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1. (WO2019045807) ASSAY DEVICES AND METHODS OF USE THEREOF
Nota: O texto foi obtido por processos automáticos de reconhecimento ótico de caracteres.
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Claims

What is claimed is:

1. A method for amplifying nucleic acids in a sample comprising:

providing a container comprising a multifunction chamber and a first reaction zone fluidly connected to the multifunction chamber, the container comprising therein magnetic particles and reagents for sample preparation, nucleic acid recovery, and a first-stage nucleic acid amplification reaction, wherein the magnetic particles and the reagents are fluidly connected to the multifunction chamber or the first reaction zone or both;

introducing the sample into the multifunction chamber;

performing at least two steps in the multifunction chamber, wherein the steps include:

(1) contacting the sample and the magnetic beads prior to lysis,

(2) generating a lysate in the presence of the magnetic particles,

(3) binding nucleic acids with the magnetic particles,

(4) isolating the magnetic particles from the lysate,

(5) performing at least one wash on the magnetic particles isolated from the lysate, and

(6) amplifying nucleic acids in a first-stage nucleic acid amplification reaction.

2. The method of claim 1 wherein the multifunction chamber is provided with lysis particles.

3. The method of claim 1 or 2, wherein the steps performed in the multifunction chamber are selected from the group of:

steps (1) and (2);

steps (1), (2), and (3);

steps (1) - (4);

steps (2) and (3); and

steps (2), (3), and (4).

4. The method of claim 1 or 2, wherein the steps performed in the multifunction chamber are selected from the group of:

steps (3) and (4);

steps (3), (4), and (5);

steps (4), (5), and (6); and

steps (5) and (6).

5. The method of claim 1 wherein the performing step includes step (3) and step (3) further comprises applying heat to the sample while generating the lysate.

6. The method of claim 1 wherein the performing step includes step (2) wherein the lysate is generated under conditions for binding the nucleic acids to the magnetic particles.

7. The method of claim 1 wherein the performing step includes step (2) wherein the lysate is generated at a lysis temperature and the nucleic acids are recovered from the lysate at a controlled temperature, wherein the lysis temperature is in the range of 40-100°C, preferably 50-100°C, or more preferably 70-100°C, and wherein the controlled temperature is below the lysis temperature and is in the range of 0-60°C, preferably 0-50°C, or more preferably 0-40°C.

8. The method of claim 1 or 2, further comprising:

wherein the performing step includes steps (3) and (4) and step (4) further comprises moving the magnetic particles from the multifunction chamber to the first reaction zone,

wherein the performing step includes step (5) and step (5) is performed in the first reaction zone, wherein the wash includes injecting a wash buffer into the first reaction zone, dispersing the magnetic beads in the wash buffer, recapturing the magnetic beads, and expelling the wash buffer, and

wherein the performing step includes step (6) and step (6) is performed in the first reaction zone.

9. The method of claim 8, wherein the lysis particles remain in the multifunction chamber after performing steps (3) and (4).

10. The method of claim 8, further comprising adding nucleic acid amplification reagents to the first reaction zone after step (5), and wherein there is no eluting step prior to step (6).

11. The method of claim 1, the performing step including steps (3), (4), and (5), wherein:

steps (3) and (4) further comprise expelling the lysate to a waste chamber, and step (5) is performed in the multifunction chamber, wherein step (5) includes injecting a wash buffer into the multifunction chamber, dispersing the magnetic beads in the wash buffer, recapturing the magnetic beads, and expelling the wash buffer.

12. The method of claim 11 wherein step (6) is a first-stage multiplex nucleic acid amplification reaction in the multifunction chamber, wherein there is no eluting step prior to the amplifying step.

13. The method of claim 1 wherein the container further comprises a second-stage reaction zone fluidly connected to the multifunction chamber, the second-stage reaction zone comprising a plurality of second-stage reaction wells, each second-stage reaction well comprising a pair of primers configured for further amplification of the sample, the second-stage reaction zone configured for contemporaneous thermal cycling of all of the plurality of second-stage reaction chambers.

