CN105874084A - Methods and apparatus for point-of-care nucleic acid amplification and detection - Google Patents
Methods and apparatus for point-of-care nucleic acid amplification and detection Download PDFInfo
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- CN105874084A CN105874084A CN201480070428.1A CN201480070428A CN105874084A CN 105874084 A CN105874084 A CN 105874084A CN 201480070428 A CN201480070428 A CN 201480070428A CN 105874084 A CN105874084 A CN 105874084A
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502738—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by integrated valves
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/52—Containers specially adapted for storing or dispensing a reagent
- B01L3/523—Containers specially adapted for storing or dispensing a reagent with means for closing or opening
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6846—Common amplification features
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/16—Reagents, handling or storing thereof
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/04—Closures and closing means
- B01L2300/041—Connecting closures to device or container
- B01L2300/044—Connecting closures to device or container pierceable, e.g. films, membranes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0672—Integrated piercing tool
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0809—Geometry, shape and general structure rectangular shaped
- B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/0874—Three dimensional network
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- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/18—Means for temperature control
- B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
- B01L2300/1827—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using resistive heater
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
- B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/06—Valves, specific forms thereof
- B01L2400/0605—Valves, specific forms thereof check valves
- B01L2400/0616—Ball valves
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/06—Valves, specific forms thereof
- B01L2400/0633—Valves, specific forms thereof with moving parts
- B01L2400/065—Valves, specific forms thereof with moving parts sliding valves
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
Abstract
Methods and apparatus are provided for point-of-care nucleic acid amplification and detection. One embodiment of the invention comprises a fully integrated, sample-to-answer molecular diagnostic instrument that optionally may be used in a multiplexed fashion to detect multiple target nucleic acid sequences of interest and that optionally may be configured for disposal after one-time use. The instrument preferable utilizes an isothermal nucleic acid amplification technique, such as loop-mediated isothermal amplification (LAMP), to reduce the instrumentation requirements associated with nucleic acid amplification. Detection of target amplification may be achieved, for example, via detection of a color shift or fluorescence in a dye added to the amplification reaction. Such detection may be performed visually by an operator or may be achieved utilizing an imaging technique, e.g., spectrophotometric imaging.
Description
Quoting of related application
This application claims the U.S. Patent Application No. 61/894,392 and April 25 in 2014 submitted on October 22nd, 2013
The priority of the U.S. Patent Application No. 14/262,683 that day submits to and applying date rights and interests, full text both this is here cited and is added
Enter.
Quote addition
Addition is all here cited in all publications and patents application mentioned in this specification, reaches just as each is single
Publication or patent application are specifically and individually pointed out to be cited the same degree of addition.
Technical field
The present invention relates to the method and apparatus for nucleic acid amplification and detection.More particularly, the present invention relate to scene
(point-of-care) nucleic acid amplification and the method and apparatus of detection.
Background of invention
Polymerase chain reaction (PCR) is considered as the goldstandard of nucleic acid amplification and detection, because the specificity of PCR and spirit
Sensitivity is more much higher than what similar elisa (" ELISA ") was tested.But, PCR system is the most expensive
And need the cleanest sample.On-the-spot (POC) PCR system is frequently not disposable completely, is not suitable for unskilled
Librarian use, needs suitable ability and/or containing complicated process and reading.Therefore, PCR Conventional cap is formed on aboundresources
, centralized laboratory environment.
In view of afore-mentioned, it is provided that overcome heretofore known method and apparatus defect for site nucleic acid amplification and inspection
The method and apparatus surveyed will be desirable.
Accompanying drawing explanation
Several embodiments of the present invention combine annexed drawings consider described in detail below after will be apparent from, in institute
State the most similar quotation mark in accompanying drawing and refer to similar component, and wherein:
Fig. 1 is the schematic diagram of an embodiment of sample divider；
Fig. 2 A-2C is for preparing sample and from the sample divider of Fig. 1, sample being transferred to site nucleic acid amplification and inspection
The equipment of measurement equipment and the isometric view of method and side view；
Fig. 3 A and 3B is for preparing sample and from the sample divider of Fig. 1, sample being transferred to site nucleic acid amplification and inspection
The replacement of measurement equipment and the side view of method and isometric view；
Fig. 4 A-4D is for preparing sample and from the sample divider of Fig. 1, sample being transferred to site nucleic acid amplification and inspection
The other replacement of measurement equipment and the side sectional view of method and isometric view；
Fig. 5 is site nucleic acid amplification and the exploded view of detection equipment of Fig. 2-4；
Fig. 6 is the site nucleic acid amplification of Fig. 2-5 and the detection passage of equipment and the bottom view of room element；
Fig. 7 A is site nucleic acid amplification and the isometric view of detection equipment of the Fig. 2-6 with heating element heater thermal communication, and schemes
7B is the isometric view of the optional detection sensor being used together with the equipment of Fig. 7 A and method；
Fig. 8 A-8E is the alternative embodiment of the method and apparatus expanding for site nucleic acid and detecting of Fig. 2-7
Isometric view, top view, bottom view and side sectional view；
Fig. 9 A-9J is the equidistant of another alternative embodiment for site nucleic acid amplification and the method and apparatus of detection
View, top view, bottom view, assembled view, lateral section detailed view and translucent detailed isometric view；
Figure 10 A-10G be for site nucleic acid amplification and detection method and apparatus another alternative embodiment etc.
Away from top view, isometric bottom view, equidistant detailed view, translucent detailed view and lateral section detailed view；With
Figure 11 A-11J is for site nucleic acid amplification and the side of the another alternative embodiment of the method and apparatus of detection
View, side sectional view, isometric view and translucent isometric view.
Detailed description of the invention
Although the disclosure is detailed and accurately so that those skilled in the art can implement disclosed technology, but this
Physical embodiments disclosed in literary composition merely illustrates various aspects of the invention, and it can be implemented with other ad hoc structures.Though
So describe preferred implementation, but details can be changed without departing from the present invention being defined by the claims.
The present invention relates to the method and apparatus for nucleic acid amplification and detection.More particularly, the present invention relate to scene
Nucleic acid amplification and the method and apparatus of detection.This equipment and method optionally can be with multiple form (multiplexed
Fashion) make for detecting multiple target nucleic acid sequence (the target nucleic acid sequence that such as, detection at least two is interested interested
Row), and this equipment optionally can be configured to abandon after a single use.
This instrument preferably employs isothermal amplification (such as ring mediated isothermal amplification (" LAMP ")) to be reduced and nucleic acid
The instrument requirements that amplification is relevant.The detection of target amplification such as can add one or more dyes in amplified reaction to by detection
The color displacement of material (as green in hydroxynaphthol blue, picogreen and/or SYBR) and/or fluorescence, or changed by turbidity come real
Existing.Such colorimetric detection, fluoroscopic examination and/or Turbidity measurement can visually be carried out by operator and/or can use imaging
Technology (such as spectrophotometric imaging and/or fluorescence imaging) realizes, as described below.
