BIOLOGICAL SPECIMEN COLLECTION AND TRANSPORT SYSTEM

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.-37. (canceled)

38. An aqueous composition that maintains fidelity of a nucleic acid sequence contained with a biological sample when mixed with the sample and maintained for a period of time without refrigeration, comprising components that upon mixing sterilize the sample, and denature proteins and inactivate nucleases of the sample, wherein the fidelity of the nucleic acid sequence as determined by PCR before and after mixing is within three CTs, and the period of time is at least 7 days.

39. The composition of claim 38, wherein the sample is selected from the group consisting of blood, plasma, serum, sputum, urine, stool, buccal swabs, throat swabs, vaginal swabs, urethral swabs, cervical swabs, rectal swabs, lesion swabs, abscess swabs, nasopharyngeal swabs, tears, mucus, saliva, semen, vaginal fluids, lymphatic fluid, amniotic fluid, spinal fluid, cerebrospinal fluid, peritoneal effusions, pleural effusions, exudates, punctates, epithelial smears, biopsies, bone marrow samples, fluid from a cyst or abscess, synovial fluid, vitreous or aqueous humor, eye washes or aspirates, pulmonary lavage or lung aspirates, an organ or tissue, and any combination thereof.

40. The composition of claim 38, which comprises a chaotrope, a detergent; a reducing agent, a chelator, and a buffer.

41. The composition of claim 40, wherein the chaotrope comprises from about 0.5 M to about 6M, the detergent comprises from about 0.1% to about 1% (wt./vol.), the reducing agent comprises from about 0.5 mM to about 0.3 M, the chelator comprises from about 0.5 mM to about 50 mM, and the buffer comprises from about 1 mM to about 1 M.

42. The composition of claim 41, which further comprises a surfactant and a short-chain alcohol.

43. The composition of claim 42, wherein the surfactant comprises from about 0.0001% to about 0.3% (wt./vol.), and the short-chain alkanol comprises from about 1% to about 25% (vol./vol.).

44. The composition of claim 38, wherein the period of time is at least 14 days.

45. The composition of claim 38, wherein the period of time is at least 21 days.

46. The composition of claim 38, wherein the period of time is at least 28 days.

47. The composition of claim 38, wherein the period of time is several months.

48. The composition of claim 38, which is transported for analyzing fidelity of nucleic acids.

49. The composition of claim 38, wherein the fidelity of the nucleic acid sequence before and after mixing is within two CTs.

50. The composition of claim 38, wherein the fidelity of the sequence is within three CTs for an RNA sequences and/or within two CTs for a DNA sequence.

51. The mixture comprising the composition of claim 38 and a biological sample.

52. The mixture of claim 51, which is safe for handling and transportation.

53. The mixture of claim 51, which is maintained at room temperature.

54. The mixture of claim 51, wherein room temperature is from 15 C. to 30 C.

55. The mixture of claim 51, wherein room temperature is from 18 C. to 28 C.

56. An aqueous composition that maintains fidelity of a nucleic acid sequence contained with a biological sample when mixed with the sample and maintained for a period of time without refrigeration, comprising components that upon mixing sterilize the sample, and denature proteins and inactivates nucleases of the sample, wherein the fidelity of the nucleic acid sequence as determined by PCR before and after mixing is within three CTs for an RNA sequence and within two CTs for a DNA sequence, and the period of time is at least 7 days.

57. The composition of claim 56, wherein the period of time is at least 14 days.

58. The composition of claim 56, wherein the period of time is at least 21 days.

59. The composition of claim 56, wherein the period of time is at least 28 days.

60. The composition of claim 56, wherein the period of time is several months.

Description

DESCRIPTION OF THE DRAWINGS

[0060] For promoting an understanding of the principles of the invention, reference will now be made to the embodiments, or examples, illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. Any alterations and further modifications in the described embodiments, and any further applications of the principles of the invention as described herein are contemplated as would normally occur to one of ordinary skill in the art to which the invention relates.

[0061] The following drawings form part of the present specification and are included to demonstrate certain aspects of the present invention. The invention may be better understood by reference to the following description taken in conjunction with the accompanying drawings, in which like reference numerals identify like elements, and in which:

[0062] FIG. 1 shows the extraction efficiency of PrimeStore (ver. 1). PrimeStore (ver. 1 [depicted here as One-step+]) compared to the Lysis Solution provided in the RNaqueous-Micro Kit (Ambion, Cat#AM1931) using a standard amount of whole influenza A virus. For the comparison either the one-step formulation or the Lysis Solution provided in the kit was used for viral RNA lyses and then extracted according to manufacturer protocols. Replicate reactions were processed and analyzed by real-time RT-PCR (rRT-PCR) using an ABI 7500 sequence detection system;

[0063] FIG. 2 shows the extraction efficiency of PrimeStore Solution (ver. 1) compared to commercial kits. Homogenized cotton rat nose(*) challenged with influenza A (H3N2) or a human clinical influenza A (H1N1) samples collected during the 2006-07 season were lysed in the PrimeStore Solution or lysed using the respective lyses solution, protocol, and extraction procedure from three commercially available kits: RNaqueous-Micro (Ambion Cat#AM1931), QiaAmp Viral Mini Kit (Qiagen), and AI/NCD MaxMag (Ambion) Kit. Extraction efficiency was evaluated using the ABI 7500 with the comparative CT method. The relative CT scores and viral copies detected were optimal when PrimeStore (depicted as the one-step formulation) was utilized in place of the respective lyses buffer for each commercial kit;

[0064] FIG. 3 shows the preservation of naked RNA in PrimeStore Solution vs. Ambion RNA Storage Solution Single-stranded Avian H5 RNA was stored in PrimeStore solution, RNA storage solution (Ambion), or water at ambient temperature (22-24 C.) for 96 hours. A total of 5 pg of RNA was extracted using the RNaqueous-Micro Kit (Ambion, Cat#AM1931) according to manufacturer recommendations and analyzed using real-time RT-PCR on an ABI 7500 (Applied Biosystems). Values are given as cycle thresholds (CT) using the absolute quantification method;

[0065] FIG. 4A shows an example of a PrimeStore packaging format for clinical diagnostic collection;

[0066] FIG. 4B Directions of sample collection using a clinical collection swab (Copan Diagnostics);

[0067] FIG. 4C a 5 mL collection tube containing 1.5 mL of PrimeStore Solution;

[0068] FIG. 4D a schematic demonstration of the manipulation of the system;

[0069] FIG. 4E a schematic demonstration of the manipulation of the system;

[0070] FIG. 5 shows an exemplary commercial PrimeStore Collection Solution. Three exemplary commercial collection solution formats: 25 mL bottle, and the 5 mL and 1.5 mL tube formats;

[0071] FIG. 6 illustrates the ability of PrimeStore Solution to rapidly kill microorganisms. Shown is a comparison of cell growth of MRSA either in culture medium (TSB), or in a solution of PS. After 10 seconds in PrimeStore Solution, no viable bacterial pathogens were detected.

