WO2022197637A1 - Compositions et procédés de traitement d'échantillons biologiques - Google Patents

Compositions et procédés de traitement d'échantillons biologiques Download PDF

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WO2022197637A1
WO2022197637A1 PCT/US2022/020256 US2022020256W WO2022197637A1 WO 2022197637 A1 WO2022197637 A1 WO 2022197637A1 US 2022020256 W US2022020256 W US 2022020256W WO 2022197637 A1 WO2022197637 A1 WO 2022197637A1
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composition
detergent
biological sample
sample
nucleic acid
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Anne-Laure SHAPIRO
James T. Tuggle
David Opalsky
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Gen Probe Inc
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Gen Probe Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis

Definitions

  • This disclosure relates to the field of biotechnology. More particularly, the disclosure concerns methods and compositions for processing biological samples, for example in preparation for use in subsequent analytical procedures.
  • nucleic acid purification may isolate all nucleic acids present in a sample, isolate different types of nucleic acids based on physical characteristics, or isolate specific nucleic acids from a sample. Many methods involve complicated procedures, use harsh chemicals or conditions, or require a long time to complete the nucleic acid isolation. Some methods involve use of specialized oligonucleotides, each specific for an intended target nucleic acid which adds complexity to the design, optimization and performance of methods, particularly if isolation of more than one target nucleic acid is desired or if the sequence of the desired target nucleic acid is unknown. Some methods isolate target nucleic acids without requiring a particular target sequence but do not isolate sequences efficiently under all circumstances. Sample transport media are commonly used to preserve and prepare samples for further analysis during the time after collection from the sample source.
  • Samples containing solid or viscous matter can be particularly challenging.
  • solid or viscous matter e.g., mucus; clots
  • Such samples can complicate liquid transfer steps, including those performed by automated platforms, that are useful for efficiently processing and analyzing the sample. Reducing viscosity and/or dissolving or otherwise liquefying such samples can reduce or eliminate such difficulties.
  • Improved sample transport media could meet this need or provide other benefits.
  • Embodiment 1 is a composition comprising at least two degradative enzymes, a detergent, a buffer, and a divalent cation chelator.
  • Embodiment 2 is the composition of embodiment 1, wherein the degradative enzymes comprise a lipase.
  • Embodiment 3 is the composition of embodiment 1 or 2, wherein the degradative enzymes comprise a protease.
  • Embodiment 4 is the composition of any one of the preceding embodiments, wherein the degradative enzymes comprise an amylase.
  • Embodiment 5 is the composition of any one of the preceding embodiments, wherein the degradative enzymes comprise a lipase, protease, and amylase.
  • Embodiment 6 is the composition of any one of the preceding embodiments, wherein the degradative enzymes comprise a carbohydrase.
  • Embodiment 7 is the composition of any one of the preceding embodiments, wherein the degradative enzymes comprise a lipase, protease, amylase, and carbohydrase.
  • Embodiment 8 is the composition of any one of the preceding embodiments, wherein at least one degradative enzyme is present at about 0.01-100 mg per liter, or about 0.05-5 mg per liter, or about 0.1-1 mg per liter.
  • Embodiment 9 is the composition of any one of the preceding embodiments, wherein each degradative enzyme is present at about 0.01-100 mg per liter, or about 0.05- 5 mg per liter, or about 0.1-1 mg per liter.
  • Embodiment 10 is the composition of any one of the preceding embodiments, wherein the detergent comprises an anionic detergent.
  • Embodiment 11 is the composition of any one of the preceding embodiments, wherein the detergent comprises an alkyl sulfate.
  • Embodiment 12 is the composition of the immediately preceding embodiment, wherein the detergent comprises a dodecyl sulfate.
  • Embodiment 13 is the composition of any one of the preceding embodiments, wherein the detergent comprises a sodium salt.
  • Embodiment 14 is the composition of any one of the preceding embodiments, wherein the detergent comprises a lithium salt.
  • Embodiment 15 is the composition of any one of the preceding embodiments, wherein the detergent comprises a non-ionic or zwitterionic detergent.
  • Embodiment 16 is the composition of any one of the preceding embodiments, wherein the composition comprises a detergent builder or complexing agent.
  • Embodiment 17 is the composition of the immediately preceding embodiment, wherein the detergent builder or complexing agent comprises zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates, or layered silicates, optionally wherein the layered silicates are Hoechst SKS-6.
  • the detergent builder or complexing agent comprises zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates, or layered silicates, optionally wherein the layered silicates are Hoechst SKS-6.
  • Embodiment 18 is the composition of any one of the preceding embodiments, wherein the detergent is present at less than about 10% by weight of the composition, optionally wherein the detergent is present at about 0.5-10% by weight of the composition, or about 2-6% by weight of the composition.
  • Embodiment 19 is the composition of any one of the preceding embodiments, wherein the composition further comprises a bleaching system.
  • Embodiment 20 is the composition of the immediately preceding embodiment, wherein the bleaching system comprises a source of H 2 O 2 .
  • Embodiment 21 is the composition of the immediately preceding embodiment, wherein the source of H 2 O 2 comprises perborate or percarbonate.
  • Embodiment 22 is the composition of embodiment 20 or 21, wherein the source of H202 comprises a peracid-forming bleach activator.
  • Embodiment 23 is the composition of the immediately preceding embodiment, wherein the peracid-forming bleach activator comprises tetraacetylethylenediamine or nonanoyloxybenzenesulfonate.
  • Embodiment 24 is the composition of any one of the preceding embodiments, wherein the composition further comprises a stabilizing agent.
  • Embodiment 25 is the composition of the immediately preceding embodiment, wherein the stabilizing agent comprises a polyol, sugar, sugar alcohol, lactic acid, boric acid, or a boric acid derivative.
  • Embodiment 26 is the composition of the immediately preceding embodiment, wherein the boric acid derivative comprises a borate ester, an aromatic borate ester, a phenylboronic acid optionally wherein the phenyl is a substituted phenyl, or 4- formylphenylboronic acid.
  • the boric acid derivative comprises a borate ester, an aromatic borate ester, a phenylboronic acid optionally wherein the phenyl is a substituted phenyl, or 4- formylphenylboronic acid.
  • Embodiment 27 is the composition of any one of the preceding embodiments, wherein the composition further comprises one or more of a clay, foam booster, suds suppressor, anti-corrosion agent, soil-suspending agent, anti-soil redeposition agent, bactericide, tarnish inhibitor, or hydrotrope, optionally wherein the hydrotrope comprises urea, tosylate, cumenesulfonate or xylenesulfonate.
  • Embodiment 28 is the composition of any one of the preceding embodiments, wherein the composition further comprises a surfactant.
  • Embodiment 29 is the composition of the immediately preceding embodiment, wherein the surfactant is present at less than about 10% by weight of the composition, optionally wherein the detergent is present at about 0.5-10% by weight of the composition, or about 2-6% by weight of the composition.
  • Embodiment 30 is the composition of any one of embodiments 1-29, wherein the composition comprises the components of an enzymatic detergent product chosen from Endozime AW Triple Plus with APA, Endozime Xtreme Power, Elementum AW, and Endozime BioClean.
  • an enzymatic detergent product chosen from Endozime AW Triple Plus with APA, Endozime Xtreme Power, Elementum AW, and Endozime BioClean.
  • Embodiment 31 is the composition of embodiment 30, wherein the components of the enzymatic detergent product are diluted in a solution comprising one or more of water; a phosphate buffer; an anionic detergent; and a divalent cation chelator.
  • Embodiment 32 is the composition of embodiment 31, wherein the components of the enzymatic detergent product are diluted to about 20% to 80% of their original concentration.
  • Embodiment 33 is the composition of embodiment 31, wherein the components of the enzymatic detergent product are diluted to about 40% to 70% of their original concentration.
  • Embodiment 34 is the composition of embodiment 31, wherein the components of the enzymatic detergent product are diluted to about 50% to 65% of their original concentration.
  • Embodiment 35 is the composition of any one of embodiments 31-34, wherein the components of the enzymatic detergent product are diluted in a solution comprising water; a phosphate buffer; an anionic detergent; and a divalent cation chelator.
  • Embodiment 36 is the composition of any one of the preceding embodiments, wherein the composition has a pH of 3 to 14.
  • Embodiment 37 is the composition of any one of the preceding embodiments, wherein the composition has a pH of 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, or 13 to 14.
  • Embodiment 38 is the composition of any one of the preceding embodiments, wherein the buffer is a phosphate buffer or an alkali salt thereof.
  • Embodiment 39 is the composition of any one of the preceding embodiments, wherein the buffer comprises sodium phosphate.
  • Embodiment 40 is the composition of any one of the preceding embodiments, wherein the divalent cation chelator comprises a magnesium chelator and/ or a calcium chelator.
  • Embodiment 41 is the composition of any one of the preceding embodiments, wherein the divalent cation chelator comprises EDTA or EGTA.
  • Embodiment 42 is the composition of any one of the preceding embodiments, further comprising a biological sample.
  • Embodiment 43 is the composition of the immediately preceding embodiment, wherein the biological sample comprises at least one viscous polymer.
  • Embodiment 44 is the composition of the immediately preceding embodiment wherein the at least one viscous polymer includes a polysaccharide.
