Molecular diagnostics system and methods

• US 20100291666A1
• Filed: 12/21/2005
• Published: 11/18/2010
• Est. Priority Date: 12/23/2004
• Status: Active Grant

• Current Assignee: Abbott Point Of Care Incorporated (Abbott Laboratories)
• Original Assignee: Abbott Point Of Care Incorporated (Abbott Laboratories)
• Inventors: Miller, Cary James, Collier, Gordon Bruce, MacLeod, Jason Andrew, Dicke, William Charles, Wood, John Allister, Nemeth, Attila Csaba
• Granted Patent: US 8,883,487 B2
• Time in Patent Office: Days
• US Class Current: 435/287.2
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/502769 characterised by multiphase flow arrangements
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B03C 1/01 by addition of magnetic adjuvants
• CPC Class Codes: B03C 1/0332 using permanent magnets
• CPC Class Codes: B03C 1/0335 using coils
• CPC Class Codes: B03C 1/288 disposed at the outer circumference of a recipient
• CPC Class Codes: B03C 1/30 Combinations with other devices, not otherwise provided for
• CPC Class Codes: B03C 2201/18 Magnetic separation whereby the particles are suspended in a liquid
• CPC Class Codes: B03C 2201/26 for use in medical or biological applications
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12N 15/1013 by using magnetic beads
• CPC Class Codes: C12Q 1/6844 : Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6853 : using modified primers or templates
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]

Abstract

The present invention relates to automated devices and methods for the extraction of nucleic acids from cells, the amplification of segments of nucleic acid and the detection of nucleic acids, all in a convenient and portable manner. The invention is particularly suited for use in point-of-care medical diagnostic testing.

Claims

1. An integrated single-use device for performing a nucleic acid analysis, comprising:

a housing,

an entry port for accepting a sample suspected of containing a target nucleic acid, a first chamber operably connected to the entry port containing a reagent for extracting the target nucleic acid,

a first conduit permitting passage of extracted nucleic acid into an amplification chamber, said housing containing an amplification reagent that is capable of incorporating a detectable label into an amplified nucleic acid target,

the amplification chamber having a heating means and a temperature sensing means for controlling amplification conditions,

the amplification chamber being operably linked to a second conduit containing a sensing region with an immobilized capture oligonucleotide,

said housing containing a means for moving the amplified target to the sensing region to permit binding of said amplified target to said capture oligonucleotide, said second conduit operably attached to a holding chamber containing a fluid able to substantially displace uncaptured amplified target from the sensing region and permitting sensing of said detectable label retained in said sensing region.

2. The device of claim 1, wherein the label moiety is selected from the group consisting of fluorocein, dinitrophenol, biotin, cholesterol, digoxigenin and estrialdiol.

3. The device of claim 1, wherein the sensing region is interrogated by an optical detector.

4. The device of claim 1, wherein the sensing region is interrogated by an integrated optical detector.

5. The device of claim 1, wherein the sensing region is interrogated by an integrated electrochemical detector.

6. A device of claim 1, wherein amplification reagent is located in said first conduit.

7. A device of claim 1, wherein amplification reagent is located in said amplification chamber.

8. A device of claim 1, wherein amplification reagent is located in a holding chamber operably connected to said amplification chamber.

9. A device of claim 1, wherein amplification reagent is located in a holding chamber operably connected to said first conduit.

10. A device of claim 1, wherein said fluid is a wash fluid.

11. A device of claim 1, wherein said means for moving the amplification target is pneumatic.

12. An integrated single-use device for performing a nucleic acid analysis, comprising:

a housing,

an entry port for accepting a sample suspected of containing a target nucleic acid,

a first chamber operably connected to the entry port containing a reagent for extracting the target nucleic acid onto magnetic beads that are located in the first chamber,

said first chamber also containing a wax for forming a substantially contiguous wax layer in the region of the first chamber and a first conduit, wherein magnetic beads that are associated with extracted target nucleic acid may pass through the wax layer into the first conduit by means of an applied magnetic field,

the first conduit permitting passage of target nucleic acid and beads into an amplification chamber, said housing containing an amplification reagent that is capable of incorporating a detectable label into an amplified nucleic acid target,

the amplification chamber having a heating means and a temperature sensing means for controlling amplification conditions,

the amplification chamber being operably linked to a second conduit containing a sensing region with an immobilized capture oligonucleotide,

said housing containing a means for moving the amplified target to the sensing region to permit binding of said amplified target to said capture oligonucleotide,

said second conduit operably attached to a holding chamber containing a fluid able to substantially displace uncaptured amplified target from the sensing region and permitting optical sensing of the detectable label retained in the sensing region.

