METHODS AND APPARATUS FOR POINT-OF-CARE NUCLEIC ACID AMPLIFICATION AND DETECTION

• US 20120264116A1
• Filed: 04/14/2012
• Published: 10/18/2012
• Est. Priority Date: 04/14/2011
• Status: Active Grant

• Current Assignee: Corporos Inc.
• Original Assignee: Corporos Inc.
• Inventors: Michlitsch, Kenneth J.
• Granted Patent: US 8,911,941 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 2547/101 by confinement to a single tube/container
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode

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. Optionally, sample preparation is fully or partially achieved via heat treatment and/or using a filter paper, such as a chemically treated filter paper, e.g., an FTA card. 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.

Claims

1. Apparatus for point-of-care amplification and detection of a target nucleic acid sequence, the apparatus comprising:

a sample collector; and

a nucleic acid amplification instrument,

wherein the nucleic acid amplification instrument comprises a sensor, a heating element, a display, and at least one reaction chamber having nucleic acid amplification reagents,

wherein the sample collector is configured to collect and deliver to the at least one reaction chamber a sample matrix that may contain the target nucleic acid sequence,

wherein the heating element is configured to heat the nucleic acid amplification reagents after delivery of the sample matrix to the at least one reaction chamber,

wherein the nucleic acid amplification reagents are configured to amplify the target nucleic acid sequence within the sample matrix upon heating of the reagents with the heating element,

wherein the sensor is configured to detect amplification of the target nucleic acid sequence, and

wherein the display is configured to output detection results obtained with the sensor.

2. The apparatus of claim 1, wherein the sample collector further comprises filter paper that is configured to collect the sample matrix.

3. The apparatus of claim 2, wherein the filter paper comprises chemically treated filter paper configured to at least partially prepare the sample matrix for nucleic acid amplification.

4. The apparatus of claim 1, wherein the nucleic acid amplification instrument further comprises at least one sample preparation element.

5. The apparatus of claim 1, wherein the heating element is configured to prepare the sample matrix via heat treatment.

6. The apparatus of claim 1, wherein the nucleic acid amplification reagents comprise isothermal nucleic acid amplification reagents.

7. The apparatus of claim 1, wherein the at least one reaction chamber comprises at least one temporary sequestration vessel configured to temporarily sequester at least one nucleic acid amplification reagent prior to amplification.

8. The apparatus of claim 7, wherein the at least one temporary sequestration vessel comprises at least one thermal encasement material configured to melt, become porous or otherwise release the at least one temporarily sequestered nucleic acid amplification reagent upon heating.

9. The apparatus of claim 2, wherein the nucleic acid amplification instrument further comprises at least one punch element that is configured to form and transfer at least one punch of the sample matrix-collecting filter paper to the at least one reaction chamber.

10. The apparatus of claim 9, wherein the nucleic acid amplification instrument further comprises at least one sealing element configured to seal the at least one reaction chamber upon transfer of the at least one punch to the at least one reaction chamber.

11. The apparatus of claim 9, wherein the at least one reaction chamber comprises at least one temporary sequestration vessel configured to temporarily sequester at least one nucleic acid amplification reagents, and wherein the at least one punch element is configured to puncture the at least one temporary sequestration vessel, thereby releasing the at least one nucleic acid amplification reagent from temporary sequestration.

12. The apparatus of claim 1, wherein the nucleic acid amplification reagents comprise at least one dye configured to change wavelength or intensity upon amplification of the target nucleic acid sequence.

13. The apparatus of claim 12, wherein the sensor is configured to detect changes in the wavelength or intensity of the at least one dye, thereby detecting amplification of the target nucleic acid sequence.

14. The apparatus of claim 1, wherein the nucleic acid amplification instrument further comprises a logic chip, a power supply, and electrical connectors,

wherein the logic chip is configured to control operation of the sensor, heating element and display,

wherein the power supply is configured to power the sensor, heating element, display and logic chip, and

wherein the electrical connectors are configured to electrically couple the logic chip to the power supply, sensor, heating element, and display.

15. The apparatus of claim 1, wherein the nucleic acid amplification instrument is chosen from the group of nucleic acid amplification instruments consisting of nucleic acid amplification instruments having a maximum dimension of less than about 15 cm, nucleic acid amplification instruments having a mass of less than about 150 g, fully disposable nucleic acid amplification instruments, and combinations thereof.

16. The apparatus of claim 1, wherein the nucleic acid amplification instrument further comprises experimental controls.

17. The apparatus of claim 1, wherein the nucleic acid amplification instrument is configured for point-of-care amplification and detection of multiple target nucleic acid sequences.

18. The apparatus of claim 1, wherein the sample collector further comprises a lateral flow strip.

19. A method for point-of-care amplification and detection of a target nucleic acid sequence, the method comprising:

collecting a sample matrix with a sample collector;

sealing the sample matrix within a reaction chamber in fluid communication with nucleic acid amplification reagents;

heating the nucleic acid amplification reagents and the sample matrix with a heating element in order to amplify the target nucleic acid sequence when contained within the sample matrix,

detecting amplification of the target nucleic acid sequence with a sensor;

displaying detection results obtained with the sensor on a display; and

disposing of the sample matrix, sample collector, reaction chamber, nucleic acid amplification reagents, heating element, sensor and display after one-time use.

20. The method of claim 19 further comprising:

controlling the sensor, heating element and display with a logic chip; and

powering the logic chip, sensor, heating element and display with a power supply,

wherein disposing of the sample matrix, sample collector, reaction chamber, nucleic acid amplification reagents, heating element, sensor and display after one-time use further comprises disposing of the logic chip and power supply after one-time use.