Biological specimen collection and transport system
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.
1. A stock solution comprising components with concentrations as follows:
a chaotrope present in an amount from 0.5 M to 6M;
a detergent present in an amount from 0.1% to 1% (wt./vol.);
a reducing agent in an amount from 0.5 mM to 0.3 M;
a chelator present in an amount from 0.01 mM to 50 mM;
a surfactant present in an amount from 0.0001% to 0.3% (wt./vol.);
a short-chain alkanol present in an amount from 1 to 25% (vol./vol.);
a buffer present in an amount from 1 mM to 1 M;
an acid or base present in an amount that provides a pH of from 5 to 7 to the stock solution; and
nuclease-free water,
wherein the stock solution, when combined with a biological sample suspected of containing a pathogen, denatures proteins, inactivates nucleases, and kills pathogens all without degrading nucleic acids of the sample.
2. The stock solution of claim 1 , wherein the chaotrope comprises guanidine thiocyanate, guanidine isocyanate, guanidine hydrochloride or a combination thereof.
3. The stock solution of claim 1 , wherein the detergent comprises sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (LDS), sodium taurodeoxycholate (NaTDC), sodium taurocholate (NaTC), sodium glycocholate (NaGC), sodium deoxycholate (NaDC), sodium cholate, sodium alkylbenzene sulfonate (NaABS), N-lauroyl sarcosine (NLS), salts of carboxylic acids, salts of sulfonic acids, salts of sulfuric acid, phosphoric and polyphosphoric acid esters, alkylphosphates, monoalkyl phosphate (MAP), salts of perfluorocarboxylic acids, anionic detergents or a combination thereof.
4. The stock solution of claim 1 , wherein the reducing agent is β-ME, DTT, DMSO or formamide present in an amount from 0.05 M to 0.3 M, or wherein the reducing agent is TCEP present in an amount from 0.5 mM to 30 mM.
5. The stock solution of claim 1 , wherein the chelator comprises 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 a combination thereof.
6. The stock solution of claim 1 , wherein the surfactant comprises cocoamidopropyl hydroxysultaine, alkylaminopropionic acids, imidazoline carboxylates, betaines, sulfobetaines, sultaines, alkylphenol ethoxylates, alcohol ethoxylates, polyoxyethylenated polyoxypropylene glycols, polyoxyethylenated mercaptans, long-chain carboxylic acid esters, alkonolamides, tertiary acetylenic glycols, polyoxyethylenated silicones, N-alkylpyrrolidones, alkylpolyglycosidases, silicone polymers such as Antifoam A®, or polysorbates such as Tween®, or a combination thereof.
7. The stock solution of claim 1 , wherein the short-chain alkanol comprises methanol, ethanol, propanol, butanol, pentanol, hexanol or a combination thereof.
8. The stock solution of claim 1 , wherein the buffer comprises 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 a combination thereof.
9. The stock solution of claim 1 , wherein the pH is 6.7±0.25.
10. The stock solution of claim 1 , wherein the pathogen comprises a virus or a bacterium.
11. The stock solution of claim 1 , wherein the pathogen is hepatitis virus, papillomavirus, HIV, biological agent of SARS, corona virus, rotavirus, Influenza virus, Ebola virus, methicillin-resistant Staphylococcus , or M. tuberculosis.
12. The stock solution of claim 1 , further comprising an internal positive control.
13. The stock solution of claim 1 , further comprising an anti-microbial agent, an anti-viral, agent, an antifungal agent, or a combination thereof.
14. The stock solution of claim 1 , further comprising betadine, bovine serum albumin, an osmolyte or a combination thereof.
15. A method for detecting a nucleic acid sequence that is indicative of the presence of the pathogen in the sample comprising, at an ambient temperature, contacting the biological sample with an amount of the stock solution of claim 1 forming a composition which is effective to kill pathogens and inactivate nucleases in the sample without degrading or modifying nucleic acid of the sample, so that the nucleic acid sequence of the pathogen is detectable by PCR amplification.
16. The method of claim 15 , wherein the pathogen comprises bacteria, virus, or a fungus; optionally, wherein the bacteria is tuberculosis or the virus is influenza.
17. A diagnostic kit containing the stock solution of claim 1 .
18. The diagnostic kit of claim 17 , which does not require refrigeration.
19. The diagnostic kit of claim 17 , further comprising one or more nucleic acid extraction devices.
20. The diagnostic kit of claim 17 , wherein the stock solution is contained in one vessel and further comprising a separate vessel containing one or more additional reagents, buffers, or compounds.