Biological specimen collection and transport system and methods of use

• US 8,293,467 B2
• Filed: 12/20/2011
• Issued: 10/23/2012
• Est. Priority Date: 10/01/2007
• Status: Active Grant

• Current Assignee: Longhorn Vaccines and Diagnostics, LLC
• Original Assignee: Longhorn Vaccines and Diagnostics, LLC
• Inventors: Fischer, Gerald W., Daum, Luke T.
• Primary Examiner(s): HENLEY III, RAYMOND J
• Application Number:
  US13/332,204
• Publication Number:
  US 20120100529A1
• Time in Patent Office: 308 Days
• Field of Search: 435/5, 435/6.12
• US Class Current: 435/5
• 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 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer

Abstract

Disclosed are compositions for isolating populations of nucleic acids from biological, forensic, and environmental samples. Also disclosed are methods for using these compositions as one-step formulations for killing pathogens, inactivating nucleases, and releasing polynucleotides from other cellular components within the sample, and stabilizing the nucleic acids prior to further processing or assay. The disclosed compositions safely facilitate rapid sample collection, and provide extended storage and transport of the samples at ambient or elevated temperature without contamination of the sample or degradation of the nucleic acids contained therein. This process particularly facilitates the collection of specimens from remote locations, and under conditions previously considered hostile for preserving the integrity of nucleic acids released from lysed biological samples without the need of refrigeration or freezing prior to molecular analysis.

Claims

1. A method for denaturing proteins, inactivating nucleases and killing pathogens in one step without degrading nucleic acid of a biological sample containing proteins, nucleases, and nucleic acid, and suspected to contain pathogens, comprising:

providing a mixture containing one or more chaotropes, one or more detergents, one or more reducing agents, one or more chelators, and one or more buffers, together present in an amount sufficient to denature proteins, inactivate nucleases, kill pathogens, and not degrade nucleic acid;

contacting the biological sample with the mixture which denatures proteins, inactivate nucleases, kill pathogens, and does not degrade nucleic acid of the biological sample; and

detecting the presence and identity of or absence of the pathogens in the biological sample.

2. The method of claim 1, wherein the one or more chaotropes are present in an amount from about 0.5 M to about 6 M, the one or more detergents are present in an amount from about 0.1% to about 1% (wt./vol.);

the one or more reducing agents are present in an amount from about 0.5 mM to about 0.3 M;

the one or more chelators are present in an amount from about 0.01 mM to about 1 mM; and

the one or more buffers are present in an amount from about 1 mM to about 1 M.

3. The method of claim 2, wherein the one or more chaotropes comprise guanidine thiocyanate, guanidine isocyanate, guanidine hydrochloride, or any combination thereof.

4. The method of claim 2, wherein the one or more detergents comprise sodium dodecyl sulfate, lithium dodecyl sulfate, sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, sodium cholate, sodium alkylbenzene sulfonate, N-lauroyl sarcosine, or any combination thereof.

5. The method of claim 2, wherein the one or more reducing agents comprise 2-mercaptoethanol, tris(2-carboxyethyl)phosphine, dithiothreitol, dimethylsulfoxide, tris(2-carboxyethyl)phosphine, or any combination thereof.

6. The method of claim 2, wherein the one or more chelators comprise ethylene glycol tetra acetic acid, hydroxyethylethylenediaminetriacetic acid, diethylene triamine penta acetic acid, N,N-bis(carboxymethyl)glycine, ethylenediaminetetraacetic, citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, diammonium citrate, ferric ammonium citrate, lithium citrate, or any combination thereof.

7. The method of claim 2, wherein the one or more buffers comprise tris(hydroxymethyl)aminomethane, citrate, 2-(N-morpholino)ethanesulfonic acid, N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 1,3-bis(tris(hydroxymethyl)methyl amino)propane, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, 3-(N-morpholine)propanesulfonic acid, bicarbonate, phosphate, or any combination thereof.

8. The method of claim 2, wherein the mixture further comprises one or more short-chain alkanols present in an amount from about 1% to about 25% (vol./vol.).

9. The method of claim 8, wherein the one or more short-chain alkanols comprise methanol, ethanol, propanol, butanol, pentanol, hexanol, or any combination thereof.

10. The method of claim 2, wherein the mixture further comprises one or more surfactants or defoaming agents present in an amount from about 0.0001% to about 0.3% (wt./vol.).

11. The method of claim 10, wherein the one or more surfactants or defoaming agents comprise silicone polymer, polysorbate, antifoam A, Tween, or any combination thereof.

12. The method of claim 2, wherein the pathogens are influenza virus particles or influenza-infected cells, bacteria that causes tuberculosis or tuberculosis-infected cells.

13. The method of claim 1, wherein the mixture further comprises added naked RNA or DNA present in an amount of about 1 pg/mL to 1 μ

g/mL.