Fig. 1 illustrates an embodiment of the sample divider 10 (known per se) for collecting nucleic acid samples S.Sample
Catcher 10 can such as comprise sponge, foam or swab (swab).Sample divider 10 can be such as by inert polymer system
Make.Various sample substrates include but not limited to food, urine, saliva, mucus, Excreta, blood, seminal fluid, tissue, cell,
DNA, RNA, protein, plant material, animal substance, liquid, solution, solid, gas and other sample substrates can be made
Deposit on sample divider 10 for sample S.
In order to collect sample S with sample divider 10, sample divider can such as immerse or insert one interested
Or in several samples substrate.In the method using sample divider 10, sample divider can be placed in mouth one section
Time is to collect saliva sample S.Additionally or alternatively, one or more sample substrates interested of one or many are permissible
Such as it is placed in or deposits on sample divider.As another alternative, sample divider 10 can such as wiping or
Smeared one or more sample substrates interested or surface.
After collecting sample S, this sample can transfer to site nucleic acid amplification and detection equipment from sample divider 10
100.Optionally, this sample can before transfer, or be prepared in transfer process after the transfer, such as by will
Sample S connects with cracking performance chemical fluids.Sample divider 10 optionally can comprise the cracking performance chemistry preparing sample S
Material.Additionally or alternatively, sample S can be prepared by heat treatment.Such as, sample S can be heated to above isothermal
Expand temperature required temperature, such as, higher than the temperature needed for ring mediated isothermal amplification (" LAMP ").At some embodiments
In, sample S can comprise whole blood, its can such as at about 99 DEG C heat treatment e.g., from about 10 minutes prepare realizing sample.Separately
Other places or alternatively, other preparation methoies known per se can be used.In some embodiments, sample S is made without system
Standby.In some embodiments, sample S mixes with water, buffer agent and/or dye solution and may be enough to prepare sample for nucleic acid
Amplification.
Fig. 2 illustrates for sample S is transferred to site nucleic acid amplification and the side of detection equipment 100 from sample divider 10
Method and an embodiment of equipment.As seen in figure 2 a, sample divider 10 can be placed in and have Luer lock (luer lock)
In the sample divider receiving element 20 of 22.Receiving element 20 include inner chamber that sample divider 10 can be placed in one or every
Room.As seen in Fig. 2 B, after sample divider 10 is placed in receiving element 20, the cap 24 with Luer lock 26 can be attached
To sample divider receiving element 20.Receiving element 20 and sample divider 10 may then pass through (the convex or volume) Shandong making cap 24
Your lock 26 coordinates with (concave or convex) Luer lock 32 of syringe 30 and to be attached to syringe 30.
As seen in figure 2 c, syringe 30 and the receiving element 20 with sample divider 10 can be by making receiving element
(convex or recessed) Luer lock 22 of 20 coordinates with (concave or convex) Luer lock 102 of equipment 100 and sets with site nucleic acid amplification and detection
Standby 100 couplings.Syringe 30 can contain liquid L (such as water, buffer and/or colorimetric or the solution of other dyestuffs) to be passed through
By lower plunger 34, sample S is eluted to equipment 100 from sample divider 10.The Luer lock 102 of equipment 100 (and/or be used for
Deliver the syringe of sample S or other substituting delivery apparatus) optionally can be included in nucleic acid amplification and detection during anti-
The check valve of backflow.
Fig. 3 illustrates alternative method and equipment for sample S transfers to equipment 100 from sample divider 10.As
Seen in Fig. 3 A, the Luer lock 22 of receiving element 20 can couple with the Luer lock 42 of the second syringe 40.Can be by hemostasis
Liquid L and sample S is eluted to the second syringe 40 by the plunger 34 of device 30 from sample divider 10.Transfer to set at sample
Before standby 100, liquid L and sample S is eluted in the second syringe 40 can strengthen liquid and is transferring to equipment 100 with sample
Mixing before.Additionally, sample S optionally can collect in multiple times and/or be eluted in the second syringe 40, transfer to afterwards
Equipment 100.
As seen in Fig. 3 B, after sample S and liquid L collects in the second syringe 40, the second syringe 40 can be with
Syringe 30, receiving element 20 and sample divider 10 depart from.Second syringe 40 may then pass through and makes Luer lock 42 and Shandong
You come and equipment 100 coupling in lock 102 cooperation.Force in sample S and liquid L access arrangement 100 by lower plunger 44.
Fig. 4 illustrates for sample S being transferred to the other alternative method of equipment 100 from sample divider 10 and setting
Standby.As seen in figure 4 a, the sample divider 10 containing sample S can be by departing to come directly with syringe by plunger 34 temporarily
It is placed in syringe 30.Optionally, liquid L can be placed in syringe 30 together with the sample divider 10 containing sample S, though
So it should be understood that liquid L or can omit.After sample divider 10 is placed in syringe 30, plunger 34 can be attached again
On syringe, as in Fig. 4 B.Syringe 30 may then pass through and makes Luer lock 32 coordinate with Luer lock 102 and and equipment
100 couplings, as in Fig. 4 C.As in Fig. 4 D, make sample divider 10 compress by lower plunger 34 and make sample S be forced into
In equipment 100.
Referring now to Fig. 5, the sample of the fully-integrated integration expanding for site nucleic acid and detecting is described to result formula
(sample-to-answer) the first embodiment of molecular diagnostic apparatus 100.Equipment 100 includes being connected to passage and room unit
The Luer lock 102 of part 110.Element 110 can such as be manufactured by polypropylene.
Channel cover 104 is such as connected to the bottom of element 110 by binding agent or screw, and chamber cap 106 is such as by viscous
Mixture or screw are connected to the top of element 110.Lid 104 and 106 can such as include binder film or band.Chamber cap 106 (with appoint
The channel cover 104 of choosing) the most translucent or transparent, in order to the content of the reative cell 112 of visual inspection element 110.
Equipment 100 can also include the top cover 120 with air filter 122 and the room window 124 alignd with the room 112 of element 110.
In some embodiments, each room 112 can have the volume of less than about 100 microlitres, such as, the body of about 30 microliters
Long-pending.
As best seen from figures 5 and 6, the reative cell 112 of element 110 is connected to by (preferably equal length) microchannel 114
Entrance 116.Sample S is collected and such as by just pressing syringe plunger and via Luer lock 102 as described in Fig. 2-4 before this
It is forced in equipment 100.Sample S such as forces sample S from entrance 116 warp by being consecutively pressed extruding continuously of syringe plunger
Entered in room 112 by microchannel 114.Each room 112 is containing the reagent 130 for carrying out nucleic acid amplification.Reagent 130 can be with example
As comprised enzyme and main mixture (master mix).When carrying out nucleic acid amplification by LAMP, enzyme can such as comprise Bst DNA
Polymerase, Bst2.0WarmStart archaeal dna polymerase and/or Bsm archaeal dna polymerase (with optional reverse transcription).Main mixture
Can such as comprise primer, dNTP, MgSO4, glycine betaine and/or excipient (such as mannitol, trehalose and/or dextrin).
