EP3060683A1 - Procédés et appareil permettant l'amplification et la détection d'acides nucléiques au point d'intervention - Google Patents

Procédés et appareil permettant l'amplification et la détection d'acides nucléiques au point d'intervention

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Publication number
EP3060683A1
EP3060683A1 EP14856081.6A EP14856081A EP3060683A1 EP 3060683 A1 EP3060683 A1 EP 3060683A1 EP 14856081 A EP14856081 A EP 14856081A EP 3060683 A1 EP3060683 A1 EP 3060683A1
Authority
EP
European Patent Office
Prior art keywords
sample
nucleic acid
reaction chamber
chamber
sample matrix
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP14856081.6A
Other languages
German (de)
English (en)
Other versions
EP3060683A4 (fr
Inventor
Jane P. Bearinger
Scott Castanon
Kenneth J. Michlitsch
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Corporos Inc
Original Assignee
Corporos Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US14/262,683 external-priority patent/US9469871B2/en
Application filed by Corporos Inc filed Critical Corporos Inc
Publication of EP3060683A1 publication Critical patent/EP3060683A1/fr
Publication of EP3060683A4 publication Critical patent/EP3060683A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • B01L3/502738—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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • 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
    • 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
    • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
    • 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
    • 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
    • B01L2300/18—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
    • 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
    • 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
    • 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
    • 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

Definitions

  • the present invention relates to methods and apparatus for nucleic acid
  • the present invention relates to methods and apparatus for point-of-care nucleic acid amplification and detection.
  • PCR Polymerase Chain Reaction
  • ELISA Enzyme-Linked Immuno-Sorbent Assay
  • Figure 1 is a schematic view of one embodiment of a sample collector
  • Figures 2A-2C are isometric and side views of apparatus and methods for preparing and transferring sample from the sample collector of Figure 1 to point-of-care nucleic acid amplification and detection apparatus;
  • Figures 3A and 3B are side and isometric views of alternative apparatus and methods for preparing and transferring sample from the sample collector of Figure 1 to point-of-care nucleic acid amplification and detection apparatus;
  • Figures 4A-4D are side-sectional and isometric views of additional alternative apparatus and methods for preparing and transferring sample from the sample collector of Figure 1 to point-of-care nucleic acid amplification and detection apparatus;
  • Figure 5 is an exploded assembly view of the point-of-care nucleic acid amplification and detection apparatus of Figures 2-4;
  • Figure 6 is a bottom view of a channel and chamber element of the point-of- care nucleic acid amplification and detection apparatus of Figure 2-5;
  • Figure 7A is an isometric view of the point-of-care nucleic acid amplification and detection apparatus of Figures 2-6 in thermal communication with a heating element
  • Figure 7B is an isometric view of an optional detection sensor for use with the apparatus and method of Figure 7A
  • Figures 8A-8E are isometric, top, bottom and side-sectional views of an alternative embodiment of the methods and apparatus for point-of-care nucleic acid amplification and detection of Figures 2-7;
  • Figures 9A-9J are isometric, top, bottom, assembly, side-sectional detail, and translucent isometric views of another alternative embodiment of methods and apparatus for point-of-care nucleic acid amplification and detection;
  • Figures 10A-10G are isometric top, isometric bottom, isometric detail, translucent detail and side-sectional detail views of another alternative embodiment of methods and apparatus for point-of-care nucleic acid amplification and detection;
  • Figures 1 A- 1 1 J are side, side-sectional, isometric, and translucent isometric views of yet another alternative embodiment of methods and apparatus for point-of-care nucleic acid amplification and detection.
  • the present invention relates to methods and apparatus for nucleic acid amplification and detection. More particularly, the present invention relates to methods and apparatus for point-of-care nucleic acid amplification and detection.
  • the apparatus and methods optionally may be used in a multiplexed fashion to detect multiple target nucleic acid sequences of interest (e.g., to detect at least two target nucleic acid sequences of interest), and the apparatus optionally may be configured for disposal after one-time use.
  • the apparatus preferable utilizes an isothermal nucleic acid amplification technique, e.g., loop-mediated isothermal amplification ("LAMP)", to reduce the instrumentation requirements associated with nucleic acid amplification.
  • LAMP loop-mediated isothermal amplification
  • Detection of target amplification may be achieved, for example, via detection of a color shift and/or fluorescence in one or more dyes, such as hydroxynaphthol blue, picogreen, and/or SYBR green, added to the amplification reaction, or via a change in turbidity.
  • Such colorimetric, fluorescent and/or turbidity detection may be performed visually by an operator and/or may be achieved utilizing an imaging technique, such as spectrophotometric and/or fluorescence imaging, as described below.
  • Figure 1 illustrates one embodiment of sample collector 10, per se known, for collecting a nucleic acid sample S.
  • Sample collector 10 may, for example, comprise a sponge, foam or swab.
  • Sample collector 10 may, for example, be fabricated from an inert polymer.
  • sample matrices - including, but not limited to, food, urine, saliva, mucous, feces, blood, semen, tissue, cells, DNA, RNA, protein, plant matter, animal matter, liquids, solutions, solids, gases, and other sample matrices - may be deposited onto sample collector 10 as sample S.