13. The device of claim 12, wherein the detectable label is selected from the group consisting of fluorocein, dinitrophenol, biotin, cholesterol, digoxigenin and estrialdiol.

14. The device of claim 12, wherein the sensing region is interrogated by an optical detector.

15. The device of claim 12;

wherein the sensing region is interrogated by an integrated optical detector.

16. A device of claim 12, wherein amplification reagent is located in said first conduit.

17. A device of claim 12, wherein amplification reagent is located in said amplification chamber.

18. A device of claim 12, wherein amplification reagent is located in a holding chamber operably connected to said amplification chamber.

19. A device of claim 12, wherein amplification reagent is located in a holding chamber operably connected to said first conduit.

20. A device of claim 12, wherein said fluid is a wash fluid.

21. A device of claim 12, wherein said means for moving the amplification target is pneumatic.

22. An integrated single-use device for performing a nucleic acid analysis, comprising:

a housing,

an entry port for accepting a sample suspected of containing a target nucleic acid,

a first chamber operably connected to the entry port containing a reagent for extracting the target nucleic acid onto magnetic beads that are located in the first chamber,

said first chamber also containing a wax for forming a substantially contiguous wax layer in the region of the first chamber and a first conduit, whereby magnetic beads associated with extracted target nucleic acid may pass through the wax layer into the first conduit by means of an applied magnetic field,

the first conduit permitting passage of target nucleic acid and beads into an amplification chamber, said housing containing an amplification reagent that is capable of incorporating a detectable label into an amplified nucleic acid target,

the amplification chamber having a heating means and a temperature sensing means for controlling amplification conditions,

the amplification chamber being operably linked to a second conduit containing a sensing region with an immobilized capture oligonucleotide and a dissolvable signaling reagent capable of binding to the detectable label, and an electrochemical sensor,

said housing containing a means for moving the amplified target to the sensing region to permit binding of the amplified target to the capture oligonucleotide, the second conduit operably attached to a holding chamber containing a detection reagent which substantially displaces from the sensing region any uncaptured amplified target and signaling reagent not bound to the detectable label,

said detection reagent capable of reacting with the signaling reagent bound to the detectable label and generate a signal at the sensor.

23. A device of claim 22, wherein amplification reagent is located in said first conduit.

24. A device of claim 22, wherein amplification reagent is located in said amplification chamber.

25. A device of claim 22, wherein amplification reagent is located in a holding chamber operably connected to said amplification chamber.

26. A device of claim 22, wherein amplification reagent is located in a holding chamber operably connected to said first conduit.

27. A device of claim 23, wherein said means for moving the amplification target is pneumatic.

28. The integrated single-use device of claim 22, adapted for insertion into a reader comprising means for actuating the magnetic beads, one or more mechanical actuating means, a means for measuring and controlling the temperature of the amplification chamber, a means for activating the sensor and applying the sensor signal to an algorithm, and displaying a result related to the presence of target nucleic acid in the sample.

29. The device of claim 22, further comprising a wash fluid holding chamber and a waste chamber, both operably attached to the first conduit and permitting the wash fluid to pass over the beads held in the first conduit to the waste chamber, to effect washing of the beads.

30. The device of claim 22, further comprising a wash fluid holding chamber and a waste chamber, both operably attached to the first chamber, permitting wash fluid to pass from the wash fluid holding chamber over beads held in the first chamber to the waste chamber, to effect washing of the beads.

31. The device of claim 22, where the amplification chamber comprises a first reversible sealing means at the ingress.

32. The device of claim 22, where the amplification chamber comprises a second reversible sealing means at the egress.

33. The device of claim 22, where a second waste chamber is attached to the second conduit, beyond the sensor.

34. The device of claim 22, where the amplification reagent comprises a DNA polymerase, and a modified primer containing a sequence of bases complimentary to a first region of the target nucleic acid, a polymerase blocking region attached thereto, a single stranded hybridization region attached to the polymerase blocking region, and the detectable label.