[0072] FIG. 7A shows the inactivation of chicken cloacal specimens in PrimeStore Solution (Ver. 1). PrimeStore Solution inactivates microbial agents in 1 hr. Four original chicken cloacal samples were immersed in PrimeStore Solution (top row) or water (bottom row) and subsequently plated on blood agar plates;

[0073] FIG. 7B demonstrates that PrimeStore inhibits RNA base hydrolysis for 30 days at room temperature. RNA was incubated at room temperature (22-26 C.) in PrimeStore (gel lane 1 and 3) and water (gel lane 2 and 4), and subsequently RT-PCR amplified (1500 base pairs) at Day 0 and Day 30. PrimeStore preserved collected RNA, and prevented RNA/DNA degradation at room temperature up to 30 days;

[0074] FIG. 8A depicts the real-time RT-PCR analysis of naked influenza A avian H5 RNA template preserved in PrimeStore Solution after incubation in RNA/DNA nucleases. H5 cRNA (2 ng) was incubated with ribonuclease A and T1, and DNAseI for 1 hour @ 37 C. and extracted using the RNAaqueous-Micro Kit (Ambion). Triplicate reactions were included for each reaction condition. Real-time RT-PCR Cycle threshold (CT) values of naked RNA preserved in PrimeStore with added nucleases (average CT: 22.88) were similar to an equal quantity of template cRNA control (average CT: 23.70). Template cRNA reactions subjected to nuclease digestion without PrimeStore were almost completely degraded (average CT 39.58);

[0075] FIG. 8B depicts the real-time RT-PCR analysis and gel electrophoresis of naked influenza A avian H5 RNA template preserved in PrimeStore Solution after incubation in RNA/DNA nucleases @ 37 C. for 7 days. Two nanograms of H5 cRNA was incubated with RNase A and T1, and DNase I, then extracted using the RNAaqueous-Micro Kit (Ambion) after 7 days. Duplicate reactions were included for each reaction. Real-time RT-PCR Cycle Threshold (CT) values of naked RNA preserved in PrimeStore with added nucleases (average CT: 33.51) were detected after 7 days. Template cRNA reactions subjected to nuclease digestion without PrimeStore were completely degraded and similar to NTC reactions.

[0076] FIG. 8C demonstrates that PrimeStore is impervious to nuclease digestion. Gel electrophoresis of post-amplified product. Lane 3 is the PCR product from template RNA+PrimeStore at 37 C. for 7 days, and Lane 5 amplification of positive control RNA. Lane 5 (no amplification) is RNA without PrimeStore Lane 2 and 6 are 100 bp ladder, and NTC reactions, respectively.

[0077] FIG. 9 illustrates that PrimeStore preservation is superior to other solutions. PrimeStore (Ver. 2 and Ver. 2.2) Preservation of RNA from influenza A virus compared to Qiagen AVL buffer, ethanol, and Viral Transport Media (VTM) at ambient temperature (22-25 C.) for 30 days.

[0078] FIG. 10 shows the extraction efficiency of Influenza A virus preserved in PrimeStore (Ver. 2.2) for 30 days at various temperatures. Environmental (21-37 C.); Freeze-thawed (25 C.; 32); ambient temperature (22-26 C.); and Lane 5: refrigerated (4 C.).

[0079] FIG. 11 is a graph of critical threshold vs. molar concentration using whole influenza A virus with TCEP as the reducing agent;

[0080] FIG. 12 is a graph of the critical threshold vs. molar concentration using H5N1 Avian influenza ssRNA with TCEP as the reducing agent;

[0081] FIG. 13A and FIG. 13B show the comparison between TCEP and -ME as reducing agent components of the PrimeStore Solution compositions, using a water only control. In FIG. 13A, H5 avian influenza RNA was employed, while in FIG. 13B, whole virus were used.

[0082] FIG. 14A shows the results of a study employing PrimeStore solution in preserving nucleic acids from blood. PrimeStore Extraction Efficiency of whole blood spiked with RNA compared to the lysis solution in the QIAamp DNA Blood Mini Kit. 0.1 pg and 1 pg of influenza A RNA were spiked and extracted using PrimeStore or AL Lysis buffer. At both RNA concentrations, PrimeStore produced superior results as evident by real-time RT-PCR CT scores;

[0083] FIG. 14B tabulates data from the study shown in FIG. 14A involving the extraction of naked H5 avian influenza ssRNA from blood tubes. PrimeStore Extraction Efficiency of whole blood spiked with RNA compared to the lysis solution in the QIAamp DNA Blood Mini Kit using different blood anticoagulants. PrimeStore was superior compared to the AL Lysis Buffer from Qiagen using Blood spiked with RNA in common anticoagulant blood-collection tubes; and

[0084] FIG. 14C tabulates data from the study shown in FIG. 14A involving the comparison of in PrimeStore Solution vs. a commercial extraction kit (Qiagen). Shown is the Extraction Efficiency of PrimeStore for whole blood spiked with RNA compared to the lysis solution in the QIAamp DNA Blood Mini Kit. 0.1 pg and 1 pg of Influenza A viral RNA were spiked and extracted using PrimeStore or AVL Lysis buffer. At both RNA concentrations, PrimeStore produced superior results as evident by real-time RT-PCR CT scores.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0085] Illustrative embodiments of the invention are described below. In the interest of clarity, not all features of an actual implementation are described in this specification. It will of course be appreciated that in the development of any such actual embodiment, numerous implementation-specific decisions must be made to achieve the developers' specific goals, such as compliance with system-related and business-related constraints, which will vary from one implementation to another. Moreover, it will be appreciated that such a development effort might be complex and time-consuming, but would be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure.

[0086] The extended stabilization, collection, transport, and preservation imparted by the disclosed formulations are particularly advantageous when a sample or specimen is located in a geographical region that is remote from a testing facility. Remote locations, also referred to as field sites, encompass a variety of environments where diagnostic testing is typically not performed. These sites include doctors' offices, triage centers, airports, border crossings, outbreak areas, and a variety of outdoor locations. The disclosed compositions and methods for their use offer particular advantages in locations where there is no access to electricity and/or refrigeration, or where access is inconsistent. Because of the extended stability at room temperature, a sample can be taken from any remote location, for example without limitation at a malarial outbreak site in Africa, and the sample can be shipped to the United States or Europe for diagnostic analysis in a laboratory. Because the disclosed collection formulations are stable at room temperature or below, and preferably even at tropical or subtropical temperatures for a time, they can routinely be taken into the field without worry that the component reagents (such as RNA controls) themselves will degrade until a sample can be analyzed, typically at a remote location from the collection.

[0087] The compositions of the invention may be any suitable aqueous formulation as described herein, including but not limited to a solution, suspension (incl. colloidal suspension), slurry, emulsion, homogenate, or the like. A preferred aqueous formulation is a solution, and therefore the term solution has been used in the exemplary sense throughout the detailed description of the preferred embodiments to refer to any of the aqueous compositions of the invention.

Specimen Collection for Clinical Diagnostic Laboratories

[0088] Collection is first step in diagnostic platforms or molecular protocols requiring the detection of potentially minute amounts of nucleic acids from pathogens including viruses. To facilitate the dynamic advancements in nucleic acid based detection strategies and their integration into the mainstream diagnostic laboratories there is a colossal need for reliable, robust, and standardized collection systems developed specifically with the intent of being utilized for downstream nucleic acid based detection such as the aforementioned platforms. The invention can alternatively be adapted for transport of nucleic acids from a doctor's office or operating room, or alternatively transported to a regional center, such as a hospital.

[0089] A clinical or veterinary specimen or a forensic or environmental sample collection system may include one or more collection tools and one or more reagents for efficiently: 1) obtaining a high yield of suitable specimen beyond what is currently available in the art; 2) inactivating potentially infectious biological pathogens so that they are no longer viable and can be handled; shipped, or transported with minimal fear of pathogen release or contamination; or 3) effectively stabilizing and preserving lysed naked RNA/DNA polymers from hydrolysis or nuclease degradation for prolonged periods at ambient temperatures until samples can be processed at a diagnostic laboratory, and preferably for achieving two or more, or all three, of these goals.

[0090] The collection/transport solutions of the present invention can provide a number of improvements and benefits over those presently available in the art. Exemplary benefits include, without limitation, one or more of the following:

[0091] Inactivation, killing, and/or lysis of microbes, viruses, or pathogens;

[0092] Destruction and/or inactivation of exogenous or endogenous nucleases, including, without limitation, RNase and/or DNase;

[0093] Compatibility with a variety of conventional nucleic acid extraction, purification, and amplification systems;

[0094] Preservation of RNA and/or DNA integrity within the sample;

[0095] Facilitation of transport and shipping at ambient temperatures, even over extended periods of time, or extreme temperature variations; and

[0096] Suitability for short- (several hours to several days), intermediate- (several days to several weeks), or long- (several weeks to several months) term storage of the isolated nucleic acids.