  • Embodiment 45 is the composition of embodiment 43, wherein the at least one viscous polymer includes an extracellular nucleic acid.
  • Embodiment 46 is the composition of any one of embodiments 42-45, wherein the biological sample comprises a lipid.
  • Embodiment 47 is the composition of any one of embodiments 42-46, wherein the biological sample comprises a lipid phase or a lipid emulsion.
  • Embodiment 48 is the composition of any one of embodiments 42-47, wherein the biological sample comprises sputum, mucus, or blood.
  • Embodiment 49 is the composition of any one of embodiments 42-48, wherein the biological sample is of plant or animal origin.
  • Embodiment 50 is the composition of any one of embodiments 42-49, wherein the biological sample comprises particulate matter.
  • Embodiment 51 is the composition of any one of embodiments 42-50, wherein the biological sample is a suspension.
  • Embodiment 52 is the composition of any one of embodiments 42-51, wherein the biological sample is of mammalian origin.
  • Embodiment 53 is the composition of any one of embodiments 42-52, wherein the biological sample is of human origin.
  • Embodiment 54 is the composition of any one of embodiments 1-53, further comprising a target sample.
  • Embodiment 55 is the composition of embodiment 54, wherein the target sample comprises a nucleic acid.
  • Embodiment 56 is the composition of embodiment 55, wherein the nucleic acid is RNA.
  • Embodiment 57 is the composition of embodiment 55, wherein the nucleic acid is DNA.
  • Embodiment 58 is the composition of any one of embodiments 42-53, wherein the biological sample comprises or is suspected of comprising a respiratory pathogen.
  • Embodiment 59 is the composition of embodiment 58, wherein the respiratory pathogen is a virus.
  • Embodiment 60 is the composition of embodiment 58, wherein the respiratory pathogen is a coronavirus, parainfluenza virus, Influenza A virus, Influenza B virus, Respiratory Syncytial Virus A, or Respiratory Syncytial Virus B.
  • the respiratory pathogen is a coronavirus, parainfluenza virus, Influenza A virus, Influenza B virus, Respiratory Syncytial Virus A, or Respiratory Syncytial Virus B.
  • Embodiment 61 is the composition of embodiment 58, wherein the respiratory pathogen is a bacterium.
  • Embodiment 62 is the composition of any one of embodiments 42-61, wherein sample type is chosen from sputum, nasal mucous, nasal discharge, and nasopharyngeal swab sample.
  • Embodiment 63 is a method of processing a biological sample, the method comprising contacting the biological sample with the composition of any one of embodiments 1-62.
  • Embodiment 64 is the method of embodiment 63, wherein the biological sample comprises at least one viscous polymer.
  • Embodiment 65 is the method of embodiment64, wherein the at least one viscous polymer includes a polysaccharide.
  • Embodiment 66 is the method of embodiment 64, wherein the at least one viscous polymer includes an extracellular nucleic acid.
  • Embodiment 67 is the method of any one of embodiments 63-66, wherein the biological sample comprises a lipid.
  • Embodiment 68 is the method of any one of embodiments 63-67, wherein the biological sample comprises a lipid phase or a lipid emulsion.
  • Embodiment 69 is the method of any one of embodiments 63-68, wherein the biological sample comprises sputum, mucus, or blood.
  • Embodiment 70 is the method of any one of embodiments 63-69, wherein the biological sample is of plant or animal origin.
  • Embodiment 71 is the method of any one of embodiments 63-70, wherein the biological sample is of mammalian origin.
  • Embodiment 72 is the method of any one of embodiments 63-71, wherein the biological sample is of human origin.
  • Embodiment 73 is the method of any one of embodiments 63-72, further comprising a target sample.
  • Embodiment 74 is the method of embodiment 73, wherein the target sample comprises a nucleic acid.
  • Embodiment 75 is the method of embodiment 74, wherein the nucleic acid is
  • Embodiment 76 is the method of embodiment 74, wherein the nucleic acid is DNA.
  • Embodiment 77 is the method of any one of embodiments 63-76, wherein the biological sample is a suspension.
  • Embodiment 78 is the method of any one of embodiments 63-77, wherein the biological sample comprises particulate matter.
  • Embodiment 79 is the method of any one of embodiments 63-78, further comprising or is suspected of comprising a respiratory pathogen.
  • Embodiment 80 is the method of embodiment 79, wherein the respiratory pathogen is a virus.
  • Embodiment 81 is the method of embodiment 79, wherein the respiratory pathogen is a coronavirus, parainfluenza virus, Influenza A virus, Influenza B virus, Respiratory Syncytial Virus A, or Respiratory Syncytial Virus B.
  • the respiratory pathogen is a coronavirus, parainfluenza virus, Influenza A virus, Influenza B virus, Respiratory Syncytial Virus A, or Respiratory Syncytial Virus B.
  • Embodiment 82 is the method of embodiment 79, wherein the respiratory pathogen is a bacterium.
  • Embodiment 83 is the method of any one of embodiments 63-82, wherein sample type is chosen from sputum, nasal mucous, nasal discharge, and nasopharyngeal swab sample.
  • Embodiment 84 is the method of any one of embodiments 63-83, further comprising amplifying the nucleic acid.
  • Embodiment 85 is the method any one of embodiments 63-84, wherein the biological sample comprises viruses having a coat or envelope and the method further comprises releasing nucleic acid from the coat or envelope.
  • Embodiment 86 is the method of any one of embodiments 63-85, wherein the biological sample comprises at least one target nucleic acid, and the method further comprises capturing the target nucleic acid with at least one capture probe or isolating the target nucleic acid by precipitation or chromatography.
  • Embodiment 87 is a kit containing the composition of any one of embodiments 1 to 41.
  • Embodiment 88 is the kit of embodiment 87, wherein the composition is contained in a tube.
  • Embodiment 89 is the kit of embodiment 87 or 88, wherein the composition is contained in a tube comprising a cap.
  • Embodiment 90 is the kit of any one of embodiments 87 to 89, wherein the kit contains from 1 mL to 10 mL of the composition.
  • Embodiment 91 is the kit of any one of embodiments 87 to 90, wherein the kit further comprises a swab comprising a tip portion and a stem portion.
  • Embodiment 92 is the kit of embodiment 91, wherein the tip portion of the swab is a flocked tip.
  • Embodiment 93 is the kit of embodiment 91 or 92, wherein the stem portion of the swab is a flexible plastic stem.
  • Embodiment 94 is the kit of any one of embodiments 91 to 93, wherein the swab is a nasopharyngeal swab.
  • Embodiment 95 is the kit of any one of embodiments 87 to 84, wherein the kit further comprises instructions for use.
  • Embodiment 96 is the kit of embodiment 95, wherein the instructions for use are provided as a digital document.
  • Embodiment 97 is the kit of embodiment 95 or 96, wherein the instructions for use include directions for biological sample collection.
  • Embodiment 98 is the kit of embodiment 95 to 97, wherein the instructions for use include directions for nasopharyngeal sample collection.
  • Embodiment 99 is a use of the kit of any one of embodiments 87 to 98 for collection of a biological sample for diagnostic testing.
  • Embodiment 100 is the use of embodiment 99, wherein the biological sample is collected for nucleic acid diagnostic testing.
  • Embodiment 101 is the use of embodiment 99 or 100, wherein the biological sample is collected for diagnostic testing to determine the presence or absence of a pathogen.
  • Embodiment 102 is the use of embodiment 99 to 101 wherein the biological sample comprises one or more of protein, mucin, sputum, saliva, nasal discharge, mucoidal secretions, nasal mucus, pulmonary secretions, urine, blood, plasma, or serum
  • Bio sample includes any specimen that may contain a target material (e.g., nucleic acid, virus, or other biomolecule), such as any tissue or material derived from or containing a living or dead organism or that may contain target material derived therefrom, including, e.g., peripheral blood, plasma, serum, lymph node, gastrointestinal tissue, cerebrospinal fluid, sputum, mucus, stool, urine, semen, vaginal secretion, saliva, biopsy material, other body fluids or materials, soil, sludge, or microbial growth such as a bacterial, archaeal, fungal, or protist colony or biofilm.
  • a target material e.g., nucleic acid, virus, or other biomolecule
  • the biological sample may be treated to physically or mechanically disrupt tissue or cell structure, thus releasing intracellular components into a solution or suspension, and/ or combined with a buffer or other components.
  • biological samples include crude samples as discussed below and processed samples, such as those obtained from passing samples over or through a filtering device, or following centrifugation, or by adherence to a medium, matrix, or support.
  • a “crude sample” is a biological sample obtained directly from a living or dead organism, food, the environment, or the like, in contrast to extracts, lysates, filtrates, or eluates. Merely dissolving, suspending, or otherwise mixing crude material in or with a liquid does not convert it to a processed sample.
  • a liquid is “aqueous” if it is water, a mixture of water and a liquid miscible with water where the water is present in more than a trace amount, or a solution in which the solvent is any of the foregoing.
  • an aqueous liquid is at least 50% water by weight.
  • Porate matter includes any material, excluding whole cells and cellular debris, of sufficient solidity to partially or completely obstruct the aperture of a pipet or micropipette tip. A solute dissolved in a solution is not particulate.
  • a “suspension” is a liquid containing undissolved solid or particulate matter.
  • “Aliquot” is used simply to denote some or all of a sample. First and second portions of an aliquot may or may not represent the entire aliquot.
