media, it is more beneficial for salmonella propagation relative to other bacteriums.Exemplary salmonella Special media includes bismuth sulfite agar (BS), xylose lysine deoxycholic acid agar (XLD), the training of selenium bright green sulfanilamide (SN) Nutrient solution (SBG) etc., but not limited to this.It is described culture the duration can be about 0.5 hour to 10 hours, for example, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, it is about 5.5 small When, about 6 hours, about 6.5 hours, about 7 hours, about 7.5 hours, about 8 hours, about 8.5 hours, about 9 hours, about 9.5 hours or About 10 hours.In some embodiments, the culture duration is no more than about 7 hours, for example, no more than about 6.5 is small When, no more than about 6 hours, no more than about 5.5 hours, no more than about 5 hours, no more than about 4.5 hours, no more than about 4 hours, No more than about 3.5 hours, no more than about 3 hours, no more than about 2.5 hours, no more than about 2 hours, no more than about 1.5 hours, No more than about 1 hour or no more than about 0.5 hour.

In some embodiments, before the biological sample experienced the culture duration under the conditions of enrichment culture And/or afterwards, one or more of computer processors can individually or generally be programmed to enter the biological sample Row centrifugation, to obtain the supernatant and precipitation comprising biological sample, or obtains supernatant and the precipitation comprising biological sample.

In some embodiments, the biological sample experienced under the conditions of enrichment culture culture the duration it Afterwards, one or more of computer processors can individually or generally be programmed to buffer the biological sample with cracking Liquid is mixed, and is not suffered from selective enrichment, is plated on differentiation culture medium, and/or expected property biomedicine identification.

In some embodiments, the biological sample experienced under the conditions of enrichment culture culture the duration it Afterwards, one or more of computer processors can individually or generally be programmed to mix lysis buffer added to described Compound.The lysis buffer can be alkaline.For example, the lysis buffer can include NaOH.The lysis buffer PH can be about 7 to about 14, such as about 8 to about 13, about 9 to about 12, about 10 to about 11.For example, the pH of the lysis buffer can It is about 7,7.5,8,8.5,9,9.5,10,10.5,11,11.5,12,12.5,13,13.5 or 14.

In some embodiments, one or more of computer processors can individually or generally be programmed to make The mixture of the biological sample and the lysis buffer adds under conditions of DNA or ribonucleic acid (RNA) extraction is not undergone Add in reaction vessel.In some cases, one or more of computer processors can individually or be generally programmed To make the mixture be added under conditions of not undergoing purifying in reaction vessel.In some cases, it is one or many Individual computer processor can individually or generally be programmed to make the mixture not undergo the condition of DNA or RNA concentrations It is added in reaction vessel down.The reaction vessel can be to include the reaction vessel of the reagent needed for for carrying out nucleic acid amplification.

In some embodiments, one or more of computer processors can individually or generally be programmed to by The biological sample obtained from subject or the mixture obtained as described herein from the biological sample, supernatant or homogeneous system Standby thing with for providing to obtain reactant mixture together with the reagent needed for nucleic acid amplification is carried out in reaction vessel.The reaction Container can be any reaction vessel specifically described herein.

In the system of the present invention, identification module can also be included, for recognizing that the system with the present invention is used together The information that kit is included.For example, the kit is marked with unique identifiers.The unique identifiers can be bar code. The bar code can be one-dimensional bar code or two-dimensional bar.The bar code can allow to extract by scanning on the examination The information of agent box.The unique identifiers can relate to contactless (contactless) technology.The contactless technology allows to pass through The kit is placed near Poul Dorset Sheep device to extract the information on the kit.The Poul Dorset Sheep device can Using radio frequency identification (RFID, radio frequency identification) technology information is extracted from the kit. In some embodiments, the unique identifiers can be RFID marker.

In some embodiments, the identification module can include bar code scanner module, for scanning on kit Bar code is to extract information.In some embodiments, the identification module can be comprising RFID module (for example, Poul Dorset Sheep Device), for extracting information from the kit using REID.In some embodiments, the identification module bag Containing both the bar code scanner module and the RFID module.

The identification module can be operably connected or communicate with one or more other modules of the present invention, so that One or more other modules are given by the information transfer extracted.Described information can be the correlation for carrying out primer extension reaction Parameter.For example, the identification module can communicate with amplification module of the present invention, so that the amplification module to be performed The relevant parameter of primer extension reaction is transferred to the amplification module.The parameter is upon receipt of, the amplification module can root The primer extension reaction is performed automatically according to the parameter.The parameter can be, such as the serial number of described primer extension reaction, Period, Denaturing, extension condition, one or more primer sets, report agent, oligonucleotide probe of each series etc., but Not limited to this.

In the system of the present invention, one or more of computer processors can individually or generally be programmed to inspection Survey amount and/or the presence or absence of the amplified production.In some cases, one or more of computer processors can be independent Ground is generally programmed to indicate that the amount of the amplified production and/or the information of presence or absence are exported to recipient.

In the system of the present invention, detection module, the amount for detecting amplified production can also be included.The detection module Any detection method specifically described herein can be used to detect amplified production.For example, the detection module can detect amplified reaction The detectable signal of middle generation.For example, the detection module can detect the detectable signal that report agent specifically described herein is produced. The detectable signal may indicate that amplified production whether there is.For example, the existence or non-existence of detectable signal may indicate that amplification Product whether there is.The intensity of detectable signal can be proportional to the amount of amplified production.In some cases, when amplified production by When being generated with the different types of nucleic acid of target nucleic acid that initially expands, the intensity of detectable signal can be with the target nucleic acid that initially expands Amount it is proportional.

In some embodiments, the detection module is optical detecting module.The optical detecting module is detectable to be expanded Increase in reaction and produce optical signalling.The optics letter that the optical signalling can produce for any optical activity dyestuff specifically described herein Number.Produced and adjoint optical signalling for example, the optical signalling can be due to amplified production.For example, the optical signalling It can be due to the optical signalling that oligonucleotide probe is produced in fracture.For example, the optical signalling can be molecular beacon The optical signalling produced when hybridizing with amplified production.

In some embodiments, the optical signalling can be fluorescence signal, and the detection module can be fluoroscopic examination Module.For example, the detectable fluorescence signal that any fluorescent dye is produced herein of the fluoroscopic examination module.For example, described glimmering Light detection module can detect the fluorescence signal for the fluorescent dye generation that one or more are selected from the group：FAM, TET, ROX, JOE, HEX, TAMRA, VIC, NED, PET, texas Red etc..

In some embodiments, the detection module can a variety of detectable signals of Parallel testing.For example, it is described it is a variety of can Detection signal is produced by a variety of report agent, and the detectable signal that a variety of report agent are produced is different from each other.It is described many Each planted in detectable signal may indicate that its corresponding amplified production whether there is.It is every in a variety of detectable signals A kind of intensity can corresponding amplified production amount it is proportional.For example, the detection module can Parallel testing FAM fluorescence and ROX fluorescence.In the case, a kind of corresponding detectable signal of amplified production can be FAM fluorescence, and another amplified production pair The detectable signal answered can be ROX fluorescence, so that can the different detectable signal of Parallel testing.Parallel testing it is different can Detection signal may be such that the presence or absence that can compare different amplified productions and amount.Or, the different detectable letter of Parallel testing Number may be such that can determine the amount of amplified production for internal reference thing.