[0097] The disclosed compositions are particularly well suited for point-of-care, field studies, in-home health care or testing, triage/emergency and casualty assessment(s), mobile forensics, pathology, epidemiological sampling, crime scene investigation, paternity testing, pre- and post-pregnancy genetic screening, rape/incest testing and family counseling, confidential screening and testing for sexually transmitted diseases, including, without limitation, HIV, syphilis, Chlamydia, gonorrhoeae, or other venereal diseases and the like, and may be of particular value during the monitoring, etiology, and control of epidemic or pandemic diseases in both human and animal populations domestically and abroad. The compositions may be of particular relevance in collecting and analyzing Influenzavirus samples, including without limitation to predict and help manage shift and drift and to manage an imminent or ongoing pandemic.

[0098] In certain embodiments, the nucleic acid(s) isolated by the methods of the present invention may serve as a template in one or more subsequent molecular biological applications, assays, or techniques, including, without limitation, genetic fingerprinting; amplified fragment length polymorphism (AFLP) polymerase chain reaction (PCR); restriction fragment length polymorphism analysis (RFLP); allele-specific oligonucleotide analysis (ASOA); microsatellite analysis; Southern hybridization; Northern hybridization; variable number of tandem repeats (VNTR) PCR; dot-blot hybridization; quantitative real-time PCR; polymerase cycling assembly (PCA); nested PCR; quantitative PCR (Q-PCR); asymmetric PCR; DNA footprinting; single nucleotide polymorphism (SNP) genotyping; reverse transcription PCR (RT-PCR); multiplex PCR (m-PCR); multiplex ligation-dependent probe amplification (MLPA); ligation-mediated PCR (LmPCR); methylation specific PCR (MPCR); helicase-dependent amplification (HDA); overlap-extension PCR (OE-PCR); whole-genome amplification (WGA); plasmid isolation; allelic amplification; site-directed mutagenesis; high-throughput genetic screening; or the like, or any combination thereof.

[0099] The compositions of the present invention provide clinical/environmental collection solutions that efficiently achieve at least three, and preferably all four of the following: 1) kill or inactivate potentially-infectious pathogens, so that they are non-viable and can be safely handled, shipped or transported; 2) lyse cells to release RNAs and/or DNAs from the biological specimen contained in the collection system; 3) protect the released or naked polynucleotides in the sample from further hydrolysis or enzymatic degradation, modification, or inactivation; and 4) prolong the conventional time-frame for storage and transportation of the processed sample under a variety of ambient, sub- or supra-optimal temperature conditions to maintain the fidelity and integrity of the released polynucleotides until the biological material can be further processed or analyzed at a diagnostic facility or analytical laboratory.

[0100] In one exemplary embodiment, the methods and formulations maintain at least substantial stability of the nucleic acids in the sample for an extended period of time, e.g., for up to about 15 days, preferably up to about 30 days, or more preferably up to about 60 days or more, without refrigeration or freezing of the sample, and even when stored at room temperature, or ambient environmental conditions including those of temperate, sub-tropical or tropical climates and the like. In other embodiments, use of the disclosed compositions to prepare nucleic acids from a sample of biological origin is desirable to maintain at least substantial integrity and fidelity of the nucleic acids released from the sample for extended periods including, without limitation, at least about 5 to about 15 days, preferably at least about 10 to 20 days, more preferably at least about 15 to 25 days, or more preferably still, at least about 20 to 30 days or more, without a requirement for refrigerating or freezing the sample either at the time of sample collection or until the sample is further processed (or both) hours, days, weeks, or even months after originally being collected and placed into the disclosed storage/collection/transport/stabilization formulations.

[0101] Nucleic acids obtained from biological samples in the practice of the disclosed methods are advantageously compatible with a number of conventional molecular and diagnostic isolation, purification, detection, and/or analytic methodologies. The disclosed compositions facilitate recovery storage, and transport of populations of stabilized, substantially non-degraded, polynucleotides for use in a variety of subsequent methodologies, including, without limitation, nucleic acid isolation, purification, amplification, and molecular analytical and/or diagnostic testing, assay, analysis, or characterization, and the like.

Exemplary Commercial Kits of the Present Invention

[0102] The following outline provides exemplary commercial kits employing the PrimeStore compositions of the present invention (FIG. 5).

Peel-Pouch Collection System

[0103] Five-mL tube containing 1.5 mL PrimeStore Solution; Collection swab (e.g., FlockedSWABS [Copan Diagnostics, Inc., Murrieta, Calif., USA]); and instructions for collection and/or processing of samples. (packed, e.g., in 50 pouches/unit) (See FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, and FIG. 4E for a schematic demonstration of such systems).

PrimeStore Stock Solution (e.g., 25-mL Bottles)

[0104] Once formulated, PrimeStore stock solution is stable at 4 C. or below for periods of at least one year or more. Formulated PrimeStore Solution has also been shown to be stable at ambient temperature (e.g., about 20-30 C.) for periods of three to six months or more.

[0105] Once a sample is contacted with a PrimeStore formulation as disclosed herein, it can reasonably expected to be stored indefinitely at temperatures of 0 C. or below, at least one year or more under refrigeration (e.g., 4 C. and at least 30 days or more at ambient temperature (e.g., about 20-30 C.), without significant loss of nucleic acid composition, fidelity or integrity of the sample. For example, without limitation, the integrity of a population of polynucleotides obtained from the sample is at least substantially maintained, and preferably entirely maintained without detectable degradation, 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 30 days to about 60 days.

[0106] The kit may also include one or more vials including the inventive compositions and one or more extraction devices to help liberate and separate the nucleic acids to obtain at least substantially pure RNA/DNA to be analyzed.

Environmental Sample and Storage Systems

[0107] 0.1-, 0.2-, 0.5-, 1-, 2-, or 3-mL collection vials each containing 0.1 mL, 0.2 mL, 0.25 mL, 0.5 mL, 0.75 mL, or 1 mL PrimeStore solution; and instructions for collection and/or processing of samples (packed, e.g., 10 vials/unit). The collection vials may be sized larger as needed depending on the proposed collection method.

Definitions

[0108] The terms about and approximately as used herein, are interchangeable, and should generally be understood to refer to a range of numbers around a given number, as well as to all numbers in a recited range of numbers (e.g., about 5 to 15 means about 5 to about 15 unless otherwise stated). Moreover, all numerical ranges herein should be understood to include each whole integer within the range.

[0109] As used herein, the term carrier is intended to include any solvent(s), dispersion medium, coating(s), diluent(s), buffer(s), isotonic agent(s), solution(s), suspension(s), colloid(s), inert(s) or such like, or a combination thereof that is pharmaceutically acceptable for administration to the relevant animal or acceptable for a diagnostic purpose, as applicable. The use of one or more delivery vehicles for chemical compounds in general, and peptides and epitopes in particular, is well known to those of ordinary skill in the pharmaceutical arts. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the diagnostic, prophylactic, and therapeutic compositions is contemplated. One or more supplementary active ingredient(s) may also be incorporated into, or administered in association with, one or more of the disclosed immunogenic compositions.

[0110] As used herein, the term nucleic acid includes one or more types of: polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases (including abasic sites). The term nucleic acid, as used herein, also includes polymers of ribonucleosides or deoxyribonucleosides that are covalently bonded, typically by phosphodiester linkages between subunits, but in some cases by phosphorothioates, methylphosphonates, and the like. Nucleic acids include single- and double-stranded DNA, as well as single- and double-stranded RNA. Exemplary nucleic acids include, without limitation, gDNA; hnRNA; mRNA; rRNA, tRNA, micro RNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snORNA), small nuclear RNA (snRNA), and small temporal RNA (stRNA), and the like, and any combination thereof.

[0111] As used herein, the terms protein, polypeptide, and peptide are used interchangeably, and include molecules that include at least one amide bond linking two or more amino acid residues together. Although used interchangeably, in general, a peptide is a relatively short (e.g., from 2 to about 100 amino acid residues in length) molecule, while a protein or a polypeptide is a relatively longer polymer (e.g., 100 or more residues in length). However, unless specifically defined by a chain length, the terms peptide, polypeptide, and protein are used interchangeably.