  • viscous polymer is a polymer that is dissolved in sufficient quantity to detectably increase the viscosity of a liquid upon dissolution.
  • viscous polymers can comprise a plurality of hydrogen bond donors and acceptors, such as hydroxyls, amines, oxos (carbonyl oxygens), etc., such that polymer molecules increase viscosity by exerting sufficient intermolecular attractive forces on a plurality of other polymer molecules and solvent molecules.
  • Polysaccharides, polypeptides, nucleic acids, and various synthetic polymers can all be viscous polymers. Any suitable approach for measuring viscosity may be used. Exemplary instruments for determining viscosity include a capillary viscometer, Zahn cup, vibrational viscometer, and rotational viscometer.
  • a “polysaccharide” includes any linear or branched chain of sugars (including but not limited to amino sugars and sugar alcohols as well as true carbohydrates), which may or may not be conjugated (e.g., to a lipid or polypeptide) or derivatized (e.g., phosphorylated, acetylated, alkylated, or the like). Polysaccharide chains may use glycosidic or other bonds (e.g., phosphodiesters in certain polysaccharides such as poly- N-acetylmannosamine phosphate).
  • Lipids include fatty acids, triglycerides, phospholipids, sterols, steroids, waxes, and oils.
  • Food includes any material intended or suitable for consumption, including solids, suspensions, emulsions, and gels (thus including gelatin, milk, ice cream, fruit smoothies, emulsified cheese dips, fruit puree, nut butter, processed and/ or textured protein, and the like as well as bread, fruit, vegetables, meat, etc., but not including clear, monodisperse solutions or water).
  • Nucleic acid refers to a multimeric compound comprising two or more covalently bonded nucleosides or nucleoside analogs having nitrogenous heterocyclic bases, or base analogs, where the nucleosides are linked together by phosphodiester bonds or other linkages to form a polynucleotide.
  • Nucleic acids include RNA, DNA, and combinations and analogs thereof such as “peptide nucleic acids” or PNAs (see, e.g., WO 95/32305) and “locked nucleic acids” (LNA), in which one or more nucleotide monomers have a bicyclic furanose unit locked in an RNA mimicking sugar conformation (see, e.g., Vester et al., Biochemistry 43:13233-41, 2004).
  • a nucleic acid “backbone” may be made up of a variety of linkages, including one or more of sugar- phosphodiester linkages, peptide-nucleic acid bonds as in PNA, phosphorothioate linkages, methylphosphonate linkages, or combinations thereof.
  • Sugar moieties of the nucleic acid may be either ribose or deoxyribose, or similar compounds having known substitutions such as, for example, 2’-mcthoxy substitutions and 2’-halide substitutions (e.g., 2’-F).
  • Nitrogenous bases may be conventional bases (A, G, C, T, U), analogs thereof (e.g., inosine, 5-methylisocytosine, isoguanine; see, e.g., The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992; Abraham et al., 2007, BioTechniques 43: 617-24), which include derivatives of purine or pyrimidine bases (e.g., N 4 -methyl deoxygaunosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases having substituent groups at the 5 or 6 position, purine bases having an altered or replacement substituent at the 2, 6 and/or 8 position, etc., (see generally U.S.
  • polynucleotide denotes a nucleic acid chain.
  • a “nucleotide” is a subunit of a nucleic acid having a phosphate group, a 5-carbon sugar, and a nitrogenous base.
  • the term also includes analogs of such subunits, such as a methoxy group at the 2’ position of the ribose (also referred to herein as “2’-O-Me” or “2’-methoxy”).
  • a “target” material such as a target nucleic acid; other embodiments of target materials include cells, viruses, and other biomolecules
  • target nucleic acids may be DNA or RNA as described herein, and may be either single- stranded or double-stranded.
  • the target nucleic acid may include other sequences besides the target sequence, which may not be detected or quantified.
  • oligomer refers to a nucleic acid having generally less than 1,000 nucleotide (nt) residues, including polymers in a range having a lower limit of about 5 nt residues and an upper limit of about 500 to 900 nt residues.
  • oligonucleotides are in a size range having a lower limit of about 12 to 15 nt and an upper limit of about 50 to 600 nt, and other embodiments are in a range having a lower limit of about 15 to 20 nt and an upper limit of about 22 to 100 nt.
  • An oligonucleotide may serve one or more of various different functions, e.g., as a primer and/ or promoter, detection probe, capture oligomer, etc.
  • “Amplifying” or “amplification” refers to any known procedure for obtaining multiple copies of a target nucleic acid sequence or its complement or fragments thereof. The multiple copies may be referred to as amplicons or amplification products, which can be double-stranded or single- stranded and can include DNA, RNA, or both.
  • Amplification of “fragments” refers to production of an amplified nucleic acid that contains less than the complete target nucleic acid or its complement, e.g., produced by using an amplification oligonucleotide that hybridizes to, and initiates polymerization from, an internal position of the target nucleic acid.
  • amplification methods include, for example, replicase-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand-displacement amplification (SDA), helicase-dependent amplification, and transcription-mediated or transcription-associated amplification.
  • Replicase-mediated amplification uses self-replicating RNA molecules, and a replicase such as QB-replicase (see.
  • PCR amplification uses a DNA polymerase, pairs of primers, and thermal cycling to synthesize multiple copies of two complementary strands of dsDNA or from a cDNA (see. e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,800,159; each incorporated by reference herein).
  • LCR amplification uses four or more different oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation (see. e.g., U.S. Pat. Nos.
  • SDA uses a primer that contains a recognition site for a restriction endonuclease and an endonuclease that nicks one strand of a hemimodified DNA duplex that includes the target sequence, whereby amplification occurs in a series of primer extension and strand displacement steps (see, e.g., U.S. Pat. Nos. 5,422,252; 5,547,861; and 5,648,211; each incorporated by reference herein).
  • Helicase-dependent amplification uses a helicase to separate the two strands of a DNA duplex generating single- stranded templates, followed by hybridization of sequence-specific primers hybridize to the templates and extension by DNA polymerase to amplify the target sequence (see, e.g., U.S. Pat. No. 7,282,328, incorporated by reference herein). Amplification may be linear or exponential.
  • Detection probe “detection oligonucleotide,” “probe oligomer,” and “detection probe oligomer” are used interchangeably to refer to a nucleic acid oligomer that hybridizes specifically to a target sequence in a nucleic acid, or in an amplified nucleic acid, under conditions that promote hybridization to allow detection of the target sequence or amplified nucleic acid. Detection may either be direct (e.g, a probe hybridized directly to its target sequence) or indirect (e.g., a probe linked to its target via an intermediate molecular structure). Examples include invasive probes and primary probes, discussed below.
  • Detection probes may be DNA, RNA, analogs thereof or combinations thereof (e.g., DNA/RNA chimerics) and they may be labeled or unlabeled.
  • a detection probe generally refers to a smaller nucleic acid sequence region within a larger nucleic acid sequence that hybridizes specifically to at least a portion of a probe oligomer by base pairing.
  • a detection probe may comprise target- specific sequences and other sequences that contribute to the three-dimensional conformation of the probe (see. e.g., U.S. Pat. Nos. 5,118,801; 5,312,728; 6,849,412; 6,835,542; 6,534,274; and 6,361,945; and US Patent Application Pub. No. 20060068417; each incorporated by reference herein).
  • label refers to a moiety or compound joined directly or indirectly to a probe that is detected or leads to a detectable signal.
  • Direct labeling can occur through bonds or interactions that link the label to the probe, including covalent bonds or non-covalent interactions, e.g., hydrogen bonds, hydrophobic and ionic interactions, or formation of chelates or coordination complexes.
  • Indirect labeling can occur through use of a bridging moiety or “linker” such as a binding pair member, an antibody or additional oligomer, which is either directly or indirectly labeled, and which may amplify the detectable signal.
  • Labels include any detectable moiety, such as a radionuclide, ligand (e.g., biotin, avidin), enzyme or enzyme substrate, reactive group, or chromophore (e.g., dye, particle, or bead that imparts detectable color), luminescent compound (e.g., bioluminescent, phosphorescent, or chemiluminescent labels), or fluorophore.
  • Labels may be detectable in a homogeneous assay in which bound labeled probe in a mixture exhibits a detectable change different from that of an unbound labeled probe, e.g., instability or differential degradation properties.
  • Capture probe “capture oligonucleotide,” “capture oligomer,” “target capture oligomer,” and “capture probe oligomer” are used interchangeably to refer to a nucleic acid oligomer that specifically hybridizes to a target sequence in a target nucleic acid by standard base pairing and joins to a binding partner on an immobilized probe to capture the target nucleic acid to a support.
  • a capture oligomer includes two binding regions: a sequence-binding region (e.g., target-specific portion) and an immobilized probe-binding region, usually on the same oligomer, although the two regions may be present on two different oligomers joined together by one or more linkers.
  • a capture oligomer uses a target-sequence binding region that includes random or non-random poly-GU, poly-GT, or poly-U sequences to bind non-specifically to a target nucleic acid and link it to an immobilized probe on a support.
  • target material e.g., target nucleic acids
  • aqueous solution phase which may also include suspended material, cellular fragments, proteins, carbohydrates, lipids, and other nucleic acids.