  • sample collector 10 In order to collect sample S with sample collector 10, the sample collector may, for example, be dipped or placed into one or more sample matrices of interest. In one method of using sample collector 10, the sample collector may be placed in a person's mouth for a period of time in order to collect a saliva sample S. Additionally or alternatively, one or more drops of one or more sample matrices of interest may, for example, be placed or deposited onto the sample collector. As yet another alternative, sample collector 10 may, for example, be swabbed or wiped across one or more sample matrices or surfaces of interest.
  • sample collector 10 After collection of sample S, the sample may be transferred from sample collector 10 to point-of-care nucleic acid amplification and detection apparatus 100.
  • the sample may be prepared before, during or after transfer, e.g., via placement of sample S in fluid communication with lysis chemicals.
  • Sample collector 10 optionally may comprise lysis chemicals that prepare sample S.
  • sample S may be prepared via heat treatment. For example, sample S may be heated to a temperature higher than that required for isothermal amplification, e.g., higher than that required for loop-mediated isothermal amplification ("LAMP").
  • sample S may comprise whole blood, which may, for example, be heat treated at about 99°C, e.g., for about 10 minutes, to achieve sample preparation. Other preparation methods, per se known, additionally or alternatively may be used.
  • sample S may not require preparation.
  • mixing of sample S with water, buffer and/ or dye solution may be sufficient to prepare the sample for nucleic acid amplification.
  • Figures 2 illustrate one embodiment of methods and apparatus for transferring sample S from sample collector 10 to point-of-care nucleic acid amplification and detection apparatus 100.
  • sample collector 10 may be placed within sample collector containment element 20 having luer lock 22.
  • Containment element 20 comprises a lumen or compartment in which sample collector 10 may be placed.
  • cap 24 having luer lock 26 may be attached to sample collector containment element 20 after placement of sample collector 10 within the containment element 20.
  • Containment element 20 and sample collector 10 then may be attached to syringe 30 via mating of (male or female) luer lock 26 of cap 24 with (female or male) luer lock 32 of syringe 30.
  • syringe 30 and containment element 20 with sample collector 10 may be coupled to point-of-care nucleic acid amplification and detection apparatus 100 by mating of (male or female) luer lock 22 of containment element 20 with (female or male) luer lock 102 of apparatus 100.
  • Syringe 30 may contain liquid L (e.g., water, buffer and/or colorimetric or other dye solution) for eluting sample S from sample collector 10 into apparatus 100 via depression of plunger 34.
  • Luer lock 102 of apparatus 100 optionally may comprise a one-way valve that prevents backflow during nucleic acid amplification and detection.
  • Figures 3 illustrate alternative methods and apparatus for transferring sample S from sample collector 10 to apparatus 100.
  • luer lock 22 of containment element 20 may be coupled to luer lock 42 of second syringe 40.
  • Plunger 34 of syringe 30 may be depressed to elute liquid L and sample S from sample collector 10 into second syringe 40. Elution of liquid L and sample S into second syringe 40 before transfer of the sample to apparatus 100 may enhance mixing of the liquid and the sample before transfer to apparatus 100.
  • sample S optionally may be collected and/or eluted multiple times into second syringe 40 before transfer to apparatus 100.
  • second syringe 40 may be detached from syringe 30, containment element 20 and sample collector 10. Second syringe 30 then may be coupled to apparatus 100 by mating of luer lock 42 to luer lock 102. Depression of plunger 44 forces sample S and liquid L into apparatus 100.
  • Figures 4 illustrate additional alternative methods and apparatus for transferring sample S from sample collector 10 to apparatus 100.
  • sample collector 10 having sample S may be placed directly within syringe 30 by temporarily detaching plunger 34 from the syringe.
  • liquid L may be placed within syringe 30 along with sample collector 10 having sample S, though it should be understood that liquid L alternatively may be omitted.
  • plunger 34 may be reattached to the syringe, as in Figure 4B.
  • Syringe 30 then may be coupled to apparatus 100 by mating of luer lock 32 with luer lock 102, as in Figure 4C. Depression of plunger 34, as in Figure 4D, compresses sample collector 10 and expresses sample S into apparatus 100.
  • Apparatus 100 comprises luer lock 102 that is connected to channel and chamber element 1 10.
  • Element 1 10 may, for example, be fabricated from polypropylene.
  • Channel cover 104 connects to the bottom of element 1 10, e.g., via adhesive or screws, while chamber cover 106 connects to the top of element 1 10, e.g., via adhesive or screws.
  • Covers 104 and 106 may, for example, comprise an adhesive film or tape.
  • Chamber cover 106 (and, optionally, channel cover 104) preferably is translucent or transparent to facilitate visual inspection of the contents of reaction chambers 1 12 of element 1 10.
  • Apparatus 100 also may comprise top cover 120 with air filter 122, as well as chamber windows 124 that align with chambers 1 12 of element 1 10.
  • each chamber 1 12 may have a volume less than about 100 microliters, e.g., a volume on the order of about 30 microliters.
  • reaction chambers 1 12 of element 1 10 are connected to inlet 1 16 via (preferably equal length) microfluidic channels 1 14.