[0112] As used herein, sample includes anything containing or presumed to contain a substance of interest. It thus may be a composition of matter containing nucleic acid, protein, or another biomolecule of interest. The term sample can thus encompass a solution, cell, tissue, or population of one of more of the same that includes a population of nucleic acids (genomic DNA, cDNA, RNA, protein, other cellular molecules, etc.). The terms nucleic acid source, sample, and specimen are used interchangeably herein in a broad sense, and are intended to encompass a variety of biological sources that contain nucleic acids, protein, one or more other biomolecules of interest, or any combination thereof. Exemplary biological samples include, but are not limited to, whole blood, plasma, serum, sputum, urine, stool, white blood cells, red blood cells, buffy coat, swabs (including, without limitation, buccal swabs, throat swabs, vaginal swabs, urethral swabs, cervical swabs, rectal swabs, lesion swabs, abscess swabs, nasopharyngeal swabs, and the like), urine, stool, sputum, tears, mucus, saliva, semen, vaginal fluids, lymphatic fluid, amniotic fluid, spinal or cerebrospinal fluid, peritoneal effusions, pleural effusions, exudates, punctates, epithelial smears, biopsies, bone marrow samples, fluid from cysts or abcess contents, synovial fluid, vitreous or aqueous humor, eye washes or aspirates, pulmonary lavage or lung aspirates, and organs and tissues, including but not limited to, liver, spleen, kidney, lung, intestine, brain, heart, muscle, pancreas, and the like, and any combination thereof. In some embodiments, the sample may be, or be from, an organism that acts as a vector, such as a mosquito, or tick, or other insect(s).

[0113] Tissue culture cells, including explanted material, primary cells, secondary cell lines, and the like, as well as lysates, homogenates, extracts, or materials obtained from any cells, are also within the meaning of the term biological sample, as used herein. Microorganisms (including, without limitation, prokaryotes such as the archaebacteria and eubacteria; cyanobacteria; fungi, yeasts, molds, actinomycetes; spirochetes, and mycoplasmas); viruses (including, without limitation the Orthohepadnaviruses [including, e.g., hepatitis A, B, and C viruses], human papillomavirus, Flaviviruses [including, e.g., Dengue virus], Lyssaviruses [including, e.g., rabies virus], Morbilliviruses [including, e.g., measles virus], Simplexviruses [including, e.g., herpes simplex virus], Polyomaviruses, Rubulaviruses [including, e.g., mumps virus], Rubiviruses [including, e.g., rubella virus], Varicellovirus [including, e.g., chickenpox virus], rotavirus, coronavirus, cytomegalovirus, adenovirus, adeno-associated virus, baculovirus, parvovirus, retrovirus, vaccinia, poxvirus, and the like), algae, protozoans, protists, plants, bryophytes, and the like, and any combination of any of the foregoing, that may be present on or in a biological sample are also within the scope of the invention, as are any materials obtained from clinical or forensic settings that contain one or more nucleic acids are also within the scope of the invention. The ordinary-skilled artisan will also appreciate that lysates, extracts, or materials obtained from any of the above exemplary biological samples are also within the scope of the invention.

[0114] As used herein, the term buffer includes one or more compositions, or aqueous solutions thereof, that resist fluctuation in the pH when an acid or an alkali is added to the solution or composition that includes the buffer. This resistance to pH change is due to the buffering properties of such solutions, and may be a function of one or more specific compounds included in the composition. Thus, solutions or other compositions exhibiting buffering activity are referred to as buffers or buffer solutions. Buffers generally do not have an unlimited ability to maintain the pH of a solution or composition; rather, they are typically able to maintain the pH within certain ranges, for example from a pH of about 5 to 7.

[0115] As used herein, the term biological molecule refers to any molecule found within a cell or produced by a living organism, including viruses. This may include, but is not limited to, nucleic acids, proteins, carbohydrates, and lipids. As used herein, a cell refers to the smallest structural unit of an organism that is capable of independent functioning and is comprised of cytoplasm and various organelles surrounded by a cell membrane. This may include, but is not limited to, cells that function independently such as bacteria and protists, or cells that live within a larger organism such as leukocytes and erythrocytes. As defined herein, a cell may not have a nucleus, such as a mature human red blood cell.

[0116] Samples in the practice of the invention can be used fresh, or can be used after being stored for a period of time, or for an extended period of time, including for example, cryopreserved samples and the like, and may include material of clinical, veterinary, environmental or forensic origin, may be isolated from food, beverages, feedstocks, potable water sources, wastewater streams, industrial waste or effluents, natural water sources, soil, airborne sources, pandemic or epidemic populations, epidemiological samples, research materials, pathology specimens, suspected bioterrorism agents, crime scene evidence, and the like.

[0117] As used herein, the term patient (also interchangeably referred to as host or subject) refers to any host that can serve as a source of one or more of the biological samples or specimens as discussed herein. In certain aspects, the donor will be a vertebrate animal, which is intended to denote any animal species (and preferably, a mammalian species such as a human being). In certain embodiments, a patient refers to any animal host, including but not limited to, human and non-human primates, avians, reptiles, amphibians, bovines, canines, caprines, cavines, corvines, epines, equines, felines, hircines, lapines, leporines, lupines, murines, ovines, porcines, racines, vulpines, and the like, including, without limitation, domesticated livestock, herding or migratory animals or birds, exotics or zoological specimens, as well as companion animals, pets, and any animal under the care of a veterinary practitioner. The invention may also be used to monitor disease outbreak, progression, and epidemiological statistics for a variety of global populations, including, without limitation, wasting disease in ungulates, tuberculosis, ebola, SARS, and avian influenzas. In certain embodiments, the samples will preferably be of mammalian origin, and more preferably of human origin.

[0118] The term chaotrope or chaotropic agent as used herein, includes substances that cause disorder in a protein or nucleic acid by, for example, but not limited to, altering the secondary, tertiary, or quaternary structure of a protein or a nucleic acid while leaving the primary structure intact.

[0119] The term e.g., as used herein, is used merely by way of example, without limitation intended, and should not be construed as referring only those items explicitly enumerated in the specification.

[0120] The term substantially free or essentially free, as used herein, typically means that a composition contains less than about 10 weight percent, preferably less than about 5 weight percent, and more preferably less than about 1 weight percent of a compound. In a preferred embodiment, these terms refer to less than about 0.5 weight percent, more preferably less than about 0.1 weight percent or even less than about 0.01 weight percent. The terms encompass a composition being entirely free of a compound or other stated property, as well. With respect to degradation or deterioration, the term substantial may also refer to the above-noted weight percentages, such that preventing substantial degradation would refer to less than about 15 weight percent, less than about 10 weight percent, preferably less than about 5 weight percent, etc., being lost to degradation. In other embodiments, these terms refer to mere percentages rather than weight percentages, such as with respect to the term substantially non-pathogenic where the term substantially refers to leaving less than about 10 percent, less than about 5 percent, etc., of the pathogenic activity.

[0121] In accordance with long standing patent law convention, the words a and an when used in this application, including the claims, denotes one or more.

EXAMPLES

[0122] The following examples are included to demonstrate illustrative embodiments of the invention. It should be appreciated by those of ordinary skill in the art that the techniques disclosed in the examples that follow represent techniques discovered to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of ordinary skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1Formulation of Exemplary Storage Solutions

[0123] The present example provides a general formulation of the PrimeStore (PanFlu) compositions of the present invention. Exemplary formulations are also detailed in Examples 2-5.

Materials

[0124] Guanidine thiocyanate, sodium citrate, Antifoam A Concentrate, and N-lauroylsarcosine, sodium salt, were all purchased from Sigma Chemical Co. (St. Louis, Mo., USA). Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) was obtained from Soltec Ventures Inc. (Beverly, Mass., USA). 2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS) was obtained from Applied Biosystems/Ambion (Austin, Tex., USA). 2-[2-(Bis(carboxymethyl)amino)ethyl-(carboxymethyl)amino]acetic acid (EDTA) GIBCO Ultra Pure was obtained from Invitrogen Corp. (Carlsbad, Calif., USA). All other reagents are available commercially from Sigma-Aldrich or USB Corporation.

