COMPOSITIONS AND METHODS FOR OBTAINING NUCLEIC ACIDS FROM SPUTUM

• US 20170152545A1
• Filed: 11/07/2016
• Published: 06/01/2017
• Est. Priority Date: 06/07/2002
• Status: Active Grant

• Current Assignee: DNA Genotek Inc. (Orasure Technologies Incorporated)
• Original Assignee: DNA Genotek Inc. (Orasure Technologies Incorporated)
• Inventors: Birnboim, H. Chaim
• Granted Patent: US 10,619,187 B2
• Time in Patent Office: Days
• CPC Class Codes: B01L 2300/042 Caps; Plugs
• CPC Class Codes: B01L 2300/046 Function or devices integrated in the closure
• CPC Class Codes: B01L 2300/047 Additional chamber, reservoir
• CPC Class Codes: B01L 2300/0672 Integrated piercing tool
• CPC Class Codes: B01L 2300/0832 cylindrical, tube shaped
• CPC Class Codes: B01L 2400/0683 mechanically breaking a wall or membrane within a channel or chamber
• CPC Class Codes: B01L 3/502 with fluid transport, e.g. in multi-compartment structures centrifugal-type cuvettes G01N21/07; analysis by separation into components G01N30/00; automatic analysers G01N35/00
• CPC Class Codes: B01L 3/5082 Test tubes per se
• 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: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2527/119 pH
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer

Abstract

The present invention relates to compositions and methods for preserving and extracting nucleic acids from saliva. The compositions include a chelating agent, a denaturing agent, buffers to maintain the pH of the composition within ranges desirable for DNA and/or RNA. The compositions may also include a reducing agent and/or antimicrobial agent. The invention extends to methods of using the compositions of the invention to preserve and isolate nucleic acids from saliva as well as to containers for the compositions of the invention.

Claims

1. A device for storage of nucleic acid from a biological sample, said device comprising:

(a) a container having a barrier capable of disestablishment, said barrier separating a reagent compartment from a sample receiving compartment; and

(b) a nucleic acid storage composition within said reagent compartment, wherein upon disestablishment of said barrier there is fluid communication between said sample receiving compartment and said reagent compartment to facilitate mixing of said biological sample with said nucleic acid storage composition.

68. The device of claim 1, wherein the nucleic acid storage composition comprises a chelating agent, a denaturing agent, and at least one additional reagent selected from a reducing agent, an antimicrobial agent, an antioxidant free-radical scavenger, an inhibitor of ribonuclease, or a protease, and the pH of the composition is greater than 5.0.

2-12. -12. (canceled)

13. A method of preserving a nucleic acid from a biological sample comprising:

(a) placing a nucleic acid storage composition into a reagent compartment of a storage container;

(b) placing a biological sample into a sample receiving compartment of said storage device, said sample receiving compartment separated from said reagent compartment by a barrier;

(c) closing said storage container; and

(d) disestablishing said barrier such that said composition contacts said biological sample to form a mixture thereby preserving the nucleic acid from said biological sample.

69. The method of claim 13, wherein the nucleic acid storage composition comprises a chelating agent, a denaturing agent, and at least one additional reagent selected from a reducing agent, an antimicrobial agent, an antioxidant free-radical scavenger, an inhibitor of ribonuclease, or a protease, and the pH of the composition is greater than 5.0.

14-23. -23. (canceled)

24. A sample receiving container comprising:

(a) a sample receiving compartment;

(b) a reagent storage compartment;

(c) a barrier separating said sample receiving compartment from said reagent storage compartment and capable of disestablishment; and

(d) means for closing said container that comprises means for disestablishing said barrier to allow fluid communication between said sample receiving compartment and said reagent storage compartment.

70. The sample receiving container of claim 24, wherein the reagent storage compartment comprises a composition comprising a chelating agent, a denaturing agent, and at least one additional reagent selected from a reducing agent, an antimicrobial agent, an antioxidant free-radical scavenger, an inhibitor of ribonuclease, or a protease, and the pH of the composition is greater than 5.0.

25-29. -29. (canceled)

30. A device for receiving and preserving nucleic acid in a biological sample, said device comprising:

a. one or more walls defining a containment vessel having a top having an opening, and a closed bottom having a sample receiving area for holding said biological sample, said opening for receiving a liquid sample and for sealably receiving a closing means, said top having an opening for receiving a biological sample from the mouth of a user and further comprising at least one marking on said one or more walls which corresponds to a fluid volume in the sample receiving area;

b. a reagent compartment having a barrier, said barrier sealing and containing reagents in said reagent compartment and capable of permanent disestablishment upon disruption to release said reagents into the sample receiving area;

c. reagents in the reagent compartment for preserving nucleic acids potentially present in the sample wherein said reagents comprise a denaturing agent, a chelator and a buffer agent; and

,

d. closing means and disruption means, said disruption means for engaging said barrier whereby when sealably closing said opening with said closing means, said disrupting means permanently and mechanically disestablishes said barrier to release said reagents to form a mixture of reagents and said biological sample wherein said buffering agent maintains a pH of said mixture equal to or above 5.0 to preserve nucleic acids potentially present in the sample.