  • Sample S is collected and expressed into apparatus 100 through luer lock 102, e.g., via depression of a syringe plunger as described previously with respect to Figures 2-4.
  • a syringe plunger as described previously with respect to Figures 2-4.
  • Sample S is collected and expressed into apparatus 100 through luer lock 102, e.g., via depression of a syringe plunger as described previously with respect to Figures 2-4.
  • reagents 130 may, for example, comprise enzyme and master mix.
  • the enzyme may, for example, comprise Bst DNA polymerase, Bst 2.0 WarmStart DNA Polymerase, and/or Bsm DNA polymerase (and, optionally, a reverse transcriptase).
  • the master mix may, for example, comprise primers, dNTPs, MgS0 4 , betaine and/or excipients (e.g., mannitol, trehalose and/or dextrin).
  • Reagents 130 also may comprise water, TE buffer, isothermal buffer and/or other buffers, which optionally may be delivered to chambers 1 12 via microfluidic channels 1 14, e.g., before, during and/or after delivery of sample S, e.g., as liquid L.
  • Reagents 130 also may comprise one or more dyes to facilitate detection of nucleic acid amplification, such as hydroxynaphthol ("HNB") blue. Detection of target amplification may be achieved, for example, via detection of a color shift in the colorimetric dye in the presence of amplicon, e.g., due to a shift in free magnesium (Mg 2+ ) concentration during LAMP amplification. Such colorimetric detection may be performed visually by an operator or may be achieved utilizing spectrophotometric imaging, as described below. In addition or as an alternative to colorimetric amplification detection with a colorimetric dye, a fluorescent dye, such as picogreen or SYBR green, may be utilized to detect amplification via fluorescence.
  • a fluorescent dye such as picogreen or SYBR green
  • One or more of the reagents 130 preferably are lyophilized, e.g., to facilitate long-term storage. Additionally or alternatively, one or more of the reagents temporarily may be sequestered from one or more of the other reagents prior to nucleic acid amplification. Such temporary reagent sequestration may facilitate long-term storage of the reagents and/or may forestall reagent mixing (and, thereby, nucleic acid amplification) until desired, e.g., until the reagents have been exposed to sample S. For example, the enzyme may be sequestered from the master mix.
  • one or more of the reagents 130 may be temporarily sequestered within one or more temporary sequestration vessels.
  • the temporary sequestration vessel(s) may, for example, comprise one or more thermal encasement materials that are configured to melt, become porous or otherwise release the sequestered reagent(s) 130 upon heating, e.g., during nucleic acid amplification.
  • the thermal encasement material(s) may, for example, comprise polycaprolactone, and/or phase change materials such as paraffin or wax.
  • the temporary sequestration vessel(s) may comprise one or more blister packs or other containers such as gel caps that may be punctured or otherwise opened to release the sequestered reagent(s) 130. When the temporary sequestration vessel(s) comprise gel caps, they optionally may be opened via hydrolysis in addition or as an alternative to puncturing.
  • each reagent-containing chamber 1 12 is configured to amplify a nucleic acid target sequence of interest, if contained in the sample S.
  • Different chambers 1 12 optionally may utilize different primers to facilitate amplification and detection of different target sequences of interest (i.e., to facilitate multiplexed nucleic acid amplification and detection) in different chambers.
  • a fraction of the chambers 1 12 may serve as positive controls (e.g., may be preloaded with one or more target nucleic acid sequences of interest that are expected to amplify during nucleic acid amplification). Additionally or alternatively, a fraction of the chambers 1 12 may serve as negative controls (e.g., may comprise reagents 130 but may not be connected to microfluidic channels 1 14 such that they do not contain sample S).
  • the chambers may be heated, e.g., isothermally heated, to amplify the one or more target nucleic acid sequences of interest.
  • the contents of chambers 1 12 may be heated in the range of about 60°C-65°C for about 5-70 minutes.
  • the contents of chambers 1 12 may be heated via a heating element 200 that is thermally coupled to apparatus 100.
  • Such heating may be achieved utilizing any of variety of techniques, including (but not limited to) electrical, chemical and/or electrochemical techniques.
  • Heating element 200 may, for example, comprise a resistive heater connected to a power supply, such as one or more batteries or a wall outlet connection, and an optional temperature controller for resistively heating the contents of chambers 1 12. Additionally or alternatively, heating element 200 may comprise a diamond/tungsten heater, an inductive heater, a chemical heater (e.g., an exothermic chemical heater, such as a supersaturated sodium acetate heater, a cellulose/iron/water/activated carbon/vermiculite/salt heater, an iron oxide heater, an iron/magnesium salt heater, a catalytic burner, a fuel cell heater, etc.). Heating element 200 may be reusable or may be configured for disposal after one-time use. Optionally, heating element 200 may be integrally connected to apparatus 100.
  • a power supply such as one or more batteries or a wall outlet connection
  • heating element 200 may comprise a diamond/tungsten heater, an inductive heater, a chemical heater (e.g., an exothermic chemical heater, such as a supersaturated sodium
  • Heating element 200 may be fully automated or may comprise controls that, e.g, allow the user to set a target temperature and duration of heating.
  • heating element 200 may comprise a phase change material, such as paraffin, for maintaining a desired temperature for an extended period of time.