TABLE-US-00001 TABLE 1 FORMULATION RANGES OF EXEMPLARY COMPONENTS FOR THE PREPARATION OF PRIMESTORE COMPOSITIONS REAGENT COMPONENT FINAL CONCENTRATION RANGES 1. A chaotrope, e.g.: Guanidine thiocyanate about 0.5M to about 6M or Guanidine hydrochloride about 0.5M to about 6M or Guanidine isocyanate about 0.5M to about 6M 2. An anionic detergent, e.g.: N-lauroyl sarcosine (inter alia Na salt) about 0.15% to about 1% (wt./vol.) or Sodium dodecyl sulfate, Same Lithium dodecyl sulfate, Same Sodium glycocholate, Same Sodium deoxycholate, Same Sodium taurodeoxycholate, or Same Sodium cholate about 0.1% to about 1% (wt./vol.) 3. A reducing agent, e.g.: TCEP about 0.5 mM to about 30 mM or -ME, DTT, formamide, or DMSO about 0.05M to about 0.3M 4. A chelator, e.g.: Sodium citrate about 0.5 mM to about 50 mM or EDTA, EGTA, HEDTA, DTPA, NTA, or APCA about 0.01 mM to about 1 mM 5. A buffer (e.g., TRIS, HEPES, MOPS, MES, Bis-Tris, etc.) about 1 mM to about 1M 6. An acid (e.g., HCl or citric acid) q.s. to adjust to a pH of about 6 to 7, preferably 6.4 to 6.8 7. Nuclease-free water q.s. to desired final volume Optionally one or more of: 8. A surfactant/defoaming agent, e.g.: Antifoam A or Tween about 0.0001% to about 0.3% (wt./vol.) 9. An alkanol (e.g., methanol, ethanol, propanol, etc.) about 1% to about 25% (vol./vol.) 10. RNA or DNA about 1 pg to about 1 g/mL

Example 2Formulation of an Exemplary Storage Solution

[0125] The present example describes a first exemplary formulation of the compositions of the invention. This formulation has also been alternatively referred to by the inventors as PrimeStore Solution or PSS version 1.

TABLE-US-00002 TABLE 2 PREPARATION OF PRIMESTORE COMPOSITION (VER. 1) Reagent Final Concentration Guanidine thiocyanate 4M Sodium citrate 30 mM Sodium dodecyl sulfate 0.25% (wt./vol.) N-lauroyl sarcosine, sodium salt 0.25% (wt./vol.) 2-mercaptoethanol (-ME) 0.1M Antifoam A 0.1% (wt./vol.) Citric acid q.s. to adjust pH to 6.5 Nuclease-free water 11.82 mL

Example 3Preparation of a Second Exemplary Storage Solution

[0126] The present example describes the preparation of another exemplary storage solution according to the present invention. This formulation has also been alternatively referred to by the inventors as PrimeStore version 2.

TABLE-US-00003 TABLE 3 PREPARATION OF PRIMESTORE COMPOSITION (VER. 2) FINAL REAGENT QUANTITY CONCENTRATION Guanidine thiocyanate 35.488 gm 3M TCEP 0.02867 gm 1 mM Sodium citrate 0.2931 gm 10 mM N-lauroyl sarcosine, 0.5 gm 0.5% sodium salt (NLS) Antifoam A 200 L 0.002% (10% solution) TRIS (1M) 10 mL 100 mM EDTA (0.5M) 20 L 0.1 mM Hydrochloric acid (HCl) q.s. to adjust pH to 6.7 Nuclease-free water q.s. to 100 mL

Example 4Preparation of a Third Exemplary Storage Solution

[0127] The present example describes the preparation of another exemplary storage solution according to the present invention. This formulation has also been alternatively referred to by the inventors as PrimeStore version 2.2.

TABLE-US-00004 TABLE 4 PREPARATION OF PRIMESTORE COMPOSITION (VER. 2.2) FINAL REAGENT QUANTITY CONCENTRATION Guanidine thiocyanate 35.488 gm 3M TCEP 0.02867 gm 1 mM Sodium citrate 0.2931 gm 10 mM N-lauroyl sarcosine, 0.5 gm 0.5% sodium salt (NLS) Antifoam A 200 L 0.002% (10% solution) TRIS (1M) 10 mL 100 mM EDTA (0.5M) 20 L 0.1 mM Ethanol, molecular grade 23 mL 23% (vol./vol.) (96-100%) Hydrochloric acid (HCl) q.s. to adjust pH to 6.7 Nuclease-free water q.s. to 100 mL

Exemplary Protocol for Preparation of PrimeStore Solution (Ver. 2.2)

[0128] 1. Add 40 mL of nuclease-free water to a clean beaker with a stir bar. [0129] 2. Place beaker on a hot plate/stirrer and adjust temperature to 60-65 C. Set stirring speed to medium. [0130] 3. Add 35.488 gm of guanidine thiocyanate slowly to the water allowing it to dissolve as added. [0131] 4. Add 0.0287 gm of TCEP to beaker and increase stirrer speed to help dissolve crystals. [0132] 5. Add 0.2931 gm of sodium citrate to the beaker. [0133] 6. Add 0.5 gm of NLS to the solution. Increase stirrer speed to create a vortex in the beaker. This will pull the NLS into the solution and help dissolve the reagent. [0134] 7. Vortex a prepared 10% Antifoam A solution (1 mL Antifoam A Concentrate+9 mL nuclease-free water). Pipette 200 L of the 10% Antifoam A into the solution. [0135] 8. Pipette 10 mL of 1 M TRIS into the solution. [0136] 9. Pipette 20 L of 0.5 M EDTA into the solution. [0137] 10. Increase the temperature to bring the solution to 75-80 C. and stir for 3-5 minutes. [0138] 11. Remove beaker from heat and allow solution to cool to room temperature (22-25 C.). [0139] 12. Add 23 mL of ethanol to the solution and mix thoroughly. [0140] 13. Adjust pH to 6.7 with HCl. [0141] 14. Pour solution into a clean 100 mL graduated cylinder. [0142] 15. Add nuclease-free water to bring total volume to 100 mL. [0143] 16. Transfer solution to a labeled sterile container. Store at room temperature (22-25 C.). [0144] *Note: Preferably, make sure each reagent is completely dissolved before adding the next.

Example 5Comparison of PrimeStore Solutions to Conventional Formulations

[0145] A sample of homogenized nasal tissue from a cotton rat (Sigmodon hispidus) challenged with influenza A (H3N2) or a human clinical influenza A (H1N1) sample collected as a human clinical nasal wash during the 2006-07 season were placed in PrimeStore Solution (Ver. 1) and tested compared to the respective lysis formulation and protocol, and extraction procedure, from three commercially available kits: RNAqueous-Micro (Ambion, Austin, Tex., USA), QIAamp Viral RNA Mini Kit (Catalogue #52904, Qiagen, Valencia, Calif., USA), and MagMax AI/ND Viral RNA Isolation Kit (Catalogue #AM1929, Ambion). Extraction efficiency was evaluated using the ABI 7500 sequence detection system with the comparative threshold cycle (C.sub.T) method (See FIG. 2). In FIG. 2, delta Rn represents the fluorescent reporter signal minus a baseline amount. As shown in FIG. 1 and FIG. 2, the relative C.sub.T scores and viral copies detected were optimal when the fixing formulation was used in place of the respective conventional lysis buffer for each commercial kit. In these two sample types, the compositions of the invention worked better than the two conventional Kits for extraction purposes. The PrimeStore Solution (ver. 1) composition was also shown to be readily compatible with commercially available nucleic acid extraction kits. FIG. 1 illustrates RNA extraction results where the version 1 of PrimeStore Solution was used in conjunction with three commercially available kits: Qiagen Viral Mini, Ambion RNAqueous Mini, and Ambion Al/NCD MagMax. As illustrated by FIG. 1, when the lysis buffer of the extraction kit was replaced with the fixing formulation (denoted on the figure as One-Step+), superior nucleic acid extraction was achieved when compared to extraction using kits according to standard protocol (denoted on the figure as One-Step-. Extraction efficiency was measured by real time (r) reverse transcription (RT) polymerase chain reaction (PCR) [rRT-PCR].

