Compositions and methods for obtaining nucleic acids from sputum

• US 10,619,187 B2
• Filed: 11/07/2016
• Issued: 04/14/2020
• 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
• Primary Examiner(s): Merkling, Sally A
• Application Number:
  US15/345,420
• Publication Number:
  US 20170152545A1
• Time in Patent Office: 1,254 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 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 sealing cap, 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 disestablishment 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. the sealing cap, whereby the device is configured such that, when sealably closing said opening with said sealing cap, the barrier mechanically disestablishes 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.

2. The device of claim 1, wherein the biological sample comprises a bodily fluid.

3. The device of claim 1, wherein the biological sample comprises sputum.

4. The device of claim 1, wherein the biological sample comprises saliva.

5. The device of claim 1, wherein the denaturing agent comprises dodecyl sulfate.

6. The device of claim 1, wherein the chelator comprises ethylenediamine tetraacetic acid (EDTA).

7. The device of claim 1, wherein the reagents further comprise an antimicrobial agent.

8. The device of claim 1, wherein the reagents further comprise an antioxidant free-radical scavenger.

9. The device of claim 1, wherein the barrier comprises a septum.

10. The device of claim 9, wherein the septum is configured to be punctured or pierced to release the reagents to form a mixture of the released reagents and the biological sample.

11. The device of claim 10, wherein the biological sample comprises saliva.

12. The device of claim 11, wherein the denaturing agent comprises dodecyl sulfate.

13. The device of claim 12, wherein the chelator comprises ethylenediamine tetraacetic acid (EDTA).

14. The device of claim 13, wherein the buffering agent comprises TRIS.

15. The device of claim 14, wherein the buffering agent maintains pH of the mixture from about 7.0 and about 10.0.

16. The device of claim 15, wherein the reagents further comprise an antimicrobial agent.

17. The device of claim 16, wherein the reagents further comprise an antioxidant free-radical scavenger.

18. The device of claim 1, wherein the device is configured such that, upon sealably closing said opening with said sealing cap, the barrier is capable of permanent disestablishment.

19. The device of claim 1, wherein the device is configured such that, upon sealably closing said opening with said sealing cap, the barrier is permanently disestablished.

20. The device of claim 1, wherein the device is configured such that, upon sealably closing said opening with said sealing cap, the barrier displaces.

21. The device of claim 20, wherein the barrier is configured to disestablish when displaced by a linear actuator.

22. The device of claim 21, wherein the linear actuator comprises a plunger.

23. The device of claim 1, wherein sealably receiving the sealing cap comprises engaging a thread on the sealing cap and the opening.

24. The device of claim 23, wherein engaging the thread on the sealing cap and the opening comprises exerting a force on the barrier, wherein the force is perpendicular to a direction of rotation of the sealing cap.

25. The device of claim 24, wherein exerting the force on the barrier comprises displacing the barrier.

26. The device of claim 24, wherein a linear actuator exerts the force on the barrier.

27. The device of claim 1, wherein the barrier remains in a disestablished position while the opening is sealably closed by the sealing cap.

28. The device of claim 27, wherein the barrier is capable of returning to a pre-disestablished position when the sealing cap is not sealably closing the opening.

29. The device of claim 27, wherein the barrier is configured to be displaced from a pre-disestablished position to a disestablished position when sealably closing the opening with the sealing cap.

30. The device of claim 1, wherein the barrier remains intact when disestablished.

31. The device of claim 1, wherein sealably receiving the sealing capa comprises engaging the containment vessel and the barrier.

32. The device of claim 31, wherein engaging the containment vessel and the barrier comprises engaging the sealing cap and a plunger, thereby engaging the plunger and the barrier.

33. The device of claim 1, wherein the barrier is configured to disestablish at room temperature.

34. The device of claim 1, wherein the sealing cap associates with the containment vessel to create a fluid-tight seal.

35. The device of claim 1, wherein at least partially dissociating the sealing cap from the containment vessel causes the barrier to close.

36. The device of claim 10, wherein the device further comprises a piercing member and wherein the piercing member is configured to puncture or pierce the septum to release the reagents to form a mixture of the released reagents and the biological sample.