14. The method of claim 13, wherein the second-stage reaction zone is fluidly connected to the multifunction chamber via the first reaction zone.

15. The method of claim 14 wherein the performing step include step (6) and further comprising combining a portion of the first- stage nucleic acid amplification reaction with reagents for a second-stage nucleic acid amplification reaction to form a second-stage nucleic acid amplification mixture, filling each of the second- stage reaction wells with the second- stage nucleic acid amplification mixture, and performing a second- stage nucleic acid amplification reaction in plurality of second- stage reaction wells of the second- stage reaction zone to generate one or more amplicons.

16. The method of claim 16 further comprising identifying one or more organisms, if present in the sample, using the one or more amplicons.

17. The method of claim 1 wherein the flexible container further comprises a sample receiving chamber in fluid communication with the multifunction chamber.

18. The method of claim 17 further comprising collecting a sample with a sample swab, inserting the swab into the sample receiving chamber and sealing the sample receiving chamber with the swab therein, dispersing the sample in the sample receiving chamber with a sample lysis buffer injected into the sample receiving chamber, and transferring the sample and the sample lysis buffer into the multifunction chamber.

19. The method of claim 1 wherein the magnetic particles and reagents for sample preparation, nucleic acid recovery, and first- stage nucleic acid amplification are provided in one or more fluid-filled reagent blisters, in one or more dry reagent blisters, or a combination thereof.

20. The method of claim 1 wherein the performing step includes step (2) and further comprising subsequent to step (2), sequestering the lysis particles in the multifunction chamber away from the lysate.

21. The method of claim 1 wherein the performing step includes step (3) and further comprising sequestering the magnetic particles in the multifunction chamber subsequent to step

(3).

22. A container for performing nucleic acid amplification on a sample in a closed system, the container comprising:

a first layer and a second layer defining a multifunction chamber therebetween, the container provided with magnetic particles and reagents for sample preparation, nucleic acid recovery, and a first- stage nucleic acid amplification reaction, wherein the magnetic particles are provided in a chamber that is fluidly connected to the multifunction chamber or are provided in the multifunction chamber, and wherein the reagents are provided in chambers that are fluidly connected to the multifunction chamber; and

a second-stage reaction zone disposed between the first layer and the second layer and fluidly connected to the multifunction chamber, the second- stage reaction zone comprising a plurality of second-stage reaction chambers, each second-stage reaction chamber comprising a pair of primers configured for further amplification of the sample, the second-stage reaction zone configured for contemporaneous thermal cycling of all of the plurality of second-stage reaction chambers.

23. The container of claim 22, wherein the multifunction chamber is provided with cell lysis components and the magnetic particles.

24. The container of claim 23 wherein the cell lysis components comprise lysis particles or lysis particles.

25. The container of claim 22, wherein the container further comprises a sample lysis chamber in fluid communication with the multifunction chamber, wherein the sample lysis chamber is provided with cell lysis components and the magnetic particles, and the multifunction chamber is provided with magnetic bead wash components and first-stage nucleic acid amplification components.

26. The container of claim 25 further comprising a filter positioned between the sample lysis chamber and the multifunction chamber.

27. The container of claim 26, wherein the filter has a porosity sized to retain the lysis particles and allow the magnetic beads to pass through.

28. The container of claim 26, wherein a channel connects the sample lysis chamber and the multifunction chamber and the filter is positioned between the first layer and the second layer across the channel.

29. The container of claim 28, wherein the filter is wider than the channel.

30. The container of claim 26, wherein a channel extends between the sample lysis chamber and the multifunction chamber, and the filter is located in the sample lysis chamber adjacent the channel.

31. The container of claim 30, wherein the channel is smaller than the sample lysis chamber and the filter is wider than the channel.