Reagent 130 can also comprise water, TE buffer agent, isothermal buffer agent and/or other buffers, and it optionally can such as deliver
Such as it is delivered to via microchannel 114 as liquid L before sample S, during delivery sample S and/or after delivering sample S
Room 112.
Reagent 130 also can comprise one or more dyestuffs and be beneficial to detect nucleic acid amplification, such as hydroxyl naphthols (" HNB ")
Blue.The detection of target amplification can be such as by detecting in the presence of amplicon such as due to free magnesium (Mg2+) concentration LAMP expand
Change in increasing process and color displacement in the colorimetric dye that produces realize.This colorimetric detection can be visual by operator
Carry out or spectrophotometric imaging can be used to realize, as described below.Except use colorimetric dye colorimetric augmentation detection in addition to or
Person is as the alternative of the colorimetric augmentation detection using colorimetric dye, and fluorescent dye (as picogreen or SYBR is green) is permissible
For being expanded by fluoroscopic examination.
One or more reagent 130 preferably lyophilizing, such as, it is beneficial to long term storage.Additionally or alternatively, a kind of
Or plurality of reagents before nucleic acid amplification can temporarily with one or more other reagent isolate.This provisional reagent every
From being conducive to the long term storage of reagent and/or reagent mixing (and being therefore prevented from nucleic acid amplification) can be stoped in advance until the phase
Prestige reagent mixes, such as until reagent is exposed to sample S.Such as, enzyme can be isolated with main mixture.
In some embodiments, one or more reagent 130 can provisional be isolated in one or more provisional every
In container.In some embodiments, provisional spacing container can such as comprise one or more hot packing materials, described
Hot packing material is configured to when heated (such as in amplification process) and melts, becomes porous or otherwise
The reagent 130 of release isolation.Hot packing material can such as comprise polycaprolactone and/or phase-change material such as paraffin or wax.One
In a little embodiments, described provisional spacing container can include one or more blister package or other containers such as Capsule (gel
Cap), it can pierce through or otherwise open with the reagent 130 of release isolation.Capsule is included at provisional spacing container
Time, they optionally can be opened by the hydrolysis outside piercing through or as the alternative pierced through.
When sample S is delivered to room 112 via microchannel 114, each is configured to expand interested containing room 112 of reagent
Nucleic acid target sequence (if being included in sample S).Different rooms 112 optionally can use different primers with favorably
In expanding and detect different target sequences interested (such as, beneficially multiple nucleic acid amplification and detection) in different rooms.Room
The part of 112 can serve as positive control (for example, it is possible to pre-loaded it is contemplated that in amplification process amplification interested
One or more target nucleic acid sequences).Additionally or alternatively, a part for room 112 can be served as negative control and (such as, may be used
Microchannel 114 can be free of attachment to so that they do not contain sample S) to comprise reagent 130.
After sample S is delivered to room 112, (such as isothermal heating) room can be heated to expand interested one or more
Target nucleic acid sequence.When carrying out isothermal nucleic acid amplification by LAMP, the content of room 112 can be in the range of about 60 DEG C-65 DEG C
Heat about 5-70 minute.As seen in figure 7 a, the content of room 112 can be by the heating element heater 200 with equipment 100 thermal coupling
Heating.Such heating can use the multiple technologies including, but is not limited to power technology, chemical technology and/or electrochemical techniques
In any one realize.Heating element heater 200 can such as include being connected to power supply (such as one or more batteries or wall outlet
Connector) resistance heater, and the temperature controller of the optional content heating described room 112 for resistance.Additionally
Ground or alternatively, heating element heater 200 can include diamond/tungsten heater, sensing heater, chemical heater (such as, heat release
Chemical heater, such as supersaturation sodium acetate heater, cellulose/ferrum/water/activated carbon/Vermiculitum/salt heater, ferrum oxide heating
Device, ferrum/magnesium salt heater, catalytic burner, fuel cell heating device etc.).Heating element heater 200 can be reusable,
Or can be configured to abandon after a single use.Optionally, heating element heater 200 can be integrally attached to equipment 100.Heating
Element 200 can be full automatic, or can include such as allowing user's target setting temperature and the duration of heat
Control.Optionally, heating element heater 200 can comprise phase-change material (such as paraffin) so that desired temperature maintains the time extended
Section.
As it was previously stated, the detection of target amplification optionally can be by detection one or more dyestuffs in the presence of amplicon
Color displacement (i.e. wavelength shift) and/or fluorescence (i.e. strength offsets) realize.Such colorimetric detection and/or fluoroscopic examination can
Visually to be carried out by operator and/or imaging technique (such as spectrophotometric imaging and/or fluorescence imaging) can be used to realize.At figure
In the embodiment of 7B, the close room 112 of sensor 300 (such as spectrophotometric CMOS or ccd imaging sensor 300) is to detect color
Some other changes of skew, fluorescence, turbidity or instruction target nucleic acid sequence amplification.Chamber cap 106 (seeing Fig. 5) is preferably transparent
With the change in profit detection reative cell.In some embodiments, sensor 300 can be integrated and connected element 110 and
Room 112 can be covered so that chamber cap 106 becomes unnecessary.
Sensor 300 optionally can comprise coating, and such as indium tin oxide (" ITO ") coating, it can be at heating element heater
Replacement outside 200 or as heating element heater 200 uses, and realizes target nucleic acid so that resistance heats the content of each room 112
Amplification.Coating can be placed near room 112.As it was previously stated, when carrying out isothermal duplication by LAMP, the content of room 112 can
To heat about 5-70 minute in the range of about 60 DEG C-65 DEG C.
Imaging sensor 300 can be at the pre-test reagent 130 of isothermal heating and the baseline color of sample S and isothermal heating
The final color (such as, after isothermal heating) of reagent afterwards.Owing to the reagent 130 in each reative cell 112 can be such as
Be included in target nucleic acid sequence amplification time color such as from purple be displaced to blueness colorimetric (or fluorescence) dyestuff, these indoor any this
The color displacement of sample can be arrived as the Difference test between baseline color and final color by imaging sensor 300, and
And this species diversity may indicate that target expands.As seen in fig. 7b, optional digitizer or display 310 can be by detection knots
Really (and/or instruction) output, to user, eliminates the risk that any detection implication is unclear.Although the embodiment of Fig. 7 B is illustratively
Colorimetric or fluoroscopic examination is achieved, it should be understood that this colorimetric or fluoroscopic examination can additionally or can by spectrophotometric imaging
Selection of land is visually carried out by operator.
Heating element heater 200 and/or sensor 300 can include for controlling the operation of heating element heater and/or sensor, use
In controlling nucleic acid amplification by the described room 112 of heating, for comparing the baseline obtained with sensor 300 and final color measurement
Result determine whether to have occurred and that amplification and/or for control instruction or testing result patrolling by the display of display 310
Collect chip.Logic chip can be connected to heating element heater 200, sensor 300 and/or power supply by electric wire and/or circuit board.Power supply
Can such as include one or more battery or wall outlet connector.