  • detection of target amplification optionally may be achieved via detection of a color shift (i.e. a wavelength shift) and/or fluorescence (i.e., an intensity shift) in one or more dyes in the presence of amplicon.
  • a color shift i.e. a wavelength shift
  • fluorescence i.e., an intensity shift
  • Such colorimetric and/or fluorescence detection may be performed visually by an operator and/or may be achieved utilizing an imaging technique, such as spectrophotometric and/or fluorescence imaging.
  • sensor 300 such as spectrophotometric CMOS or CCD imaging sensor 300, is in proximity to chambers 1 12 for detection of a color shift, fluorescence, turbidity or some other change indicative of target nucleic acid sequence amplification.
  • Chamber cover 106 (see Figure 5) preferably is transparent to facilitate detection of changes within the reaction chambers.
  • sensor 300 may be integrally connected to element 1 10 and may cover chambers 1 12, obviating chamber cover 106.
  • Sensor 300 optionally may comprise a coating, such as an Indium Tin Oxide (“ITO") coating, which may be utilized in addition or as an alternative to heating element 200 to resistively heat the contents of each chamber 1 12 to achieve target nucleic acid amplification.
  • ITO Indium Tin Oxide
  • the coating may be placed in proximity to chambers 1 12. As discussed previously, when conducting isothermal amplification via LAMP, the contents of chambers 1 12 may be heated in the range of about 60°C-65°C for about 5-70 minutes.
  • Imaging sensor 300 may measure a baseline color of reagents 130 and sample S prior to isothermal heating, and a final color of the reagents after isothermal heating (e.g., after isothermal heating). Since the reagents 130 within each reaction chamber 1 12 may, for example, include a colorimetric (or fluorescent) dye that shifts in color, e.g., from purple to blue, upon amplification of a target nucleic acid sequence, any such shift in color within the chambers may be detected by the imaging sensor 300 as a differential between the baseline and final color, and this differential may be indicative of target amplification.
  • a colorimetric (or fluorescent) dye that shifts in color, e.g., from purple to blue
  • optional digital readout or display 310 may output detection results (and/or instructions) to the user, removing any risk of detection ambiguity. While the embodiment of Figure 7B illustratively achieves colorimetric or fluorescence detection via spectrophotometric imaging, it should be understood that such colorimetric or fluorescence detection additionally or alternatively may be performed visually by an operator.
  • Heating element 200 and/or sensor 300 may comprise a logic chip for controlling operation of the heating element and/or the sensor, for controlling nucleic acid amplification via heating of chambers 1 12, for comparing baseline and final color measurements taken with sensor 300 to determine whether amplification has occurred, and/or for controlling the display of instructions or detection results via display 310.
  • Wires and/or a circuit board may connect the logic chip to heating element 200, sensor 300 and/or a power supply.
  • the power supply may, for example, comprise one or more batteries or a wall outlet connection.
  • element 1 10' comprises four chambers 1 12 rather than sixteen (as will be apparent to those of skill in the art, any number of chambers 1 12 may be provided).
  • Element 1 10' comprises vent channels 1 18 in fluid communication with the top of each chamber 1 12 for venting air from the chambers to the atmosphere.
  • Microfluidic channels 1 14 deliver sample S to the bottom of each chamber 1 12, and vent channels 1 18 vent overflow from the top of each chamber out of apparatus 100 through breathable membrane or one-way valve 1 19.
  • Figure 8A is an isometric view of apparatus 100. In the top view of element 1 10' seen in Figure 8B, the fluid communication of vent channels 1 18 with the tops of chambers 1 12 is visible.
  • Figures 9 provide another alternative embodiment of apparatus 100 comprising element 1 10".
  • Figure 9A provides an isometric view of apparatus 100
  • Figure 9B shows a top view of element 1 10" of the apparatus with chamber cover 106 removed
  • Figure 9C shows a bottom view of the element 1 10" with channel cover 104' removed.
  • the embodiment of apparatus 100 shown in Figures 8 comprises venting of air from chambers 1 12 to the atmosphere via vent channels 1 18 and membrane or valve 1 19 of element 1 10'
  • the embodiment of apparatus 100 shown in Figures 9 vents air from chambers 1 12 through vent channels 1 18' to one or more overflow chamber(s) 125 of element 1 10" (see Figure 9C), rather than venting to the atmosphere.
  • Overflow chamber(s) 125 preferably are sized to limit a pressure increase in the overflow chamber(s) during nucleic acid amplification to less than about 5-10 psi.
  • element 1 10" also comprises anti-backflow valves 140 that prevent cross-contamination between chambers 1 12 via backflow across microfluidic channels 1 14'. Furthermore, as best seen in Figure 9B, element 1 10" comprises flow control media 150 positioned along vent channels 1 18 between chambers 1 12 and overflow chamber(s) 125 that allow venting of air or other gases from the chambers 1 12 but not fluid, thereby ensuring equal fill of sample S in all chambers 1 12 while releasing excess pressure. [0044] Element 1 10" of Figures 9 has shorter microfluidic channels 1 14' as compared to microfluidic channels 1 14 of element 1 10' of Figures 8.
  • Element 1 10 may have a priming volume on the order of 20-50 microliters.
