MOLECULAR DIAGNOSTICS AMPLIFICATION SYSTEM AND METHODS

Abstract

The present invention relates to automated devices and methods for the amplification of segments of nucleic acid in a convenient and portable manner. A single-use nucleic acid amplification device for producing an amplicon includes a housing and an amplification chamber. The chamber includes an ingress with a first reversible seal, an egress with a second reversible seal, a sealable sample entry orifice, and a first wall forming a portion of the chamber. The first wall includes a thermally conductive material and includes an interior surface and an exterior surface. The exterior surface includes a heating circuit and a temperature sensor. The sample entry orifice permits a sample of nucleic acid to enter the amplification chamber. The ingress is connected to a first conduit along with a pneumatic pump and a fluid pouch. The egress is connected to a second conduit permitting egress of the amplicon from the amplification chamber.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects and advantages of the present invention will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments, in conjunction with the accompanying drawings, wherein like reference numerals have been used to designate like elements, and wherein:

FIG. 1 illustrates a representation of the integrated single-use DNA amplification device and its interaction with an instrument, in accordance with an exemplary embodiment of the present invention.

FIG. 2 illustrates a top view of the integrated single-use DNA amplification device, in accordance with an exemplary embodiment of the present invention.

FIGS. 3 (a)-(b) illustrate different perspectives of the integrated single-use DNA amplification device and its interaction with an instrument, in accordance with an exemplary embodiment of the present invention.

FIGS. 4 (a)-(b) illustrate the ingress and egress valves with flexible diaphragm seals and with pylon seals, respectively, in accordance with an exemplary embodiment of the present invention.

FIGS. 5 (a)-(b) illustrates the DNA swab device for collection of a buccal swab sample mating with a single-use DNA amplification device by a screw-in means, in accordance with an exemplary embodiment of the present invention.

FIGS. 6 (a)-(b) illustrates the DNA swab for collection of a buccal swab sample mating with a single-use DNA amplification device by a latch means, in accordance with an exemplary embodiment of the present invention.

FIGS. 7 (a)-(d) illustrates the silicon chip forming a wall of the amplification chamber where the exterior surface has a heating circuit and a temperature sensing circuit, in accordance with an exemplary embodiment of the present invention. FIG. 7(a) illustrates an extra rib support and a fan cooling means. FIG. 7(b) illustrates the details of FIG. 7(a) wherein a cooling fan and an associated heat sink on the heater chip is used. FIG. 7(c) illustrates a cross-sectional view of the silicon chip. FIG. 7(d) illustrates the interaction and connections from the amplification device to the silicon chip.

FIG. 8 illustrates the integrated single-use DNA amplification device interaction with an instrument, in accordance with an exemplary embodiment of the present invention.

FIG. 9 illustrates a heating cycle profile versus time applied to the amplification device and the temperature response of the temperature sensor, in accordance with an exemplary embodiment of the present invention.

FIG. 10 illustrates gel electrophoresis of amplicons for target gene 1 (in example 1) after 22, 24, 26, 28, 30 and 35 PCR amplification cycles in the amplification device, in accordance with an exemplary embodiment of the present invention.

FIG. 11 illustrates a typical chronoamperometry output for PCR with target gene 1 after 22, 24, 26, 28, 30 and 35 PCR amplification cycles in the amplification device, in accordance with an exemplary embodiment of the present invention.

FIG. 12 illustrates the cross section of a single-use DNA amplification device with respect to the clipping means of attaching the silicon heater to the amplification chamber, in accordance with an exemplary embodiment of the present invention.

FIG. 13 illustrates the cross section of a single-use DNA amplification device with respect to a staking means of attachment, in accordance with an exemplary embodiment of the present invention.

FIG. 14 illustrates a preferred reaction sequence for PCR amplification, in accordance with an exemplary embodiment of the present invention.

Claims

1. A single-use nucleic acid amplification device for producing an amplicon, comprising: a housing; andan amplification chamber, comprising: an ingress with a first reversible seal;an egress with a second reversible seal;a sealable sample entry orifice; anda first wall forming a portion of the amplification chamber, wherein the first wall comprises a thermally conductive material and includes a first surface and an second surface,wherein the second surface includes a heating circuit and a temperature sensor,wherein the sample entry orifice permits a sample of nucleic acid to enter the amplification chamber,wherein the ingress is connected to a first conduit along with a pump and a reservoir, andwherein the egress is connected to a second conduit permitting egress of the amplicon from the amplification chamber.

2. The amplification device of claim 1, wherein the pump comprises a flexible diaphragm.

3. The amplification device of claim 2, wherein the flexible diaphragm is capable of engaging and being actuated by a plunger on an instrument with which the amplification device is capable of mating.

4. The amplification device of claim 1, wherein the pump comprises a pneumatic pump.

5. The amplification device of claim 1, wherein the reservoir comprises a fluid pouch.

6. The amplification device of claim 5, wherein the fluid pouch includes a fluid for performing nucleic acid amplification.

7. The amplification device of claim 1, wherein the reservoir comprises a flexible diaphragm.

8. The amplification device of claim 7, wherein the flexible diaphragm is capable of engaging and being actuated by a plunger on an instrument with which the amplification device is capable of mating.

9. The amplification device of claim 1, wherein the first wall comprises silicon.

10. The amplification device of claim 9, wherein the silicon comprises about 30 to about 50 percent of the first surface area of the amplification chamber.