Referring now to Fig. 8, describe the alternative embodiment of equipment 100.In the embodiment of Fig. 8, element 110 ' wraps
Include four rooms 112 rather than 16 (as the most obvious, it is provided that any number of room
112).Element 110 ' includes the passing away connected for the Top fluidic with each room 112 that air is discharged to air from room
118.Sample S is delivered to the bottom of each room 112 by microchannel 114, and passing away 118 by overflow from each room
Top is discharged to outside equipment 100 via ventilated membrane or check valve 119.Fig. 8 A is the isometric view of equipment 100.Finding in the fig. 8b
Element 110 ' top view in, passing away 118 is visible with the fluid communication at the top of room 112.Seen in Fig. 8 C
Element 110 ' bottom view in, microchannel 114 is visible from entrance 116 to the extension of room 112, and film or valve 119 are also
So.The side sectional view of Fig. 8 D is the outlet intercepting through Luer lock 102 and passing away 118.The side sectional view of Fig. 8 E
Intercept through room 112, and show the fluid communication of microchannel 114 and the bottom of room and passing away 118 and room
The fluid communication at top.
Fig. 9 provides and includes element 110 " another alternative embodiment of equipment 100.Fig. 9 A provides equipment
The isometric view of 100, Fig. 9 B shows the element 110 of the equipment removing chamber cap 106 " top view, and Fig. 9 C shows shifting
Element 110 except channel cover 104 ' " bottom view.Although the embodiment of the equipment 100 of display includes air from room in Fig. 8
112 are discharged into air via passing away 118 and the film of element 110 ' or valve 119, but the enforcement of the equipment 100 of display in Fig. 9
Air is discharged into element 110 from room 112 via passing away 118 ' by mode " one or more overflow rooms (see figure
9C), rather than be discharged into air.Therefore, the equipment 100 of Fig. 9 is completely enclosed.Overflow room 125 is preferably designed and sized to
In amplification process, the pressure increase in overflow room is limited to less than about 5-10psi.
Most advantageously see in Fig. 9 C, element 110 " also include anti-reflux valve 140, it prevents from passing through between room 112
Cross-contamination via the backflow of microchannel 114 '.Additionally, most advantageously see in Fig. 9 B, element 110 " include along
The flow control medium 150 that passing away 118 between room 112 and overflow room 125 disposes, described flow control medium allows air
Or other gases discharge from room 112, but do not allow fluid to discharge from room 112, therefore ensure that sample S is equal to being had family in 112
Ground is filled, and discharges excessive pressure simultaneously.
The element 110 of Fig. 9 " microchannel 114 of the microchannel 114 ' that the has element 110 ' than Fig. 8 is shorter.Shorter
Microchannel decrease sample S and must advance to arrive the preliminary filling volume (priming volume) of room 112.Element 110 " can
There is the preliminary filling volume of approximation 20-50 microlitre.Compared with aforementioned microchannel 114, microchannel 114 ' is along element 110 "
Both top and bottom extend, and through element 110 " extend.The circuitous path of microchannel 114 ' is in further detail below
Description.
In the embodiment of Fig. 9, channel cover 104 ' includes lamilated body 160, and described lamilated body is positioned at element except covering
110 ", outside a part for the microchannel 114 ' on bottom, be also combined with anti-reflux valve 140 and work to prevent between room 112
Cross-contamination.In an embodiment seen in the exploded view of Fig. 9 D, lamilated body 160 includes double-sided adhesive
Layer 162, elastomer layer 166 and optional single-sided adhesive backing layer 168.Element 110 " include optional alignment post
(registration post) 111 is so that the layer of lamilated body 160 is attached to element 110 at lamilated body " during align.Layer
162 include the optional alignment cuts (cutout) 163 alignd with alignment post 111.Similarly, layer 166 includes optional alignment
Otch 167, and layer 168 includes optional alignment cuts 169.Layer 162 also includes the valve otch 164 around anti-reflux valve 140, and
Layer 168 includes valve otch 170.Double-sided adhesive layer 162 is attached to element 110 " and it is attached to elastomer layer 166.Optionally, single
The adhesive-backed layer in face 168 may be coupled to elastomer layer 166 to reduce the risk of lamilated body 160 layering.Fig. 9 E is to be attached with
The bottom view of the equipment 100 of channel cover 104 '.
In conjunction with Fig. 9 A-9E with reference to Fig. 9 F and 9G, describe and use the side of the embodiment of the equipment 100 of display in Fig. 9
Method.As seen in Fig. 9 F, syringe 30 (or other any sample transfer device, such as said injector device 40 or aforementioned there is appearance
Receive the syringe 30 of element 20) by making Luer lock 32 coordinate with Luer lock 102 to come and equipment 100 coupling.Syringe 30 makes sample
Product S (with optional liquid L) is forced in equipment 100 via entrance 116.Sample S is along element 110 " bottom miniflow lead to
(seeing Fig. 9 F in conjunction with Fig. 9 C) is advanced in road 114 '.Microchannel is then by element 110 " and take sample S to element 110 "
Top, be branched off into multiple microchannel 114 ' (seeing Fig. 9 F in conjunction with Fig. 9 B) afterwards.Microchannel 114 ' is then via element
110 " back extend and sample S is delivered to anti-reflux valve 140.The pressure applied by syringe 30 causes lamilated body 160
Elastomer layer 166 deflects partly and temporarily at next-door neighbour's valve 140, thus allows sample S by (seeing figure in conjunction with Fig. 9 C
9G).After sample S passes through, anti-reflux valve 140 reseals to prevent sample S from refluxing and is therefore prevented from the intersection dirt of room 112
Dye.Most advantageously seeing in Fig. 9 G of Fig. 9 C as combined, microchannel 114 ' will be brought back to by the sample S of valve 140
Element 110 " top and take in room 112.Room 112 comprises reagent 130, such as, freeze-dried reagent 130.
Passing away 118 ' extend from room 112 with air A is discharged into from room 112 overflow room 125 (in conjunction with Fig. 9 B and
9C sees Fig. 9 G).In the passage 118 ' that flow control medium 150 is located between room 112 and overflow room 125.Flow control medium
150 can such as comprise the aperture hydrophobic material allowing air to pass through but not allow fluid from.Flow control is passed through at air
After medium 150, its in passing away 118 ' from element 110 " top enter overflow room 125 via this element row.
As other all embodiments of equipment 100, in Fig. 9, the embodiment of the equipment 100 of display can wrap
Include heating element heater or with heating element heater (heating element heater 200 of such as Fig. 7) coupling to expand interested one by reagent 130
Plant or multiple target nucleic acid sequence (when being present in sample S).Target sequence amplification such as can be passed through by operator's visual detection
Visual detection visual indicators, as the color displacement in colorimetric dye or turbidity change, or via such as being referred to by vision
Show the detection of agent (color displacement, fluorescence, turbidity change etc.) to detect the sensor (such as the sensor 300 of Fig. 7 B) of amplification from
Dynamic detection.In Fig. 9, the embodiment of the equipment 100 of display schematically includes air overflow room 125 and anti-reflux valve 140.
