Composition and method for stabilizing nucleic acids in biological samples

1. A method of stabilizing nucleic acid contained in a biological sample at ambient temperature comprising the steps of:

a) obtaining a biological sample comprising nucleic acid;

b) contacting the biological sample with an aqueous composition comprising a chelating agent, wherein the chelating agent is present at a concentration of at least 150 mM, and wherein the composition has a pH of greater than 9.5, to form a mixture;

wherein the chelating agent is selected from 1,2-cyclohexanediamine tetraacetic acid (CDTA), diethylenetriamine pentaacetic acid (DTPA), tetraazacyclododecanetetraacetic acid (DOTA), tetraazacyclotetradecanetetraacetic acid (TETA), or desferioximine;

c) homogenizing the mixture of (b) to form a homogeneous mixture; and

d) storing the homogeneous mixture at ambient temperature wherein the nucleic acid is deoxyribonucleic acid (DNA).

2. The method of claim 1 , wherein the biological sample is selected from a fecal sample, a soil sample, a sewage sample, a wastewater sample, or a water sample.

3. The method of claim 2 , wherein the nucleic acid is microbial DNA and the method stabilizes a microbiome profile of the biological sample.

4. The method of claim 3 , wherein the method renders the microbiome profile of the biological sample stable for:

at least 7 days at room temperature;

at least 7 days at a temperature of from about 37° C. to about 50° C.; and/or

at least 30 days at −20° C.

5. The method of claim 3 , wherein the method renders the microbiome profile of the biological sample stable for:

at least 14 days at room temperature; and/or

at least 14 days at a temperature of from about 37° C. to about 50° C.

6. The method of claim 3 , wherein the method renders the microbiome profile of the biological sample stable for:

at least 30 days at room temperature.

7. The method of claim 3 , wherein the method renders the microbiome profile of the biological sample stable for:

at least 60 days at room temperature.

8. The method of claim 1 , wherein the biological sample is a fecal sample obtained from a mammal.

9. The method of claim 8 , wherein the nucleic acid is human DNA.

10. The method of claim 9 , wherein the method renders the human DNA stable for:

at least 7 days at room temperature;

at least 7 days at a temperature of from about 37° C. to about 50° C.; and/or

at least 30 days at −20° C.

11. The method of claim 9 , wherein the method renders the human DNA stable for:

at least 14 days at room temperature; and/or

at least 14 days at a temperature of from about 37° C. to about 50° C.

12. The method of claim 9 , wherein the method renders the human DNA stable for:

at least 30 days at room temperature.

13. The method of claim 9 , wherein the method renders the human DNA stable for:

at least 60 days at room temperature.

14. The method of claim 8 , wherein the mammal is a human.

15. The method of claim 1 , wherein the composition has a pH of from 10.5 to 11.5.

16. The method of claim 1 , wherein the composition further comprises one or more of the following:

(i) at least one buffering agent capable of buffering in the pH range of 9.5 to 11.5; and

(ii) a water-soluble organic solvent.

17. The method of claim 16 , wherein the composition comprises a water soluble organic solvent, the water-soluble organic solvent is ethanol, and the ethanol is present in the composition at a concentration of less than about 24% by volume.

18. The method of claim 16 , wherein the water-soluble organic solvent is a C 1 -C 6 alkanol.

19. The method of claim 1 , wherein the composition further comprises one or more of the following:

(i) a detergent; and

(ii) an antifoaming agent.

20. The method of claim 1 , wherein the mixture is homogenized using a homogenization means.

21. The method of claim 20 , wherein the homogenization means is at least one mixing ball.

22. The method of claim 21 , wherein the method comprises forming the mixture of the biological sample and the composition in a sample container containing the at least one mixing ball, sealing the sample container, and homogenizing the mixture by shaking the mixture in the presence of the at least one mixing ball.

23. The method of claim 22 , further comprising one or more of the following characteristics:

(i) shaking the mixture in the presence of the at least one mixing ball comprises shaking the mixture by hand; and

(ii) the at least one mixing ball is a stainless steel mixing ball or a tungsten carbide mixing ball.

24. The method of claim 22 , further comprising one or more of the following characteristics:

(i) shaking the mixture in the presence of the at least one mixing ball comprises shaking the mixture by hand; and

(ii) the at least one mixing ball is a stainless steel mixing ball having a diameter of about 5.6-11.1 mm and a density of at least about 7.6 g/cm 3 .

25. The method of claim 22 , further comprising one or more of the following characteristics:

(i) shaking the mixture in the presence of the at least one mixing ball comprises shaking the mixture by hand; and

(ii) the at least one mixing ball is a stainless steel mixing ball, wherein the stainless steel mixing ball has a diameter of about 7.1-8.7 mm, and the sample container is a round-bottom tube having an internal diameter of about 12.9 mm.

26. The method of claim 1 , wherein the biological sample is a fecal sample obtained from a mammal, the composition has a pH of from 10.5 to 11.5, and the composition comprises, consists essentially of, or consists of:

CDTA in an amount of from about 250 mM to about 350 mM;

β-alanine in an amount of from about 30 mM to about 70 mM;

ethanol in an amount of from about 21.5% to about 23.5% by volume;

sodium dodecyl sulfate in an amount of from about 0 to about 1% (w/v); and

Antifoam A in an amount of from about 0 to about 0.2% (v/v).

27. The method of claim 26 , wherein the method comprises forming the mixture of the fecal sample and the composition in a round-bottom tube having an internal diameter of about 12.9 mm and containing at least one stainless steel mixing ball having a diameter of about 5.6-11.1 mm and a density of at least about 7.6 g/cm 3 , sealing the round-bottom tube, and homogenizing the mixture by shaking the mixture by hand in the presence of the at least one stainless steel mixing ball.

28. The method of claim 27 , wherein the nucleic acid is microbial DNA, and the method stabilizes a microbiome profile of the fecal sample, wherein the microbiome profile of the fecal sample is rendered stable for:

at least 7 days at room temperature;

at least 7 days at a temperature of from about 37° C. to about 50° C.; and/or

at least 30 days at −20° C.

29. The method of claim 27 , wherein the nucleic acid is microbial DNA, and the method stabilizes a microbiome profile of the fecal sample, wherein the microbiome profile of the fecal sample is rendered stable for:

at least 14 days at room temperature; and/or

at least 14 days at a temperature of from about 37° C. to about 50° C.

30. The method of claim 27 , wherein the nucleic acid is microbial DNA, and the method stabilizes a microbiome profile of the fecal sample, wherein the microbiome profile of the fecal sample is rendered stable for:

at least 30 days at room temperature.

31. The method of claim 27 , wherein the nucleic acid is microbial DNA, and the method stabilizes a microbiome profile of the fecal sample, wherein the microbiome profile of the fecal sample is rendered stable for:

at least 60 days at room temperature.

32. The method of claim 1 , wherein the chelating agent is CDTA.

33. The method of claim 1 , wherein the concentration of the chelating agent is from about 250 mM to about 350 mM.

34. The method of claim 1 , wherein the composition has a pH of 11.

35. The method of claim 1 , wherein the biological sample is a fecal sample obtained from a mammal, the composition has a pH of from 10.5 to 11.5, and the composition comprises, consists essentially of, or consists of:

CDTA in an amount of about 300 mM;

β-alanine in an amount of about 50 mM;

ethanol in an amount of about 23.5% by volume;

sodium dodecyl sulfate in an amount of about 0.5% (w/v); and

Antifoam A in an amount of about 0.1% (v/v).