  • “Separating” or “purifying” does not connote any particular degree of purification.
  • separating or purifying reduces the amount or concentration of other sample components by at least 70%, or at least 80%, or at least 95%. In some embodiments, separating or purifying renders the target material at least 70%, or at least 80%, or at least 95% pure.
  • a composition for sample processing may comprise a degradative enzyme, a detergent, and at least one additive, such as a buffer and/ or a chelator.
  • the composition may comprise two or more degradative enzymes.
  • a degradative enzyme may be wild- type, chemically modified, or engineered. Enzymes may differ in some engineered way from a related wild-type sequence by way of amino acid substitution. For example, a nucleic acid substitution may give rise to an enzyme variant that is different in its specific activity, thermostability, or pH optimum. In some embodiments, an enzyme variant may comprise an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type enzyme.
  • the enzyme may be an amylase, arabinase, carbohydrase, cellulase, cutinase, esterase, galactanase, glucanase, lipase, mannanase, oxidase, pectinase, peroxidase, protease, or xylanase, or a combination thereof.
  • a composition may comprise a plurality (e.g., at least two, at least three, or at least four) of these exemplary enzymes.
  • a composition may comprise a lipase, protease, and/ or amylase.
  • a composition may comprise a lipase, protease, carbohydrase, and/ or amylase.
  • the enzyme may comprise one or more of the enzymes present in an enzymatic detergent product, such as Endozime AW Triple Plus with APA (Ruhof Corporation, cat no.
  • Endozime Xtreme Power (Ruhof Corporation, cat no 34530), Elementum AW (Ruhof Corporation, cat no 34511), Endozime BioClean (Ruhof Corporation, cat no 34524), Pure Enzymatic Detergent (Boston Scientific, cat no SEE-573-4), Revital-Ox Enzymatic Detergents (Steris Healthcare, cat no 2D97AW), and Metrizyme (Metrix Research LLC, cat no 10-4000).
  • the enzymes comprise each of the enzymes present in an enzymatic detergent product, such as Endozime AW Triple Plus with APA, Endozime Xtreme Power, Elementum AW, Endozime BioClean, Pure Enzymatic Detergent, Revital-Ox Enzymatic Detergents, and Metrizyme.
  • an enzymatic detergent product such as Endozime AW Triple Plus with APA, Endozime Xtreme Power, Elementum AW, Endozime BioClean, Pure Enzymatic Detergent, Revital-Ox Enzymatic Detergents, and Metrizyme.
  • the components of the enzymatic detergent product are diluted in a solution comprising one or more of water; a phosphate buffer; an anionic detergent; and a divalent cation chelator.
  • the enzymes comprise each of the enzymes present in an enzymatic detergent product, such as Endozime AW Triple Plus with APA, Endozime Xtreme Power, Elementum AW, Endozime BioClean, Pure Enzymatic Detergent, Revital-Ox Enzymatic Detergents, and Metrizyme, wherein the components of the enzymatic detergent product are diluted to about 20% to 80% of their original concentration.
  • an enzymatic detergent product such as Endozime AW Triple Plus with APA, Endozime Xtreme Power, Elementum AW, Endozime BioClean, Pure Enzymatic Detergent, Revital-Ox Enzymatic Detergents, and Metrizyme
  • the enzymes comprise each of the enzymes present in an enzymatic detergent product, such as Endozime AW Triple Plus with APA, Endozime Xtreme Power, Elementum AW, Endozime BioClean, Pure Enzymatic Detergent, Revital-Ox Enzymatic Detergents, and Metrizyme, wherein the components of the enzymatic detergent product are diluted to about 40% to 75% of their original concentration.
  • an enzymatic detergent product such as Endozime AW Triple Plus with APA, Endozime Xtreme Power, Elementum AW, Endozime BioClean, Pure Enzymatic Detergent, Revital-Ox Enzymatic Detergents, and Metrizyme
  • the enzymes comprise each of the enzymes present in an enzymatic detergent product, such as Endozime AW Triple Plus with APA, Endozime Xtreme Power, Elementum AW, Endozime BioClean, Pure Enzymatic Detergent, Revital-Ox Enzymatic Detergents, and Metrizyme, wherein the components of the enzymatic detergent product are diluted to about 50% to 65% of their original concentration.
  • an enzymatic detergent product such as Endozime AW Triple Plus with APA, Endozime Xtreme Power, Elementum AW, Endozime BioClean, Pure Enzymatic Detergent, Revital-Ox Enzymatic Detergents, and Metrizyme
  • Suitable lipases include microbial lipases of bacterial or fungal origin. Wild-type, chemically modified, and protein engineered mutants are included.
  • Examples of bacteria from which lipase may be derived from include bacteria from the genus Brochothris, Lactobacillus, and Pseudomonas. Examples of bacteria include Brochothris thermosohata, Lactobacillus curvatus, Pseudomonas cepacian, Pseudomonas plantari, and Pseudomonas stutzeri.
  • fungi from which lipases may be derived from include fungi from the genus Candida, Humicola, Rhizomucor, and Thermomyces.
  • fungi include Absidia blakesleena, Absidia corymbifera, Aureobasidiurn puliulans, Candida antartica, Coprinus cinerius, Fusarium oxysporum, Fusarium solani, Geotricum penicillatum, Hansenula anomala, Humicola insolens, Penicillum expansum, Rhizomucor miehei, Rhizopus japonicas, Rhizopus microsporus, Rhodotorula glutinis, Sporobplomyces shibatanus, Thermomyces lanuginosa, Thiarosporella phaseolina, Trichoderma harzanium, and Trichoderma reesei.
  • Absidia blakesleena Absidia corymbifera
  • Suitable proteases include proteases of animal, vegetable, or microbial origin. Wild-type, chemically modified, and protein engineered mutants are included.
  • proteases examples include serine proteases, neutral proteases, and alkaline proteases.
  • Serine protease is an enzyme that catalyzes the hydrolysis of peptide bonds and includes an essential serine residue at the active site.
  • the serine protease is porcine trypsin, bovine trypsin, or Fusarium protease.
  • the protease is derived from Aspergillus, Rhizopus, Bacillus alcalophilus, B. cereus, B. vulgatus, B. myocoide, or Nocardiopsis N. natto.
  • Neutral protease is an enzyme that catalyzes the hydrolysis of peptide bonds in a neutral, weakly acidic, or weakly alkaline environment.
  • Neutral proteases have optimal proteolytic activity in a neutral pH range of about 6 to about 8, and may be derived from bacterial, fungal, yeast, plant, or animal sources. Examples of neutral proteases include aspartate and metalloproteases.
  • the metalloprotease is Neutrase® (produced by submerged fermentation of a strain of Bacillus subtilis).
  • Alkaline protease is catalytically active in a pH that ranges from neutral to alkaline.
  • alkaline proteases include RP-II proteases and subtilisins.
  • the protease is derived from Bacillus, Nocardiopsis spe, or Nocardiopsis solonvillei.
  • the protease is a subtilisin Novo, subtilis Carlsberg, subtilisin 309, subtilisin 147 or subtilisin 168.
  • the protease is a wild-type RP-II protease or variants thereof, or wild-type subtilisin or variants thereof.
  • the subtilisin variant comprises substitutions in one or more of the following positions relative to a wild-type subtilisin amino acid sequence: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274.
  • a wild-type subtilisin amino acid sequence see US 6,605,458 B1 at Fig. 1 (BLSAVI sequence, SEQ ID NO: 10 of US 6,605,458 Bl). US 6,605,458 B1 is incorporated herein by reference.
  • a commercially available protease may be used, such as AlcalaseTM, DuralaseTM, EsperaseTM, FN2TM, FN3TM, KannaseTM, MaxacalTM, MaxapemTM, MaxataseTM, PrimaseTM, ProperaseTM, Purafect OxPTM, PurafectTM, and SavinaseTM.
  • Suitable conventional fermented commercial proteases may include, for example, Alcalase® (produced by submerged fermentation of a strain of Bacillus licheniformis), Durazyme® (a protein-engineered variant of Savinase®), Esperase® (produced by submerged fermentation of an alkalophilic species of Bacillus), Rennilase® (produced by submerged fermentation of a non-pathogenic strain of Mucor miehei), and Savinase® (produced by submerged fermentation of a genetically modified strain of Bacillus) .
  • Alcalase® produced by submerged fermentation of a strain of Bacillus licheniformis
  • Durazyme® a protein-engineered variant of Savinase®
  • Esperase® produced by submerged fermentation of an alkalophilic species of Bacillus
  • Rennilase® produced by submerged fermentation of a non-pathogenic strain of Mucor miehei
  • Savinase® produced by submerged fermentation of a genetically modified strain of Bacillus
  • a composition may comprise RP-II and subtilisin of the subtilase group I-S2 (Siezen et al., Protein Science, 6: 501-523 91997) or high alkaline subtilisins.
  • I-S2 proteases include ESPERASE®, MAXACAL®, SAVINASE®, subtilisin 147, subtilisin 309, subtilisin PB92, and alkaline elastase YaB.
  • the combination of proteases may provide an advantage for use with detergents.
  • a composition may comprise BLC and JA96 and variants thereof with SavinaseTM and variants thereof, e.g., DuralaseTM, FN2TM, FN3TM, KannaseTM, MaxacalTM, MaxapemTM, MaxataseTM, ProperaseTM, Purafect OxPTM, and PurafectTM.