It should be understood that equipment can only include anti-reflux valve alternatively or only include overflow room.
Referring now to Fig. 9 H-9J, equipment 100 optionally can include the shell 180 of receiving equipment 100.Shell 180 can wrap
Include have depression 184 cavity 182, described depression be configured to receive element 110 " anti-reflux valve 140.Heating element heater 200 is also
Cavity 182 can be placed in interior near room 112, with the content of heating chamber 112.Shell 180 also includes covering 186, and it has
It is beneficial to the visual room otch 188 of room 112, and there is the Luer lock otch 190 providing the path to Luer lock 102.Lid 186
Such as by screw or press-in coordinate (press fit) be stably attached to inner chamber 182, be placed in equipment 100 therein
Other assemblies formed shell 180.
Figure 10 provides and includes element 110 " ' another alternative embodiment of equipment 100.In Figure 10, display sets
The embodiment of standby 100 includes anti-return lock-up valve 200, and it is configured to make element 110 " ' microchannel 114 " be locked in fair
Permitted flowing through passage 114 " release position or prevent between room 112 via passage 114 " backflow and the pass of cross-contamination
In closed position.The locking of such passage can be formed as reversible or irreversible as expected.Figure 10 A provides equipment 100
Isometric top view, and Figure 10 B provides the isometric bottom view of equipment.Figure 10 C provides the equidistant detailed of anti-return lock-up valve 200
View.For the sake of clarity, chamber cap 106 and channel cover 104 show the most in Fig. 10.It should be understood, however, that they can be as above
The offer described by equipment embodiment.
As seen in Figure 10 A and 10B, anti-return lock-up valve 200 is configured to be placed in element 110 " ' room 202 in, with logical
Cross in room 202 relative to element 110 " ' slide lock valve 200 and make passage 114 " be locked in as expected open (i.e.,
Allow to flowing) or close in (that is, block flow) position.As seen in Figure 10 C, inner chamber 230 is by anti-return lock-up valve 200
And can optionally with passage 114 " align or do not line up to unlock respectively and locking channel.Lock-up valve 200 can be such as
Substrate 210 including the relative rigid or rigidity with elastomer Bao Mo (overmold) 220.Elastomer bag mould 220 can wrap
Include O element 222a and 222b, its be configured to produce to element 110 " ' liquid-tight seal.O element 222a with pass through
Locking channel 114 " prevent the locked configuration of the anti-return lock-up valve 200 of cross-contamination between room 112 to be associated.O
Element 222b and inner chamber 230 be concentrically aligned and with allow through passage 114 " the anti-return lock-up valve 200 of fluid flowing
Unlocked configuration be associated.In the alternative embodiment (not shown) of lock-up valve 200, elastomer bag mould 220 can be saved
Omit, and O element 222a and/or 222b can form or be directly attached to substrate 210.Lock-up valve 200 is preferably included in
Be beneficial to during use to handle the increase of lock-up valve terminal part 240 (i.e., it is possible to by user grip with by lock-up valve from unlocking structure
Type slides into locked configuration, and vice versa).
Figure 10 D and 10E is the translucent detailed view of the actuating that lock-up valve 200 is described.As seen in Figure 10 D, passage
114 " element 110 can be positioned at by making lock-up valve 200 " ' room 202 in make inner chamber 230 and microchannel 114 " right
It is placed in together in unlocked configuration.Optionally, lock-up valve 200 and/or room 202 can be beneficial to phase-locked for room by lubrication
Slide.In this unlocked configuration, O element 222b is at passage 114 " periphery formation fluid-tight so that sample can be from sample
Product transfer device (such as syringe) is through passage 114 " the first section, through inner chamber 230 with through passage 114 " second
Section flow is to room 112.As seen in Figure 10 E, lock-up valve 200 then can slide in room 202 so that passage 114 " place
In locked configuration so that inner chamber 230 does not aligns with microchannel.In this locked configuration, O element 222a is at passage 114 "
Periphery forms fluid-tight, is thus isolated from each other and closes each passage 114 " and prevent between room 112 by through passage
The cross-contamination of backflow.
In one embodiment, the terminal part 240 of the increase of lock-up valve 200 may be located at and the locked configuration of Figure 10 E
In element 110 " ' flush so that once lock-up valve 200 has locked passage, and user can not grip end 240 unlock logical
Road 114 ".Such irreversible lock-up valve can reduce the risk of backflow pollution or sample accidental discharge to the wind in environment
Danger.In alternative embodiment, the terminal part 240 of the increase of lock-up valve 200 can be from the element being in locked configuration
110 " ' prominent so that user can grip terminal part 240 reversibly to lock reconciliation lock access 114 with lock-up valve 200 ".
In conjunction with Figure 10 A-10E with reference to Figure 10 F and 10G, describe and use the embodiment of the equipment 100 of display in Figure 10
Method.In figure 10f, lock-up valve 200 makes passage 114 " it is positioned in Figure 10 D in the unlocked configuration of display.Syringe or its
His sample transfer device is by coordinating with Luer lock 102 to come and equipment 100 coupling.Syringe or other sample transfer device make sample
Product S (with optional liquid L) is forced in equipment 100 via entrance 116.Sample S is along element 110 " ' bottom miniflow lead to
Road 114 " interior traveling, described microchannel is branched off into multiple microchannel 114 " (seeing Figure 10 F in conjunction with Figure 10 B and 10D).Each
Microchannel then via the inner chamber 230 of lock-up valve 200 by element 110 " top of ', thus take sample S to element 110 " '
In portion and the room 112 containing reagent 130 (such as, the reagent 130 of lyophilizing).As seen in Figure 10 G, lock-up valve 200 then can phase
For element 110 " slide so that passage 114 in room 202 " be positioned in the locked configuration of Figure 10 E, wherein said passage is hindered
Disconnected.At passage 114 " when being in locked configuration, sample S cannot pass through lock-up valve 200 and refluxes, and which prevent room 112 and passes through warp
The cross-contamination of the backflow of passage.
Element 110 " ' include extending from room 112 the air A (but non-sample S) from room 112 is discharged into overflow room
The aforementioned passing away 118 ' (seeing Figure 10 G in conjunction with Figure 10 A and 10B) of 125.Flow control medium 150 is placed in room 112 and overflows
In passage 118 ' between thing room 125.Flow control medium 150 can such as comprise permission air by but do not allow fluid from
Aperture hydrophobic material.At air by after flow control medium 150, its in passing away 118 ' from element 110 " ' top
Portion is via element 110 " ' advance to overflow room 125.In Figure 10, the embodiment of the equipment 100 of display schematically includes sky
Gas overflow room 125 and anti-return lock-up valve 200.It should be understood that this equipment can only include anti-return lock-up valve alternatively or only wrap
Include overflow room.
