AU2012211365B2 - Biological specimen collection and transport system and methods of use - Google Patents

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.

Description

CLAIMS: 1. An aqueous composition that comprises: a chaotrope; a detergent; a chelator; a 5 reducing agent; nuclease-free water, and a buffer with a pH of about 5 to about 7, wherein upon contact of of the composition at ambient temperature with a biological sample suspected of containing a pathogen, nucleic acids and other macromolecules, creates an effective concentration of the composition that, in one step, disinfects said sample, inactivates nuclease of said sample, and extracts nucleic acids from the other macromolecules of the biological 10 sample such that a target sequence of the nucleic acids is detectable by a nucleic acid test. 2. The composition of claim 1, wherein the chaotrope comprises guanidine thiocyanate, guanidine isocyanate, guanidine hydrochloride, or any combination thereof. 15 3. The composition of claim 1 or claim 2, wherein an effective concentration of the chaotrope is about 0.5 M to about 6 M. 4. The composition of any one of the preceding claims, wherein the detergent comprises sodium dodecyl sulfate, lithium dodecyl sulfate, sodium taurodeoxycholate, sodium 20 taurocholate, sodium glycocholate, sodium deoxycholate, sodium cholate, sodium alkylbenzene sulfonate, N-lauroyl sarcosine, or any combination thereof. 5. The composition of any one of the preceding claims, wherein an effective concentration of the detergent is about 0.1% to about 1% (wt/vol). 25 6. The composition of any one of the preceding claims, wherein the chelator comprises ethylene glycol tetra acetic acid, hydroxyethylethylenediaminetriacetic acid, diethylene triamine penta acetic acid, NN-bis(carboxymethyl)glycine, ethylenediaminetetraacetic, citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, 30 diammonium citrate, ferric ammonium citrate, lithium citrate, or any combination thereof. 45 7. The composition of any one of the preceding claims, wherein an effective concentration of the chelator is about 0.5 mM to about 50 mM. 8. The composition of any one of the preceding claims, wherein the reducing agent 5 comprises 2-mercaptoethanol, tris(2-carboxyethyl) phosphine, dithiothreitol, dimethylsulfoxide, tris(2-carboxyethyl) phosphine, or any combination thereof. 9. The composition of any one of the preceding claims, wherein an effective concentration of the reducing agent is about 0.5 mM to about 30 mM. 10 10. The composition of any one of the preceding claims, wherein the buffer comprises tris(hydroxymethyl) aminomethane, citrate, 2-(N-morpholino)ethanesulfonic acid, NN-Bis(2 hydroxyethyl)-2 -aminoethanesulfonic acid, 1,3-bis(tris(hydroxymethyl)methyl amino)propane, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, 3-(N-morpholino) 15 propanesulfonic acid, bicarbonate, phosphate, or any combination thereof. 11. The composition of any one of the preceding claims, wherein an effective concentration of the buffer is about 1 mM to about 1 M.. 20 12. The composition of any one of the preceding claims, further comprising a short-chain alkanol. 13. The composition of claim 12, wherein the short-chain alkanol comprises methanol, ethanol, propanol, butanol, pentanol, hexanol, or any combination thereof 25 14. The composition of claim 12 or claim 13, wherein an effective concentration of the short-chain alkanol is about 1% to about 25% (vol./vol.). 15. The composition of any one of the preceding claims, further comprising a surfactant or 30 anti-foaming agent. 46 16. The composition of claim 15, wherein the surfactant or anti-foaming agent comprises a silicone polymer or a polysorbate. 17. The composition of claim 15 or claim 16, wherein an effective concentration of the 5 surfactant or anti-foam agent is about 0.0001% to about 0.3% (wt./vol.). 18. The composition of any one of the preceding claims, which is diluted prior to contact with the biological sample. 10 19. The composition of any one of the preceding claims, wherein the pH is from about 6 and about 7. 20. The composition of any one of the preceding claims, which contains a predetermined amount of a known nucleic acid sequence as an internal positive control for the nucleic acid 15 test. 21. The composition of any one of the preceding claims, wherein the other macromolecules include one or more of enzymes, lipids, proteins, polysaccharides and biomolecules. 