14. The method of claim 13, further comprising detecting the presence or absence of the added naked DNA or RNA.

15. The method of claim 2, wherein the mixture is buffered to a pH of from 5 to 7.

16. The method of claim 15, wherein the mixture is buffered to a pH of from about 6.4 to 6.9.

17. The method of claim 2, wherein the biological sample contains viral particles, virally infected cells, bacteria, bacterially infected cells, fungal organisms, animal cells, or plant cells.

18. The method of claim 17, wherein contacting comprises detectably liberating nucleic acid contained within the particles, cells or organisms of the biological sample.

19. The method of claim 18, wherein nucleic acid of the biological sample is detectable by PCR analysis.

20. The method of claim 19, wherein the nucleic acid of the biological sample remains detectable by PCR analysis after the mixture containing the biological sample is stored at a temperature of 4C or more for a period of at least 14 days.

21. The method of claim 20, wherein about 5% or less of the nucleic acids of the biological sample are degraded after the mixture containing the biological sample is stored at a temperature of 12C or more for a period of at least 14 days.

22. The method of claim 19, wherein the nucleic acid of the biological sample remains detectable by PCR analysis after the mixture containing the biological sample is stored at a temperature of 4C or more for a period of at least 30 days.

23. The method of claim 22, wherein the nucleic acid of the biological sample remains detectable by PCR analysis after the mixture containing the biological sample is stored at a temperature of 12C or more for a period of at least 30 days.

24. A method for denaturing proteins, inactivating nucleases and killing pathogens in one step without degrading nucleic acid of a biological sample containing proteins, nucleases, and nucleic acid, and suspected to contain pathogens, comprising:

providing a mixture containing one or more chaotropes, one or more detergents, one or more reducing agents, one or more chelators, one or more buffers, one or more short-chain alkanols, and one or more surfactants, together present in an amount sufficient to denature proteins, inactivate nucleases, kill pathogens, and not degrade nucleic acid;

contacting the biological sample with the mixture which denatures proteins, inactivate nucleases, kill pathogens, and does not degrade nucleic acid of the biological sample, and

detecting the presence and identity of or absence of the pathogens in the biological sample, wherein,

said one or more chaotropes are selected from the group consisting of guanidine thiocyanate, guanidine isocyanate, guanidine hydrochloride, and any combination thereof;

said one or more detergents are selected from the group consisting of sodium dodecyl sulfate, lithium dodecyl sulfate, sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, sodium cholate, sodium alkylbenzene sulfonate, N-lauroyl sarcosine, and any combination thereof;

said one or more chelators are selected from the group consisting of ethylene glycol tetra acetic acid, hydroxyethylethylenediaminetriacetic acid, diethylene triamine penta acetic acid, N,N-bis(carboxymethyl)glycine, ethylenediaminetetraacetic, citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, diammonium citrate, ferric ammonium citrate, lithium citrate, and any combination thereof; and

said one or more buffers are selected from the group consisting of tris(hydroxymethyl)aminomethane, citrate, 2-(N-morpholino)ethanesulfonic acid, N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 1,3-bis(tris(hydroxymethyl)methyl amino)propane, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, 3-(N-morpholino) propanesulfonic acid, bicarbonate, phosphate, and any combination thereof; and

said one or more short-chain alkanols are selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol, or hexanol, and any combination thereof;

which are together present in an amount sufficient to denature proteins, inactivate nucleases;

kill pathogens, and not substantially degrade nucleic acid of the biological sample upon contacting the mixture.

25. The method of claim 24, wherein:

the one or more chaotropes are present in an amount from about 0.5 M to about 6 M;

the one or more detergents are present in an amount from about 0.1% to about 1% (wt./vol.);

the one or more reducing agents are present in an amount from about 0.5 mM to about 0.3 M;

the one or more chelators are present in an amount from about 0.01 mM to about 1 mM;

the one or more buffers are present in an amount from about 0.0001% to about 0.3% (wt./vol.);

the one or more short-chain alkanols are present in an amount from about 1 to about 25% (vol./vol.); and

the one or more surfactants are present in an amount from about 0.0001% to about 0.3%.

26. The method of claim 25, wherein the mixture further contains naked RNA or DNA that has a predetermined nucleic acid sequence as an internal positive control that is detectable by PCR analysis.

27. The method of claim 25, wherein the pathogens are influenza virus particles or influenza virus-infected cells, bacteria that causes tuberculosis or tuberculosis-infected cells.

28. The method of claim 25, wherein the biological sample contains whole blood, plasma, serum, sputum, urine, stool, a white blood cell, a red blood cell, buffy coat, tears, mucus, saliva, semen, vaginal fluid, lymphatic fluid, amniotic fluid, spinal or cerebrospinal fluid, a peritoneal effusion, a pleural effusion, an exudate, a punctuate, an epithelial smear, a biopsy, a bone marrow sample, fluid from a cyst or an abscess, synovial fluid, vitreous or aqueous humor, an eye wash or aspirate, a pulmonary lavage or lung aspirate, or any combination thereof.