  • microfluidic channels 1 14' extend along both the top and the bottom of element 1 10", as well as through the element 1 10". The circuitous path of microfluidic channels 1 14' is described in more detail below.
  • channel cover 104' comprises laminate 160 that, in addition to covering the portion of microfluidic channels 1 14' positioned on the bottom of element 1 10", works in conjunction with anti-backflow valves 140 to prevent cross-contamination between chambers 1 12.
  • laminate 160 comprises double-sided adhesive layer 162, elastomer layer 166 and optional single-sided adhesive backing layer 168.
  • Element 1 10" comprises optional registration posts 1 1 1 for aligning the layers of laminate 160 during attachment of the laminate to element 1 10".
  • Layer 162 comprises optional registration cutouts 163 that align with registration posts 1 1 1 .
  • layer 166 comprises optional registration cutouts 167, while layer 168 comprises optional registration cutouts 169.
  • Layer 162 also comprises valve cutouts 164 that encircle anti-backflow valves 140, while layer 168 comprises valve cutouts 170.
  • Double-sided adhesive layer 162 is attached to element 1 10" and to elastomer layer 166.
  • single-sided adhesive backing layer 168 may be connected to elastomer layer 166 to reduce a risk of laminate 160 delaminating.
  • Figure 9E is a bottom view of apparatus 100 with channel cover 104' attached.
  • syringe 30 (or any other sample transfer device, e.g., previously described syringe 40 or previously described syringe 30 with containment element 20) is coupled to apparatus 100 via mating of luer lock 32 with luer lock 102.
  • Syringe 30 expresses sample S (and, optionally, liquid L) into apparatus 100 through inlet 1 16.
  • Sample S travels along the bottom of element 1 10" within microfluidic channel 1 14' (see Figure 9F in conjunction with Figure 9C).
  • microfluidic channel then passes through element 1 10" and takes sample S to the top of the element 1 10" before branching into multiple microfluidic channels 1 14' (see Figure 9F in conjunction with Figure 9B).
  • the microfluidic channels 1 14' then travel back through element 1 10" and deliver sample S to anti-backflow valves 140.
  • Pressure applied via syringe 30 causes elastomer layer 166 of laminate 160 to locally and temporarily deflect in the immediate vicinity of valves 140, thereby allowing passage of sample S (see Figure 9G in conjunction with Figure 9C).
  • anti-backflow valves 140 After passage of sample S, anti-backflow valves 140 reseal to prevent backflow of sample S and, thereby, cross-contamination of chambers 1 12.
  • microfluidic channels 1 14' take sample S that has passed through valves 140 back to the top of element 1 10" and into chambers 1 12.
  • Chambers 1 12 comprise reagents 130, e.g., lyophilized reagents 130.
  • Vent channels 1 18' extend from chambers 1 12 for venting of air A from chambers 1 12 to overflow chamber(s) 125 (see Figure 9G in conjunction with Figures 9B and 9C).
  • Flow control media 150 are positioned within channels 1 18' between chambers 1 12 and overflow chamber(s) 125.
  • Flow control media 150 may, for example, comprise a small pore hydrophobic material that allows passage of air but not fluid. After air passes through flow control media 150, it travels within vent channels 1 18' from the top of element 1 10" through the element to overflow chamber(s) 125.
  • the embodiment of apparatus 100 shown in Figures 9 may comprise or be coupled to a heating element (e.g., heating element 200 of Figures 7) for amplifying one or more target nucleic acid sequence(s) of interest, when present in sample S, via reagents 130.
  • Target sequence amplification may be detected visually by an operator, e.g. by visual detection of a visual indicator such as a color shift in a colorimetric dye or a turbidity change, or automatically, e.g. via a sensor (such as sensor 300 of Figure 7B) that detects amplification by detection of a visual indicator (color shift, fluorescence, turbidity change, etc.).
  • apparatus 100 optionally may comprise case 180 that contains apparatus 100.
  • Case 180 may comprise cavity 182 with indentations 184 configured to receive anti-backflow valves 140 of element 1 10".
  • Heating element 200 also may be positioned within cavity 182 in the vicinity of chambers 1 12 for heating the contents of chambers 1 12.
  • Case 180 further comprises cover 186 with chamber cutout 188 to facilitate visualization of chambers 1 12, and with luer lock cutout 190 to provide access to luer lock 102.
  • Cover 186 firmly attaches to cavity 182, e.g., via screws or a press fit, to form case 180 with the other components of apparatus 100 disposed therein.
  • Figures 10 provide another alternative embodiment of apparatus 100 comprising element 1 10"'.
  • the embodiment of apparatus 100 shown in Figures 10 comprises anti-backflow locking valve 200 that is configured to lock microfluidic channels 1 14" of element 1 10"' in either an open position that allows flow through the channels 1 14" or a closed position that prevents backflow and cross-contamination between chambers 1 12 via channels 1 14".
  • Such locking of the channels may be made reversible or irreversible, as desired.
  • Figure 10A provides an isometric top view of apparatus 100
  • Figure 10B provides an isometric bottom view of the apparatus.
  • Figure 10C provides an isometric detail view of anti-backflow locking valve 200.