32. The container of claim 22 wherein the magnetic particles are nucleic acid-binding magnetic particles.

33. The container of claim 22 further comprising a sample receiving chamber in fluid communication with the multifunction chamber.

34. The container of claim 33 wherein the sample receiving container comprises a sample collection swab.

35. The container of claim 34 wherein the sample collection swab comprises an elongate shaft, wherein the sample receiving chamber and the elongate shaft of the swab are fabricated from chemically compatible materials that can be at least partially fused with a heat seal.

36. The container of claim 22 wherein the magnetic particles and the reagents are provided in one or more reagent blisters in fluid communication with the multifunction chamber.

37. The container of claim 36 wherein one or more of the reagent blisters are fluid-filled reagent blisters and are filled at the time of manufacture of the container.

38. The container of claim 36 wherein one or more of the reagent blisters comprise dry reagents disposed in the reagent blisters.

39. The container of claim 36 further comprising an openable seal between the reagent blisters and the multifunction chamber.

40. The container of claim 39 wherein the openable seal is a burstable seal.

41. The container of claim 39 wherein the openable seal is a tacked together film seal.

42. A self-contained reaction vessel, comprising

a first reaction zone fluidly connected to a second reaction zone, wherein the second reaction zone comprises:

a first layer, a card layer with a plurality of second- stage reaction wells formed therein, and a second layer,

wherein the first layer is disposed over and seals a first end of the plurality of second-stage reaction wells, and the second layer is disposed over and seals a second, opposite side of the plurality of second-stage reaction wells,

a fill channel fluidly connecting the first reaction zone to the plurality of second-stage reaction wells of the second reaction zone;

wherein the fill channel is formed at least in part as a space between the second layer and the card layer, and the fill channel further forms a convoluted flow path into each of the plurality of second-stage reaction wells so as to suppress fluid communication between wells in the second stage reaction zone.

43. The self-contained reaction vessel of claim 42, the fill channel comprising a channel formed in the card layer, the second layer, or a combination of the card layer and the second layer, wherein the fill channel individually fluidly connects all of the second- stage reaction wells to the first-stage reaction zone.

44. The self-contained reaction vessel of claim 42, further comprising a third layer bonded to the second layer, wherein the second layer comprises a pierced layer having a plurality of piercings fluidly connected to the plurality of second- stage reaction wells, and wherein the fill channel is formed as a space between the second and third layers.

45. The self-contained reaction vessel of claim 44, wherein the convoluted flow path for each second- stage reaction well comprises an opening in the second layer adjacent to but not aligned with a second- stage reaction well and in fluid communication with a cutout in the card layer adjacent to but not aligned with a second-stage reaction well.

46. The self-contained reaction vessel of claim 44, wherein the convoluted flow path for each second- stage reaction well comprises an opening in the second layer adjacent to but not aligned with a second- stage reaction well, the opening being in fluid communication with a

substantially vertical cutout in the card layer extending adjacent to a second-stage reaction well from the second film layer to a substantially horizontal cutout in the card layer adjacent to the first layer and creating a fluid conduit from the opening to the substantially vertical cutout and into the second- stage reaction well.

47. The self-contained reaction vessel of claim 42, wherein the fill channel is heat sealable.

48. The self-contained reaction vessel of claim 47, wherein a single heat seal seals flow from a fill channel to multiple second-stage reaction wells and seals the second-stage reaction wells from each other.

49. The self-contained reaction vessel of claim 42, wherein the fill channel further comprises a dilution zone that includes a dilution well in the fill channel between the first and second reaction zones, wherein the dilution well is configured to receive a volumetric portion of a reaction product from the first reaction zone, combine the volumetric portion with a dilution medium to form a combined volumetric portion, and fill the plurality of second- stage reaction wells of the second reaction zone with the combined volumetric portion.