[0146] FIG. 3 shows preservation of naked RNA in PrimeStore Solution compared to preservation in a prior solution, with water used as a control. As illustrated in FIG. 3, detection (by fluorescence) occurred at the earliest amplification cycle for RNA stored in PrimeStore Solution (ver. 1) at all time-points assayed.

Example 6PrimeStore Solution for the Collection of Nasal Wash Specimens

[0147] A prospective clinical detection study was conducted using nasal wash specimens from: 1) symptomatic pediatric patients and 2) asymptomatic or symptomatic family members. Detection of influenza virus compared nasal wash specimens collected in PrimeStore Solution and Viral Transport Medium (VTM) by real-time RT-PCR (rRT-PCR) and traditional culture, respectively. Genetic characterization of influenza genes encoding hemagglutinin (HA), neuraminidase (NA), and matrix surface (MA) proteins were performed using select nasal wash specimens preserved in PrimeStore Solution to evaluate vaccine effectiveness and drug sensitivity within viral strains.

[0148] Influenza is a highly evolving, RNA-based respiratory virus responsible for more than 200,000 hospitalizations and about 36,000 fatalities each year in the United States. Widespread emergence of influenza drift variants among contemporary circulating human viruses prompted a change in all three vaccine components for the upcoming 2008/09 season. Increased morbidity and mortality during the 2007/08 season included 72 influenza-associated pediatric deaths and continued drug resistance (oseltamivir [TamiFlu, Roche Laboratories, Inc., Nutley, N.J., USA] and adamantadine) within circulating strains.

Materials and Methods

[0149] A total of 100 pediatric (index) patients who met the clinical case criteria for influenza infection and 126 family contacts were enrolled in the study. Nasal washings were placed into PrimeStore Solution and Universal Viral Transport Medium (Becton-Dickinson, Franklin Lakes, N.J., USA) and analyzed by rRT-PCR or culture analysis, respectively. rRT-PCR was performed using influenza type (A or B) and subtype (H3, H1, H5) specific primers/probes according to Daum et al. (2007). Further genetic characterization of selected clinical samples preserved in PrimeStore Solution was performed using standard RT-PCR and direct nucleotide sequencing of the hemagglutinin HA, NA, and MA viral proteins.

Results

[0150] Of the total samples evaluated (N=226; 100 index, 126 family contacts), 66 (29%) tested positive for influenza virus (45 H3N2, 2 H1N1 and 19 B) by rRT-PCR. rRT-PCR from nasal washings preserved in PrimeStore Solution detected influenza virus from 11 patients (9 Flu A and 2 Flu B) that were not detected by culture (Table 5 and Table 6). Of these 11 specimens, five were from patients enrolled as family contacts.

[0151] Phylogenetic analysis of influenza A and B HA genes exhibited drifting compared to the 2007/08 vaccine strains and revealed a higher genetic homology to the 2008/09 Brisbane vaccine strains. Some genetic differences in viruses were noted among family members, particularly among influenza A (H3N2) strains. MA analysis revealed adamantane resistance in all influenza A H3N2 strains, but sensitivity in both H1N1 viruses. All influenza B strains (n=18) were sensitive to the neuraminidase inhibitor drugs zanamivir (Relenza GlaxoSmithKline, Research Triangle Park, N.C., USA) and oseltamivir (Tamiflu Roche) based on the presence of an aspartic acid (D) at amino acid 197 (influenza B numbering) in the NA gene.

Real-Time Rt-PCR Vs. Culture

[0152] rRT-PCR is superior to traditional culture for the detection of influenza virus from original nasal wash specimens preserved in PrimeStore solutions: influenza was detected within 2 hours (c.f. 2 to 7 days for conventional culture methods); and the analyses were more sensitive (11 specimens; 9 Flu A and 2 Flu B detected below culture limits). Moreover, the use of molecular diagnostic methods in lieu of conventional organism culture did not propagate potentially infectious viruses, and simultaneously provided the type and subtype of the influenza virus.

Genetic Analysis

Vaccine Relatedness

[0153] H3N2 Strains: Analysis of the HA1 gene of the influenza A (H3N2) hemagglutinin (HA) revealed genetic drift including five amino acid differences in all Texas strains compared to the 2007-08 A/Wisconsin/67/2005 vaccine strain. One HA1 mutation noted in all the Texas strains (D122N) is a potential glycosylation site. All A/Texas (H3N2) strains exhibited a greater HA homology (99.0-99.7%) to the newly selected 2008-09 A/Brisbane/10/2007 strain.

H1N1 Strains:

[0154] The hemagglutinin HA1 gene of the 2 influenza A (H1N1) exhibited 7 amino acid changes compared to A/Solomon Island/3/2006 vaccine strain. Four substitutions (R90K, T145V, K210T and E290K) were within known H1 antibody combining sites. Both Texas H1N1 strains exhibited greater HA homology (98.8% and 99.4%) to the newly selected 2008-09 A/Brisbane/59/2007 vaccine strain.

Influenza B Strains:

[0155] Analysis of the HA1 hemagglutinin and neuraminidase genes revealed all Texas strains were of the B/Yamagata lineage and genetically more homologous to the 2008-09 B/Brisbane/5/2007 vaccine strain than the 2007/08 B/Malaysia/2506/2004 vaccine strain.

Family Mutation

[0156] Amino acid changes were noted in the NA, HAL M1 and M2e among family members. The HA1 Hemagglutinin showed the highest mutation of the influenza genes analyzed, with one family exhibiting five amino acid changes.

[0157] Analysis of the highly conserved 24 amino acid M2e viral surface proton pump showed some variation within families. One index patient strain contained 3 unique amino acid M2e substitutions that were wild-type within family member strains.

Antiviral Susceptibility

Adamantane:

[0158] Matrix gene (MA) genetic analysis, specifically a serine-to-asparagine substitution at position 31 (S31N), revealed adamantane resistance in all influenza A (H3N2) strains but sensitivity in both influenza A (H1N1) viruses.

Neuraminidase Inhibitors:

[0159] All Texas influenza A (H3N2) isolates were shown to be sensitive to oseltamivir through genetic analysis of E119V, R292K, and N294S substitutions in the NA gene. Genetic analysis of the influenza B NA gene revealed that all Texas strains contained an aspartic acid (D) residue at position 197, and are thus likely sensitive to oseltamivir.

The protocols and tests herein can be adapted for other microbes like tuberculosis, malaria, staphylococcus, and the like and other pathogens where there is a need to know antimicrobial susceptibility quickly.

Example 7Influenza Sample Collection Using PrimeStore Solution

[0160] The compositions of the present invention provide a single sample collection, transport, and storage reagent that facilitate: 1) procuring high quality nucleic acids from clinical or environmental specimens, 2) inactivation of potentially infectious biological pathogens for safe handling and transport of specimens, and 3) stabilization and preservation of released naked RNA/DNA preventing hydrolysis/nuclease degradation for prolonged periods at ambient temperatures. The results of one such study are presented in the following example:

TABLE-US-00005 TABLE 5 Influenza Subtype Detection: rRT-PCR vs. Culture Influenza A Influenza B Total FluA Samples rRT-PCR Culture rRT-PCR Culture Total FluB Samples (N = 47) (N = 47) (N = 40) (N = 19) (N = 17) (N = 19) Index Patients (28) 28/28 (100%) 23/28 (82%) 16/16 (100%) 14/16 (86%) Index Patients (16) Family Contacts (19) 19/19 (100%) 15/19 (79%) 3/3 (100%) 3/3 (100%) Family Contacts (3)

TABLE-US-00006 TABLE 6 Positive Influenza Detection: rRT-PCR vs. Culture Total Samples Flu Positive rRT-PCR (N = 226) (N = 66) (N = 66) Culture (N = 66) Index Patients (100) 44/100 39/39 (100%) 37/39 (94%) Family Contacts (126) 22/126 27/27 (100%) 18/27 (67%)

[0161] This example illustrates the effectiveness of the PrimeStore Solution (ver. 2.2) in killing pathogenic microbe(s).