20 22. The composition of any one of the preceding claims, wherein an effective concentration of the chaotrope is about 0.5 M to about 6 M, an effective concentration of the detergent is about 0.1% to about 1% (wt/vol); an effective concentration of the chelator is about 0.5 mM to about 50 mM, and an effective concentration of the reducing agent is about 25 0.5 mM to about 30 mM. 30 47 23. The composition of any one of the preceding claims, wherein the chaotrope comprises guanidine thiocyanate, guanidine isocyanate, guanidine hydrochloride, or any combination thereof; the detergent comprises sodium dodecyl sulfate, lithium dodecyl sulfate, sodium 5 taurodeoxycholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, sodium cholate, sodium alkylbenzene sulfonate, N-lauroyl sarcosine, or any combination thereof; the chelator comprise ethylene glycol tetraacetic acid (EGTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), diethylene triamine pentaacetic acid (DTPA), NN-bis(carboxymethyl)glycine (NTA), ethylenediaminetetraacetic (EDTA), citrate 10 anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, diammonium citrate, ferric ammonium citrate, lithium citrate, or any combination thereof; the reducing agent comprises 2-mercaptoethanol, tris(2-carboxyethyl) phosphine (TCEP), dithiothreitol (DTT), dimethylsulfoxide (DMSO), formamide, or any combination thereof. 15 24. The composition of claim 23, comprising: about 1 M to about 4 M guanidine thiocyanate; about 0.5 mM to 10 mM TCEP; about 1 mM to about 100 mM sodium citrate; about 0.1% to about 1.0% N-lauroyl sarcosine, sodium salt; about 0.001% to about 0.0001% silicone polymer; about 10 mM to about 500 mM TRIS; about 0.1 mM to about 1 mM EDTA; and about 10% to about 25% ethanol (vol./vol.). 20 25. The composition of any one of the preceding claims, which can be maintained at ambient temperatures for greater than 48 hours without reducing the detectability of the target sequence by the nucleic acid test. 25 26. The composition of any one of the preceding claims, wherein the pathogen-specific sequence is detectable in the composition at a concentration of about 0.1 ng or less. 27. The composition of any one of the preceding claims, wherein the nucleic acid test is a polymerase chain reaction (PCR), nucleic acid fingerprinting, genotyping, genetic sequencing 30 or characterization, a single-nucleotide or fragment polymorphism analysis, or a drug sensitivity or resistance determination. 48 28. The composition of claim 27, wherein the pathogen-specific sequence is detectable by the PCR at a Ct value that is lower than the Ct value of a control composition. 5 29. The composition of claim 28, wherein the control composition is water. 30. The composition of any one of the preceding claims, further comprising an anti-viral agent, an anti-microbial agent, or an anti-fungal agent. 10 31. A method of detecting a target sequence in a biological sample comprising: mixing, in one step, the biological sample with a composition at an ambient temperature creating an effective concentration of a chaotrope, a detergent, a chelator, a reducing agent, nuclease-free water, and a buffer with a pH of about 5 to about 7; that is effective to disinfect said sample, inactivate nuclease of said sample, and extract nucleic acids 15 from other macromolecules of the biological sample such that a target sequence within the nucleic acids of the biological sample is detectable by a nucleic acid test; and can be maintained at ambient temperature for a period of time of at least 2 days; performing the nucleic acid test on an aliquot of the mixture to detect the target sequence; and 20 detecting the target sequence in the biological sample. 32. The method of claim 31, wherein the detection of the target sequence is indicative of a disease, a genotype, a single-nucleotide or fragment polymorphism, or an infection of the biological sample, or a pathogen within the biological sample. 25 33. The method of claim 32, wherein the pathogen is an infectious pathogen, a virus, a bacterium, a parasite, a yeast, a fungal cell, a eukaryotic cell, a prokaryotic cell, a bioterrorism agent, a vector, or a microorganism. 30 34. The method of claim 32 or claim 33, wherein the pathogen is an influenza virus or a tuberculosis bacterium. 49 35. The method of any one of claims 31 to 34, wherein the period of time is at least 2 weeks. 5 36. The method of any one of claims 31 to 35, wherein the period of time is at least 4 weeks. 37. The method of any one of claims 32 to 36, wherein the nucleic acid test is a polymerase chain reaction (PCR), nucleic acid fingerprinting, genotyping, genetic sequencing 10 or characterization, a single-nucleotide or fragment polymorphism analysis, or a drug sensitivity or resistance determination. 38. A sample collection system for screening a patient population for the presence or absence of a pathogen comprising: a collection device; and a collection vessel comprising the 15 composition of any one of claims 1 to 30. 50