50. The self-contained reaction vessel of claim 49, the dilution zone further comprising a dilution blister in fluid communication with the dilution well, wherein the dilution blister is configured to receive the dilution medium, combine the volumetric portion with the dilution medium, and fill the plurality of second-stage reaction wells of the second reaction zone.

51. A self-contained reaction vessel, the self-contained reaction vessel comprising: a sample lysis zone configured for lysis of cells or spores present in a sample,

a first reaction zone fluidly connected to the sample lysis zone, the first reaction zone nucleic acid configured for recovering nucleic acids from a lysed sample and for first-stage amplification of nucleic acids present in the,

a second-stage reaction zone fluidly connected to the first reaction zone, the second-stage reaction zone comprising a plurality of second-stage reaction wells, each second-stage reaction well comprising a pair of primers configured for further amplification of the sample, the second-stage reaction zone configured for contemporaneous thermal cycling of all of the plurality of second- stage reaction chambers, and

a plurality of liquid reagent blisters fluidly connected to one or more of the sample lysis zone, the first reaction zone, or the second- stage reaction zone, wherein liquid reagents are provided in the liquid reagent blisters at time of manufacture.

52. The self-contained reaction vessel of claim 51 wherein the sample lysis zone is provided with lysis particles configured for lysing cells or spores located in the sample and magnetic beads configured for recovering nucleic acids from a lysate.

53. The self-contained reaction vessel of claim 52, wherein the lysis particles, magnetic beads, and a lysis buffer are provided in one or more of the liquid reagent blisters that are fluidly connected to the sample lysis zone.

54. The self-contained reaction vessel of claim 51 wherein one of the liquid reagents is provided in a liquid reagent pack within one of the liquid reagent blisters. .

55. The self-contained reaction vessel of claim 54, wherein the liquid reagent pack comprises a volume of liquid sealed between a first layer and a second layer.

56. The self-contained reaction vessel of claim 55, wherein at least one of the first layer or the second layer comprises a barrier film.

57. The self-contained reaction vessel of claim 51 further comprising an openable seal between the liquid reagent disposed in the liquid reagent blister and one or more of the sample lysis zone, the first reaction zone, or the second-stage reaction zone.

58. The self-contained reaction vessel of claim 57, wherein the openable seal is a burstable seal.

59. The self-contained reaction vessel of claim 57, wherein the openable seal is a tacked together film seal.

60. A system for performing nucleic acid amplification on a sample, the system comprising:

a reaction vessel that includes:

a first layer and a second layer defining a multifunction chamber therebetween, a second-stage reaction zone disposed between the first layer and the second layer and fluidly connected to the multifunction chamber, the second- stage reaction zone comprising a plurality of second-stage reaction chambers, each second-stage reaction chamber comprising a pair of primers configured for further amplification of the sample, the second-stage reaction zone configured for contemporaneous thermocycling of all of the plurality of second-stage reaction chambers; and

one or more reagent blisters formed between the first layer and the second layer in fluid communication with the multifunction chamber and the second- stage reaction zone, wherein magnetic particles are provided in a blister fluidly connected to the multifunction chamber or are provided in the multifunction chamber;

a thermocycling instrument that includes:

a receptacle for positioning the flexible container in the instrument; a heater/cooler positionable in the instrument for heating and/or cooling one or more of the multifunction chamber and the second-stage reaction zone;

a cell lysis component configured for generating a lysate in the reaction vessel; and

a fluid movement component configured for moving fluids in the flexible container between at least the one or more reagent blisters, the multifunction chamber, and the second-stage reaction zone.

61. The system of claim 60, wherein the multifunction chamber is provided with cell lysis components and the magnetic particles.

62. The system of claim 60 wherein the container further comprises a sample lysis chamber in fluid communication with the multifunction chamber, wherein the sample lysis chamber is provided with cell lysis components and the magnetic particles, and the multifunction chamber is provided with magnetic bead wash components and first-stage nucleic acid amplification components.