  • chamber cover 106 and channel cover 104 are not shown in Figures 10. However, it should be understood that they may be provided as described with respect to prior embodiments of the apparatus.
  • anti-backflow locking valve 200 is configured for placement inside void 202 of element 1 10"' in order to lock channels 1 14" in the open (i.e. flow-enabled) or closed (i.e., flow-blocked) position, as desired, by sliding the locking valve 200 within void 202 relative to the element 1 10"'.
  • lumens 230 pass through anti-backflow locking valve 200 and may be selectively aligned and unaligned with channels 1 14" to unlock and lock the channels, respectively.
  • Locking valve 200 may, for example, comprise relatively stiff or rigid substrate 210 with elastomeric overmold 220.
  • Elastomeric overmold 220 may comprise O-ring elements 222a and 222b that are configured to create a fluid-tight seal against element 1 10"'.
  • O-ring elements 222a are associated with the locked configuration of anti-backflow lock 200 that prevents cross- contamination between chambers 1 12 by blocking channels 1 14".
  • O-ring elements 222b are concentrically aligned with lumens 230 and are associated with the unlocked configuration of anti-backflow locking valve 200 that allows fluid flow through channels 1 14".
  • elastomeric overmold 220 may be omitted, and O-ring elements 222a and/or 222b may be formed or attached directly to substrate 210.
  • Locking valve 200 preferably comprises enlarged end 240 that facilitates manipulation of the locking valve during use (i.e., that may be grasped by the user for sliding the locking valve from the unlocked to the locked configuration, or vice versa).
  • Figures 10D and 10E are translucent detail views that illustrate actuation of locking valve 200.
  • channels 1 14" may be placed in the unlocked configuration by positioning locking valve 200 within void 202 of element 1 10"' such that lumens 230 are aligned with microfluidic channels 1 14".
  • locking valve 200 and/or void 202 may be lubricated to facilitate sliding of the lock relative to the void.
  • O-ring elements 222b create fluid seals around the perimeters of channels 1 14" such that sample may flow from a sample transfer device (e.g., a syringe) through the first section of channels 1 14", through lumens 230 and through the second section of the channels 1 14" to chambers 1 12.
  • a sample transfer device e.g., a syringe
  • locking valve 200 then may be slid within void 202 to place channels 1 14" in the locked configuration such that lumens 230 are out of alignment with the microfluidic channels.
  • O-ring elements 222a create fluid seals around the perimeters of channels 1 14", thereby isolating and blocking each channel 1 14" from the others and preventing cross-contamination between chambers 1 12 via backflow through the channels.
  • enlarged end 240 of locking valve 200 may sit flush with element 1 10"' in the locked configuration of Figure 10E, such that the user is unable to grasp end 240 and unlock channels 1 14" once locking valve 200 has blocked the channels. Such an irreversible locking valve may reduce a risk of backflow contamination or of accidental venting of sample to the environment.
  • enlarged end 240 of locking valve 200 may protrude from element 1 10"' in the locked configuration, such that the user may grasp end 240 for reversible locking and unlocking of channels 1 14" with locking valve 200.
  • FIG. 10F locking valve 200 positions channels 1 14" in the unlocked configuration shown in Figure 10D.
  • a syringe or other sample transfer device is coupled to apparatus 100 via mating with luer lock 102.
  • the syringe or other sample transfer device expresses sample S (and, optionally, liquid L) into apparatus 100 through inlet 1 16.
  • Sample S travels along the bottom of element 1 10"' within microfluidic channel 1 14", which branches into multiple microfluidic channels 1 14" (see Figure 10F in conjunction with Figures 10B and 10D).
  • Each microfluidic channel then passes through element 1 10"' via a lumen 230 of locking valve 200, thereby taking sample S to the top of the element 1 10"' and into chambers 1 12 having reagents 130 (e.g., lyophilized reagents 130).
  • locking valve 200 then may be slid within void 202 relative to element 1 10" in order to position channels 1 14" in the locked configuration of Figure 10E wherein the channels are blocked. Sample S cannot flow back through locking valve 200 when channels 1 14" are in the locked configuration, which prevents cross-contamination of chambers 1 12 via backflow through the channels.
  • Element 1 10"' comprises previously described vent channels 1 18' that extend from chambers 1 12 for venting of air A (but not sample S) from the chambers 1 12 to overflow chamber(s) 125 (see Figure 10G in conjunction with Figures 10A and 10B).
  • Flow control media 150 are positioned within channels 1 18' between chambers 1 12 and overflow chamber(s) 125.
  • Flow control media 150 may, for example, comprise a small pore hydrophobic material that allows passage of air but not fluid. After air passes through flow control media 150, it travels within vent channels 1 18' from the top of element 1 10"' through the element 1 10"' to overflow chamber(s) 125.
  • the embodiment of apparatus 100 shown in Figures 10 illustratively comprises both air overflow chambers 125 and anti- backflow locking valve 200. It should be understood that the apparatus alternatively may comprise only the anti-backflow lock or only the overflow chambers.
  • the embodiment of apparatus 100 shown in Figures 10 may comprise or be coupled to a heating element (e.g., heating element 200 of Figures 7) for amplifying one or more target nucleic acid sequence(s) of interest, when present in sample S, via reagents 130.