31. The device of claim 30, wherein the biological sample comprises a bodily fluid.

32. The device of claim 30, wherein the biological sample comprises sputum.

33. The device of claim 30, wherein the biological sample comprises saliva.

34. The device of claim 30, wherein the denaturing agent comprises dodecyl sulfate.

35. The device of claim 30, wherein the chelator comprises ethylenediamine tetraacetic acid (EDTA).

36. The device of claim 30, wherein the reagents further comprise an antimicrobial agent.

37. The device of claim 30, wherein the reagents further comprise an antioxidant free-radical scavenger.

38. The device of claim 30, wherein the barrier comprises a septum.

39. The device of claim 38, wherein the disruption means punctures or pierces the septum to release the reagents to form a mixture of the released reagents and the biological sample.

40. The device of claim 39, wherein the biological sample comprises saliva.

41. The device of claim 40, wherein the denaturing agent comprises dodecyl sulfate.

42. The device of claim 41, wherein the chelator comprises ethylenediamine tetraacetic acid (EDTA).

43. The device of claim 42, wherein the buffering agent comprises TRIS.

44. The device of claim 43, wherein the buffering agent maintains pH of the mixture from about 7.0 and about 10.0.

45. The device of claim 44, wherein the reagents further comprise an antimicrobial agent.

46. The device of claim 45, wherein the reagents further comprise an antioxidant free-radical scavenger.

47. A method of preserving nucleic acid in a biological sample, comprising the steps of:

a. providing a device for receiving said biological sample, said device having;

i. one or more walls defining a containment vessel having a top having an opening, and a closed bottom having a sample receiving area for holding said biological sample, said opening for receiving a liquid sample and sealably receiving a closing means said top having an opening for receiving a biological sample from the mouth of a user and further comprising at least one marking on said one or more walls which corresponds to a fluid volume in the sample receiving area;

ii. a reagent compartment having a barrier, said barrier sealing and containing reagents in said reagent compartment and capable of permanent disestablishment upon disruption to release said reagents into the sample receiving area;

iii. reagents in the reagent compartment for preserving nucleic acids potentially present in the sample wherein said reagents comprise a denaturing agent, a chelator and a buffering agent; and

,

iv. closing means and disruption means, said disruption means for engaging said barrier whereby when sealably closing said opening with said closing means, said disrupting means permanently and mechanically disestablishes said barrier to release said reagents to preserve nucleic acids potentially present in said biological sample.;

b. receiving a biological sample in said top having an opening, and

c. affixing closing means to seal the sample in said containment vessel and simultaneously permanently and mechanically disestablishing said barrier to release said reagents to form a mixture of said reagents and said biological sample wherein said buffering agent maintains the pH of said mixture equal to or above 5.0 to preserve nucleic acids in said biological sample.

48. The method of claim 47, wherein the biological sample comprises a bodily fluid.

49. The method of claim 47, wherein the biological sample comprises sputum.

50. The method of claim 47, wherein the biological sample comprises saliva.

51. The method of claim 47, wherein the denaturing agent comprises dodecyl sulfate.

52. The method of claim 47, wherein the chelator comprises ethylenediamine tetraacetic acid (EDTA).

53. The method of claim 47, wherein the reagents further comprise an antimicrobial agent.

54. The method of claim 47, wherein the reagents further comprise an antioxidant free-radical scavenger.

55. The method of claim 47, wherein the barrier comprises a septum.

56. The method of claim 55, wherein the disruption means punctures or pierces the septum to release the reagents to form a mixture of the released reagents and the biological sample.

57. The method of claim 56, wherein the biological sample comprises saliva.

58. The method of claim 57, wherein the denaturing agent comprises dodecyl sulfate.

59. The method of claim 58, wherein the chelator comprises ethylenediamine tetraacetic acid (EDTA).

60. The method of claim 59, wherein the buffering agent comprises TRIS.

61. The method of claim 60, wherein the buffering agent maintains pH of the mixture from about 7.0 and about 10.0.

62. The method of claim 61, wherein the reagents further comprise an antimicrobial agent.

63. The method of claim 62, wherein the reagents further comprise an antioxidant free-radical scavenger.

64. The method of claim 63, wherein the reagents further comprise a protease.

65. A method of preserving a nucleic acid contained in sputum comprising the steps of:

a. obtaining sputum from a subject; and

b. contacting said sputum with a composition comprising a chelating agent and a denaturing agent, wherein the pH of said composition is greater than 5.0, thus preserving said nucleic acid.

66. A method of recovering a nucleic acid from sputum comprising the steps of:

a. obtaining sputum from a subject;

b. contacting said sputum with a composition comprising a chelating agent and a denaturing agent, wherein the pH of said composition is greater than 5.0, to form a mixture;

c. contacting said mixture with a protease;

and d. recovering said nucleic acid from said mixture.