  • SavinaseTM and variants thereof e.g., DuralaseTM, FN2TM, FN3TM, KannaseTM, MaxacalTM, MaxapemTM, MaxataseTM, ProperaseTM, Purafect OxPTM, and PurafectTM.
  • Suitable amylases include those of bacterial or fungal origin. Wild-type, chemically modified, and protein engineered mutants are included.
  • the amylase is an alpha amylase, which may be obtained from Bacillus, e.g., a special strain of B. licheniformis.
  • the amylase is derived from Bacillus amyloliquefaciens, Bacillus licheniformis Bacillus stearothermophilus, or Bacillus subtilis.
  • Bacillus subtilis is derived from an Aspergillus, such as Aspergillus niger or Aspergillus oryzae.
  • the amylase variant comprises substitutions in one or more of the following positions relative to a wild-type amylase amino acid sequence: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444 (e.g., the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, which are incorporated herein by reference).
  • An exemplary Xl. oryzae ⁇ -amylase sequence appears at page 7, lines 18-33, of WO 94/02597, which is incorporated herein by reference.
  • a commercially available amylase may be used, such as BANTM, Bioamylase D(G), DuramylTM, FungaMylTM, Kam’s Enzyme Plus, KemzymTM AT 9000, PurafectTM OxAm, PurastarTM HPAmL, PurastarTM St, RapidaseTM TEX, StainzymeTM, and Termamyl®.
  • BANTM Bioamylase D(G), DuramylTM, FungaMylTM, Kam’s Enzyme Plus, KemzymTM AT 9000, PurafectTM OxAm, PurastarTM HPAmL, PurastarTM St, RapidaseTM TEX, StainzymeTM, and Termamyl®.
  • a carbohydrase is an enzyme capable of degrading an oligosaccharide or polysaccharide.
  • a carbohydrase is used which is other than an amylase, e.g., a cellulase, maltase, galactosidase, lactase, pectinase, xylanase, or the like.
  • a detergent may be used to lyse cells containing target material and/ or to solubilize matter that is otherwise insoluble or poorly soluble in aqueous liquids.
  • Target materials are discussed in Section B above.
  • the detergent may comprise a concentration that is appropriate for cell lysis and desired the catalytic efficiencies of enzymes in the composition.
  • the detergent may be used in a concentrated or diluted form.
  • a detergent may be anionic, non-ionic, or zwitterionic.
  • the detergent comprises an alkyl sulfate, such as a dodecyl sulfate (also known as a lauryl sulfate) .
  • the detergent comprises a sodium salt and/ or a lithium salt.
  • an alkyl sulfate detergent with other components described herein (e.g., two or more degradative enzymes) can beneficially reduce problems with sample aspiration due to clots while also maintaining nucleic acids in a form suitable for subsequent amplification and/ or detection.
  • a detergent may comprise a surfactant that is non-ionic, anionic, amphoteric, or cationic, or any combination thereof.
  • nonionic surfactants include alkanolamides, amine oxides, block polymers, ethoxylated primary and secondary alcohols, ethoxylated alkyphenols, ethoxylated fatty esters, sorbitan derivatives, glycerol esters, propoxylated and ethoxylated fatty acids, alcohols, and alkyl phenols, glycol esters, polymeric polysaccharides, sulfates and sulfonates of ethoxylated alkylphenols, and polymeric surfactants.
  • anionic surfactants include ethoxylated amines or amides, sulfosuccinates and derivatives, sulfates of ethoxylated alcohols, sulfates of alcohols, sulfonates and sulfonic acid derivatives, phosphate esters, and polymeric surfactants.
  • amphoteric surfactants include betaine derivatives.
  • cationic surfactants include amine surfactants.
  • the detergent comprises lithium lauryl sulphate.
  • the composition comprises a detergent concentration of 0-0.5% w/v, 0.25-0.75% w/v, 0.5-1.0% w/v, 0.75-1.25% w/v, 1.0-1.5% w/v, 1.25-1.75% w/v, 1.5-2.0% w/v, 1.75-2.25% w/v, 2-2.5% w/v, 2.25-2.75% w/v, 2.5-3.0% w/v, 2.75- 3.25% w/v, 3-3.5% w/v, 3.25-3.75% w/v, 3.5-4.0% w/v, 3.75-4.25% w/v, 4.0-4.5% w/v, 4.25-4.75% w/v, 4.5-5.0% w/v, 4.75-5.25% w/v, 5.0-5.5% w/v, 5.25-5.75% w/v,
  • the detergent concentration is 2.75-3.25% w/v.
  • the detergent may be present at a concentration of less than about 10% by weight of the composition. In some embodiments, the detergent is present at about 0.5-10%, 2-6%, 0.5-1.5%, 1-2%, 1.5-2.5%, 2-3%, 2.5-3.5%, 3-4%, 3.5- 4.5%, 4-5%, 4.5-5.5%, 5-6%, 5.5-6.5%, 6-7%, 6.5-7.5%, 7-8%, 7.5-8.5%, 8-9%, or 8.5- 9.5% by weight of the composition. 3. Additives
  • compositions of the present disclosure may comprise an additive, e.g., at a concentration from about 0.1 wt % to about 2.0 wt % of the total weight of the composition.
  • additives include bactericides, biocides, biopolymer degrading agents, bleaching agents, bleaching systems, buffers, chelators, enhancing agents, fungicides, polymers, reagents, salts, and stabilizing agents.
  • Buffers may be used to maintain pH of a composition within a desired range.
  • the desired pH range may be chosen from the following pH ranges:
  • buffers examples include Good’s Buffers; alkali salts, ammonia buffer; 3-(N-morpholino)propanesulfonic acid (MOPS); bis(tris(hydroxymethyl)methylamino)propane (BIS-TRIS); borate buffer; carbonate buffer; citrate buffer; hydroxyethyl piperazineethanesulfonic acid (HEPES); phosphate buffer such as sodium phosphate; tartrate buffer; and tris (hydroxymethyl) aminomethane (TRIS) such as TRIS-hydrochloride (TRIS-HCl) and TRIS-EDTA.
  • the buffer comprises sodium phosphate.
  • the composition comprises a buffer concentration of 0-0.2% w/v, 0.1-0.3% w/v, 0.2-0.4% w/v, 0.3-0.5% w/v, 0.4-0.6% w/v, 0.5-0.7% w/v, 0.6-0.8% w/v, 0.7-0.9% w/v, 0.8-1.0% w/v, 0.9-1.1% w/v, 1.0-1.2% w/v, or more.
  • the buffer concentration is 0.4-0.5% w/v.
  • Chelators may be used to inhibit divalent cation-dependent proteases.
  • Examples of chelators include magnesium chelators and calcium chelators.
  • the chelator is ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis( ⁇ -aminocthyl ether) -N,N,N',N'-tetraacetic acid (EGTA), or a salt thereof.
  • the chelator comprises EDTA and EGTA.
  • the composition comprises a chelator concentration of at least 20 mg/L, 25 mg/L, 30 mg/L, 35 mg/L, 40 mg/L, 45 mg/L, 50 mg/L, 55 mg/L, 60 mg/L, 65 mg/L, 70 mg/L, 75 mg/L, 80 mg/L, 90 mg/L, 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L, 500 mg/L or more.
  • composition comprises a chelator concentration of 200-300 mg/L, 250-350 mg/L, 300-400 mg/L, 350-450 mg/L, 400-500 mg/L, 450-550 mg/L, 500-600 mg/L, 550-650 mg/L, 600-700 mg/L, 650-750 mg/L, 700-800 mg/L, 750-850 mg/L, 800-900 mg/L, 850-950 mg/L, 900-1000 mg/L, or more.
  • the chelator concentration is 700-800 mg/L.
  • Salts may be used to maintain osmolarity of a composition.
  • Exemplary salts used include alkali salt, sodium salt, potassium salt, chloride salt, and acetate salt.
  • the composition has an osmolarity of 0 to 50 mOsm, 20 to 50 mOsm, 50 to 100 mOsm, 100 to 200 mOsm, 200 to 300 mOsm, 300 to 400 mOsm, 400 to 500 mOsm, 500 to 600 mOsm, 600 to 700 mOsm, 700 to 800 mOsm, or 800 to 1000 mOsm.
  • Detergent builder or complexing agent 0 to 50 mOsm, 20 to 50 mOsm, 50 to 100 mOsm, 100 to 200 mOsm, 200 to 300 mOsm, 300 to 400 mOsm, 400 to 500 mOsm, 500 to 600 mOsm, 600 to 700 mOsm,
  • the composition may comprise a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g., SKS-6 from Hoechst), e.g., at a concentration less than or equal to about 65% by weight.
  • a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g., SKS-6 from Hoechst), e.g., at
  • the composition may comprise a bleaching system, which may comprise a H 2 O 2 source such as perborate, percarbonate, or a peracid- forming bleach activator.
  • the composition may comprise a peracid-forming bleach activator such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate.
  • the bleaching system may comprise peroxyacids of e.g., the amide, imide, or sulfone type.
  • Stabilizing agents may be used to stabilize enzymes of the composition or target material, such as RNA or DNA.