35. The device of claim 22, where the second conduit contains an exonuclease capable of substantially removing unused amplification reagent containing a primer.

36. The device of claim 22, where the second conduit has a temperature sufficient to substantially convert an amplification reagent containing a self-annealing primer from open form to self-annealed form.

37. The device of claim 22, where the second conduit contains an immobilized oligonucleotide proximal to the egress of the amplification chamber which can specifically hybridize with the amplification reagent to substantially remove unused primer from the amplified target prior to entering the sensing region.

38. The device of claim 22, where the second conduit contains an electrophoretic element proximal to the egress of the amplification chamber capable of electrophoresing the amplified target and substantially removing unused amplification reagent from the amplified target prior to entering the sensing region.

39. The device of claim 22, where the sensor is an electrochemical sensor.

40. The device of claim 22, where the sensor is amperometric.

41. The device of claim 22, where the detectable label is biotin.

42. The device of claim 22, where the signaling reagent is streptavidin-labeled alkaline phosphatase.

43. The device of claim 22, where the detection reagent is p-aminophenol phosphate and the signal at the sensor is related to the formation of p-aminophenol.

44. The device of claim 22, where the extraction reagent comprises a chelating agent and a chaotropic agent.

45. The device of claim 22, where the magnetic beads have a diameter in the range of 2 to 5 um and are coated with a cross-linked polystyrene having a hydrophilic glycidyl ether layer with carboxylic acid groups on the surface.

46. The device of claim 22, where the wax is selected from the group consisting of heneicosane, tricosaheneicosane, docosane and tricosane.

47. The device of claim 22, where the amplification chamber comprises one or more walls formed of plastic and at least one wall formed of silicon.

48. The device of claim 22, where the extraction chamber comprises one or more walls formed of plastic and at least one wall formed of silicon.

49. The device of claim 22, where the amplification reagent contains at least one primer for amplification of the target and at least one primer which incorporates a PEG blocking region attached to a single stranded oligonucleotide complimentary to the capture oligonucleotide.

50. The device of claim 22, wherein the amplification reagent contains a reagent selected for rolling circle amplification, helicase amplification, polymerase chain reaction amplification or strand displacement amplification.

51. The device of claim 22, which is able to determine the presence or absence of a target nucleic acid in a sample.

52. The device of claim 22, which is able to quantify the amount of a target nucleic acid in a sample.

53. An integrated single-use device for performing a nucleic acid analysis, comprising:

a housing,

an entry port for accepting a sample suspected of containing a target nucleic acid,

an extraction chamber operably connected to the entry port containing a reagent for extracting the target nucleic acid onto a filter material located in the extraction chamber, said extraction chamber also containing extraction reagents for promoting extraction of nucleic acid,

the housing also comprising a wash fluid holding chamber containing a wash fluid, and a waste chamber, both operably connected to said extraction chamber and permitting wash fluid to pass over the filter material to the waste chamber, thereby washing the filter,

said extraction chamber also having a heating means and a temperature sensing means for controlling the temperature of the extraction chamber to effect conditions suitable for desorption of the target nucleic acid from the filter,

where the extraction chamber is operably connected to a first conduit that is operably connected to an amplification chamber,

said housing containing an amplification reagent capable of incorporating a detectable label into an amplified target,

said amplification chamber also having a heating means and a temperature sensing means for controlling the temperature of the amplification chamber to effect conditions suitable for amplification of the target nucleic acid,

said amplification chamber operably attached to a second conduit containing a sensing region and an immobilized capture oligonucleotide, a dissolvable signaling reagent capable of binding to said detectable label, and an electrochemical sensor,

said housing containing a means for moving the amplified target to the sensing region to permit binding of the amplified target to the capture oligonucleotide, and said signaling reagent to said detectable label,

the second conduit operably attached to a holding chamber containing a detection reagent which can substantially displace from the sensing region any uncaptured amplified target and signaling reagent not bound to the detectable label,

said detection reagent capable of reacting with said signaling reagent bound to the detectable label to generate a signal at the sensor.

54. A device of claim 53, wherein amplification reagent is located in said first conduit.

55. A device of claim 53, wherein amplification reagent is located in said amplification chamber.

56. A device of claim 53, wherein amplification reagent is located in a holding chamber operably connected to said amplification chamber.