Detection module can include an imaging sensor or multiple images sensor.Imaging sensor be able to can be carried out Optical detection.Imaging sensor can include charge coupling device (CCD) sensor, and it includes cooled CCD.Imaging sensor CMOS active pixel sensor (APS), such as COMS or NMOS sensors can be included.Detection module can include laser sensor. Detection module can include photodiode, such as avalanche photodide.Detection module can include photomultiplier (PMT).Sensor can include single sensor or same type or different types of multiple sensors.

Detection module can be imaged to detection object (detection zone described in other parts such as herein).Detection Module can in a visual field to it is described detection object at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%th, 90%, 95% or 100% it is imaged.Detection module can herein described in other parts device or system (example Such as nucleic acid amplifier or system) operation before, during or after detection object is imaged.Detection module can be in dress Put or be imaged in real time when system is operated, for example, be imaged in real time when carrying out nucleic acid amplification reaction using thermal cycler.

Detection module can record single still image or can record video.Detection module can be with sampling per minute extremely Few about 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27, 28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、 53rd, 54,55,56,57,58,59,60,90,120,150,180,210,240,270 or 300 times.Detection module can be with per minute Sampling at most about 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25, 26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、 51st, 52,53,54,55,56,57,58,59,60,90,120,150,180,210,240,270 or 300 times.Detection module can be with It is per minute sampling about 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24, 25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、 50th, 51,52,53,54,55,56,57,58,59,60,90,120,150,180,210,240,270 or 300 times.Detection module can With by least about 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0,1.1,1.2,1.3,1.4,1.5,1.6, 1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0、3.1、3.2、3.3、3.4、3.5、 3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5.0、6、7、8、9、10、15、20、 25th, 30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,105,110,115 or 120Hz speed is carried out Sampling.Detection module can by most about 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0,1.1,1.2,1.3, 1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0、3.1、3.2、 3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5.0、6、7、 8th, 9,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,105,110,115 or 120Hz speed is sampled.Detection module can by about 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0, 1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、 3.0、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、 4.9、5.0、6、7、8、9、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、 105th, 110,115 or 120Hz speed is sampled.

Detection module can have specific resolution ratio or sensitivity.Detection module can include at least about in one side 100th, 200,500,1000,2000,3000,4000,5000,6000,7000,8000,9000 or 10000 pixels.Detect mould Block can include at least about 0.5,1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5, 8.0、8.5、9.0、9.5、10.0、10.5、11.0、11.5、12.0、12.5、13.0、13.5、14.0、14.5、15.0、15.5、 16.0、16.5、17.0、17.5、18.0、18.5、19.0、19.5、20.0、21、22、23、24、25、26、27、28、29、30、 31st, 32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49 or 50,000,000 pixels.Detection module Can have at least about 10mm, 9mm, 8mm, 7mm, 6mm, 5mm, 4mm, 3mm, 2mm, 1mm, 0.9mm, 0.8mm, 0.7mm, 0.6mm、0.5mm、0.4mm、0.3mm、0.2mm、0.1mm、0.09mm、0.08mm、0.07mm、0.06mm、0.05mm、 0.04mm、0.03mm、0.02mm、0.01mm、0.009mm、0.008mm、0.007mm、0.006mm、0.005mm、0.004mm、 0.003mm、0.002mm、0.001mm、900nm、800nm、700nm、600nm、500nm、400nm、300nm、250nm、200nm Or 100nm resolution ratio.Detection module can have at most about 10mm, 9mm, 8mm, 7mm, 6mm, 5mm, 4mm, 3mm, 2mm, 1mm、0.9mm、0.8mm、0.7mm、0.6mm、0.5mm、0.4mm、0.3mm、0.2mm、0.1mm、0.09mm、0.08mm、 0.07mm、0.06mm、0.05mm、0.04mm、0.03mm、0.02mm、0.01mm、0.009mm、0.008mm、0.007mm、 0.006mm、0.005mm、0.004mm、0.003mm、0.002mm、0.001mm、900nm、800nm、700nm、600nm、 500nm, 400nm, 300nm, 250nm, 200nm or 100nm resolution ratio.Detection module can have about 10mm, 9mm, 8mm, 7mm、6mm、5mm、4mm、3mm、2mm、1mm、0.9mm、0.8mm、0.7mm、0.6mm、0.5mm、0.4mm、0.3mm、0.2mm、 0.1mm、0.09mm、0.08mm、0.07mm、0.06mm、0.05mm、0.04mm、0.03mm、0.02mm、0.01mm、0.009mm、 0.008mm、0.007mm、0.006mm、0.005mm、0.004mm、0.003mm、0.002mm、0.001mm、900nm、800nm、 700nm, 600nm, 500nm, 400nm, 300nm, 250nm, 200nm or 100nm resolution ratio.

Detection module can include light source.Light source can be integrated into detection module housing or electrophoretic apparatus housing.Light Source can include lamp, such as incandescent lamp, Halogen lamp LED, fluorescent lamp, gas-discharge lamp, arc lamp or LED.Light source can include swashing Light device.Light source can produce specific wavelength or wave-length coverage, such as UV.Light source can include filter, for control one or Multiple output wavelengths.Light source can include the multiple light sources of identical or different type, and it can be used separately or in combination.Light Source can be by the housing of their own or by electrophoretic apparatus housings close.

Detection module can include various optical elements, including but not limited to filter, lens, collimater, reflective mirror, anti- Emitter, beam splitter and diffusing globe.Detection zone can be exposed to light, such as light from light source.Light can pass through light source and base One or more optical elements between matter, or light directly can advance to detection zone from light source.Detection module can connect Receive the light from detection zone.Light can be through detection zone and detection module one or more optical elements, or Light directly can advance to detection module from detection zone.Light can be collimation.Light can be evenly distributed in detection zone One or more dimensions on.One or more dimensions can be illuminated using the light for being substantially identical intensity.Can be by Light is guided substantially perpendicular to the mode of detection zone upper surface.Can also be according to being not substantially perpendicular in detection zone upper table The mode in face guides light.Light can be multidirectional.Light can include a wavelength, narrowband wavelength, broad band wavelength or all band Spectrum.Light can include visible wavelength.Light can include ultraviolet wavelength.Light can include IR wavelength.Light can be by Lens focus.The image produced by detection module can include bright vision image.The image produced by detection module can include Dark field image.The image produced by detection module can include fluoroscopic image.

Detection module can include one or more filters, including but not limited to wavelength filter (for example, colour filter, Ultraviolet filter, infrared filter), dichroic filter and polarizing filter.Filter can include same type or Different types of multiple filters, it can be used separately or in combination.

Detection module can include one or more lens.Lens can be pack-shot lens or " close-shot " lens.Lens can To be zoom lens.Lens can be Infrared Lens.Lens can be ultraviolet lens.Lens can be wide-angle lens, including not It is limited to wide-angle lens, extrawide angle lens and fish-eye lens.Lens can include same type or different types of multiple lens, its It can be used separately or in combination.

Detection module can include the device for being used to remove pattern distortion or aberration, such as removing barrel distortion or fish Eye distortion, pincushion distortion, beard shape distort, monochromatic aberration (for example, piston, tilt, defocus, spherical aberration, coma, astigmatism, as Field bend, pattern distortion) or aberration (for example, axial direction, longitudinal direction, lateral, horizontal aberration) device.Such device It can include being programmed to realize the computer system for the partially or even wholly instruction of correcting image distortion.For example, can To use Blang's distortion model (Brown ' s distortion model) or Blang-Joseph Conrad model (Brown-Conrady Model correct radial distortion and tangential distortion) are come.