As other all embodiments of equipment 100, in Figure 10, the embodiment of the equipment 100 of display can wrap
Include heating element heater or with heating element heater (heating element heater 200 of such as Fig. 7) coupling, to expand interested by reagent 130
One or more target nucleic acid sequences (when being present in sample S).Target sequence amplification can be the most logical by operator's visual detection
Cross visual detection visual indicators, as the color displacement in colorimetric dye or turbidity change, or via such as passing through vision
The detection of indicator (color displacement, fluorescence, turbidity change etc.) detects the sensor (such as the sensor 300 of Fig. 7 B) of amplification and comes
Automatically detection.Optionally, heating element heater, element 110 " ' and/or some other aspect of equipment 100 can be at lock-up valve 200
In allowing flow through passage 114 " open configuration in time, including stoping element 110 " ' with the geometry of heating element heater coupling
Or other constraints.Such constraint can reduce at passage 114 " it is locked in the risk of sample amplification before closing in configuration,
The thus risk of the cross-contamination that the backflow of reduction room 112 causes.
The embodiment of equipment 100 described so far by sample S via the microchannel making sample S distribute between the chambers
It is delivered to reative cell 112.In the embodiment of the equipment 100 shown in fig. 11, sample S is not using the feelings of microfluidic device
It is delivered to reative cell under condition.In Figure 11, the embodiment of the equipment 100 of display illustratively includes single reative cell, it should be understood that
Equipment 100 can include the reative cell of any desired quantity.
With reference to Figure 11 A, equipment 100 includes reative cell 400 and punch elements 500.Punch elements 500 includes convex (male) unit
Part 502, described convex element is configured to be press-fit to recessed (female) element of reative cell 400 before nucleic acid amplification and detection
In 402 and enclosed reaction chamber 400.
As seen in Figure 11 B and 11D, the female element 402 of reative cell 400 includes that can be press-fit to reagent therein inserts
Enter part 410.Reagent insert 410 includes cutting element 412 and reagent chamber 414.Reagent 130 is positioned at reagent chamber 414.Reagent
130 can e.g. solution or liquid form.Or, reagent 130 can be lyophilizing, as in Figure 10.
Reagent insert 410 is sealed in the female element 402 of reative cell 400 by sealing 404 (see for example Figure 11 C).
Seal 404 and can such as include metal forming or plastic foil.The sealing of reative cell 400 can be conducive to reagent 130 before use
Long term storage and/or may insure that freeze-dried reagent 130 before use keep be dried.
As seen in Figure 11 B, 11E and 11F, the convex element 502 of punch elements 500 includes being press-fit to therein
Liquid insert 510.Liquid insert 510 includes cutting element 512 and liquid chamber 514.Liquid chamber 514 is with sealing 516 sealings.
Seal 516 and can such as include metal forming or plastic foil.Liquid L (such as water and/or TE buffer) is sealed in liquid chamber 514
In.Dyestuff, MgSO4, glycine betaine and/or isothermal buffer agent can additionally or alternatively be sealed in room 514.
Equipment 100 also includes and the heating element heater 200 of reative cell 400 thermal coupling.(it is the most permissible for heating element heater 200
Abandon together with the remainder of equipment 100 after a single use) it is configured to the content of reacting by heating room 400 to realize core
Acid amplification, such as isothermal nucleic acid amplification, such as LAMP.Heating element heater 200 can include such as resistance heater, and it includes being encapsulated in
Etched foil element between two layers of polyimide film.Heating element heater can also include power supply, such as battery or and standard wall
The connection of seat, and for the thermocouple of temperature monitoring in the feedback loop have temperature controller, described temperature controller is used
In the temperature monitored according to expectation regulation to realize nucleic acid amplification.
The most transparent or semitransparent visualization being beneficial to reative cell of reative cell 400, and detect target nucleus interested
The amplification of acid sequence.Nucleic acid amplification can by the color displacement of colorimetric dye, risen by turbidity or by fluorescence etc. come
Detection.Detection can with bore hole and/or by can with after the optional sensor 300 that abandons realize.Testing result can show
Can with after on the display that abandons.
Sample S can be placed directly within reative cell 400 and/or punch elements 500, seals reaction by punch elements afterwards
Room.Or, the sample divider 10 comprising sample S can be placed between reative cell 400 and punch elements 500 so that convex unit
Sample S is placed in reative cell 400, as shown in figure 11 by part 502 with coordinating of female element 402.In the embodiment of Figure 11, sample
Product catcher 10 can such as include filter paper, such as chemically treated filter paper, such as available from Whatman (GE Healthcare's
Part) Flinders Technology Associates (" FTA ") card.Various sample substrates include but not limited to food
Thing, urine, saliva, mucus, Excreta, blood, seminal fluid, tissue, cell, DNA, RNA, protein, plant material, animal substance,
Solution, solid and other sample substrates can deposit on sample divider 10 that (other sample substrate will be obvious
).By this way, sample divider 10 can collect sample S via filter paper.
In order to collect sample S with sample divider 10, filter paper can such as immerse or insert interested one or more
In substrate.Additionally or alternatively, one or many one or more sample substrates interested can such as be placed in or deposit
On filter paper.Additionally or alternatively, filter paper can such as wiping or smeared one or more sample substrates interested or table
Face.
Referring now to Figure 11 G-11J, the method describing the embodiment using equipment 100 seen in fig. 11.Such as Figure 11 G
Seen in, reative cell 400 and punch elements 500 are close so that the convex element 502 of punch elements and the female element 402 of reative cell
Coordinate to come enclosed reaction chamber.The cutting element 512 of liquid insert 510 pierces through sample divider 10, and convex element 502 is from sample
Product catcher 10 removes the sample S of a punching press, thus makes sample S be placed in reative cell 400.
As seen in Figure 11 H, reative cell 400 continues close to the cutting causing liquid insert 510 with punch elements 500
Element 512 pierces through the sealing 404 of reative cell 400, thus provides the path of reagent insert 410.As seen in Figure 11 I, more
Further close to causing the cutting element 412 of reagent insert 410 to pierce through the sealing 516 of liquid insert 510, thus cause
Liquid L trickle room 514 and in Jin Shuo reagent chamber 414.As seen in Figure 11 J, reative cell 400 is complete with punch elements 500
Complete close to making nucleic acid amplification and the necessary all material (sample S, reagent 130 and optional liquid L) of detection be positioned at reagent chamber
In 414.
After reative cell and punch elements are close, the content that heating element heater 200 heats reagent chamber 414 is interested to realize
The nucleic acid amplification of target nucleic acid sequence (when being present in sample S).Detection can be by bore hole and/or by sensor 300
Realize.
The equipment 100 of Figure 11 optionally can serve as the co-pending U.S. Patent submitted to before this on April 14th, 2012
Patent application serial numbers 13/447, the part of the instrument 40 described in 218, the full text of this application here cites addition.Specifically, Tu10Zhong
The reative cell 400 of equipment 100 and punch elements 500 can substitute for punch elements 90 He of the instrument 40 of display in the application of ' 218
Room 70.