  • Stabilizing agents may be chosen from boric acid, a boric acid derivative such as an aromatic borate ester or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, calcium ions, glycerol, lactic acid, magnesium ions, polyethylene glycol, propylene glycol, sodium borate, sugar, sugar alcohol, and suitable enzymes.
  • the enzyme(s) of the composition may be stabilized, e.g., using a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in e.g., WO 92/19709 and WO 92/19708.
  • a polyol such as propylene glycol or glycerol
  • a sugar or sugar alcohol lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid
  • the stabilizing agent comprise one or more ribonuclease (RNase) inhibitors.
  • RNase inhibitors may inhibit RNase A, RNase B, and/ or RNase C.
  • RNase inhibitors include small molecules, enzymes, or antibodies that are used to inhibit RNases from degrading RNA molecules.
  • RNAse inhibitor enzymes and antibodies may be of eukaryotic origin.
  • Exemplary RNase inhibitors include ribonucleosside-vanadyl complex, guanadine thiocyanate, polyvinylsulfonic acid, and human placenta RNase inhibitor.
  • RNase inhibitors that may be used include RNeasy, SUPERase-InTM, RNaseOUTTM, Ambion ® RNase Inhibitor, Ambion ® RNA secureTM, RNasin ® , RNasin ® Plus, RIBOPROTECT, RiboLock, and RiboSafe.
  • RNase inhibitors include RNeasy, SUPERase-InTM, RNaseOUTTM, Ambion ® RNase Inhibitor, Ambion ® RNA secureTM, RNasin ® , RNasin ® Plus, RIBOPROTECT, RiboLock, and RiboSafe.
  • the composition may comprise one or more surfactants which may include non-ionic, anionic, amphoteric, cationic, or a combination of surfactants.
  • the composition may comprise nonionic surfactants such as alkanolamides, amine oxides, block polymers, ethoxylated primary and secondary alcohols, ethoxylated alkyphenols, ethoxylated fatty esters, sorbitan derivatives, glycerol esters, propoxylated and ethoxylated fatty acids, alcohols, and alkyl phenols, glycol esters, polymeric polysaccharides, sulfates and sulfonates of ethoxylated alkylphenols, and polymeric surfactants.
  • the composition may comprise anionic surfactants such as ethoxylated amines or amides, sulfosuccinates and derivatives, sulfates of ethoxylated alcohols, sulfates of alcohols, sulfonates and sulfonic acid derivatives, phospohate esters, and polymeric surfactants.
  • the composition may comprise amphoteric surfactants such as betaine derivatives.
  • the composition may comprise cationic surfactants such as amine surfactants.
  • the concentration of surfactants may vary based on the enzymatic activity of the enzymes, the exposure time of the detergent to the biofilm, whether the detergent is in solid, liquid, spray, or gel form, the medical instrument exposed with the detergent, and the cleaning system utilized.
  • the surfactants may be present in the composition at a concentration of less than about 10% of the total weight of the composition (i.e., wt/wt or wt %).
  • the surfactant is present at about 0.5-10%, 2-6%, 0.5-1.5%, 1-2%, 1.5-2.5%, 2-3%, 2.5-3.5%, 3-4%, 3.5-4.5%, 4- 5%, 4.5-5.5%, 5-6%, 5.5-6.5%, 6-7%, 6.5-7.5%, 7-8%, 7.5-8.5%, 8-9%, or 8.5-9.5% by weight of the composition.
  • the concentration of surfactants may vary based on the enzymatic activity of the enzymes, the exposure time of the detergent to the biofilm, whether the detergent is in solid, liquid, spray, or gel form, the medical instrument exposed with the detergent, and the cleaning system utilized. h) Polymers
  • the detergent may comprise one or more polymers, such as carboxymethylcellulose, poly(vinylpyrrolidone), poly(ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), and polycarboxylates such as polyacrylates, maleic /acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.
  • polymers such as carboxymethylcellulose, poly(vinylpyrrolidone), poly(ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), and polycarboxylates such as polyacrylates, maleic /acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.
  • the composition may also contain other ingredients such as clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil re-deposition agents, dyes, bactericides, hydrotropes, and tarnish inhibitors.
  • the hydrotropes may be chosen from urea, tosylate, cumenesulfonate, and xylenesulfonate.
  • a biological sample may comprise a body fluid obtained from a subject that may contain a target material. Examples of biological samples and target materials are provided in Section B above.
  • the biological sample is sputum, nasal mucus, nasal discharge, and/ or a nasopharyngeal swab sample.
  • the target material comprises nucleic acids and/ or respiratory pathogens.
  • a biological sample may comprise target material derived from a respiratory pathogen.
  • Many types of viruses, fungi, and bacteria can infect the respiratory tract. While respiratory infections may share similar symptoms, such as bronchiolitis, chest pain, chills, coughing, croup, fatigue, fever, headache, hypoxia, loss of appetite, muscle or body aches, pneumonia, sore throat, stuffy or runny nose, trouble breathing, and/ or whooping cough, treatment can be different depending on the causative agent. It can, therefore, be useful to determine the respiratory pathogen behind the infection.
  • the respiratory pathogen is a virus, e.g., an influenza virus.
  • Influenza viruses can cause seasonal epidemic of disease known as the flu season, as well as flu pandemics.
  • influenza vims include influenza A, influenza B, influenza C, and influenza D.
  • the respiratory pathogen is a coronavirus.
  • Coronaviruses can cause respiratory infections and have been responsible for regional and global pandemics. Coronaviruses are classified into four subtypes - alpha, beta, gamma, and delta. Examples of coronaviruses include 229E, HKU1, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), NL63, OC43, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).
  • MERS-CoV Middle East Respiratory Syndrome Coronavirus
  • SARS-CoV Severe Acute Respiratory Syndrome Coronavirus
  • SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2
  • the respiratory pathogen is a paramyxovirus.
  • paramyxoviruses include respiratory syncytial virus (RSV), including RSV include RSV subtypes A and B; human parainfluenza viruses (HPIV), including HPIV subtypes 1 to 4; and human metapneumovirus (HMPV) .
  • RSV respiratory syncytial virus
  • HPIV human parainfluenza viruses
  • HPIV human metapneumovirus
  • the respiratory pathogen is an adenovirus.
  • adenoviruses can also cause diarrhea and conjunctivitis.
  • examples of adenovirus include mammalian adenoviruses (mastadenoviruses) and avian adenoviruses (aviadenoviruses) .
  • adenovirus type A (serotypes 12, 18, and 31), B (serotypes 3, 7, 11, 14, 16, 21, 34, and 35), C (serotypes 1, 2, 5, and 6), D (serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-36, and 42-47), E (serotype 4), and F (serotypes 40 and 41).
  • the respiratory pathogen is a rhinovirus.
  • examples of rhinoviruses include Rhinoviruses A, B, and C.
  • the respiratory pathogen is an enterovirus.
  • enteroviruses include EV-D68 and EV-71.
  • the respiratory pathogen is a Bordetella species, such as Bordetella pertussis, which causes whooping cough or pertussis, or Bordetella parapertussis.
  • the respiratory pathogen is Streptococcus pneumoniae. Streptococcus pneumoniae infections can range from respiratory tract, ear, and sinus infections to pneumonia and bloodstream infections.
  • the respiratory pathogen is a mycobacterium. This genus includes pathogens known to cause serious disease in mammals, including tuberculosis and leprosy in humans. Examples of mycobacterium include Mycobacterium tuberculosis.
  • the respiratory pathogen is an Aspergillus species, such as Aspergillus fumigatus. Infection by Aspergillus species may cause aspergillosis.
  • the respiratory pathogen is a Blastomyces species, such as Blastomyces dermatitidis. Infection by Blastomyces dermatitidis may cause blastomycosis.
  • the respiratory pathogen is a Coccidioides species, such as Coccidioides immitis. Infection by Coccidioides immitis may cause coccidioidomycosis, also called valley fever.
  • the respiratory pathogen is a Cryptococcus species, such as Cryptococcus neoformans. Infection by Cryptococcus neoformans may cause Cryptococcosis.
  • the respiratory pathogen is a Histoplasma species, such as Histoplasma capsulatum. Infection by Histoplasma capsulatum may cause histoplasmosis.
  • the respiratory pathogen is a Rhizopus species or Mucor species. Infection by Rhizopus species or Mucor species may cause mucormycosis.
  • the respiratory pathogen is a Pneumocystis species, such as Pneumocystis jirovecii. Infection by Pneumocystis jirovecii may cause Pneumocystis pneumonia.
  • a biological sample may comprise target material derived from a gastrointestinal pathogen.
  • target material derived from a gastrointestinal pathogen.
  • Many types of viruses, parasites, and bacteria can cause gastrointestinal infections. While gastrointestinal infections may share similar symptoms, such as nausea, vomiting, diarrhea, abdominal pain, fatigue, fever, headache, loss of appetite, and/ or muscle or body aches, treatment can be different depending on the causative agent. It can, therefore, be useful to determine the gastrointestinal pathogen behind the infection.
  • the gastrointestinal pathogen is a Yersinia species, such as Yersinia enterocolitica. Infection by Yersinia enterocolitica may cause yersiniosis.
  • the gastrointestinal pathogen is a Vibrio species, such as Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae. Infection by Vibrio species may cause vibriosis.
  • the gastrointestinal pathogen is an Escherichia species, such as Escherichia coli 0157, Escherichia coli Stxl, and Escherichia coli Stx2.