63. The system of claims 60 or 62 wherein the instrument further comprises a magnet deployable in the instrument for isolating the magnetic beads in a portion of the multifunction chamber.

64. The system of claim 60 wherein the heater/cooler comprises a first heater adjacent to a first portion of the multifunction chamber for adjusting a first portion of a sample to a first temperature, and a second heater adjacent to a second portion of multifunction chamber for adjusting a second portion of a sample to a second temperature, the second temperature being different from the first temperature.

65. The system of claim 64 wherein the heater/cooler is mechanically associated with a wiper element that moves the first portion of the sample to the second portion of the multifunction chamber while moving the second portion of the sample to the first portion of the multifunction chamber such that portions of the sample are under control of each of the heaters simultaneously.

66. The system of claim 65 wherein the wiper element repeatedly moves portions of the sample to opposite portions of the multifunction chamber to thermocycle the sample.

67. The system of claim 64 wherein the instrument further comprises a translator mechanically coupled to at least one of the receptacle, the flexible container, or the heater/cooler to laterally align at least one portion of the multifunction chamber, the second- stage reaction zone, or both relative to the first and second heater elements of the heater/cooler such that the at least one portion of the multifunction chamber or the second-stage reaction zone is under temperature control of at least one of the first or the second heater elements.

68. The system of claim 67 wherein the instrument is configured to repeatedly align the at least one portion with the first heater element and then the second heater element for thermocycling a fluid sample in the at least one portion.

69. An array assembly comprising:

a plurality of wells arranged in an array;

a fluid fill channel in fluid communication with each of the plurality of wells, at least one of the wells of the array comprising a reaction well with a first diameter and a sub- well recessed below a first surface of the array assembly with a second, smaller diameter, wherein the recessed sub-well is not fluidly connected to the fluid fill channel.

70. The array assembly of claim 69, the fluid fill channel comprising a plurality of branch channels in fluid communication with each of the plurality of wells, wherein the recessed sub-well is not fluidly connected to its branch channel.

71. The array assembly of claim 69 the sub-well being recessed below a second surface of the array, wherein the second surface of the array is opposite the first surface.

72. A method for spotting an array comprising:

providing an array assembly that includes a plurality of wells that are arranged in an array, wherein the array assembly and the plurality of wells do not include a backing layer prior to spotting reagents and/or reaction components to the plurality of wells;

positioning the array assembly in a spotting apparatus relative to a spotting assembly that includes a plurality of cannulae arranged in an array that corresponds to the wells of the array assembly, wherein each cannula is fluidly connected to a fluid reagent reservoir;

delivering a droplet of fluid to an end of each cannula;

contacting the droplets and the wells of the array to transfer the droplets to the array assembly; and

evaporating fluid from the droplets to yield an array assembly having dried reagents in the wells.

73. The method of claim 72 wherein the spotting apparatus includes alignment pins, stops, a frame, or a combination thereof positioned to align the array assembly relative to the cannulae.

74. The method of claim 72 wherein two or more cannulae are fluidly connected to the same fluid reagent reservoir.

75. The method of claim 72 wherein each cannula is fluidly connected to a different fluid reagent reservoir.

76. The method of claim 72 further comprising:

extending the cannulae through the wells of the array assembly prior to the delivering step; and

wherein the contacting step comprises retracting the cannula to contact the droplets and the wells of the array to transfer the droplets to the array assembly.

77. The method of claim 72 further comprising:

moving the array assembly relative to the cannulae to extend the cannulae through the wells of the array assembly prior to the delivering step; and

wherein the contacting step includes retracting the array assembly relative to the cannulae to contact the droplets and the wells of the array to transfer the droplets to the array assembly.

78. A method of making a reaction pouch comprising:

providing an array assembly spotted according the method or one of claims 72-77;

inserting the array assembly into a preformed pocket between two or more film layers; sealing the film layers to the array assembly to seal the film layers to a top and a bottom surface of the array assembly; and

sealing the preformed pocket.