The method and apparatus of Fig. 1-11 provides and is being suitable for by the most unskilled user at the environment of limited resources
Completely self-contained (fully in (abandoning after optionally using, such as abandon after being intended for single use) equipment of middle use
Contained), sample prepares to the nucleic acid samples of result formula, (the most multiple) target expands and detection.
Conclusion
Although described above is the preferred illustrated embodiment of the present invention, but the brightest
Aobvious is it can be made various change and be modified without departing from the present invention.Such as, although the various assemblies of equipment
Coordinate and have been described as the cooperation that connected by Luer lock, it should be understood that can also use Luer slip joint (luer slip),
Press-in coordinates or other fit connection known per se.Although additionally, some embodiments described of equipment are illustratively
Use one or more syringe that sample S transfers to nucleic acid amplification and detection equipment, it should be understood that any replacing can also be used
For property sample transfer device, including the transfer device manufactured for specific purpose.
Further, although equipment 100 and correlation technique are described with regard to nucleic acid amplification and detection, it should be understood that
This equipment and correlation technique can include and/or be used for keeping and analyze sample without amplification and/or detection sample alternatively
Nucleic acid in product.In such embodiment, equipment 100 can include that the nucleic acid being used in analysis or other samples maintain
Sample holder 100 in reative cell, described reative cell can serve as observation ward and/or analysis room.Analysis can include such as removing
Outside nucleic acid amplification and detection or one or more technology of alternative as nucleic acid amplification and detection, as microscopy,
Hybridization and/or protein analysis.
When equipment 100 includes sample holder, keep sample with the method being analyzed can include collect sample substrate,
Transfer at least one reaction/observation/analysis room, optionally heat institute via at least one microchannel by described sample substrate
State the sample substrate part as analytical technology, and (such as in heating process) prevents described sample substrate from described at least one
Reflux via at least one microchannel described in individual room.Anti-backflow can prevent cross-contamination when providing multiple room.Prevent
Backflow can by the check valve in described reaction/observation/analysis room and/or by described sample substrate is transferred to described
Block described microchannel behind room to realize.
Appended claims are intended to cover the true spirit falling into the present invention and the whole such change in scope and change
Dynamic.
Claims (30)
1., for on-the-spot amplification and the method for detection of target nucleic acid sequence, described method includes:
Collect sample substrate；
Described sample substrate is transferred at least one reative cell containing nucleic acid amplification agents via at least one microchannel；
Heat described nucleic acid amplification agents to expand described target nucleus when described target nucleic acid sequence is included in described sample substrate
Acid sequence；
Prevent described sample substrate in heating process from least one reative cell described via at least one microchannel described
Backflow；With
The amplification of described target nucleic acid sequence is detected when described target nucleic acid sequence is present in described sample substrate.
2. the method described in claim 1, wherein prevent backflow further include at described sample substrate transfer to described at least
During one reative cell, make described sample substrate by least one check valve.
3. the method described in claim 1, wherein prevent backflow further include at described sample substrate transfer to described at least
After one reative cell, block at least one microchannel described.
4. the method described in claim 1, it also includes discharging overflow from least one reative cell described.
5. the method described in claim 1, after it is additionally included in first use, abandons described sample substrate, at least one miniflow
Passage, at least one reative cell and nucleic acid amplification agents.
6. the method described in claim 1, wherein heating farther includes:
With nucleic acid amplification agents described in heating element heats；And
Abandon described heating element heater after a single use.
7. the method described in claim 1, wherein detection amplification farther include detect colorimetric dye color displacement, detect turbid
Degree rises or detection fluorescence.
8. the method described in claim 1, wherein transfer farther includes described sample substrate via at least one branch
Microchannel shifts, and the microchannel of described branch distributes described sample substrate between multiple reative cells.
9. the method described in claim 8, wherein preventing refluxes farther includes to prevent the intersection between the plurality of reative cell
Pollute.
10. the method described in claim 8, it is additionally included in different reative cells and expands and detect different target nucleic acid sequences
Row.
11. 1 kinds of on-the-spot amplifications for target nucleic acid sequence and the method for detection, described method includes:
Collect sample substrate；
Described sample substrate is transferred at least one reative cell containing nucleic acid amplification agents from sample divider；
Heat described nucleic acid amplification agents to expand described target nucleus when described target nucleic acid sequence is included in described sample substrate
Acid sequence；
Overflow is discharged from least one reative cell described in heating process；With
Detect the amplification of described target nucleic acid sequence.
Method described in 12. claim 11, wherein transfer farther includes to shift described sample via at least one microchannel
Product substrate.
Method described in 13. claim 11, wherein collects and farther includes to collect described sample substrate with sample divider, and
And wherein transfer farther includes at least one punching of described sample divider to be placed at least one reative cell described.
Method described in 14. claim 11, wherein discharges overflow and farther includes to be discharged into the spilling of at least one isolation
Thing room.
Method described in 15. claim 11, wherein discharge overflow further include at prevent fluid discharge while via
Flow control medium discharges gas.
Method described in 16. claim 11, it also includes preventing described sample substrate from described at least one in heating process
Individual reative cell refluxes via at least one microchannel described.
17. expand and the equipment of detection for the on-the-spot of target nucleic acid sequence, and described equipment includes:
Sample divider, described sample divider is configured to collect sample substrate；
There is at least one microchannel, at least one reative cell containing nucleic acid amplification agents and the device of at least one valve；
With
Heating element heater,
At least one microchannel wherein said be configured to by described sample substrate from described sample divider be transported to described to
A few reative cell,
Wherein said heating element configuration becomes be transported to by described sample substrate described at least one reative cell post-heating described
Nucleic acid amplification agents to expand described target nucleic acid sequence when described target nucleic acid sequence is included in described sample substrate, and
At least one valve wherein said is configured to prevent described sample substrate from least one reative cell described in heating process
Reflux via at least one microchannel described.
Equipment described in 18. claim 17, wherein said device also includes for discharging from least one reative cell described excessive
Go out at least one isolation room of thing.
Equipment described in 19. claim 18, wherein said device also includes for discharging gas from least one reative cell described
Body but the flow control medium of not drain.
Equipment described in 20. claim 17, wherein said valve is selected from check valve, lock-up valve, reversal valve, irreversible valve and group thereof
Close.
Equipment according to any one of 21. claim 17-19, wherein said valve includes slider bar valve, described slider bar valve energy
Enough selectively slide to stop described fluid communication between at least one reative cell and at least one microchannel described
Closed position, or slide into the described fluid communication between at least one reative cell and at least one microchannel described of permission
Release position.
Equipment described in 22. claim 21, wherein has multiple reative cell and multiple microchannel and in the closed position
Described slider bar valve stop the fluid communication between multiple corresponding reative cells and microchannel.
The equipment of 23. field assays being used for fluid sample, described equipment includes:
Sample divider, described sample divider is configured to collect fluid sample；With
There is at least one microchannel, at least one analysis room and the device of slider bar valve, at least one miniflow wherein said
Passage is configured to from described sample divider, described fluid sample is transported at least one analysis room described, and wherein said cunning
Dynamic rod valve is configured to optionally allow for or stop described between at least one microchannel and at least one analysis room described
Fluid communication.
