# US 2014/0193804 A1 · Biological Specimen Collection and Transport System and Method of Use

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- Number: US 2014/0193804 A1
- Document ID: US20140193804A1
- Status: Published application
- Kind: A1
- Application number: 14/149,278
- Filed: 2014-01-07
- Published: 2014-07-10
- Earliest claimed: 2007-10-01
- Assignees: Longhorn Vaccines and Diagnostics, LLC
- Applicants: Longhorn Vaccines and Diagnostics, LLC
- Inventors: Gerald W Fischer; Luke T. Daum
- Web page: https://patentgrep.com/patent/US20140193804A1

## CPC

- C12Q 1/70: involving virus or bacteriophage {(immunoassay for viruses G01N33/56983)}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## 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 formations 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 presenting the integrity of nucleic acids released from lysed biological samples without the need of refrigeration or freezing prior to molecular analysis.

## Claims

### Claim 1

A aqueous composition comprising: a) one or more chaotropes; b) one or more detergents; c) one or more reducing agents; d) one or more chelators; and e) one or more buffers, each present in an amount sufficient to denature one or more proteins, inactivate one or more nucleases, kill one or more pathogens, or prevent one or more nucleic acids from degrading in a sample suspected of containing nucleic acids, when the sample is contacted with the composition.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the one or more reducing agents comprise 2-mercaptoethanol, tris(2-carboxyethyl)phosphine, dithiothreitol, dimethylsulfoxide, or any combination thereof.

### Claim 3 (depends on 2)

The composition of claim 2, wherein the one or more reducing agents comprise tris(2-carboxyethyl)phosphine.

### Claim 4 (depends on 1)

The composition of claim 1, comprising: a) one or more chaotropes, each present in an amount from about 0.5 M to about 6 M; one or more detergents, each present in an amount from about 0.1% to about 1% (wt./vol.); c) one or more reducing agents, each present in an amount from about 0.05 M to about 0.3 M; d) one or more chelators, each present in an amount from about 0.01 mM to about 1 mM; and e) one or more buffers, each present in an amount from about 0.0001% to about 0.3% (wt./vol.).

### Claim 5 (depends on 1)

The composition of claim 1, wherein the one or more chaotropes comprise guanidine thiocyanate, guanidine isocyanate, guanidine hydrochloride, or any combination thereof.

### Claim 6 (depends on 1)

The composition of claim 1, 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.

### Claim 7 (depends on 1)

The composition of claim 1, wherein a) the one or more chelators comprise ethylene glycol tetraacetic acid, hydroxyethylethylenediaminetriacetic acid, diethylene triamine pentaacetic 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; or b) the one or more surfactants comprise a silicone polymer, a polysorbate, or any combination thereof.

### Claim 8 (depends on 1)

The composition of claim 1, wherein the at least 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 3-(N-morpholino) propanesulfonic acid, bicarbonate, phosphate, or any combination thereof.

### Claim 9 (depends on 8)

The composition of claim 8, wherein each of the buffers is present it the composition in an amount from about 1 mM to about 1 M.

### Claim 10 (depends on 1)

The composition of claim 1, further comprising one or more short-chain alkanols.

### Claim 11 (depends on 10)

The composition of claim 10, wherein said short-chain alkanols comprise methanol, ethanol, propanol, butanol, pentanol, or hexanol, or any combination thereof.

### Claim 12 (depends on 11)

The composition of claim 11, wherein each of the short-chain alkanols is present in an amount from about 1 to about 25% (vol./vol.).

### Claim 13 (depends on 1)

The composition of claim 1, buffered to a pH of about 6.4 to 6.8.

### Claim 14 (depends on 1)

The composition of claim 1, at least substantially free of RNAse or DNAse activity.

### Claim 15 (depends on 1)

The composition of claim 1, further comprising a defoaming agent that comprises a silicone polymer or a polysorbate.

### Claim 16 (depends on 1)

The composition of claim 1, further comprising a population of isolated polynucleotides that comprises RNA, DNA, or a combination thereof.

### Claim 17 (depends on 1)

The composition of claim 1, comprising: a) about 4 M guanidine thiocyanate; b) about 30 mM sodium citrate; c) about 0.25% (wt./vol.) sodium dodecyl sulfate; d) about 0.25% (wt./vol.) N-lauroyl sarcosine, sodium salt; e) about 0.1 M 2-mercaptoethanol; and f) about 0.1% silicone polymer (wt./vol.).

### Claim 18 (depends on 1)

The composition of claim 1, comprising: a) about 3 M guanidine thiocyanate; b) about 1 mM TCEP; c) about 10 mM sodium citrate; d) about 0.5% N-lauroyl sarcosine; e) about 0.0002% silicone polymer; f) about 100 mM 2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS); and 2) about 0.1 mM EDTA.