Detection module can include communication equipment, and such as wired (for example, USB) or wireless (for example, Wi-Fi, bluetooth) leads to Believe equipment.Communication equipment can include the equipment for radio.Communication equipment can include being used for Free Space Optics (FSO) The equipment of communication, such as visible light communication or infrared (IR) communicate.Communication equipment can include the equipment for wire communication, bag Include but be not limited to USB (USB), optical fiber, Peripheral Component Interconnect (PCI), PCI Express (PCIe) or Thunderbolt.Communication equipment can include the equipment for Wi-Fi, such as IEEE 802.11a, b, g or n Wi-Fi.It is logical Believe equipment can include for cellular data service equipment, such as GSM, CDMA, GPRS, 3G (for example, W-CDMA, EDGE and ) or 4G (for example, Long Term Evolution (LTE), mobile WiMAX) CDMA2000.Communication equipment can include being used for mobile satellite communication Equipment.Communication equipment can include the equipment for Bluetooth communication.Communication equipment can include polytype communication equipment, Such as USB and Wi-Fi, or bluetooth and Wi-Fi.

Communication equipment can transmit the information for carrying out self-detection module, and the image such as recorded by detection module is (for example, detection The image in region).Communication equipment can transmit the information of such as image etc in real time.Communication equipment can be with electrophoretic apparatus And/or the communication of its control system.Communication equipment can communicate with remote computer system, the remote computer system such as Desktop computer, laptop computer, tablet PC, smart phone device or server.Communication equipment can be with such as hand Hold the display device communication of formula display device etc.Communication equipment can be transmitted to the user at independent position or facility to be believed Breath, such as image.

In some embodiments, the detection module can be any of detection module, include but is not limited to herein Detector described in middle other parts.

In some cases, before the primer extension reaction is carried out, one or more of computer processors can Individually or generally it is programmed to the target nucleic acid molecule is undergone one or more Denaturings.It is one or more of to become Property condition may be selected from denaturation temperature distribution and denaturant.

In some cases, before the primer extension reaction is carried out, one or more of computer processors can Individually or generally it is programmed to preheat the biological sample under about 90 DEG C to about 100 DEG C of pre-heating temperature and does not surpass The pre-add thermal endurance for spending about 10 minutes.In some embodiments, the pre-add thermal endurance is no more than 1 minute.

Any aspect in the multiple aspect, one or more of computer processors can individually or generally It is programmed to carry out the primer extension reaction of multiple circulations.Any an appropriate number of circulation can be carried out.For example, the period carried out Can with less than about 100,90,80,70,60,50,40,30,20,10 or 5 circulations.

In some embodiments, one or more of computer processors can individually or generally be programmed to make Reactant mixture undergoes multiple serial primer extension reactions.Single series in the multiple series may include multiple circulations Specific primer extension, the reaction is characterised by, for example, specific denaturation as described elsewhere herein and extension bar Part.Generally, for example, for Denaturing and/or extension condition, each single series is different from the multiple series extremely Few other single series.For example, when continuing with regard to oblique variable Rate, denaturation temperature, denaturation duration, elongating temperature and extension Between in any one, two, three, four or all for five, single series may differ from the multiple series Another single series.In addition, multiple series may include any number of single series, for example, at least about or about 2,3,4, 5th, 6,7,8,9,10 or more a single series.

For example, one or more of computer processors can individually or generally be programmed to include first and be Multiple serial primer extension reactions of row and second series.First Series, for example, it may include the primer more than ten circulations prolongs Reaction is stretched, reactant mixture is incubated and is no more than by wherein each circulation of First Series including (i) at about 92 DEG C to about 95 DEG C 30 seconds, reactant mixture was incubated no more than about one minute by subsequent (ii) at about 35 DEG C to about 65 DEG C.Second series, for example, It may include the primer extension reaction more than ten circulations, each circulation of wherein second series exists reactant mixture including (i) It is incubated and is no more than 30 seconds at about 92 DEG C to about 95 DEG C, reactant mixture is incubated by subsequent (ii) at about 40 DEG C to about 60 DEG C is not surpassed Spend about 1 minute.In this instantiation, the first and second series are different in their elongating temperature condition.However, the reality Example is not intended to limit, because any combination of different extensions and Denaturing can be used.

In some embodiments, one or more of computer processors can individually or generally be programmed to make Target nucleic acid is subjected to Denaturing before primer extension reaction startup.In the case of multiple serial primer extension reactions, institute Stating one or more computer processors can individually or generally be programmed to make target nucleic acid perform the multiple series Before be subjected to Denaturing, or Denaturing can be subjected between the multiple series.For example, one or more of computers Processor can individually or generally be programmed to make to pass through between First Series and second series of the target nucleic acid in multiple series By Denaturing.The non-limiting examples of these Denaturings include denaturation temperature distribution (for example, one or more become warm-natured Degree) and denaturant.

In some embodiments, one or more of computer processors can individually or be generally programmed to Carry out preheating biological sample before primer extension reaction.Preheat the temperature (for example, pre-heating temperature) of biological sample and hold The continuous time (for example, pre-add thermal endurance) can change according to the particular organisms sample for example analyzed.In some instances, Can by biological sample preheating no more than about 60 minutes, 50 minutes, 40 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 Minute, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, 45 seconds, 30 seconds, 20 seconds, 15 Second, 10 seconds or 5 seconds.In some instances, biological sample can be preheated at a temperature of about 80 DEG C to about 110 DEG C.In some realities In example, biological sample can be preheated at a temperature of about 90 DEG C to about 100 DEG C.In some instances, can be at about 90 DEG C to about 97 Biological sample is preheated at a temperature of DEG C.In some instances, biological sample can be preheated at a temperature of about 92 DEG C to about 95 DEG C Product.In other example, can 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, preheat at a temperature of 99 DEG C or 100 DEG C it is biological Sample.

In some embodiments, system of the invention may include the electronic display of the user interface with display graphic element Display screen, the graphic element can be accessed by the user, to perform to expand the amplification scheme of the target nucleic acid in biological sample.The system It may also include computer processor (including any suitable dress with computer processor as described elsewhere herein Put), it is coupled to the electronic display and is programmed to perform the amplification scheme when user selects the graphic element.The expansion Increasing scheme may include：Make to undergo multiple systems comprising biological sample and for the reactant mixture of the reagent needed for progress nucleic acid amplification The primer extension reaction of row, to generate amplified production.The amplified production may indicate that the presence of biological sample target nucleic acid.In addition, The primer extension reaction of each series may include two or more following circulations：With denaturation temperature and denaturation duration Incubation reaction mixture under the Denaturing being characterized, then in the extension bar being characterized with elongating temperature and extension duration Incubation reaction mixture under part.For Denaturing and/or extension condition, single series may differ from the multiple series At least one other single series.

In some embodiments, target nucleic acid can be related to disease.For example, the disease can be with RNA virus or DNA virus phase Close.The example of virus is provided in elsewhere herein.In some embodiments, the disease can be with malignant bacteria (e.g., tuberculosis point Branch bacillus) or pathogenic protozoa (for example, such as plasmodium in malaria) (including these pathogen described elsewhere herein Example) it is related.In some embodiments, the amplification scheme may point to the presence based on amplified production to determine the disease Presence.