Equipment described in 24. claim 23, wherein has multiple reative cell and multiple microchannel, and described slider bar valve
It is configured to optionally allow for or stop the fluid communication between multiple corresponding reative cell and microchannel.
Equipment according to any one of 25. claim 23-24, wherein said slider bar valve can be selectively described in permission
Slide between release position and the closed position stoping described fluid communication of fluid communication.
Equipment according to any one of 26. claim 23-25, wherein said analysis room is reative cell.
Equipment according to any one of 27. claim 23-26, wherein said analysis includes amplification and the inspection of target nucleic acid sequence
Survey.
Equipment according to any one of 28. claim 23-27, its heating element heater also including heating described fluid sample.
Equipment according to any one of 29. claim 23-28, wherein said analysis room is included in described fluid sample and is provided
To the reagent being combined with described fluid sample during described analysis room.
Equipment according to any one of 30. claim 23-29, it also includes that imaging sensor is to capture in described analysis room extremely
The image of few a part of described fluid sample.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201361894392P | 2013-10-22 | 2013-10-22 | |
| US61/894,392 | 2013-10-22 | ||
| US14/262,683 | 2014-04-25 | ||
| US14/262,683 US9469871B2 (en) | 2011-04-14 | 2014-04-25 | Methods and apparatus for point-of-care nucleic acid amplification and detection |
| PCT/US2014/061808 WO2015061480A1 (en) | 2013-10-22 | 2014-10-22 | Methods and apparatus for point-of-care nucleic acid amplification and detection |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105874084A true CN105874084A (en) | 2016-08-17 |
Family
ID=52993515
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201480070428.1A Pending CN105874084A (en) | 2013-10-22 | 2014-10-22 | Methods and apparatus for point-of-care nucleic acid amplification and detection |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP3060683A4 (en) |
| JP (1) | JP2016539633A (en) |
| CN (1) | CN105874084A (en) |
| CA (1) | CA2928406A1 (en) |
| WO (1) | WO2015061480A1 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106010961A (en) * | 2016-08-03 | 2016-10-12 | 珠海百瑞生物科技有限公司 | Device and method for identifying food-borne pathogens by high-throughput loop-mediated isothermal amplification |
| CN107129930A (en) * | 2017-06-09 | 2017-09-05 | 北京百康芯生物科技有限公司 | A kind of fully integrated detection of nucleic acids micro-fluidic chip and its application method |
| CN110198784A (en) * | 2017-01-02 | 2019-09-03 | 极小微技术股份公司 | Bracket for reagent carrier elements |
| CN111278540A (en) * | 2017-11-06 | 2020-06-12 | 赛多利斯司特蒂姆生物工艺公司 | Filter module and method for detecting microorganisms |
| CN112852928A (en) * | 2021-01-14 | 2021-05-28 | 广东东阳光药业有限公司 | Multiplex nucleic acid amplification product detection method and kit |
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| WO2018175424A1 (en) * | 2017-03-22 | 2018-09-27 | The Board Of Trustees Of The University Of Illinois | System for rapid, portable, and multiplexed detection and identification of pathogen specific nucleic acid sequences |
| US11596939B2 (en) | 2017-04-04 | 2023-03-07 | Selfdiagnostics Deutschland Gmbh | Lamp component distribution in a microfluid cell |
| WO2018187687A1 (en) * | 2017-04-07 | 2018-10-11 | Arizona Board Regents On Behalf Of Arizona State University | Integrated diagnostic devices having embedded biomolecular computing systems and uses thereof |
| CN108642148B (en) * | 2018-07-09 | 2024-01-30 | 南京岚煜生物科技有限公司 | Nucleic acid amplification detection micro-fluidic chip and detection method thereof |
| US20220235394A1 (en) * | 2021-01-25 | 2022-07-28 | Sandra Janeth Perdomo Lara | Reverse transcription polymerase chain reaction diagnostic testing device |
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| CA2788113A1 (en) * | 2010-02-03 | 2011-08-11 | President And Fellows Of Harvard College | Devices and methods for multiplexed assays |
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- 2014-10-22 EP EP14856081.6A patent/EP3060683A4/en not_active Withdrawn
- 2014-10-22 WO PCT/US2014/061808 patent/WO2015061480A1/en active Application Filing
- 2014-10-22 CA CA2928406A patent/CA2928406A1/en not_active Abandoned
- 2014-10-22 CN CN201480070428.1A patent/CN105874084A/en active Pending
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| CN102939543A (en) * | 2010-02-12 | 2013-02-20 | 西北大学 | Assay card for sample acquisition, treatment and reaction |
| US20120264116A1 (en) * | 2011-04-14 | 2012-10-18 | Michlitsch Kenneth J | Methods and apparatus for point-of-care nucleic acid amplification and detection |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106010961A (en) * | 2016-08-03 | 2016-10-12 | 珠海百瑞生物科技有限公司 | Device and method for identifying food-borne pathogens by high-throughput loop-mediated isothermal amplification |
| CN106010961B (en) * | 2016-08-03 | 2019-11-22 | 珠海百瑞生物科技有限公司 | High-throughput ring mediated isothermal amplification identification food borne pathogens body device and detection method |
| CN110198784A (en) * | 2017-01-02 | 2019-09-03 | 极小微技术股份公司 | Bracket for reagent carrier elements |
| CN110198784B (en) * | 2017-01-02 | 2021-07-20 | 极小微技术股份公司 | Holder for a reagent carrier element |
| CN107129930A (en) * | 2017-06-09 | 2017-09-05 | 北京百康芯生物科技有限公司 | A kind of fully integrated detection of nucleic acids micro-fluidic chip and its application method |
| CN107129930B (en) * | 2017-06-09 | 2019-11-26 | 北京百康芯生物科技有限公司 | A kind of fully integrated detection of nucleic acids micro-fluidic chip and its application method |
| CN111278540A (en) * | 2017-11-06 | 2020-06-12 | 赛多利斯司特蒂姆生物工艺公司 | Filter module and method for detecting microorganisms |
| CN111278540B (en) * | 2017-11-06 | 2022-07-26 | 赛多利斯司特蒂姆生物工艺公司 | Filter module and method for detecting microorganisms |
| US11541357B2 (en) | 2017-11-06 | 2023-01-03 | Sartorius Stedim Biotech Gmbh | Filter module and method for detecting microorganisms |
| CN112852928A (en) * | 2021-01-14 | 2021-05-28 | 广东东阳光药业有限公司 | Multiplex nucleic acid amplification product detection method and kit |
Also Published As
| Publication number | Publication date |
|---|---|
| EP3060683A4 (en) | 2017-08-09 |
| WO2015061480A1 (en) | 2015-04-30 |
| JP2016539633A (en) | 2016-12-22 |
| EP3060683A1 (en) | 2016-08-31 |
| CA2928406A1 (en) | 2015-04-30 |
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