57. A device of claim 53, wherein amplification reagent is located in a holding chamber operably connected to said first conduit.

58. A device of claim 53, wherein said means for moving the amplification target is pneumatic.

59. An integrated single-use device for performing a nucleic acid analysis, comprising;

a housing,

an entry port for accepting a sample suspected of containing a target nucleic acid,

an extraction chamber operably connected to the entry port containing a reagent for extracting the target nucleic acid onto a filter located in the extraction chamber,

the housing also comprising a wash fluid holding chamber containing a wash fluid and a waste chamber, both operably attached to said extraction chamber and permitting wash fluid to pass over the filter to the waste chamber, to effect washing of the filter,

the extraction chamber having heating means and temperature sensing means for controlling the temperature to effect conditions suitable for desorption of the target nucleic acid from the filter,

the extraction chamber operably attached to a first conduit that is operably attached to an amplification chamber permitting transfer of the target nucleic acid from the extraction chamber to the amplification chamber,

said housing containing an amplification reagent capable of incorporating a detectable label into an amplified target,

the amplification chamber having a heating means and a temperature sensing means for controlling the temperature to effect conditions suitable for amplification of the target nucleic acid,

said amplification chamber operable attached to a second conduit containing a sensing region comprising an immobilized capture oligonucleotide,

the housing containing means for moving the amplified target to the sensing region to permit binding of the amplified target to the capture oligonucleotide,

the second conduit operably attached to a second wash chamber capable of delivering a fluid into the second conduit to substantially displace uncaptured amplified target from the sensing region to permit optical sensing of the detectable label retained in the sensing region.

60. A device of claim 59, wherein amplification reagent is located in said first conduit.

61. A device of claim 59, wherein amplification reagent is located in said amplification chamber.

62. A device of claim 59, wherein amplification reagent is located in a holding chamber operably connected to said amplification chamber.

63. A device of claim 59, wherein amplification reagent is located in a holding chamber operably connected to said first conduit.

64. A device of claim 59, wherein said means for moving the amplification target is pneumatic.

65. A method for performing a nucleic acid analysis, comprising:

adding a sample suspected of containing a target nucleic acid to a reagent for extracting said target nucleic acid onto magnetic beads,

applying a magnetic field to said beads to draw them through a substantially contiguous wax layer,

adding an amplification reagent to the beads which is capable of incorporating a detectable label into an amplified target,

heating said mixture of beads and amplification reagent to a temperature that promotes formation of said amplified target,

adding the amplified target to a signaling reagent capable of binding to the detectable label and to an immobilized capture oligonucleotide in the region of an electrochemical sensor to form a conjugate of the amplified target and the capture oligonucleotide, and of said signaling reagent and detectable label,

applying a wash fluid to substantially displace unconjugated amplified target and signaling reagent from the conjugate,

adding to the conjugate a detection reagent capable of reacting with the signaling reagent bound to the detectable label and generating a signal at the sensor related to the presence or absence of the target nucleic acid.

66. The method of claim 65, where said heating is from about 88°

C. to 99°

C. for about 1 to 2 seconds and then from about 60°

C. to 72°

C. for about 4 to 60 seconds, repeated for twenty to forty cycles.

67. The method of claim 65, performed in an integrated single-use device, where an amplification chamber is heated to about 97°

C. for about 1 to 2 seconds and then about 68°

C. for about 12 seconds, and repeated for about thirty cycles.

68. The method of claim 65, performed in an integrated single-use device, wherein the amplification reagents comprise reagents selected for rolling circle amplification, helicase amplification, polymerase chain reaction amplification and strand displacement amplification.

69. The method of claim 65, performed in an integrated single-use device with magnetic beads, where said beads are washed prior to contacting the amplification reagent.

70. A method for performing a nucleic acid analysis, comprising:

adding a sample suspected of containing a target nucleic acid to a reagent for extracting said target nucleic acid onto magnetic beads,

applying a magnetic field to said beads to draw them through a substantially contiguous wax layer,

adding an amplification reagent to the beads which is capable of incorporating a detectable label into an amplified target,

heating said mixture of beads and amplification reagent to a temperature that promotes formation of said amplified target,

adding the amplified target to an immobilized capture oligonucleotide in a sensing region, to form a conjugate of said amplified target and said capture oligonucleotide,

applying a wash fluid to substantially displace unconjugated amplified target,

optically detecting a signal from said label in the sensing region and relating the signal to the presence or absence of target nucleic acid in the sample.