In some cases, user interface can be graphic user interface.In addition, user interface may include it is one or more Graphic element.Graphic element may include image and/or text message, such as picture, icon and text.Graphic element is in user interface On can be of different sizes and direction.In addition, electronic display can be any suitable electronic console, including herein its The example of his place description.The non-limiting examples of electronic display include monitor, mobile device screen, laptop computer Screen, TV, portable video game system screen and calculator screen.In some embodiments, electronic display may include to touch Shield (for example, condenser type or resistive touch screen) so that the graphic element shown in the user interface of electronic display can be through Selected by user's touch electronic display.

In some embodiments, the amplification scheme can further comprise selecting primer sets for target nucleic acid.Situations such as this Under, primer sets can be for expand target nucleic acid molecule one or more sequences and specially designed primer sets.In some implementations In scheme, the amplification scheme can further comprise that selection has specific report to one or more sequences of target nucleic acid molecule Agent (for example, other kinds of report agent described in oligonucleotide probe or elsewhere herein comprising optical activity species). In addition, in some embodiments, the reagent may include as described elsewhere herein for appointing necessary to nucleic acid amplification What suitable reagent, such as DNA (DNA) polymerase, the primer sets for target nucleic acid and (optionally) reverse transcription Enzyme.

In some embodiments, user interface can show multiple graphic elements.Each graphic element can be with multiple amplifications Given amplification scheme in scheme is associated.Each scheme in the multiple amplification scheme may include serial primer extension reaction Various combination.However, in some cases, user interface can show the multiple pattern primitives associated with same amplification scheme Element.In some embodiments, each graphic element and/or can be associated with disease, and in the multiple amplification scheme Given amplification scheme may point to determine the presence of the disease of subject.Therefore, in tiiese cases, graphic element may be selected in user To run an amplification scheme (or a series of amplification scheme) so as to analyze specific disease.In some embodiments, The disease can be with virus (for example, any RNA virus or DNA virus, including these viral realities described elsewhere herein Example) it is related.The non-limiting examples of virus include human immunodeficiency virus I (HIV I), human immunodeficiency virus II (HIV II), orthomyxovirus, Ebola virus, dengue virus, influenza virus (for example, H1N1 is viral, H3N2 virus, H7N9 virus or H5N1 virus), hepatitis viruse, hepatitis A virus, hepatitis type B virus, HCV (for example, tool first the types of RNA- third Hepatitis viruse (RNA-HCV)), Hepatitis D virus, HEV, HGV RNA, Epstein-Barr virus, monocytosis Syndrome virus, cytomegalovirus, SARS virus, west nile fever virus, poliovirus, measles virus, herpes simplex virus, Variola virus, adenovirus (for example, 55 type adenovirus (ADV 55), 7 type adenovirus (ADV 7)) and varicella virus.In some realities Apply in scheme, the disease can be with malignant bacteria (for example, mycobacterium tuberculosis) or pathogenic protozoa (for example, the malaria in malaria Protozoon) (example for including these pathogen described elsewhere herein) correlation.

In many aspects, the system includes input module, and the input module receives amplification and is present in directly from subject User's request of target nucleic acid (for example, target RNA, target DNA) in the biological sample of acquisition.Can be used can receive these users please Any suitable module asked.The input module may include, for example, the device comprising one or more processors.Include processing The non-limiting examples of the device of device (for example, computer processor) include：Desktop computer, laptop computer, flat board meter Calculation machine (for example, iPad、Galaxy Tab), cell phone, smart phone (for example, iPhone、The phone of support), personal digital assistant (PDA), video game console, TV, Music player devices (for example,IPod), video playback apparatus, pager and calculator.Processor can with one or Other units of multiple controllers, computing unit and/or computer system are associated, or when needed in implantation firmware.Such as Fruit is implemented in software, then routine (or program) can be stored in any computer-readable memory such as RAM, ROM, flash storage In device, disk, laser disk or other storage mediums.Similarly, the software can be transported to dress via any of transfer approach Put, this method includes, for example, through communication channel, such as telephone wire, internet, local Intranet, wireless connection, or via Portable medium, such as computer readable disk, flash disc drives.Each step can be used as various district's groups, operation, instrument, mould Block or technology are implemented, and the latter can implement in hardware, firmware, software or its any combination again.When implementing within hardware, Some or all of these district's groups, operation, technology etc. can be in such as customization integrated circuit (IC), application specific integrated circuits (ASIC), implement in FPGA (FPGA), programmable logic array (PLA) etc..

In some embodiments, input module is configured to receive the user's request for carrying out target nucleic acid amplification.Input mould Block can directly (for example, by input equipment, the keyboard such as operated by user, mouse or touch-screen) or indirectly (for example, By wired or wireless connection, including through internet) receive user's request.Input module can be via output electronic device to amplification Module provides the request of user.In some embodiments, input module may include user interface (UI), such as graphic user interface (GUI), the user interface is configured to the request for making user provide amplification target nucleic acid.GUI may include text, figure and/ Or audio frequency component.GUI can be provided on an electronic display, and the electronic console includes the aobvious of the device containing computer processor Show device.These displays may include resistance-type or capacitive touch screen.

The non-limiting examples of user include therefrom obtaining the subject, medical worker, clinician's (example of biological sample Such as, doctor, nurse, Laboratory Technician), lab assistant (such as hospital laboratory technical staff, Research Scientist, pharmacy Scientist), the clinical observer of clinical test, or other users in health care industry etc..

In many aspects, the system includes amplification module, and the amplification module is used in response to being received by input module User asks and carries out nucleic acid amplification reaction to target nucleic acid or part thereof.The amplification module can be able to carry out as described herein Where method, and can include fluid treating device, one or more thermal cyclers, for receiving one or more reaction vessels The device in (for example, hole of the hot block of thermal cycle), can detect amplified production detector (for example, fluorescence detector, spectrum examine Survey device, electrochemical detector) and for recipient export on amplified production (DNA product of amplification) presence and/or In the device of the information (for example, information of initial data, processed data or any other type as described herein) of amount Any device.In some cases, the amplification module may include with computer processor as described elsewhere herein Device, and can with can be analyzed under the auxiliary of Suitable software Autonomous test acquisition initial data.In addition, in some implementations In scheme, the amplification module may include to input electronic device necessary to receiving instruction from input module and may include and export Module exports electronic device necessary to being communicated.

In some embodiments, material, amplification of nucleic acid, detection amplified production and output information are provided to reaction vessel One or more steps can be by amplification module automation mechanized operation.In some embodiments, automation mechanized operation may include to use one Individual or multiple fluid processors and associated software.These processes can be run using some commercially available fluid handling systems Automation mechanized operation.The non-limiting examples of these fluid processors include coming from Perkin-Elmer, Caliper Life Sciences, Tecan, Eppendorf, Apricot Design and Velocity 11 fluid processor.