  • Target sequence amplification may be detected visually by an operator, e.g.
  • the heating element, element 1 10"' and/or some other aspect of apparatus 100 may comprise a geometric or other constraint that precludes coupling of element 1 10"' to the heating element when locking valve 200 is positioned in the open configuration allowing flow through channels 1 14". Such a constraint may reduce a risk of sample amplification before locking of channels 1 14" in the closed configuration, thereby reducing a risk of backflow-induced cross-contamination of chambers 1 12.
  • Embodiments of apparatus 100 described thus far have delivered sample S to reaction chambers 1 12 via microfluidic channels that distribute the sample S across the chambers.
  • sample S is delivered to a reaction chamber without microfluidics.
  • the embodiment of apparatus 100 shown in Figures 1 1 illustratively comprises a single reaction chamber, but it should be understood that apparatus 100 may comprise any desired number of reaction chambers.
  • apparatus 100 comprises reaction chamber 400 and punch element 500.
  • Punch element 500 comprises male element 502, which is configured to press fit into female element 402 of reaction chamber 400 and seal the reaction chamber 400 in advance of nucleic acid amplification and detection.
  • female element 402 of reaction chamber 400 comprises reagent insert 410 that may be press fit therein.
  • Reagent insert 410 comprises cutting element 412 and reagent chamber 414.
  • Reagents 130 are positioned within reagent chamber 414.
  • the reagents 130 may, for example, be in solution or liquid form. Alternatively, the reagents 130 may be lyophilized, as in Figures 10.
  • Reagent insert 410 is sealed within female element 402 of reaction chamber 400 via seal 404 (see, e.g., Figure 1 1 C).
  • Seal 404 may, for example, comprise a metal foil or plastic film. Sealing of reaction chamber 400 may facilitate long-term storage of reagents 130 prior to use and/or may ensure that lyophilized reagents 130 remain dry prior to use.
  • male element 502 of punch element 500 comprises liquid insert 510 that may be press fit therein.
  • Liquid insert 510 comprises cutting element 512 and liquid chamber 514.
  • Liquid chamber 514 is sealed with seal 516.
  • Seal 516 may, for example, comprise a metal foil or plastic film.
  • Liquid L such as water and/or TE buffer, is sealed within liquid chamber 514.
  • Dye, MgS04, betaine and/or isothermal buffer additionally or alternatively may be sealed within chamber 514.
  • Apparatus 100 further comprises heating element 200, which is in thermal communication with the reaction chamber 400.
  • Heating element 200 which optionally may be disposed of after single use along with the rest of apparatus 100, is configured to heat the contents of reaction chamber 400 to achieve nucleic acid amplification, e.g., isothermal nucleic acid amplification such as LAMP.
  • Heating element 200 may comprise, for example, a resistive heater comprising an etched foil element encapsulated between two layers of polyimide film.
  • the heating element further may comprise a power supply, such as batteries or connection to a standard wall outlet, as well as a thermocouple for temperature monitoring in a feedback loop with a temperature controller for adjusting the monitored temperature as desired to achieve nucleic acid amplification.
  • Reaction chamber 400 preferably is transparent or translucent to facilitate visualization of the reaction chamber in order to detect amplification of a target nucleic acid sequence of interest.
  • Nucleic acid amplification may be detected via a color shift in a colorimetric dye, via an increase in turbidity, via fluorescence, etc.
  • Detection may be achieved with the naked eye and/or via optional sensor 300, which may be disposable. Detection results may be shown on a display, which may be disposable.
  • Sample S may be placed directly into reaction chamber 400 and/or punch element 500 prior to sealing of the reaction chamber with the punch element.
  • sample collector 10 comprising sample S may be positioned between the reaction chamber 400 and the punch element 500 such that mating of male element 502 with female element 402 places sample S within the reaction chamber 400, as shown in Figures 1 1 .
  • sample collector 10 may, for example, comprise a filter paper, such as a chemically treated filter paper, e.g., Flinders Technology Associates ("FTA”) cards available from Whatman (part of GE Healthcare).
  • FTA Flinders Technology Associates
  • sample matrices including, but not limited to, food, urine, saliva, mucous, feces, blood, semen, tissue, cells, DNA, RNA, protein, plant matter, animal matter, solutions, solids, and other sample matrices - may be deposited onto sample collector 10 (additional sample matrices will be apparent). In this manner, sample collector 10 may collect sample S via the filter paper.
  • the filter paper may, for example, be dipped or placed into one or more sample matrices of interest. Additionally or alternatively, one or more drops of one or more sample matrices of interest may, for example, be placed or deposited onto the filter paper. Additionally or alternatively, the filter paper may, for example, be swabbed or wiped across one or more sample matrices or surfaces of interest.
  • FIG. 1 1 G-1 1 J a method of using the embodiment of apparatus 100 seen in Figures 1 1 is described.
  • reaction chamber 400 and punch element 500 are approximated, such that male element 502 of the punch element mates with female element 402 of the reaction chamber to seal the reaction chamber.
  • Cutting element 512 of liquid insert 510 pierces sample collector 10, and male element 502 removes a punch of sample S from sample collector 10, thereby placing sample S within reaction chamber 400.