29. A method for denaturing proteins, inactivating nucleases and killing pathogens in one step without degrading nucleic acid of a biological sample containing proteins, nucleases, and nucleic acid comprising:

providing a mixture containing one or more chaotropes, one or more detergents, one or more reducing agents, one or more chelators, and one or more buffers, together present in an amount sufficient to denature proteins, inactivate nucleases, kill pathogens, and not degrade nucleic acid;

providing the biological sample suspected to contain pathogens;

contacting the biological sample with the mixture which liberates nucleic acid of the pathogens for PCR analysis;

PCR amplifying the nucleic acid, if present; and

determining the presence or absence of the pathogens in the biological sample.

30. The method of claim 29, wherein the one or more chaotropes are present in an amount from about 0.5 M to about 6 M, the one or more detergents are present in an amount from about 0.1% to about 1% (wt./vol.);

the one or more reducing agents are present in an amount from about 0.5 mM to about 0.3 M;

the one or more chelators are present in an amount from about 0.01 mM to about 1 mM;

the one or more buffers are present in an amount from about 100 mM to about 1M and the pH of the mixture is about 6.2 to 6.9.

31. A method for denaturing proteins, inactivating nucleases and killing pathogens in one step without degrading nucleic acid of a biological sample containing proteins, nucleases, and nucleic acid, and suspected to contain pathogens, comprising:

providing a mixture containing one or more chaotropes, one or more detergents, one or more reducing agents, one or more chelators, and one or more buffers, together present in an amount sufficient to denature proteins, inactivate nucleases, kill pathogens, and not degrade nucleic acid, wherein the one or more chaotropes are present in an amount from about 0.5 M to about 6 M, the one or more detergents are present in an amount from about 0.1% to about 1% (wt./vol.);

the one or more reducing agents are present in an amount from about 0.5 mM to about 0.3 M;

the one or more chelators are present in an amount from about 0.01 mM to about 1 mM; and

the one or more buffers are present in an amount from about 1 mM to about 1M.

32. The method of claim 31, wherein the one or more chaotropes comprise guanidine thiocyanate, guanidine isocyanate, guanidine hydrochloride, or any combination thereof;

the one or more detergents comprise sodium dodecyl sulfate, lithium dodecyl sulfate, sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, sodium cholate, sodium alkylbenzene sulfonate, N-lauroyl sarcosine, or any combination thereof;

the one or more reducing agents comprise 2-mercaptoethanol, tris(2-carboxyethyl)phosphine, dithiothreitol, dimethylsulfoxide, tris(2-carboxyethyl)phosphine, or any combination thereof;

the one or more chelators comprise ethylene glycol tetra acetic acid, hydroxyethylethylenediaminetriacetic acid, diethylene triamine penta acetic acid, N,N-bis(carboxymethyl)glycine, ethylenediaminetetraacetic, citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, diammonium citrate, ferric ammonium citrate, lithium citrate, or any combination thereof;

the one or more buffers comprise tris(hydroxymethyl)aminomethane, citrate, 2-(N-morpholino)ethanesulfonic acid, N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 1,3-bis(tris(hydroxymethyl)methyl amino)propane, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, 3-(N-morpholino) propanesulfonic acid, bicarbonate, phosphate, or any combination thereof; and

the mixture is buffered to a pH of from about 5 to 7.

33. The method of claim 31, wherein the mixture is buffered to a pH of from about 6.4 to 7.0.

34. The method of claim 31, further comprising contacting the biological sample with the mixture which denatures proteins, inactivate nucleases, kill pathogens, and does not degrade nucleic acid of the biological sample.

35. The method of claim 34, wherein contact with the composition is performed in one vessel and, after contact, the biological sample is non-infectious for transport.

36. The method of claim 35, wherein the contacted biological sample does not require refrigeration for subsequent detection of the presence or absence of pathogens.

37. The method of claim 36, wherein detection of the presence and identity of or absence of the pathogens can be determined for at least 7 days after contact with the mixture.

38. The method of claim 35, wherein the contacted biological sample is at an ambient temperature for a period of time prior to the detection of the presence or absence of pathogens.

39. The method of claim 38, wherein the period of time is at least 48 hours.

40. The method of claim 31, wherein the mixture further comprises one or more short-chain alkanols present in an amount from about 1% to about 25% (vol./vol.);

one or more surfactants or defoaming agents present in an amount from about 0.0001% to about 0.3% (wt./vol.); and

added naked RNA or DNA present in an amount of about 1 pg/mL to 1 μ

g/mL.

41. The method of claim 40, wherein the one or more short-chain alkanols comprise methanol, ethanol, propanol, butanol, pentanol, hexanol, or any combination thereof;

and the one or more surfactants or defoaming agents comprise silicone polymer, polysorbate, antifoam A, Tween, or any combination thereof.

42. The method of claim 40, further comprising detecting the presence or absence of the added naked DNA or RNA.