In some embodiments, amplification module may include real-time detector device.The non-limiting examples bag of these instruments Include real-time PCR thermal cyclers, ABI7000 sequence detection systems, ABI7700 sequence detection systems, The real-time PCR systems of Applied Biosystems 7300, the real-time PCR systems of Applied Biosystems 7500, Applied Biosystems 7900HT Fast real-time PCR systems (are all from Applied Biosystems)；LightCycler^TMSystem (Roche Diagnostics GmbH)；Mx3000P^TMReal-time PCR system, Mx3005P^TMReal-time PCR system and Multiple quantitative PCR system ( Multiplex Quantitative PCR System)(Stratagene,La Jolla,Calif.)；And intelligent circulation instrument system (Smart Cycler System) (Cepheid, by Fisher Scientific is distributed).In some embodiments, amplification module may include another self-reacting device, for example, AmpliPrep/System (Roche Molecular Systems), TIGRIS DTS systems (Hologic Gen-Probe, San Diego, CA), PANTHER systems (Hologic Gen-Probe, San Diego,CA)、BD MAX^TMSystem (Becton Dickinson), GeneXpert systems (Cepheid),(BioFire Diagnostics) system, iCubate systems, IDBox systems (Luminex), EncompassMDx^TM(Rheonix) system, Liat^TMAanlyzer (IQuum) system, Biocartis Molecular Diagnostic Platform systems,ML systems (Enigma Diagnostics),System (T2Biosystems)、System (NanoSphere), Great Basin Diagnostic System, Unyvero^TMSystem (Curetis), PanNAT systems (Micronics) or Spartan^TMRX systems (Spartan Bioscience)。

In all fields, the system includes the output module for being operably coupled to amplification module.In some embodiment party In case, the output module can include the device with the processor as described above for input module.The output module can be wrapped Input equipment as described herein is included, and/or may include the input electronic device for being communicated with amplification module.At some In embodiment, the output module can be electronic console, in some cases, and the electronic console includes UI.At some In embodiment, the output module is operably coupled to the communication interface of computer network such as internet.In some implementations In scheme, any suitable telecommunication media (including computer network, wireless network, local Intranet can be used in the output module Or internet) by information to being sent to the recipient in Local or Remote position.In some embodiments, the output module The data received from amplification module can be analyzed.In some cases, the output module includes that report can be generated and will reported The Report Builder for being sent to recipient is accused, wherein this report includes the amount on amplified production as described elsewhere herein And/or any information existed.In some embodiments, the output module may be in response to the information received from amplification module Information is automatically transmitted, such as with initial data or in the form of the data analysis of the software progress included in amplification module. Or, the output module can transmit information after user instruction is received.The information transmitted by output module can be through electronically Checked or printed by printer.

One in input module, biological sample processing module, identification module, detection module, amplification module and output module Kind or it is a variety of can be included in same device, or one or more identical components can be included.For example, amplification module can also be wrapped Containing input module, biological sample processing module, identification module, detection module, output module, or one or more therein. In other examples, the device comprising processor, which both can be included in input module, can also reside in output module.User can make Asked to expand target nucleic acid with the device, and can also use the apparatus as by the information transmission of amplified production extremely The instrument of recipient.In some cases, the device comprising processor can be included in all six kinds of modules so that should include place The device of reason device can also be used for instrument of the control included in amplification module or any other module (for example, thermal cycler, incubation Device, detector, fluid treating device), instruction is provided the instrument, and receives the information returned from the instrument.Six kinds of moulds Each of block can further include one or more computer processors specifically described herein, and/or can perform described one kind Or the function that a variety of computer processors are programmed to execute.

Example system for expanding target nucleic acid according to methods described herein is shown in Figure 1.The system includes both filling When again a part for input module may act as the computer 101 of a part for output module.User will include and be ready for nucleic acid The reaction vessel 102 of the reactant mixture of amplification is put into amplification module 104.The amplification module includes thermal cycler 105 and inspection Survey device 106.Input module 107 includes computer 101 and related input equipment 103 (for example, keyboard, mouse etc.), and input is set Standby 103 can receive the request that user is expanded to the target nucleic acid in reactant mixture.Input module 107 is by the request of user Amplification module 104 is communicated to, and nucleic acid amplification starts in thermal cycler 105.As amplification is carried out, the detection of module is expanded Device 106 is detected to amplified production.Information (for example, the initial data obtained by detector) on amplified production is from detection Device 106 sends back computer 101, and computer 101 also functions as the component of output module 108.Computer 101 is received from amplification mould The information of block 104, carries out any extra operation to the information, subsequently generates the report for including the information through processing.Once report Accuse generation, computer 101 then via computer network interface 110 by computer network (for example, Intranet, internet), It will be reported with hard copy form or via the electronic console 112 for being operably coupled to computer 101 via printer 111 It is sent to its final recipient 109.On the one hand, the invention provides a kind of computer-readable comprising machine executable code Medium, when it is executed by one or more processors, implements the method according to any method disclosed herein.On the other hand, The invention provides a kind of computer-readable medium for including machine executable code, it is by one or more computer processors During execution, implement amplification and be present in the direct method from the target nucleus ribosomal ribonucleic acid (RNA) in the biological sample that subject obtains, the party Method includes：(a) provide comprising biological sample and for abreast carrying out reverse transcription amplification institute with DNA (DNA) amplification The reaction vessel of required reagent, to obtain reactant mixture, the reagent include (i) reverse transcriptase, (ii) archaeal dna polymerase, and (iii) target RNA primer sets are directed to；And (b) makes the primer extend of the multiple circulations of reactant mixture experience in reaction vessel anti- Should, to generate the DNA amplification product for indicating there is target RNA, each circulation incubates reactant mixture including (i) under denaturation temperature Educate the denaturation duration less equal than 60 seconds, reactant mixture is incubated by subsequent (ii) under elongating temperature to be less equal than The extension duration of 60 seconds, so as to expand target RNA.

On the other hand, the invention provides a kind of computer-readable medium for including machine executable code, it is by one When individual or multiple computer processors are performed, implement amplification and be present in directly from the target nucleus sugar in the biological sample that subject obtains The method of nucleic acid (RNA), this method includes：(a) biological sample obtained from subject is received；(b) provide comprising biological sample Product and the reaction vessel that necessary reagent is expanded for progress reverse transcription amplification and optional DNA (DNA), with Reactant mixture is obtained, the reagent is directed to target RNA primer sets comprising (i) reverse transcriptase and (ii)；(c) pass through reactant mixture The primer extension reaction of multiple circulations is gone through, is produced with the DNA amplification for producing the detectable amount for indicating to exist in biological sample target RNA Thing；(d) amount of the DNA product of detection (c)；And (e) exports the information of the amount on DNA product to recipient, wherein being used for (a)-(e) time quantum is completed less than or equal to about 30 minutes.

On the one hand, the invention provides a kind of computer-readable medium for including machine executable code, its by one or When multiple computer processors are performed, implement amplification and be present in from the target nucleus ribosomal ribonucleic acid in the biological sample that subject obtains (RNA) method, this method includes：(a) provide comprising biological sample and for the anti-of reagent necessary to progress nucleic acid amplification Container is answered, to obtain reactant mixture, the reagent includes (i) archaeal dna polymerase and optional reverse transcriptase, and (ii) is directed to target nucleus The primer sets of acid；And (b) makes the reactant mixture in reaction vessel undergo multiple serial primer extension reactions with by target nucleic acid Amplified production is generated, each series includes two or more following circulations：(i) with denaturation temperature and denaturation duration Incubation reaction mixture under the Denaturing being characterized, subsequent (ii) is in prolonging for being characterized with elongating temperature and extension duration Incubation reaction mixture under the conditions of stretching, wherein for Denaturing and/or extension condition, single series is different from the multiple At least one other single series in series.