11. The amplification device of claim 1, wherein the amplification chamber includes a second wall comprising a plastic material.

12. The amplification device of claim 11, wherein the second wall comprises a wall thickness in the range of about 0.2 mm to about 5 mm, and wherein the second wall includes one or more additional rib supports.

13. The amplification device of claim 1, wherein the internal volume of the amplification chamber is in the range of about 5 uL to about 50 uL.

14. The amplification device of claim 1, wherein an amplification chamber surface to an amplification chamber volume ratio is in the range of about 50 to about 200 square mm for the amplification chamber surface and to about 5 to about 30 cubic mm for the amplification chamber volume.

15. The amplification device of claim 1, wherein an internal shape of the amplification chamber comprises one of a substantially rectangular structure, a substantially rectangular shape with rounded corners, a cylinder, and a cylindrical structure with a substantially oval cross-section.

16. The amplification device of claim 1, wherein the second surface of the first wall comprises a heating circuit.

17. The amplification device of claim 16, wherein the heating circuit comprises a resistive electrical path fabricated on the second surface with a first and second connecting pad for contacting an external circuit for providing current flow through the path.

18. The amplification device of claim 1, wherein the second surface of the first wall comprises a temperature sensor.

19. The amplification device of claim 18, wherein the temperature sensor comprises one of a thermistor and a thermocouple fabricated on the second surface with a first and second connecting pad for contacting an external circuit for connecting to the one of the thermistor and the thermocouple.

20. The amplification device of claim 1, wherein the sample entry orifice is capable of mating with a sample introduction element.

21. The amplification device of claim 20, wherein the sample introduction element comprises: a wand, wherein the wand comprises: a first end with an absorbent pad capable of collecting and retaining a nucleic acid sample; anda second end forming a handle, wherein the first end is capable of passing through the sample entry orifice into the amplification chamber, andwherein the wand includes an engaging structure between the first and second ends for engaging and sealing the wand in the sample entry orifice.

22. The amplification device of claim 21, wherein the engaging structure comprises a male screw structure on the wand and a female screw structure on the sample entry orifice.

23. The amplification device of claim 21, wherein the engaging structure comprises a male collar locking structure on the wand and a female collar locking structure on the sample entry orifice.

24. The amplification device of claim 1, wherein the amplification chamber comprises a sugar glass coating on at least a portion of the first surface of the first wall.

25. The amplification device of claim 1, wherein the amplification chamber is capable of a temperature increase ramp rate in the range of about 10 to about 50 degrees centigrade per second.

26. The amplification device of claim 1, wherein the amplification chamber is capable of a temperature decrease ramp rate in the range of about 4 to about 50 degrees centigrade per second.

27. The amplification device of claim 1, wherein the amplification chamber comprises an optical window.

28. The amplification device of claim 1, wherein the second surface of the first wall comprises a Peltier circuit with a first and second connecting pad for contacting an external circuit.

29. The amplification device of claim 1, wherein the first reversible seal comprises a flexible diaphragm.

30. The amplification device of claim 29, wherein the flexible diaphragm is capable of actuation into a closed position by an applied force and an open position by the absence of the applied force.

31. The amplification device of claim 29, wherein the flexible diaphragm is capable of actuation into a closed position by an applied force provided by an engaged instrument with a pin mating with the flexible diaphragm.

32. The amplification device of claim 1, wherein the second reversible seal comprises a flexible diaphragm.

33. The amplification device of claim 32, wherein the flexible diaphragm is capable of actuation into a closed position by an applied force and an open position by the absence of the applied force.

34. The amplification device of claim 32, wherein the flexible diaphragm is capable of actuation into a closed position by an applied force provided by an engaged instrument with a pin mating with the flexible diaphragm.

35. The amplification device of claim 1, wherein the second conduit comprises a mating feature for engaging a device for detection of the amplicon.

36. The amplification device of claim 1, wherein the first conduit comprises a chip insert with a fluid detection sensor.

37. The amplification device of claim 1, wherein the first surface comprises an interior surface, and wherein the second surface comprises an exterior surface.

38. A method of nucleic acid amplification for producing an amplicon in a single-use device, comprising the steps of: a.) introducing a nucleic acid sample into an amplification chamber through a sample entry orifice;b.) sealing the orifice;c.) transferring a fluid from a reservoir through a reversibly sealable ingress to the amplification chamber;d.) sealing the ingress and an egress of the amplification chamber;e.) mixing the fluid with the sample to form a mixture comprising nucleic acid, a buffer, a polymerase and one or more primers;f.) cycling the temperature of the amplification chamber between first and second temperatures for a predetermined time and for a predetermined number of cycles to form an amplicon;g.) opening the ingress and egress of the chamber; andh.) applying a pneumatic force to the ingress to move the amplicon from the chamber through the egress.

39. A method of nucleic acid amplification for producing an amplicon in a single-use device, comprising the steps of: a.) introducing a nucleic acid sample into an amplification chamber through a sample entry orifice;b.) sealing the orifice;c.) transferring a fluid from a reservoir through a reversibly sealable ingress to the amplification chamber;d.) sealing the ingress and an egress of the chamber;e.) mixing the fluid with the sample to form a mixture comprising nucleic acid, a buffer, a polymerase and one or more primers;f.) increasing the temperature of the chamber to an isothermal amplification temperature for a predetermined time to form an amplicon;g.) opening the ingress and the egress of the amplification chamber; andh.) applying a pneumatic force to the ingress to move the amplicon from the chamber through the egress.