  • the gastrointestinal pathogen is a Plesiomonas species, such as Plesiomonas shigelloides.
  • the gastrointestinal pathogen is a Campylobacter species, such as Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis. Infection by Campylobacter species may cause campy lobacterio sis.
  • the gastrointestinal pathogen is a Shigella species such as Shigella boydii, Shigella dysenteriae, Shigella flexneri, and Shigella sonnei. Infection by Shigella species may cause shigellosis. 2. Viruses
  • the gastrointestinal pathogen is a vims such as norovirus, rotavirus, astrovirus, and sapovirus.
  • a biological sample may comprise target material derived from a parasite. While parasitic infections may share similar symptoms, treatment can be different depending on the causative agent. It can, therefore, be useful to determine the parasite behind the infection.
  • the parasite is a Giardia species, such as Giardia intestinalis. Infection by Giardia species may cause giardiasis.
  • the parasite is a Cryptosporidium species, such as Cryptosporidium parvum. Infection by Cryptosporidium species may cause cryptosporidiosis.
  • the parasite is an Entamoeba species, such as Entamoeba histolytica. Infection by Entamoeba hystolytica may cause amebiasis.
  • the parasite is a Cyclospora species, such as Cyclospora cayetanensis. Infection by Cyclospora cayetanensis may cause cyclosporiasis.
  • the bloodborne pathogen is Babesia species such as Babesia microti. Infection by Babesia species may cause babesiosis, which is spread by the bite of infected Ixodes scapularis ticks.
  • the parasite is Plasmodium species, such as Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi. Infection by Plasmodium may cause malaria.
  • methods disclosed herein use a swab associated with an aliquot of a biological sample.
  • methods according to the disclosure can comprise contacting the biological sample with the swab, wherein an aliquot of the biological sample and the swab become associated.
  • the association between the sample aliquot and the swab should be temporary or reversible at least in part, so that at least some analyte (e.g., cells or nucleic acid) can be released into a liquid.
  • One skilled in the art can choose an appropriate manner of contacting the sample with the swab so as to achieve a suitable association of the sample aliquot with the swab, e.g., dipping, wiping, poking, or spinning the swab into, on, or across the sample.
  • Any swab suitable for adsorbing and releasing biological material can be used.
  • a swab is used that comprises a stem with absorbent and/ or adsorbent material (e.g., sponge or fibers, which can be, e.g., cotton, alginate, silk, carbon fiber, or polymeric fibers, such as rayon, polyester, or polyamide) wrapped around, bonded, or adhered to an end to form a tip, the absorbent or adsorbent material being suitable for adsorbing and/ or absorbing the sample to be collected.
  • the tip has a thickness (measured perpendicularly to the stem axis) of 0.5 to 3 mm.
  • the tip comprises fibers having a linear density of 1 to 4 mg per 10 meters.
  • the swab is a nasal swab.
  • a flocked swab comprising an elongate support body and a plurality of flocked fibers at an end of the support.
  • exemplary flocked swabs are described in US 2006/0142668, which is incorporated herein by reference. Swabs can have elongate stems generally made of plastic materials, for example polystyrene.
  • Any biological sample that can become associated with a swab can be used in methods according to the disclosure.
  • Biological samples used in methods according to the disclosure can be emulsions, suspensions, or solutions (or a combination thereof) and can comprise various types of impurities or contaminants, such as viscous polymers or particulate matter.
  • Viscous polymers can include polysaccharides (e.g., mucopolysaccharides, lipopolysaccharides, proteoglycans, starch, glycogen, or soluble cellulose), extracellular nucleic acids, and polypeptides (including glycoproteins and lipopolypeptides) .
  • Particulate matter can include material derived from food, solid lipids, inorganic solids such as phosphates (e.g., calcium or iron phosphate), and insoluble fibrous matter (e.g., cellulose fibers).
  • a sample can comprise a lipid phase or a lipid emulsion.
  • a sample can comprise cellular debris, which depending on its size, solubility, and hydrophobicity may occur at least in part as particulate matter, viscous polymers, a lipid phase or emulsion, or a combination thereof.
  • Samples can be crude or may have undergone some initial purification, such as filtration, extraction, or fractionation (e.g., chromatographic) .
  • a crude sample is diluted, suspended, or at least partially dissolved with a suitable liquid such as water or a solution, which may be buffered, saline, or isotonic with the sample.
  • sample types that may include particulate matter, viscous polymers, or emulsions are sputum, saliva, mucus such as nasal mucus, blood, soil, sludge, stool, or a microbial growth.
  • Sludge and soil include natural and artificial versions thereof, such as mud, potting mix, chemical waste, etc.
  • Microbial growths include bacterial, archaeal, protist, or fungal growths, such as colonies, biofilms, mycelia, etc.
  • the sample is a food sample.
  • a sample comprises or is suspected of comprising a pathogen, virus, fungus, or bacterium.
  • the pathogen is a respiratory tract pathogen. Examples of respiratory pathogens are discussed in Section E above.
  • any volume of material sufficient to give an adequate second portion for the intended downstream use can be associated with the swab.
  • a volume of 20 to 350 microliters of sample such as 20 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, or 300 to 350 microliters or 20 to 75, 75 to 125, 125 to 175, 175 to 225, 225 to 275, or 275 to 350 microliters becomes associated with the swab.
  • sample material including cells, nucleic acids, or polypeptides; capture, amplification, and detection of targets; target bacterial or pathogen sequences
  • viruses or cells released into the liquid are disrupted or lysed, e.g., by heating, sonication, vortexing, osmolysis, proteolysis, exposure to alkali and/ or detergent, or a combination thereof. This can occur in the liquid, either commencing immediately upon transferring the swab into the liquid such as in the case of the liquid being a lysis solution, or afterward, e.g., as in the case of further manipulation by addition of reagents or use of equipment following release of the portion of the aliquot into the liquid.
  • nucleic acids are isolated from the lysate, e.g., by chromatography or precipitation, or binding to a binding agent, such as at least one capture oligomer.
  • nucleic acids released from the swab into the carrier liquid or from cells or viruses released and lysed as discussed above are further isolated or purified.
  • further isolation or purification can be performed using chromatography or precipitation, or by binding nucleic acid to a binding agent, such as at least one capture oligomer.
  • a binding agent such as at least one capture oligomer.
  • At least one capture oligomer can be sequence-specific (i.e., it comprises a target-hybridizing region configured to specifically hybridize to a site in the target nucleic acid to form a duplex).
  • a capture oligomer includes two binding regions: a sequence-binding region (e.g., target-specific portion) and an immobilized probe-binding region, usually on the same oligomer, although the two regions may be present on two different oligomers joined together by one or more linkers.
  • capture oligomers with randomized, repeating, or non-specific sequences for hybridization to a target can be used, such as poly-(k) and poly-(r) capture oligomers and combinations thereof, which are described, e.g., in US 2013/0209992 and in US 2020/0165599, which are incorporated herein by reference.
  • a capture oligomer uses a target-sequence binding region that includes random or non- random poly-GU, poly-GT, or poly-U sequences to bind non-specifically to a target nucleic acid.
  • a capture oligomer uses a target-sequence binding region that includes random or non-random poly-GA, sequences to bind non-specifically to a target nucleic acid.
  • a combination of capture oligomers is used in which a first capture oligomer comprises random or non-random poly-GA, sequences and the second capture oligomer comprises random or non-random poly-GU or poly-GT sequences.
  • the capture oligomers can further comprise a sequence or moiety that binds an immobilized probe on a support.
  • a method further comprises determining the presence or absence of at least one macromolecule in the sample.
  • nucleic acids isolated from the lysate can be subjected to one or more analytical procedures such as detection of the presence or absence of one or more target sequences (e.g., using one or more probes, such as probe oligomers, for the target regions, which can follow amplification of the target sequences or can be performed on isolated nucleic acid directly, as in Southern, slot, or dot blotting); sequencing; electrophoresis; molecular cloning; or microarray analysis.
  • an amplification reaction is performed such as PCR, TMA, or any other type of amplification including those discussed above.
  • a probe oligomer or FRET cassette is used that comprises a label.
  • Particularly suitable labels include compounds that emit a detectable light signal, e.g., fluorophores or luminescent (e.g., chemiluminescent) compounds that can be detected in a homogeneous mixture. More than one label, and more than one type of label, may be present on a particular probe, or detection may rely on using a mixture of probes in which each probe is labeled with a compound that produces a detectable signal (see. e.g., U.S. Pat. Nos. 6,180,340 and 6,350,579, each incorporated by reference herein) .
  • Labels may be attached to a probe by various means including covalent linkages, chelation, and ionic interactions, but in some embodiments the label is covalently attached.
  • a detection probe has an attached fluorescent or chemiluminescent label such as, e.g., an acridinium ester (AE) compound (see. e.g., U.S. Pat. Nos. 5,185,439; 5,639,604; 5,585,481; and 5,656,744; each incorporated by reference herein), which in typical variations is attached to the probe by a non-nucleotide linker (see. e.g., U.S. Pat. Nos.
  • AE acridinium ester
  • a probe oligomer is labeled with an interactive pair of detectable labels.
  • detectable labels that are members of an interactive pair of labels include those that interact with each other by FRET or non-FRET energy transfer mechanisms.