71. The method of claim 70, wherein the signal is used to determine the amount of target nucleic acid in the sample.

72. A method for performing a nucleic acid analysis, comprising:

adding a sample suspected of containing a target nucleic acid to a reagent for extracting said target nucleic acid, thereby extracting it onto a filter,

washing the filter to substantially remove sample residue, heating the filter in the presence of an aqueous fluid to elute the target nucleic acid from the filter into the aqueous fluid, and adding said aqueous fluid to an amplification reagent capable of incorporating a detectable label into an amplified target,

heating said mixture to a temperature to promote formation of an amplified target,

adding the amplified target to an immobilized capture oligonucleotide in a sensing region to form a conjugate of said amplified target and said capture oligonucleotide,

applying a wash fluid to substantially displace unconjugated amplified target,

optically detecting a signal from said label in the sensing region and relating the signal to the presence or absence of target nucleic acid in the sample.

73. The method of claim 72, wherein the signal is used to determine the amount of target nucleic acid in the sample.

74. A method for performing a nucleic acid analysis, comprising:

adding a sample suspected of containing a target nucleic acid to a reagent for extracting said target nucleic acid, thereby extracting it onto a filter,

washing the filter to substantially remove sample residue, heating the filter in the presence of an aqueous fluid to elute target nucleic acid from the filter into the aqueous fluid, and adding said aqueous fluid to an amplification reagent capable of incorporating a detectable label into an amplified target,

heating said mixture to a temperature to promote formation of an amplified target,

adding the amplified target to an immobilized capture oligonucleotide and a signaling reagent in the region of an electrochemical sensor to form a conjugate of said amplified target and said capture oligonucleotide, and of said signaling reagent and said label,

applying a wash fluid to substantially displace unconjugated amplified target and signaling reagent from said conjugate,

adding to said conjugate a detection reagent capable of reacting with the signaling reagent bound to the label and generating a signal detectable at said electrochemical sensor that is related to the presence or absence of the target nucleic acid.

75. A single-use device for performing nucleic acid amplification and detection, comprising:

a housing,

said housing comprising an entry port for accepting an extracted sample suspected of containing a target nucleic acid into a first conduit attached to an amplification chamber,

said amplification chamber also being attached to an amplification, reagent holding chamber, said reagent capable of incorporating a detectable label into an amplified target,

said housing having a means capable of moving the sample and the amplification reagent into the amplification chamber, said amplification chamber also having a heating means and a temperature sensing means for controlling the temperature to effect conditions suitable for amplification of the target nucleic acid,

said amplification chamber attached to a second conduit containing a sensing region comprising an immobilized capture oligonucleotide, a dissolvable signaling reagent capable of binding said detectable label and an electrochemical sensor,

said housing containing a means for moving the amplified target to the sensing region to permit binding of amplified target to the capture oligonucleotide and said signaling reagent to said detectable label,

said second conduit attached to a detection reagent holding chamber capable of delivering the detection reagent into the second conduit to substantially displace uncaptured amplified target and unbound signaling reagent from the sensing region,

wherein the detection reagent is capable of reacting with the signaling reagent bound to the label and generate a signal at the sensor.

76. The single-use device of claim 75, wherein said entry port is capable of matting with a nucleic acid extraction device to effect transfer of extracted target nucleic acid into said single-use device.

77. The single-use device of claim 75, wherein said entry port is capable of matting with a nucleic acid extraction device to effect transfer of extracted target nucleic acid into the single-use device,

said extraction device comprising a chamber for receiving a sample suspected of containing a target nucleic acid, said chamber containing a reagent comprising a chelating agent and a chaotropic agent, for extracting said target nucleic acid onto magnetic beads located in said chamber,

said chamber containing a contiguous wax layer in the region of said chamber and a conduit,

wherein magnetic beads that are associated with said target nucleic acid are capable of passing through said wax layer into said conduit by means of an applied magnetic field, said first conduit capable of permitting transfer of said beads to said entry port.

78. An single-use device of claim 77, where said magnetic beads are capable of being held in said conduit to permit a wash fluid to pass from a wash fluid holding chamber over said beads to a waste chamber, to effect washing of said beads.