Claims (18)

1. A method of detecting a target sequence in a biological sample comprising: mixing the biological sample with a nuclease-free aqueous composition buffered to a pH of about 5 to about 7 at an ambient temperature creating an effective concentration,. collectively, of a chaotrope, a detergent, a chelator and a reducing agent, wherein the effective concentration is effective to disinfect said sample, inactivate nuclease of said sample, and extract nucleic acids from other niacromolecules of the biological sample such that a target sequence within the nucleic acids of the biological sample is detectable by a nucleic acid test and can be maintained at ambient temperature for a period of time of at least 2 days: performing the nucleic acid test on an aliquot of the mixture to detect the target sequence; and. detecting the target sequence in the biological sample.

2. The method of claim I, wherein the detection of the target sequence is indicative of a disease, a genotype, a single-nucleotide or fragment polymorphism, or an infection of the biological sample, or a pathogen within the biological sample.

3. The method of claim 2, wherein the pathogen is an infectious pathogen, a virus, a bacterium, a parasite, a yeast, a fingal cell, a eukaryotic cell, a prokaryotic cell, a bioterrorism agent, a vector, or a microorganism.

4. The method of claim 2 or claim 3, wherein the pathogen is an influenza virus or a tuberculosis bacterium.

5. The method of any one of claims 2 to 4, wherein the target sequence is a pathogen specific sequence that is detectable in the composition at a concentration of about 0.Ing or less.

6. The method of any one of claims 1 to 5, wherein the period of time is at least 2 weeks.

7. The method of any one of claims I to 6, wherein the period of time is at least 4 weeks. 45

8. The method of any one of claims I to 7, wherein the nucleic acid test is a polymerase chain reaction (PCR), nucleic acid fingerprinting, genotyping, genetic sequencing or characterization, a single-nucleotide or fragment polymorphism analysis, or a drug sensitivity or resistance determination.

9. The method of claim 8, wherein the target sequence is detectable by the PCR at a Ct value that is lower than the Ct value of a control composition. 10, The method of claim 9, wherein the control composition is water.

11. The method of any one of the claims 1 to 10, wherein the composition is diluted prior to contact with the biological sample.

12. The method of any one of the claims 1 to 11, wherein the composition contains a predetermined amount of a known nucleic acid sequence as an internal positive control for the nucleic acid test. 1.3. The method of any one of the claims 1 to 12, wherein after the mixture has been maintained at ambient temperatures for at least 2 hours, the detectability of the target sequence by the nucleic acid test is not substantially reduced.

14. The method of any one of the claims 1 to 13, wherein the chaotrope comprises guanidine thiocyanate, guanidine isocyanate, guanidine hydrochloride, or any combination thereof; the detergent comprises sodium dodecyl sulfate, lithium dodecyl sulfate, sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate, sodium deoxycholatc, sodium cholate, sodium alkylbenzene sulfonate, N-lauroyl sarcosine, or any combination thereof; the chelator comprise ethylene glycol tetraacetic acid (ECTA), hydroxyethy lethylenediaminetri acetic acid (HEIDTA), diethylene triamine pentaacetic acid (DTP.A), N,N-bis(carboxymethyl)glycine (NTA'), ethylenediaminetetraacetic (EDTA), citrate anhydrous, sodium citrate, calcium citrate, anunonium citrate, ammonium bicitrate., citric acid, diammonium citrate, ferric ammonium citrate, lithium citrate, or any combination thereof; the reducing agent comprises 2-mercaptoethanol, tris(2-carboxyethyl) phosphine 46 (TCEP), dithiothreitol (DTT), diinethylsulfoxide (DMSO), formamide, or any combination thereof.

15. The method of any one of claims 1 to 14, wherein the composition further comprises a short-chain alkanol.

16. The method of claim 15, wherein the short-chain alkanol comprises methanol, ethanol, propanol, butanol, pentanol, hexanol, or any combination thereof

17. The method of claim 15 or claim 16, wherein an effective concentration of the short chain alkanol is about 1% to about 25% (vol,/vol.),

18. The method of claim 17, wherein the composition comprises: about I M to about 4 M guanidine thiocyanate; about 0.5 mM to 10 mM TCEP; about 1 mM to about 100 mM sodium citrate; about 0.1% to about 1.0% N-lauroyl sarcosine, sodium salt; about 0.001% to about 0.0001% silicone polymer; about 10 mM to about 500 mM TRIS; about 0.1 mM to about 1 mM EDTA; and about 10% to about 25% ethanol (vol./vol).

19. A method for screening a patient population for the presence or absence of a pathogen, the method comprising obtaining a biological sample from a patient and detecting the presence of a pathogen specific sequence according to -the method of any one of claims 2 to 5.

20. A sample collection system when used for screening a patient population for the presence or absence of a pathogen according to the method of claim 19: wherein the sample collection system, comprises: a collection device; and a collection vessel comprising an effective concentration of a chaotrope, a detergent, a chelator, a reducing agent, nuclease-free water, and a buffer with a pH of about 5 to about 7 that is effective to disinfect said biological sample, inactivate nuclease of said biological sample, and extract nucleic acids from other macromolecules of the biological sample such that a pathogen specific target sequence within the nucleic acids of the biological sample is detectable by a nucleic acid test; and can be maintained at ambient temperature for a period of time of at least 2 days. 47