Computer-readable medium can take many forms, include but is not limited to, tangible (or non-transitory) storage medium, load Ripple medium or physical transmission medium.Non-volatile memory medium includes, for example, appointing in CD or disk, such as any computer What storage device etc., such as available for calculation procedure, process step is implemented.Volatile storage medium includes dynamic memory The main storage of device, such as computer.Tangible transmission media includes coaxial cable；Wrapped in copper cash and optical fiber, including computer system Wire containing bus.Carrier wave transmission media can take electric signal or electromagnetic signal or sound wave or light wave such as radio frequency (RF) and infrared (IR) form of those produced in data communication process.Therefore, the common form of computer-readable medium includes, for example：It is soft It is disk, flexible disk (flexible disk), hard disk, tape, any other magnetic medium, CD-ROM, DVD or DVD-ROM, any Other optical mediums, perforated paper tape, any other physical storage medium with hole patterns, RAM, PROM and EPROM, FLASH-EPROM, any other storage chip or cassette, transmission data or instruction carrier wave, transmit these carrier waves cable or Link (link) or computer can therefrom program code read and/or data any other medium.These computer-readable Jie Many in matter form, which may participate in, to be carried one or more sequences of one or more instructions to processor to perform.

Embodiment

Embodiment 1：The amplification and detection of nucleic acid in viral stock sample and biological sample

PCR reagent obtains (Coyote One-Step PCR from Kayoudi Biotechnology (Beijing) Co., Ltd. Reagent).Virus is detected in artificial synthesized imitated clinical sample using digital pcr and qPCR.The sample includes serum With HBV analogs (Kayoudi Biotechnology (Beijing) Co., Ltd.).The imitated clinical sample in part uses Qiagen QIAamp DNA Mini Kit carry out nucleic acid purification, and other imitated clinical samples are without nucleic acid purification.Purified sample is in this experiment As control.

By the imitated clinical sample (10IU/ reactions) through nucleic acid purification and without nucleic acid purification be placed in different test tubes with Coyote One-Step PCR Reagent are mixed.Method formation droplet of the reactant mixture according to described in this specification. Specifically, on the basis of the use HFE7500 and the preparation of surfactant that are known in the art, Tween 60 is with the addition of, And optional Fluorinert FC-70 (Sigma Aldrich) are used to form droplet.Be on the inside of the droplet comprising The aqueous solution of the reactant mixture (including HBV viruses), outside is wrapped up by oil reservoir.

After droplet is formed, the droplet is analyzed with quantitative PCR and digital pcr.It is direct with PCR amplifications As described in this description, wherein denaturation temperature may be up to 100 DEG C to detection HBV method, and period may be up to 80 circulations.

Experimental result is shown in Fig. 2-3.Fig. 2A-D show the droplet prepared using the method for the present invention, and it is right respectively The droplet that Ying Yu is prepared using following preparations：2%Tween 60, HFE7500,2% glycolic polyoxyethanyl bay ether (A)； 0.1%Tween 60, HFE7500,2% glycolic polyoxyethanyl bay ether (A)；FC-70, HFE7500,2% glycolic polyoxy Ethyl Lauroyl ether (C)；And FC-70, HFE7500,2% glycolic polyoxyethanyl bay ether (D).

As can be seen, the droplet prepared using the preparation containing Tween 60 has bad droplet stability and small liquid Uniformity between drop.On the contrary, using containing FC-70 preparation prepare droplet have good droplet stability and Uniformity between droplet.

The further addition Tween 20, and be optionally followed by being vortexed in reagent so that droplet more stablizes (number According to not showing).

Quantitative PCR and digital pcr have been carried out using these droplets to react, and find these droplets at high temperature (100 DEG C) keep stable, and be resistant to longer circulation time (up to 80 circulations), and do not deform or droplet each other it Between merging.

Quantitative PCR and the digital pcr reaction carried out using these droplets successfully detected in imitated clinical sample Serum in the HBV that contains.Fig. 3 shows the HBV (A containing different potency：High-titer HBV；B：Medium potency HBV；C：It is poorly efficient Valency HBV；D：Negative control) droplet fluoroscopic examination result.As a result the amplified reaction that display is carried out using these droplets Virus can delicately be detected.Control sample (purified nucleic acid samples) also by similar method using quantitative PCR and Digital pcr detected viral (result is not shown).

Therefore, One-Step PCR Reagent are included into electric liquid (such as FC-70) and/or based on list with of the invention The novel formulation combination of the emulsifying agent (such as Tween 60) of stearate can simplify by PCR method (such as quantitative PCR and numeral PCR the flow of the infectious disease detection) carried out.

Embodiment 2：For droplet or the system of biology sample detection

Referring to Fig. 4, which depict the top view of the detection unit 400 in the detecting system of the present invention.As illustrated, detection Unit 400 includes fluorescence detector 401, and it is configured to detect multiple droplets along optical window in given detection zone Subset or one or more of subset biological sample.In one embodiment, fluorescence detector can be caught including image Obtain equipment, such as camera.In one embodiment, the fluorescence detector can include CCD camera.In an implementation In scheme, the CCD camera can be focused light on CCD by camera lens (including camera lens).Then, CCD is filtered Color, photosensitive (photoelectric conversion) etc. are operated, and reference object has been resolved into pixel one by one with certain arrangement mode.With Afterwards, the pixel of analog picture signal form is converted into data signal by using analog-digital converter, and be sent at image Reason device is handled, to obtain real image.These images are possibly stored in storage medium for use as other after compression Possible application, for example, such as analyze according to the digital pcr of the present invention.

Further, one or more filters 402 are also included according to the detection unit of the present invention.As illustrated, described one Individual or multiple filters are arranged on filter wheel, and desired optical filtering is selected so as to manually or automatically rotating filtering wheel Device.In one embodiment, filter can optionally transmit the light in a wavelength range or frequency range, you can with It is configured to transmit the light of different colours, while stopping the light of undesirable remainder.In other words, the filter can be with It is configured to select the light of the given frequency or frequency range detected by the fluorescence detector, prevents or filter out optical detection The frequency of the undesirable detection of device or the light of frequency range.Therefore in this way, there is fluorescence detector detection to expect wavelength Or the light of frequency, for example detect the exciting light by irradiating droplet or biological sample and obtaining.

In one embodiment, detection unit also include at least one excitaton source, one for example gone out as shown in the figure or Multiple light emitting diodes (LED) 403 and the radiator 404 being arranged on one or more of LED.In some cases, when One or more of the subset or the subset of multiple droplets to be detected biological sample is placed on sample holder simultaneously And along single plane keep when, the exciting light (such as illumination light) that the excitaton source can be arranged to be produced is guided into single The given detection zone in multiple detection zones in plane, thus to give detection zone in multiple droplets subset or Biological sample in subset is irradiated, can be by optics to make the biological sample in the subset or subset of multiple droplets launch The exciting light of detector detection.In some cases, at least one excitaton source can be multiple excitaton sources, so as to produce a variety of face The light of color, many of color can be at least three kinds, four kinds, five kinds or more kind colors.