79. The single-use device of claim 77, wherein said entry port is capable of matting with a nucleic acid extraction device to effect transfer of extracted target nucleic acid into the single-use device, said extraction device comprising a housing containing a filter capable of absorbing nucleic acid liberated from a sample and capable of desorbing said nucleic acid when exposed to an aqueous solution at a temperature of above about 75°

C. to permit said transfer.

80. A single-use device for performing nucleic acid amplification and detection comprising:

a housing comprising an entry port for accepting an extracted sample suspected of containing a target nucleic acid into a first conduit attached to an amplification chamber, said amplification chamber being attached to an amplification reagent holding chamber, said amplification reagent capable of incorporating a detectable label into an amplified target,

said housing having a means capable of moving said sample and said amplification reagent into said amplification chamber,

said amplification chamber having a heating means and a temperature sensing means for controlling the temperature of said amplification chamber to effect conditions suitable for amplification of said target nucleic acid,

said amplification chamber attached to a second conduit containing a sensing region comprising an immobilized capture oligonucleotide,

said housing containing a means for moving said amplified target to said sensing region to permit binding of said amplified target to said capture oligonucleotide,

said second conduit attached to a wash fluid holding chamber capable of delivering said wash fluid into said second conduit to substantially displace uncaptured amplified target from the sensing region, to permit optical sensing of said moiety retained in said sensing region.

81. An integrated single-use device for performing a nucleic acid extraction and amplification, comprising:

a housing comprising an entry port for accepting a sample suspected of containing a target nucleic acid,

a first chamber connected to said entry port containing a reagent for extracting said target nucleic acid onto magnetic beads located in said first chamber,

said first chamber containing a wax for forming a substantially contiguous wax layer in the region of said chamber and a first conduit,

wherein magnetic beads associated with target nucleic acid are capable of passing through said wax layer into said first conduit by means of an applied magnetic field,

said first conduit attached to an amplification chamber, said housing containing an amplification reagent capable of incorporating a detectable label into an amplified target, said beads and said amplification reagent being capable of moving into said amplification chamber, said amplification chamber also having a heating means and a temperature sensing means for controlling the temperature of said amplification chamber to effect conditions suitable for amplification of said target nucleic acid,

said amplification chamber attached to a second conduit permitting egress of the amplified target from said integrated device.

82. An integrated device of claim 81, where said amplified target is introduced into a second housing comprising a sensing region comprising an immobilized capture oligonucleotide, a dissolvable signaling reagent capable of binding to said label and a sensor,

said housing containing a means for moving said amplified target to said sensing region to permit binding of said amplified target to said capture oligonucleotide and said signaling reagent to said label,

said second housing having a detection reagent holding chamber capable of delivering a detection reagent into said sensing region to substantially displace uncaptured amplified target and signaling reagent not bound to said label from the sensing region,

said detection reagent capable of reacting with said signaling reagent bound to said label to generate a signal detectable at said sensor.

83. An integrated single-use device for performing a nucleic acid analysis, comprising:

a housing comprising an entry port for accepting a sample suspected of containing a target nucleic acid,

an extraction chamber connected to said entry port containing a reagent for extracting said target nucleic acid onto a filter located in said extraction chamber, said chamber also containing extraction reagents for promoting extraction of nucleic acid,

said housing comprising a wash fluid holding chamber containing a wash fluid and a waste chamber, both attached to said extraction chamber, permitting said wash fluid to pass from the wash fluid holding chamber over the filter to said waste chamber, to effect washing of said filter,

said extraction chamber having a heating means and a temperature sensing means for controlling the temperature of said extraction chamber and to effect conditions suitable for desorption of said target nucleic acid from said filter,

said extraction chamber attached to a first conduit, said first conduit attached to an amplification chamber, where said conduit permits transfer of said target nucleic acid from said extraction chamber to said amplification chamber,

said housing containing an amplification reagent, said amplification reagent capable of incorporating a label into an amplified target,

said amplification chamber having a heating means and a temperature sensing means for controlling the temperature of said amplification chamber and to effect conditions suitable for amplification of said target nucleic acid,

said amplification chamber attached to a second conduit permitting egress of the amplified target from said integrated device.