  • Fluorescence resonance energy transfer involves the radiationless transmission of energy quanta from the site of absorption to the site of its utilization in the molecule, or system of molecules, by resonance interaction between chromophores, over distances considerably greater than interatomic distances, without conversion to thermal energy, and without the donor and acceptor coming into kinetic collision.
  • the “donor” is the moiety that initially absorbs the energy
  • the “acceptor” is the moiety to which the energy is subsequently transferred.
  • FRET fluorescence transfer
  • At least one probe system is used, wherein the oligomers of the system function together to facilitate detection of a target sequence.
  • an invasive oligomer which can also but does not necessarily function as an amplification oligomer, e.g., primer
  • a primary probe e.g., primer
  • FRET cassette e.g., FRET cassette
  • INVADER and INVADER PLUS assays are discussed in detail, e.g., in US 2018/0163259.
  • a detection probe oligomer in accordance with the present disclosure can comprise a non-target-hybridizing sequence.
  • probes exhibiting at least some degree of self-complementarity are desirable to facilitate detection of probe:target duplexes in a test sample without first requiring the removal of unhybridized probe prior to detection.
  • Specific embodiments of such detection probes include, for example, probes that form conformations held by intramolecular hybridization, such as conformations generally referred to as hairpins.
  • Particularly suitable hairpin probes include a “molecular torch” (see. e.g., U.S. Pat. Nos. 6,849,412; 6,835,542; 6,534,274; and 6,361,945, each incorporated by reference herein) and a “molecular beacon” (see. e.g.,
  • a detection probe is a linear oligomer that does not substantially form conformations held by intramolecular bonds.
  • an amplification and detection procedure is performed such as an INVADER or INVADER PLUS assay, a TaqMan assay, or a TMA reaction with probe hybridization and detection (e.g., using at least one probe oligomer or FRET cassette comprising a fluorophore and a FRET partner and exhibiting hybridization- dependent and/or degradation-dependent fluorescence).
  • probe hybridization and detection e.g., using at least one probe oligomer or FRET cassette comprising a fluorophore and a FRET partner and exhibiting hybridization- dependent and/or degradation-dependent fluorescence.
  • detection is in real time during the amplification reaction. In some embodiments, detection occurs near, at, or after the end of the amplification reaction. In some embodiments, at least one target sequence is quantified, e.g., based on a standard curve and/ or one or more calibration standards.
  • the method can comprise determining the presence or absence of at least one nucleic acid characteristic of the pathogen.
  • polypeptides isolated from the lysate can be subjected to one or more analytical procedures such as detection of the presence or absence of one or more target or other forms of characterization, which may include one or more of an antibody or aptamer-binding assay (e.g., ELISA or Western blot); electrophoresis; amino acid sequencing; or mass spectrometry.
  • at least one polypeptide is quantified, e.g., based on a standard curve and/ or one or more calibration standards.
  • cells released into the carrier liquid are fixed, e.g., with an aldehyde or other cross-linking agent.
  • the cells are stained, e.g., with a fluorescent dye or chromophore, which is optionally linked to a binding agent such as a nucleic acid probe (e.g., FISH probe), antibody, or aptamer.
  • the cells are subjected to flow cytometry, microscopy, or other optical analysis.
  • kits containing any one of the compositions described herein.
  • the composition is contained within a tube, e.g., a tube comprising a cap.
  • the kit further comprises a swab comprising a tip portion and a stem portion.
  • the tip portion of the swab is a flocked tip.
  • the stem portion of the swab is a flexible plastic stem.
  • the swab is a nasopharyngeal swab.
  • the kit contains from 1 mL to 10 mL of any one of the compositions described herein.
  • the kit further comprises instructions for use.
  • the instructions for use are provided as a digital document.
  • the instructions for use include directions for biological sample collection.
  • the instructions for use include directions for nasopharyngeal sample collection.
  • kits described herein for collection of a biological sample for diagnostic testing.
  • the biological sample is collected for nucleic acid diagnostic testing.
  • the biological sample comprises one or more of protein, mucin, sputum, saliva, nasal discharge, mucoidal secretions, nasal mucus, pulmonary secretions, urine, blood, plasma, or serum.
  • the biological specimen is collected for diagnostic testing to determine the presence or absence of a pathogen.
  • the biological sample is collected for diagnostic testing to determine the presence or absence of a respiratory pathogen.
  • Example 1 Effect of Multi-Enzyme Detergent and Sample Transport Media on Sample Viscosity and the Amplification and Detection of Target Nucleic Acids
  • the recipe for STM is as follows: 2.07 g/L sodium phosphate, monobasic, monohydrate; 30 g/L lithium lauryl sulphate; 0.372 g/L EDTA, disodium, dihydrate; 0.38 g/L EGTA, free acid; and 2.13 g/L sodium phosphate, dibasic.
  • Target nucleic acid extraction, amplification, and detection was performed using the Panther Fusion Flu A/B/RSV assay generally according to manufacturer’s instructions (Hologic, Inc., cat no. PRD-04328).
  • target nucleic acids were successfully amplified and detected in all samples in STM (Table 3). These results were consistent with results from positive control reactions without clots (Table 4), in which the target nucleic acids for Influenza A (FAM Flu A), Influenza B virus (ROX Flu B), and Respiratory Syncytial Virus A/B (HEX RSV) were detected in all the reactions that were performed at about 0, 12, and 16 hours after they were spiked with IVT.
  • FAM Flu A Influenza A
  • ROX Flu B Influenza B virus
  • HEX RSV Respiratory Syncytial Virus A/B
  • Table 3 Detection of Influenza A virus (FAM Flu A), Influenza B virus (ROX Flu B), and Respiratory Syncytial Virus A/B (HEX RSV) in Collected Samples.
  • Table 4 Detection of Influenza A virus (FAM Flu A), Influenza B virus (ROX Flu B), and Respiratory Syncytial Virus A/B (HEX RSV) in Control Samples.
  • Example 2 Aspiration of Viscous Samples and/or Samples Containing Non- Homogenous Content Following Collection into Several Sample Transport Media
  • the purpose of this example was to reduce aspiration failure associated with specimen containing mucoid or other viscous or non-homogenous material.
  • Clinical specimens from thirty healthy volunteers were collected using nasal swabs. Following collection, the swabs were each individually placed into 1 ml of STM.
  • the recipe for STM is as described above in Example 1. Further, each collected clinical specimen in a specimen condition was agitated and then centrifuged at 200 RCF for 1 minute. Additionally, each ml of STM contained about 6.9 copies/ ⁇ L of IVT for each of Influenza A, Influenza B, and respiratory syncytial virus target nucleic acid.
  • the collected specimens were inspected for the presence of blood and ranked as -Blood, +Blood, or + +Blood, depending on whether the specimen contained no blood, trace amounts of blood, or visible blood clots, respectively.
  • the total volume was then brought to 2.9 mL using additional STM or a combination of STM and Endozime AW Triple Plus with APA, a multi-enzymatic detergent.
  • the resulting sample transport media contained 0% Endozime AW Triple Plus with APA (i.e., STM only), 50% Endozime AW Triple Plus with APA, or 65% Endozime AW Triple Plus with APA (Table 5).
  • Example 3 Amplification and Detection of Viscous Samples and/or Samples Containing Non-Homogenous Content Following Collection into Several Sample Transport Media
  • Results from this example are presented in Tables 6-8. Conditions used and the presence/ absence of blood in the sample are indicated. These results show that target nucleic acids were amplifiable and detectable in all three of the sample transport media conditions. These results also show roughly equivalent Counts (Cts) but lower standard deviations for the specimen processed in the 50% Endozime AW Triple Plus with AP and the 65% Endozime AW Triple Plus with APA compared to those processed in the STM.
  • a further set of assays were performed using the STM, 50% Endozime AW Triple Plus with APA, and 65% Endozime AW Triple Plus with APA conditions as described above in Example 2 and Table 6 except that patient specimens (nasal swabs from volunteers) was not included in the samples.
  • Target nucleic acid extraction, amplification, and detection was performed on a Panther Fusion system using the Panther Fusion Flu A/B/RSV assay according to manufacturer’s instructions.
  • results show equal or lower Cts for target nucleic acids spiked in to the 50% or 65% Endozime AW Triple Plus with APA, compared with those spiked into the 0% Endozime AW Triple Plus with APA (i.e., STM in Table 9), indicating that use of a multi-enzymatic detergent such as Endozime AW Triple Plus with APA in combination with STM did not negatively impact assay performance.
  • Table 9 Detection of Influenza A virus (FAM Flu A), Influenza B virus (ROX Flu B), and Respiratory Syncytial Virus A/B (HEX RSV).

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Abstract

L'invention concerne des procédés, des appareils et des systèmes pour traiter un échantillon biologique. Des exemples de procédés comprennent le transfert d'un écouvillon associé à une aliquote de l'échantillon biologique dans un milieu de transport d'échantillon. Les procédés peuvent permettre un transfert efficace d'un matériau cible tel que des cellules ou un acide nucléique dans le milieu de transport d'échantillon pour l'extraction, l'amplification et la détection du matériau cible.
PCT/US2022/020256 2021-03-15 2022-03-14 Compositions et procédés de traitement d'échantillons biologiques Ceased WO2022197637A1 (fr)

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