In some embodiments of the present invention, excitaton source can include in one or more LED, and the plurality of LED Each LED launch different frequency light.In some cases, multiple LED can produce white light.In this case, multiple LED For example it can drive what is lighted with red, yellow, and green, green grass or young crops, five kinds of different colors of blue total, and by the rotation of actuating mechanism Five LED produce mixed white light.By irradiating white light to droplet or biological sample, fluorescence detector can obtain small liquid The white image of drop or biological sample.In some embodiments, in the case where not coming from specific LED pseudomorphism, it will can be somebody's turn to do White image is used for the reference that total droplet is counted, so as to obtain total droplet number.In some embodiments, it is described One or more LED can have certain transmitting optical range, and such as LED transmitting boundaries can be received at 390 nanometers (nm) to 700 In the range of rice.It is appreciated that transmitting optical range here is merely exemplary that those skilled in the art are according to the present invention Teaching, the LED with other transmitting optical ranges can also be selected as excitaton source.Unlike the prior art, it is of the invention Embodiment is imaged by using the LED being circular layout, and without using usually used spectroscope.

In one embodiment, the radiator for being placed on LED (or being attached at LED heating surface) can be radiating Piece (or radiating fin).The fin is the heat dissipation element of passivity, with good heat conductivility and with compound Heat exchange pattern radiates, and thus reduces temperature during LED operation, and extend LED working life.

In some embodiments, at least one excitaton source also includes one or more optical units, for swashing described It is luminous to guide into and/or focus in the given detection zone.In some cases, one or more optical units can be wrapped Condenser lens or speculum are included, so that exciting light is converged into given detection zone.

In one embodiment, the position of at least one excitaton source can be selected so that at least one excitaton source can be with It is oriented to and guides or be introduced only into exciting light into optical window, so as to the detection of fluorescence detector.In some cases, at least one Individual excitaton source is oriented to guides exciting light along first axle into, wherein the fluorescence detector is oriented to along flat not with first axle The second capable axle detects the subset or one or more of biological samples of the multiple droplet.

In some embodiments, detection unit of the invention includes at least one actuator, such as stepper motor 405.In one embodiment, the stepper motor 405 is electric impulse signal to be transformed into angular displacement to control rotor The motor of rotation.By the rotation of rotor, shown fluorescence detector can carry out in vertical direction above and below (such as along perpendicular Straight Z axis) movement so that close to or away from droplet or biological sample to be measured, be achieved in fluorescence detector (for example Camera) auto-focusing.

In some embodiments, detection unit herein shown also additionally include at least one actuator or with extremely Few actuator coupling, so that the operation of actuator makes the optical window of fluorescence detector along single planar movement, for example, edge X-axis and Y-axis movement in plane right-angle coordinate.Using foregoing stepper motor, detection unit of the invention can be in X, Y Move, moved so as to realize along three degree of freedom under the three-dimensional coordinate constituted with Z axis.In some embodiments, detection unit Can be along the planar movement parallel to described single plane, so as to the droplet or biological sample that are placed in single plane Detected.By comparison according in other embodiments, detection unit can be along the plane for being orthogonal to the single plane It is mobile.

Fig. 5 is the schematic diagram for describing the detecting system 500 of the present invention.As illustrated, the detecting system is included as in Fig. 4 Shown detection unit 400, including fluorescence detector 401, one or more filters 402, one or more luminous Diode (LED) 403 and the radiator 404 being arranged on one or more of LED.In addition, being further illustrated that optics The lens or lens group 406 of detector (such as camera), for example, can include convex lens, concavees lens or its combination, to realize Good light control and imaging.

Further figure 5 illustrates sample holder 407, it can detect the subset or the son of multiple droplets During one or more of collection biological sample, the multiple droplet 408 is kept along single plane.In some embodiments In, at least one actuator can also be coupled to sample holder, so as to realize the relative movement of sample holder.

Fig. 6 be describe the present invention Fig. 4 detection unit 400 bottom view, illustrated therein is fluorescence detector 401, One or more filters 402, one or more light emitting diodes (LED) 403 and it is arranged on one or more of LED Radiator 404.As shown in FIG., all LED are focused on for fluorescence detector 401.

Fig. 7 is the schematic diagram for the lightray propagation for describing the detecting system of the present invention.As shown in Figure 7, by as sharp The illumination light that the LED 403 of light emitting source is launched incides the droplet or biological sample 408 positioned at sample holder 407 On, so as to produce reflected light 409 and the exciting light 410 obtained by irradiation droplet or biological sample.In this embodiment, As fluorescence detector camera will detect the exciting light 410 so that the droplet or biological sample are imaged with Perform such as dPCR analyses.In the present embodiment, due to the angle theta 1 and reflected light between illumination light 411 and exciting light 410 Angle theta 2 between 409 and exciting light 410 is equal, so that reflected light 409 can't interfere with the imaging of camera.In some realities Apply in scheme, the angle can be with the range of substantially 30 degree~50 degree, it is therefore preferable to substantially 45 degree.

In some embodiments, detecting system of the invention can be used in two LED of symmetric position as sharp Light emitting source is imaged to droplet or biological sample, so as to minimizing the distortion of imaging, to avoid to droplet or The interference of such as dPCR analyses of biological sample.In some embodiments, the excitation source can be that 180 degree is arranged symmetrically, Five-five ten LED being arranged symmetrically for example in Fig. 4 are shown.

According to embodiment of the present invention, detecting system can use fluorescence detector to be collected from the multiple detection zone At least one illumination correction chart picture, and come from the multiple inspection using at least one illumination correction chart picture to compensate or correct The illumination heterogeneity for the multiple droplets surveyed in region.For example, the detecting system of the present invention can collect at least one illumination Correction chart picture, so as to correct the illumination heterogeneity in dPCR analyses.

Fig. 8 is the schematic diagram of the motion track for the optical window for describing the detecting system of the present invention.As shown in Figure 8 , droplet or biological sample to be detected is divided into 20 sub- droplets or subsample, and according to the square of 5 rows 4 row in figure Formation formula is placed on sample holder.Then, detecting system of the invention, the specially optical window of fluorescence detector will be Under the driving of one or more actuators, according to the direction shown in arrow in figure in order from the 1st sub- droplet or subsample Set out and start detection until sequence detection complete 20th sub- droplet or subsample.In this process, the illumination light sent by LED The sub- droplet or subsample from the 1st to the 20th will be irradiated according to same order, thus by detection unit Lai Detect the exciting light launched from droplet.That is, detection unit of the invention can cause exciting light and optical window for many Individual detection zone is relatively moved simultaneously.Using the track of the relative movement or pattern (i.e. along multiple translations of multiple detection zones Vector), exciting light can cause the warm-up movement of droplet or biological sample, and the track be also minimize droplet movement and Optical window moves the optimal path of (or sample holder movement) distance.

According to the detecting system of the present invention, due to the LED illumination light of five kinds of colors can be used, for intensity difference For digital pcr, very high multiplicity (multiplexing) will be obtained.For example, when droplet or biological sample to be analyzed During in the presence of 7 kinds of concentration, then the analyte that multiplicity is planted in 35 (5 × 7) can be obtained.

Although the preferred embodiments of the invention have been shown and described herein, to those skilled in the art It is readily apparent that what these embodiments were only provided in an illustrative manner.Those skilled in the art are without departing substantially from this hair In the case of bright, a large amount of changes are will now occur, changes and substitutes.It should be appreciated that invention described herein embodiment is each Alternative solution is planted to can be used for implementing the present invention.Purpose is, limits the scope of the present invention with following claims, thus covers Method and structure and its equivalent in these rights.

Claims

1. a kind of be used for the system of droplet or biology sample detection, it includes：

Sample holder, it is arranged to detecting one or more of the subset or the subset of multiple droplets biology During sample, the multiple droplet is kept along single plane；

Detection unit, including：(i) at least one excitaton source, it is arranged to guide exciting light in the single plane into Given detection zone in multiple detection zones, and (ii) fluorescence detector, it is arranged in the given detection zone The middle subset or one or more of biological samples that the multiple droplet is detected along optical window；And

At least one actuator, it is arranged to make the optical window relatively move along the multiple detection zone.