84. A nucleic acid separation method, comprising:

exposing a sample with cells containing nucleic acid to an aqueous mixture comprising a lytic reagent and one or more beads capable of binding the nucleic acid released from said cells,

said aqueous mixture contacting a layer of substantially immiscible liquid,

passing said beads through said immiscible liquid layer to separate the nucleic acid from the aqueous mixture.

85. The method of claim 84, where said beads are magnetic and pass through said immiscible liquid layer by means of an applied magnetic field.

86. The method of claim 84, wherein said immiscible liquid layer is an organic liquid.

87. The method of claim 84, wherein said immiscible liquid layer is a wax.

88. The method of claim 84, wherein said immiscible liquid is heated from an ambient temperature to an elevated temperature to permit passage of said one or more beads.

89. A method of transferring nucleic acid from a first location to a second location through an intermediary layer comprising:

causing nucleic acid at a first location to associate with one or more beads to form a nucleic acid-bead complex in a liquid,

passing said nucleic acid-bead complex to a second location separated from the first location by an intermediary layer that is substantially immiscible with said liquid.

90. The method of claim 89, where nucleic acid and inhibitors of nucleic acid amplification processes are substantially soluble in said intermediary layer.

91. A device for nucleic acid separation, comprising:

a chamber with a first layer comprising an aqueous lytic reagent and one or more magnetic beads capable of binding nucleic acid to form a complex;

a second layer that is substantially immiscible with said first layer and though which said beads may pass when the second layer is in liquid form, wherein nucleic acid and inhibitors of nucleic acid amplification processes are substantially insoluble in said second layer.

92. A method of using a device for nucleic acid separation, wherein

(a) a sample containing intact biological cells is pipetted through said second layer into said first layer,

(b) incubating said device for sufficient time to permit lysis of said cells and formation of nucleic acid-bead complex,

(c) generating a magnetic field in proximity to said device sufficient to move said one or more bead from said first layer and through said second layer.

93. The method of claim 92, where said second layer is a wax and said device is heated to a sufficient temperature to permit the wax to melt and form a layer covering said first layer.

94. The method of claim 92, where said beads are transferred to a means for nucleic acid amplification.

95. A device for extraction of nucleic acid from a biological sample, comprising:

a housing comprising a first and a second location,

a biochemically inert filter between said first and second locations, wherein at least a portion of said filter is impregnated with a mixture comprising a chaotropic salt, a weak basic buffer and a chelating agent.

97. A method for extracting nucleic acid from a biological sample with a device of claim 95, comprising:

applying a biological sample to a biochemically inert filter between said first and second location in a conduit,

wherein at least a portion of said filter is impregnated with a mixture comprising a chaotropic salt, a weak basic buffer and a chelating agent,

retaining said sample on said filter for sufficient time to permit extraction of nucleic acid onto said filter,

applying a wash fluid through said conduit, said wash fluid substantially removing from said filter matter capable of interfering with a nucleic acid amplification reaction, while substantially retaining said extracted nucleic acid on said filter,

applying an aqueous solution to said filter at a temperature above about 75°

C. to elute nucleic acid from said filter.

96. A device for extraction of nucleic acid from a blood sample, comprising:

a housing comprising a first and second location,

a biochemically inert filter between said first and second location, where at least a portion of said filter is impregnated with a strong base.

98. A method of extracting nucleic acid from a blood sample with a device of claim comprising:

applying blood sample to a biochemically inert filter between said first and second location in a conduit, wherein at least a portion of said filter is impregnated with a strong base,

retaining said sample on said filter for sufficient time to permit extraction of nucleic acid onto said filter,

applying a wash fluid through said conduit, said wash fluid substantially removing from said filter matter capable of interfering with a nucleic acid amplification reaction, while substantially retaining said extracted nucleic acid on said filter,

applying an aqueous solution to said filter at a temperature above about 75°

C. to elute nucleic acid from said filter.

99. A device for separating a nucleic acid primer from higher mass amplified target nucleic acid, comprising:

a first conduit for delivering a mixture of primer and amplified target to an electrophoretic element, said element comprising a first electrode in the first conduit,

a second conduit attached to said first conduit containing an electrophoretic separation material and a second electrode.

100. A device for separating a nucleic acid primer from higher mass amplified target nucleic acid, comprising:

a first conduit for delivering a mixture of primer and amplified target to an electrophoretic element, said element comprising a first electrode in said first conduit,

a second conduit attached to said first conduit containing an electrophoretic separation material and a second electrode proximal to said first electrode and a third electrode distal from said first electrode.