Methods

[0162] Real-time RT-PCR was used to assay influenza A (H5N1) virus nucleic acid preserved in PrimeStore Solution. A time-course study at room temperature was carried out to evaluate the integrity of clinical specimens, cloacal samples, and cloned template avian influenza A virus (H5) RNA stored and extracted from PrimeStore Solution, Viral Transport Media, RNA Storage Solution, or nuclease-free water. PrimeStore Solution extraction efficiency was compared to three commercially available nucleic acid extraction kits. Furthermore, the ability of RNA contained in PrimeStore Solution to resist nuclease degradation was evaluated.

Results

[0163] PrimeStore Solution (version 2, but lacking ethanol) inactivated microbial agents while preserving released RNA/DNA from clinical material, i.e., nasal washes, throat swabs, or environmental samples. Clinical specimens or environmental samples placed in this solution were stabilized at room temperature for up to 30 days while degradation of nucleic acids occurred in other transport media. PrimeStore Solution is compatible with commercially available RNA isolation kits and produced an increased nucleic acid yield.

Example 8Killing of MRSA (ATCC33592) in PrimeStore Solution

[0164] This example illustrates the effectiveness of the PrimeStore Solution (ver. 2.2) in killing a potential bacterial contaminant. Methicillin-resistant Staphylococcus aureus (MRSA) strain ATCC33592 was diluted 10-fold and 1000-fold into PrimeStore Solution (Ver. 2.2) and quantitated (see FIG. 6).

[0165] Experimental Protocol

TABLE-US-00007 DAY PROCEDURE 0 Transfer MRSA (ATCC33592) from a Culti-loop (Remel) to 1.5 mL of TSB in a 15-ml conical test tube. Incubate at 37 C. for approximately 15 min. Gently vortex suspension and transfer 100 L to a blood-agar plate. Incubate the plate overnight at 37 C. 1 Observe heavy and uniform colony growth after 12 hr incubation. Transfer ~10% of colonies to 300 mL of tryptic soy broth (TSB) in a sterile, 1-liter flask. Place flask on shaker at 37 C. and 200 rpm. After approximately 4-6 hrs' incubation, transfer ~50 mL of bacterial suspension to new 1-liter flask containing 300 mL fresh TSB. After approximately 4-6 hrs' incubation, transfer ~100 L of culture into 900 L of TSB (1:10 control dilution). From this suspension 10 L were transferred to 990 L of TSB (1:1000 control dilution). Transfer 100 L of culture into 900 L of PrimeStore Solution (1:10 PrimeStore dilution). From this suspension 10 L were transferred to 990 L of TSB (1:1000 PrimeStore dilution). Immediately after transfer into TSB or PrimeStore Solution (ver. 2.2), the suspensions were gently vortexed and 100 L were plated from both dilutions of control and PrimeStore suspensions onto blood agar plates. The time-zero time point was actually about two minutes following addition of the bacteria to the TSB or PrimeStore Solution. The suspensions in TSB and PrimeStore Solution (ver. 2.2) were kept at room temperature. An additional 100 L was plated onto blood agar plates at 5, 15, 30, 60, 120 and 240 minutes after the preparation of the suspensions in TSB and PrimeStore Solution. Bacterial suspensions on the plates were allowed to dry, the plated inverted and the plates incubated overnight at 37 C. A titration of the shaker culture was also performed by mixing 100 L of the suspension from the shaker culture with 900 L of TSB (10.sup.1 dilution). Serial 10-fold dilutions were prepared through 10.sup.9. 100 L samples were plated onto blood agar from all dilutions except 10.sup.1. 2 Plates were observed for bacterial colonies. All plates were stored at 4 C. for later observation, if necessary.

Results

[0166] The results are presented in Table 7 and Table 8. Briefly, the bacterial suspension contained approximately 4.710.sup.9 cfu/ml. Thus, the 1:10 dilution contained approximately 4.710.sup.8 cfu/ml and the 1:1000 dilution contained 4.710.sup.6 cfu/ml. At all time points, the bacteria suspended in TSB were too numerous to count. At all time points the bacteria suspended in PrimeStore Solution and plated onto blood agar plates had no detectable colonies.

TABLE-US-00008 TABLE 7 KILLING OF MRSA (ATCC 33592) BY PRIMESTORE SOLUTION (VER. 2.2) Incubation Time In TSB In Primestore (Minutes) 1:10 1:1000 1:10 1:1000 0 TNTC TNTC 0 0 5 TNTC TNTC 0 0 15 TNTC TNTC 0 0 30 TNTC TNTC 0 0 60 TNTC TNTC 0 0 120 TNTC TNTC 0 0 240 TNTC TNTC 0 0 TNTC = too numerous to count.

TABLE-US-00009 TABLE 8 TITRATION OF MRSA ATCC33592 FROM SUSPENSION CULTURE Dilution CFU/plate CFU/ml 1.E+01 TNTC 1.E+02 TNTC 1.E+03 TNTC 1.E+04 TNTC 1.E+05 TNTC 1.E+06 TNTC 1.E+07 35 3.5 10{circumflex over ()}9 1.E+08 6 6 10{circumflex over ()}9 1.E+09 0 NOTE: CFU/ml calculations are corrected to include the plating volume of 0.1 mls Final Conc: 4.7 10{circumflex over ()}9/ml TNTC = too numerous to count. CFU = colony forming units.
An additional study was performed to determine the time of exposure necessary for killing MRSA ATCC33592 when diluted 10-fold into PrimeStore Solution (Ver. 2.2), and to determine the effect of dilution of the bacteria after exposure to PrimeStore Solution, but before plating.

Experimental Protocol

Day Procedure

[0167] 0 Transfer MRSA (ATCC33592) from TNTC plate from the study described above into 4 mL of TSB. These plates had been stored at 4 C. for approximately 48 hrs. Bacteria were vortexed gently and placed at room temperature for approximately 10 min. before use.
0.1 mL of bacterial suspension was transferred to 0.9 mL PrimeStore Solution and vortexed gently. After approximately 60 sec, the bacteria in PrimeStore were again vortexed gently and
0.1 mL of bacterial suspension was transferred into 0.3 mL of TSB (1:4 dilution).
100 L of bacteria in PrimeStore Solution (designated neat) and from the 1:4 dilution into TSB were plated onto blood agar plates (5% sheep RBCs in TSA).
This process was repeated at 5 and 15 min., and then again with dilutions made into TSB instead of PrimeStore Solution.
The liquid bacterial suspensions on the blood agar plates were allowed to dry at room temperature and then incubated overnight at 37 C.
1 After approximately 16 hrs. incubation, the plates were removed from the incubator and colonies counted.

Results

[0168] The bacterial suspension contained an unknown number of colony forming units (cfu) per mL. At all time points the bacteria suspended in tryptic soy broth (TSB) were too numerous to count (TNTC). At all timepoints the bacteria suspended in PrimeStore compositions and plated onto blood agar plates produced no colonies (Table 9).

TABLE-US-00010 TABLE 9 KILLING OF MRSA (ATCC33592) BY PRIMESTORE SOLUTION Incubation Time In TSB In TSB (minutes) neat 1:4 neat 1:4 1 TNTC TNTC 0 0 5 TNTC TNTC 0 0 15 TNTC TNTC 0 0 TNTC = too numerous to count.

Example 9Additional Studies Evaluating PrimeStore Solutions

[0169] The data in FIG. 7B illustrate the ability of PSS to inactivate microbes. Shown is a study in which chicken cloacal specimens were collected in PrimeStore Solution (Ver. 1). PrimeStore Solution inactivated the microbial agents in 1 hr. Four original chicken cloacal samples were immersed in PrimeStore Solution or water and subsequently plated on blood agar plates. These results showed that the disclosed composition could quickly kill or inactive microorganisms in the sample.