67. A method of preserving and/or recovering a nucleic acid from a bodily fluid comprising:

i. placing said bodily fluid into a first region of a container;

ii. placing a composition comprising a chelating agent and a denaturing agent, wherein the pH of said composition is greater than 5.0, into a second region of said container, said second region separated from said first region by a barrier;

iii. closing said container; and

iv. disestablishing said barrier such that said composition contacts said bodily fluid forming a mixture, thereby preserving said nucleic acid.

71. A method of reducing the viscosity of a mucin-containing bodily fluid or tissue, comprising contacting a mucin-containing bodily fluid or tissue from a subject with a composition comprising a chelating agent, a denaturing agent, and a reducing agent, wherein the pH of said composition is greater than 5.0, thus reducing the viscosity of said mucin-containing bodily fluid or tissue.

72. The method of claim 71, wherein the composition further comprises an antimicrobial agent, an antioxidant free-radical scavenger, an inhibitor of ribonuclease, or a protease.

73. The method of claim 71, which comprises the steps:

(a) placing the mucin-containing bodily fluid or tissue into a first region of a container;

(b) placing the composition into a second region of the container, the second region separated from said first region by a barrier;

(c) closing the container; and

(d) disestablishing the barrier such that the composition contacts the mucin-containing bodily fluid or tissue forming a mixture, thereby reducing the viscosity of said mucin-containing bodily fluid or tissue.

74. A device for preserving and/or isolating a nucleic acid obtained from a biological sample, which device comprises:

(a) a container that has a first region for collecting a biological sample and a second region containing a composition for preserving a nucleic acid,

(b) a barrier between said first region and said second region that keeps said biological sample and said composition separate,

(c) a means for closing said container, and

(d) a means for disturbing the integrity of said barrier such that said composition is capable of contacting said biological sample,

wherein

said first region has an opening with a diameter from 2.0 to 7.0 cm, and

said means for closing said container is coupled to said means for disturbing the integrity of said barrier such that, upon closing said container, the integrity of said barrier is disturbed to allow contact between said biological sample and said composition for preserving a nucleic acid in said biological sample.

75. The device of claim 74, wherein the composition for preserving a nucleic acid comprises a chelating agent, a denaturing agent, and at least one additional reagent selected from a reducing agent, an antimicrobial agent, an antioxidant free-radical scavenger, an inhibitor of ribonuclease, or a protease, and the pH of the composition is greater than 5.0.

76. The device of claim 74, wherein the barrier is a pivoting disc, where in a first position, the disc spans the container and separates the first and second regions, and pivoting the disc to a second position disestablishes the barrier and allows the biological sample contained in the first region to contact the composition that is contained in the second region.

77. A method of manufacturing a device for preserving a nucleic acid in a biological sample, which method comprises:

(a) providing a container that has a first region and a second region, with said first region suitable for containing a composition for preserving a nucleic acid and said second region having an opening suitable for the application of a biological sample;

(b) placing said composition into said first region; and

(c) applying a barrier to said container between said first region and said second region, with said barrier being impermeable to said composition and capable of being disestablished,

wherein said composition comprises a chelating agent, a denaturing agent, and at least one additional reagent selected from a reducing agent, an antimicrobial agent, an antioxidant free-radical scavenger, an inhibitor of ribonuclease, or a protease, and the pH of the composition is greater than 5.0.

78. The method of claim 77, wherein the barrier is a pivoting disc, where in a first position, the disc spans the container and separates the first and second regions, and pivoting the disc to a second position disestablishes the barrier and allows the biological sample contained in the second region to contact the composition that is contained in the first region.

79. A device, which device comprises:

(a) a container that has a first region for collecting a biological sample and a second region containing a composition for preserving at least a component of said biological sample,

(b) a barrier between said first region and said second region that keeps said biological sample and said composition separate,

(c) a means for closing said container, and

(d) a means for disturbing the integrity of said barrier such that said composition is capable of contacting said biological sample,

wherein

said device is constructed of a sturdy breakage-resistant plastic or a material that does not leach chemicals into said container'"'"'s contents;

said device comprises a broad mouth to allow a subject to place the required volume of fluid sample into said container;

said first region of said container is configured to collect a volume of 1-2 milliliters of a fluid sample;

said means for closing said container is a cap that is configured to lock once tightened to become tamper-resistant, or a cap that is configured to provide a liquid-tight and/or airtight seal for said container once said cap is fixed into place;

said barrier is a septum or plastic bag compartment that separates said composition from said biological sample until said septum or bag compartment is pierced or the contents otherwise released, said barrier is in the form of a pivoting partition, or said barrier is press fit, glued, or heat fit into place;

said means for closing said container is coupled to the disestablishment of said barrier; and

/or

the outside of said device is coated with an antimicrobial agent.

80. The device of claim 79, wherein the biological sample is skin, hair, fecal matter, a bodily fluid, or a tissue.

81. The device of claim 79, wherein the biological sample comprises a nucleic acid, a protein, a peptide, a toxin, a chitin, a fatty acid, or a glycogen.