101. A method for separating a nucleic acid primer from higher mass amplified target nucleic acid, comprising:

introducing into a first conduit a mixture of primer and amplified target to contact an electrophoretic element comprising a first electrode in said first conduit,

a second conduit attached to said first conduit containing an electrophoretic separation material and a second electrode,

applying a negative polarity to said first electrode and positive polarity to said second electrode to cause the primer and amplified target to separate,

reversing the polarity sufficient for said amplified target to re-enter said first conduit while substantially retaining said primer in the separation material,

removing polarity from said electrodes and removing said amplified target from said first conduit.

102. A method for separating a nucleic acid primer from higher mass amplified target nucleic acid, comprising:

introducing into a first conduit a mixture of primer and amplified target to contact an electrophoretic element comprising a first electrode in said first conduit,

a second conduit attached to said first conduit containing an electrophoretic separation material and a second electrode proximal to said first electrode and a third electrode distal from said first electrode,

applying a negative polarity to said first electrode and a positive polarity to said second electrode to cause the primer and amplified target to separate,

changing the polarity of the first electrode to positive and said second electrode to negative and applying a positive polarity to said third electrode after sufficient time for said primer to move past said second electrode,

applying new polarity for sufficient time for said amplified target to re-enter said first conduit while substantially retaining said primer in the separation material,

removing polarity from said electrodes and removing said amplified target from said first conduit.

103. A method for performing a nucleic acid amplification assay comprising:

(a) combining reagents, DNA polymerase, a target nucleic acid and an amount of a modified primer comprising a sequence of bases complimentary to a first region of said target nucleic acid, a polymerase blocking region attached to said primer, a single stranded hybridization region attached to said polymerase blocking region, and a detectable label,

(b) cycling the mixture of (a) to provide multiple copies of an amplicon incorporating said modified primer,

(c) substantially eliminating excess unincorporated modified primer from said mixture,

(d) exposing said mixture to a capture oligonucleotide complimentary to said single stranded hybridization region,

(e) hybridizing said single stranded hybridization region of said amplicon incorporating said modified primer, with said capture oligonucleotide, and

(f) detecting said label associated with said hybridization.

104. A method for performing a nucleic acid amplification assay comprising:

(a) combining reagents, DNA polymerase, a target nucleic acid and two modified primers,

said first modified primer comprising a sequence of bases complimentary to a first region of said target nucleic acid, a polymerase blocking region, and a first single stranded hybridization region attached to said polymerase blocking region,

said second modified primer comprising a sequence of bases complimentary to a second region of said target nucleic acid, a polymerase blocking region, and a second single stranded hybridization region attached to said polymerase blocking region,

where a detectable label is attached to at least one of the first and second modified primers,

(b) cycling the mixture of (a) to provide multiple copies of an amplicon incorporating said modified primers,

(c) substantially eliminating unincorporated modified primer with said detectable label from said mixture,

(d) exposing said mixture to a capture oligonucleotide complimentary to at least one of said first and second single stranded hybridization regions,

(e) hybridizing said single stranded hybridization region of said amplicon incorporating said modified primer with said capture oligonucleotide, and

(f) detecting said label associated with said hybridization.

105. A method for performing a nucleic acid amplification assay comprising;

(a) combining reagents, DNA polymerase, a target nucleic acid and two modified primers,

said first modified primer comprising a sequence of bases complimentary to a first region of said target nucleic acid, a polymerase blocking region, a first single stranded hybridization region attached to said polymerase blocking region, and an attached detectable label;

said second modified primer comprising a sequence of bases complimentary to a second region of said target nucleic acid, a polymerase blocking, a second single stranded hybridization region attached to said polymerase blocking region, and an attached detectable label,

(b) cycling the mixture of (a) to provide multiple copies of an amplicon incorporating said modified primers,

(c) substantially eliminating unincorporated modified primers from said mixture,

(d) exposing said mixture to a capture oligonucleotide complimentary to at least one of said first and second single stranded hybridization regions,

(e) hybridizing said single stranded hybridization region of said amplicon incorporating said modified primer with said capture oligonucleotide, and

(f) detecting labels associated with said hybridization.