2. system according to claim 1, wherein at least one described actuator is a part for the detection unit.

3. system according to claim 1, wherein at least one described actuator is multiple actuators.

4. system according to claim 1, wherein at least one described actuator is coupled to the detection unit.

5. system according to claim 1, wherein at least one described actuator is coupled to the sample holder.

6. the excitaton source of system according to claim 1, wherein energy is arranged to guide into the exciting light The given detection zone.

7. system according to claim 1, it also includes one or more computer processors, and it is operably coupled to The detection unit and at least one described actuator, wherein one or more of computer processors are individually or generally It is programmed for：(i) guide at least one described actuator, so as to when the multiple droplet along the single plane adjacent to During the sample holder so that the optical window is relatively moved along the multiple detection zone, wherein the relative movement The optical window is positioned in the given detection zone, (ii) guides at least one described excitaton source by the exciting light Guide the given detection zone into, and (iii) guides the fluorescence detector described in the given detection zone to detect The subset or one or more of biological samples of the multiple droplet in optical window.

8. system according to claim 7, wherein one or more of computer processors individually or generally by Program for guiding at least one described actuator so that the optical window in order along the multiple detection zone Each detection zone relative movement.

9. system according to claim 7, wherein one or more of computer processors individually or generally by Programming is used for：(i) at least one illumination correction chart picture is collected from the multiple detection zone using the fluorescence detector, and (ii) compensate or correct using at least one described illumination correction chart picture come from it is described many in the multiple detection zone The illumination heterogeneity of individual droplet.

10. system according to claim 1, wherein at least one described excitaton source is multiple excitaton sources.

11. system according to claim 10, wherein the multiple excitaton source is multiple light emitting diodes (LED).

12. system according to claim 11, wherein the multiple LED each LED launches the light of different frequency.

13. system according to claim 11, wherein the multiple LED produces white light.

14. system according to claim 10, wherein the multiple excitaton source produces the light of multiple color.

15. system according to claim 14, wherein the multiple color includes at least three kinds colors.

16. system according to claim 15, wherein the multiple color includes at least four colors.

17. system according to claim 16, wherein the multiple color includes at least five kinds colors.

18. system according to claim 1, wherein at least one described excitaton source includes light emitting diode (LED).

19. system according to claim 18, wherein the LED transmitting boundaries are at 390 nanometers (nm) to 700 nanometer ranges Interior light.

20. system according to claim 1, wherein at least one described excitaton source includes radiator.

21. system according to claim 20, wherein the radiator is fin.

22. system according to claim 1, wherein at least one described excitaton source includes one or more optical units, For guiding and/or focusing on the exciting light in the given detection zone into.

23. system according to claim 22, wherein one or more of optical units include condenser lens.

24. system according to claim 22, wherein one or more of optical units include directional mirror.

25. system according to claim 1, wherein the detection unit does not include spectroscope.

26. system according to claim 1, wherein the detection unit further comprises one or more filters, its For the given frequency or the light of frequency range for selecting to be detected by the fluorescence detector.

27. system according to claim 26, wherein one or more of filters are multiple filters.

28. system according to claim 27, wherein the multiple filter includes each optical filtering rotatably selected Device.

29. system according to claim 1, wherein the fluorescence detector includes charge coupling device (CCD) camera.

30. system according to claim 1, wherein the detection unit is arranged to move along three degree of freedom.

31. system according to claim 30, wherein the detection unit is arranged to along parallel to described single flat The planar movement in face.

32. system according to claim 30, wherein the detection unit be arranged to it is described single flat along being orthogonal to The planar movement in face.

33. system according to claim 1, further comprises fluid stream unit, it is arranged to adjacent to the sample Product supporter provides the multiple droplet.

34. system according to claim 1, wherein at least one described excitaton source is oriented to and guides the exciting light into The optical window.

35. system according to claim 34, wherein at least one described excitaton source is oriented to the exciting light only Guide the optical window into.

36. system according to claim 1, wherein at least one described excitaton source is oriented to the exciting light along One axle is guided into, wherein the fluorescence detector be oriented to it is the multiple to detect along not parallel with the first axle the second axle The subset or one or more of biological samples of droplet.

37. system according to claim 1, wherein the detection unit is arranged to so that the exciting light and described Optical window is relatively moved simultaneously for the multiple detection zone.

38. a kind of method for droplet or biology sample detection, including：

(a) detecting system is activated, the detecting system includes

A. sample holder, it is arranged to detecting that one or more of subset or the subset of multiple droplets are raw During thing sample, the multiple droplet is kept along single plane；

B. detection unit, including：(i) at least one excitaton source, it is arranged to guide exciting light in the single plane into Multiple detection zones in given detection zone, and (ii) fluorescence detector, it is arranged in the given detection zone The subset or one or more of biological samples of the multiple droplet are detected in domain along optical window；

C. at least one actuator, it is arranged to make the optical window relatively move along the multiple detection zone；With And

(b) using at least one described actuator, so as to when the multiple droplet along the single plane adjacent to the sample During product supporter so that the optical window is relatively moved along the multiple detection zone, wherein the relative movement will be described Optical window is positioned in the given detection zone,

(c) exciting light is guided into the given detection zone using at least one described excitaton source；And

(d) detect the multiple small in the optical window in the given detection zone using the fluorescence detector The subset or one or more of biological samples of drop.

39. the method according to claim 38, further comprises that there is provided adjacent to the sample holder before (b) The multiple droplet.

40. the method according to claim 38, wherein one or more of biological samples include DNA (DNA) molecule.

41. the method according to claim 38, wherein one or more of biological samples are included in template nucleic acid molecule The nucleic acid amplification product of the nucleic acid amplification reaction of upper progress.

42. method according to claim 41, further comprises before (b) so that the template nucleic acid molecule experience foot To carry out the condition of the nucleic acid amplification reaction.

43. method according to claim 42, wherein when the multiple droplet is adjacent to the sample holder, entering The row nucleic acid amplification reaction.

44. method according to claim 42, wherein the condition is included the temperature of the multiple droplet multiple Circulated between at least two temperature of temperature levels.

45. method according to claim 42, wherein the condition includes maintaining the temperature of the multiple droplet Within given temperature or given temperature range.

46. the method according to claim 38, further comprises at least one described actuator of guiding, so that described Each detection zone along the multiple detection zone of optical window in order is relatively moved.

47. the method according to claim 38, further comprises that (i) uses the fluorescence detector from the multiple detection At least one illumination correction chart picture is collected in region, and (ii) is come using at least one described illumination correction chart picture to compensate or correct From the illumination heterogeneity of the multiple droplet in the multiple detection zone.

48. the method according to claim 38, further comprises so that the exciting light and the optical window are for institute Multiple detection zones are stated to relatively move simultaneously.

49. the method according to claim 38, further comprises the subset or described for detecting the multiple droplet Intensity difference in one or more biological samples.

50. the method according to claim 38, further comprises so that the optical window is relative according to priority along track Mobile, the track includes multiple translation vectors along the multiple detection zone.

51. method according to claim 50, wherein the multiple translation vector is orthogonal to institute including first group of vector State second group of vector of first group of vector.