### Claim 19 (depends on 1)

The composition of claim 1, comprising: a) about 1 M to about 4 M guanidine thiocyanate; b) about 0.5 mM to 10 mM TCP, about 1 mM to 100 mM sodium citrate; c) about 0.1% to about 1% SDS or NLS; d) about 0.001% to about 0.0001% of a silicone polymer, e) about 10 mM to about 500 mM TRES, f) about 0.1 mM to about 1 mM APCA, EDTA, EGTA, HEDTA DTPA, NTA, or citrate; and g) about 10% to about 25% ethanol (vol./vol.).

### Claim 20 (depends on 1)

The composition of claim 1, comprising: a) about 3 M guanidine thiocyanate; b) 1 mM TCEP; about 10 mM sodium citrate; c) about 0.5% N-lauroyl sarcosine, sodium salt; d) about 0.0002% of a silicone polymer; e) about 100 mM TRIS; f) about 0.1 mM EDTA; and g) about 10% to about 25% ethanol (vol./vol.).

### Claim 21 (depends on 1)

A method for obtaining a population of polynucleotides from a sample suspected of containing nucleic acids, comprising contacting the sample with an amount of a composition in accordance with claim 1, under conditions effective to obtain a population of polynucleotides from the sample.

### Claim 22 (depends on 21)

The method of claim 21, wherein the sample is of clinical, veterinary, epidemiological, environmental, forensic, or pathological origin; or wherein the sample comprises one or mote vital, bacterial, fungal, animal, or plant cells or is suspected of containing a population of nucleic acids.

### Claim 23 (depends on 21)

The method of claim 21, wherein the sample is contacted with the composition at a temperature of from about −20° C. to about 40° C. for a period of from about 24 to about 96 hrs.

### Claim 24 (depends on 21)

The method of claim 21, wherein the integrity of a population of polynucleotides in the sample is at least substantially maintained when the composition comprising the sample is stored at a temperature of from about −20° C. to about 40° C. for a period of from about 7 to about 14 days.

### Claim 25 (depends on 21)

The method of claim 21, wherein the integrity of a population of polynucleotides in the sample is at least substantially maintained when the composition comprising the sample is stored at a temperature of from about −20° C. to about 40° C. for a period of from about 14 to about 30 days.

### Claim 26 (depends on 21)

The method of claim 21, wherein the sample further comprises one or more nucleases, at least a portion of which is at least substantially inactivated by the composition.

### Claim 27 (depends on 21)

The method of claim 21, wherein the sample further comprises one or more pathogens, at least a portion of which is killed or at least substantially inactivated by the composition.

### Claim 28 (depends on 21)

The method of claim 21, wherein one or more biological cells in the sample is at least substantially lysed to release a population of polynucleotides into the composition.

### Claim 29 (depends on 21)

The method of claim 21, wherein the composition comprising the sample is stored substantially at ambient temperature from the time of collection to the time of analyzing a population of polynucleotides therein.

### Claim 30 (depends on 21)

The method of claim 21, wherein the composition comprising the sample is stored at a temperature of from about 10° C. to about 40° C. substantially from the time of collection to the time of isolating, purifying, or characterizing a population of polynucleotides therein.

### Claim 31 (depends on 21)

The method of claim 21, wherein less than about 5% of the population of polynucleotides contained in the sample is degraded after the composition comprising the sample has been stored at a temperature of from about 10° C. to about 40° C. for a period of about 7 to about 30 days.

### Claim 32 (depends on 1)

A sample collection system that comprises: a) a collection device; and b) a collection vessel comprising the composition of claim 1.

### Claim 33 (depends on 32)

The sample collection system of claim 32, wherein the collection device comprises a swab, curette, or culture loop, or any combination thereof; and the collection vessel comprises a vial, tube or specimen cup, or any combination thereof.

### Claim 34 (depends on 1)

A method of preparing a one-step aqueous formulation of claim 1, which comprises:
combining one or more chaotropes and nuclease-free water at a temperature of about 20° C. to 90° C. in a reaction zone;
then combining the dissolved one or more chaotropes with one or more reducing agents, one or more chelators, and one or more detergents in the reaction zone to form an intermediate composition;
optionally combining a silicone polymer with the intermediate composition in an amount sufficient to minimize foaming during further preparation of the one-step aqueous formulation;
combining a sufficient amount of buffer to the intermediate composition to maintain a pH of about 6 to 6.9;
optionally combining a second chelating agent to the reaction zone;
then increasing the temperature of the second intermediate composition to about 60 to 95° C. for about 1 to 30 minutes and lowering the temperature to ambient conditions;
optionally then combining a C1-6alcohol with the contents of the reaction zone; and
optionally adjusting the pH to be about 6.4 to 6.9.

### Claim 35

A method for preparing one-step aqueous formulation adapted to obtain a population of polynucleotides from a biological sample suspected of containing nucleic acids, comprising contacting the sample with an amount of the one-step aqueous formulation effective to:
at least substantially kill or inactivate potentially-infectious pathogens in the sample;
lyse a portion of cells to release RNAs and/or DNAs from the sample; and
substantially inhibit or prevent the released polynucleotides in the sample from further hydrolysis or enzymatic degradation, modification, or inactivation,
so as to obtain the population of polynucleotides from the sample.