  • reaction chamber 400 and punch element 500 causes cutting element 512 of liquid insert 510 to puncture seal 404 of reaction chamber 400, thereby providing access to reagent insert 410.
  • still further approximation causes cutting element 412 of reagent insert 410 to puncture seal 516 of liquid insert 510, thereby causing liquid L to flow out of liquid chamber 514 into reagent chamber 414.
  • full approximation of reaction chamber 400 with punch element 500 positions all materials necessary for nucleic acid amplification and detection (sample S, reagents 130 and optional liquid L) within reagent chamber 414.
  • heating element 200 heats the contents of reagent chamber 414 to achieve nucleic acid amplification of a target nucleic acid sequence of interest when present in sample S. Detection may be achieved via the naked eye and/or via sensor 300.
  • Apparatus 100 of Figures 1 1 optionally may be used as part of instrument 40 previously described in co-pending U.S. patent application Serial No. 13/447,218, filed April 14, 2012, which is incorporated herein by reference in its entirety. Specifically, reaction chambers 400 and punch elements 500 of apparatus 100 in Figures 10 may be substituted for punch elements 90 and chambers 70 of instrument 40 shown in the '218 application.
  • Figures 1 -1 1 provide fully contained, sample- to-answer, nucleic acid sample preparation, (optionally multiplexed) target amplification and detection in (optionally disposable, e.g., single-use disposable) apparatus that is appropriate for use in limited resource settings at the point of care by relatively unskilled users.
  • apparatus 100 and associated methods have been described with respect to nucleic acid amplification and detection, it should be understood that the apparatus and associated methods alternatively may comprise and/or be used for holding and analyzing a sample without necessarily amplifying and/or detecting nucleic acid in the sample.
  • apparatus 100 may comprise sample holder 100 that maintains a nucleic acid or other sample for analysis within the reaction chamber(s), which may serve as observation and/or analysis chamber(s). Analysis may comprise, for example, one or more techniques such as microscopy, hybridization and/or protein analysis - in addition, or as an alternative, to nucleic acid amplification and detection.
  • a method of holding a sample for analysis may comprise collecting a sample matrix, transferring the sample matrix through at least one microfluidic channel to at least one reaction/observation/analysis chamber, optionally heating the sample matrix as part of an analytical technique, and preventing backflow of the sample matrix from the at least one chamber through the at least one microfluidic channel (e.g., during heating).
  • Backflow prevention may prevent cross-contamination when multiple chambers are provided.
  • Backflow prevention may be achieved via a one-way valve into the reaction/observation/analysis chamber(s) and/or via blocking of the microfluidic channel(s) after transferring of the sample matrix to the chamber(s).

Abstract

L'invention porte sur des procédés et un appareil pour l'amplification et la détection d'acides nucléiques au point d'intervention. Un mode de réalisation de l'invention comprend un instrument de diagnostic moléculaire totalement intégré de l'échantillon à la réponse qui peut éventuellement être utilisé de manière multiplexée pour détecter de multiples séquences d'acide nucléique cibles présentant de l'intérêt et qui peut éventuellement être configuré pour être jeté après un usage unique. L'instrument utilise de préférence une technique d'amplification d'acides nucléiques isotherme, telle que l'amplification isotherme induite par boucle (LAMP), pour réduire les exigences d'instrumentation associée à l'amplification d'acides nucléiques. La détection de l'amplification de la cible peut être réalisée, par exemple, par détection d'un changement de couleur ou d'une fluorescence dans un colorant ajouté à la réaction d'amplification. Une telle détection peut être effectuée visuellement par un opérateur ou peut être réalisée à l'aide d'une technique d'imagerie, par exemple l'imagerie spectrophotométrique.
EP14856081.6A 2013-10-22 2014-10-22 Procédés et appareil permettant l'amplification et la détection d'acides nucléiques au point d'intervention Withdrawn EP3060683A4 (fr)

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US201361894392P 2013-10-22 2013-10-22
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 (fr) 2013-10-22 2014-10-22 Procédés et appareil permettant l'amplification et la détection d'acides nucléiques au point d'intervention

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WO2018175424A1 (fr) * 2017-03-22 2018-09-27 The Board Of Trustees Of The University Of Illinois Système pour la détection et l'identification rapides, portables et multiplexées de séquences d'acides nucléiques spécifiques d'agents pathogènes
WO2018185143A1 (fr) * 2017-04-04 2018-10-11 Selfdiagnostics Deutschland Gmbh Distribution de composants de lampe dans une cellule à microfluide
US11547997B2 (en) 2017-04-07 2023-01-10 Arizona Board Of Regents On Behalf Of Arizona State University Integrated diagnostic devices having embedded biomolecular computing systems and uses thereof
CN107129930B (zh) * 2017-06-09 2019-11-26 北京百康芯生物科技有限公司 一种全集成核酸检测微流控芯片及其使用方法
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CN108642148B (zh) * 2018-07-09 2024-01-30 南京岚煜生物科技有限公司 一种核酸扩增检测微流控芯片及其检测方法
CN112852928A (zh) * 2021-01-14 2021-05-28 广东东阳光药业有限公司 一种多重核酸扩增产物检测方法及试剂盒
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WO2015061480A1 (fr) 2015-04-30

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