[0170] The data in FIG. 7C illustrate the ability of PSS to inhibit RNA base hydrolysis for 30 days at room temperature. RNA was incubated at room temperature (22-26 C.) in PrimeStore (gel lane 1 and 3) and water (gel lane 2 and 4), and subsequently RT-PCR amplified (1500 base pairs) at Day 0 and Day 30. PrimeStore preserved collected RNA, and prevented RNA/DNA degradation at room temperature up to 30 days (see also, e.g., Table 11).

[0171] Flu Inhibition Assay

[0172] The reagents for this assay include

[0173] Trypsin Medium:

[0174] 45 mL Sterile N/C EMEM

[0175] 3 mL stock 7.5% Na Bicarbonate (2%)

[0176] 1.5 mL SPG (1%)

[0177] 75 L Trypsin (0.05%)

[0178] 1.5 mL Fungizone (1%)

[0179] 150 L Gentamicin

[0180] Filter medium.

[0181] Crystal Violet:

[0182] 150 ml glutaric dialdehyde

[0183] 2 gm crystal violet

[0184] 2850 mL deionized waterProtocols

Preparation of Serum Samples for Assay

[0185] Thaw and vortex serum samples. For each sample, label the lid of a corresponding Spin-X tube. Combine 450 L non-complete EMEM with 50 L serum into a Spin-X tube. Warm tubes containing the sera and EMEM in a 56 C. water bath for 30 min. Centrifuge tubes at 8000 RPM for 2 min. at room temperature. Label and place samples into a 20 C. freezer until assayed.

Dilution Plates

[0186] Load 160 L of each neat compound or serum sample into wells A1 through A12. Load the remaining wells with 120 L trypsin medium. Using a multi-channel pipette, draw 40 L of neat sample from row A and dilute into the corresponding wells in row B. Repeat dilution for each row, mixing well after each transfer. At row H, after mixing the transfer from row G, draw up 40 L from each well and discard. Obtain virus stock (10.sup.6) from 80 C. freezer and thaw. Dilute virus stock in trypsin media to a 10.sup.3 dilution. After serial dilutions are completed, transfer 120 L of influenza virus (10.sup.4 TCID.sub.50 ml) to all wells in the dilution plate. This results in a total of 240 L in all wells. Incubate dilution plate(s) at room temperature for 1 hour.

MDCK Cell Plates

[0187] Sterilize and place glass reservoir, comb dispenser and tubing inside the fume hood. Inside the hood, connect the tubing to the reservoir and fill nozzle of the comb. Connect the aspirator tube to the vacuum nozzle on the comb. Place the reservoir on an elevated surface and turn on the aspirator. Put PBS into the reservoir (1 L or more may be needed depending on the number of plates). Wash the cell plates 3 with the PBS comb (aspirate the medium, then press the button for roughly 1 second to wash the wells, repeat twice). Using a multi-channel pipette, transfer 50 L from each well in column 1 of the dilution plate to columns 1 through 4 of the cell plate. Transfer 50 L from each well in column 2 of the dilution plate to columns 5 through 8 of the cell plate. Transfer 50 L from each well in column 3 to columns 9 through 12 of the cell plate. Repeat transfer to additional cell plates for remaining samples. Incubate cell plates for 1 hour at 37 C. After incubation period, add 50 L trypsin medium to all wells of the cell plates. Return plates to incubation chamber, and incubate for 4 days post-infection.

Staining

[0188] Add 100 L of Crystal Violet to all wells. Let sit for 1 hour. Rinse plates in cold running water and air dry.

TABLE-US-00011 TABLE 10 TITRATION OF TCEP USING WHOLE INFLUENZA A VIRUS 5 mM 10 mM 25 mM 35 mM 50 mM 30.353 24.58 24.52 24.14 25.9582 30.2261 22.74 24.26 22.74 26.0337 30.28955 23.66 24.39 23.44 25.99595 0.089732 1.301076 0.183848 0.989949 0.053387 Titration of TCEP Using H5 Avian ssRNA 27.2 25.25 25.63 27.3 28.3039 26.73 24.89 25.36 27.62 26.6854 26.965 25.07 25.495 27.46 27.49465 0.33234 0.254558 0.190919 0.226274 1.144452

Time-Course Study of the Long-Term Stability of PrimeStore Compositions

[0189] The following data demonstrate the effectiveness of various PrimeStore compositions in preserving nucleic acid integrity over a thirty-day period with samples stored at room temperature. PrimeStore compositions have been compared to water alone, ethanol alone, commercial buffers such as VTM and AVL.

TABLE-US-00012 TABLE 11 30-DAY TIME-COURSE COMPARISON STUDY OF VARIOUS COMPOSITIONS PS-V1 PS-V1 PS-V2.2 VTM (Year old) (new lot) PS-V2 (w/EtOH) DAY 1 PS-V1 PS-V1 PS-V2.2 VTM Water EtOH AVL (year old) (new lot) PS-V2 (w/EtOH) 27.0225 26.1403 18.4463 24.2698 24.2607 23.9524 23.4426 20.2102 24.42 25.6044 18.3206 24.4789 24.3716 23.9615 23.7387 20.063 25.72125 25.87235 18.4463 24.37435 24.31615 23.95695 23.59065 20.1366 AVG 1.840245 0.378939 0.088883 0.147856 0.0784181 0.006435 0.209374 0.10408612 STDEV DAY 6 PS-V1 PS-V1 PS-V2.2 VTM Water EtOH AVL (Year old) (new lot) PS-V2 (w/EtOH) 29.1988 29.3053 27.4058 37.9226 27.2379 27.165 24.53 22.4887 28.6799 28.7916 27.0781 40 26.4857 26.7658 24.4418 22.4676 28.93935 29.04845 27.24195 38.9613 26.8618 26.9654 24.4859 22.47815 AVG 0.366918 0.363241 0.231719 1.468944 0.5318857 0.282277 0.062367 0.01491995 STDEV PS-V1 PS-V1 PS-V2.2 VTM Water EtOH AVL (year old) (new lot) PS-V2 (w/EtOH) DAY 12 27.997 28.151 26.9011 40 30.8352 31.0478 25.8926 22.2074 28.0062 28.2211 26.2139 38.0439 30.4502 30.1935 25.3037 22.0025 28.0016 28.18605 26.5575 39.02195 30.6427 30.62065 25.59815 22.10495 AVG 0.006505 0.049568 0.485924 1.383172 0.2722361 0.604081 0.416415 0.14488618 STDEV DAY 20 27.9851 28.7713 27.1105 40 30.1844 27.193 25.7407 20.8364 28.4067 27.7929 27.0105 40 30.2465 27.2274 25.6213 20.2843 28.1959 28.2821 27.0605 40 30.21545 27.2102 25.681 20.56035 AVG 0.298116 0.691833 0.070711 0 0.0439113 0.024324 0.084429 0.39039365 STDEV DAY 30 29.23 31.9168 33.012 40 29.1993 30.2386 23.0589 20.9348 29.9067 31.3252 32.3001 40 28.827 29.6081 22.9662 20.4973 29.56835 31.621 32.65605 40 29.01315 29.92335 23.01255 20.71605 AVG 0.478499 0.418324 0.503389 0 0.2632559 0.445831 0.065549 0.30935922 STDEV PS-V1 (year old) = One-year old PrimeStore Formulation (Ver. 1). PS-V1 (new lot) = Fresh PrimeStore Formulation (Ver. 2). PS-V2 = Fresh PrimeStore Formulation (Ver 2) (without ethanol). PS-V2.2 (w/EtOH) = Fresh PrimeStore Formulation (Ver. 2.2) (i.e. with ethanol).

[0190] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of exemplary embodiments, it will be apparent to those of ordinary skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically- and physiologically-related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those of ordinary skill in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. Accordingly, the exclusive rights sought to be patented are as described in the claims below.

f refrigeration or freezing prior to molecular analysis.