79. The method of claim 78 wherein the two or more film layers are laminated together and heat formed prior to positioning the array between the film layers to define defined areas selected from the group consisting of one or more reaction blisters, one or more reagent blisters, and one or more pockets.

80. The method of claim 78 wherein the two or more film layers are laminated together and heat formed after positioning the array between the film layers to define defined areas selected from the group consisting of one or more reaction blisters, one or more reagent blisters, and one or more pockets.

81. The method of claim 78 wherein the array pocket is fluidly connected to one or upstream fluid blisters so that the array can be flooded with fluid.

82. The method of claim 78, wherein the array assembly includes a fluid channel system and a vacuum channel system fluidly connected to each well of the array assembly.

83. A reaction container comprising:

a first layer of material and at least a second layer of material defining the reaction container;

a sample introduction blister and a dilution blister formed between the first and second layers of material, wherein the dilution blister is fluidly connected to the sample introduction blister, and

one or more reaction wells fluidly connected to the sample introduction blister and one or more reaction wells fluidly connected to the dilution blister,

wherein the dilution blister is configured to make a selected dilution of an aliquot of sample from the sample introduction blister, and

wherein the one or more reaction wells include a reagent for performing an assay on the sample.

84. The reaction container of claim 83 wherein the dilution blister is provided with a selected volume fluid to perform a selected dilution of the sample added to the sample introduction blister.

85. The reaction container of claim 83 wherein reaction container further comprises a volumetric dilution well sized and dimensioned to receive a selected volume of fluid from a blister and combine it with a selected volume of a diluent to make a diluted sample.

86. The reaction container of claim 83 wherein reaction container further comprises a channel sized and dimensioned to receive a selected volume of fluid from a blister and combine it with a selected volume of a diluent to make a diluted sample.

87. The reaction container of claim 83 wherein the one or more reaction wells contain the same reagent.

88. The reaction container of claim 83 wherein the one or more reaction wells contain a different reagent.

89. The reaction container of claim 83 wherein at least one of the reaction wells comprises a reaction well with two or more co-fillable sub-wells fluidly connected to the same sample blister.

90. The reaction container of claim 83 further comprising a known standard solution blister fluidly connected to one or more reaction wells that include a reagent for performing an assay on the standard solution for providing a reference for the sample the sample introduction blister and the dilution blister.

91. An array assembly, comprising:

a plurality of wells arranged in an array;

a fluid fill channel comprising a plurality of branch channels in fluid communication with each of the plurality of wells,

wherein at least one of the wells of the array comprises a reaction well with two or more co-fillable sub-wells fluidly connected to the same branch channel.

92. The array assembly of claim 91 wherein the two or more sub-wells each comprise a separate reagent.

93. The array assembly of claim 92 wherein the separate reagents in the sub-wells are combinable when the reaction well is filled with fluid.

94. The array assembly of claim 91 further comprising a card layer having the plurality of wells formed therein.

95. A method for sealing a reaction container comprising:

providing a reaction container comprising a sample receiving chamber and a first reaction zone fluidly connected to the sample receiving zone;

providing a sample collection swab comprising an elongate shaft;

inserting the swab into the sample receiving chamber; and

applying a seal to the sample receiving chamber across the elongate shaft of the swab to seal the sample receiving chamber.

96. The method of claim 95 wherein the seal is a heat seal that at least partially fuses the sample receiving chamber and the elongate shaft of the sample collection swab at the seal.

97. The method of claim 95 wherein the shaft has a non-circular cross-section.

98. The method of claim 95 further comprising applying additional seal across the elongate shaft to divide the sample receiving chamber into a plurality of chambers that are fluidly connected to the first reaction zone.

99. The method of claim 95 wherein the sample receiving chamber and the elongate shaft of the swab are fabricated from chemically compatible materials that can be at least partially fused with a heat seal.