Compositions and methods for biological sample processing - Patents.com

Abstract

Methods, devices and systems for processing biological samples are provided. An exemplary method includes transferring a swab associated with an aliquot of the biological sample to a sample transport medium. The method can provide efficient transfer of target material (e.g., cells or nucleic acid) to the sample transport medium for extraction, amplification and detection of the target material. Embodiment 1 is a composition comprising at least one degrading enzyme and a detergent, and optionally a buffer and / or a divalent cation chelating agent.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 161,320, filed March 15, 2021, and U.S. Provisional Patent Application No. 63 / 296,804, filed January 5, 2022, the contents of which are incorporated by reference herein in their entirety.

[0002] Technical Field The present disclosure relates to the field of biotechnology. More particularly, the present disclosure relates to methods and compositions for processing biological samples, for example, in preparation for use in subsequent analytical procedures. [Background technology]

[0003] Introduction and Overview Biological samples derived from subjects are prepared using a variety of methods so that the sample is suitable for storage, processing, and analysis. Throughout sample storage and for as long as necessary during sample processing, the target analyte in the sample must be preserved, while any potentially infectious agents in the sample must be inactivated. Sample transport media are commonly used to stabilize nucleic acids for further analysis during the time following collection from the sample source. Sample transport media can also act to destroy the ability of infectious pathogens to replicate or infect a host.

[0004] Samples containing solid or viscous materials (e.g., mucus; clots) can be particularly challenging. For example, such samples can complicate the specimen transfer steps (including those performed by automated platforms) required to efficiently process and analyze the samples. In some cases, such samples can form precipitates during storage at low temperatures (e.g., 4°C, -20°C, and -70°C), which can complicate subsequent pipetting and loading of the sample into automated instruments. Reducing the viscosity of such samples and / or dissolving or otherwise liquefying them can reduce or eliminate these difficulties.

[0005] Thus, there remains a need for simple, efficient, and rapid methods for preparing samples for storage, processing, and analysis. Improved sample transport media may meet this need or may offer other advantages. Summary of the Invention [Means for solving the problem]

[0006] Thus, the following embodiments are among those provided by the present disclosure:

[0007] Embodiment 1 is a composition comprising at least one degradative enzyme and a detergent, and optionally a buffer and / or a divalent cation chelator.

[0008] Embodiment 2 is the composition of embodiment 1, wherein the at least one degradative enzyme is two degradative enzymes.

[0009] Embodiment 3 is the composition of embodiment 1 or 2, wherein the at least one degrading enzyme comprises a lipase.

[0010] Embodiment 4 is the composition of any one of the preceding embodiments, wherein the at least one degrading enzyme comprises a protease.

[0011] Embodiment 5 is the composition of any one of the preceding embodiments, wherein the degradative enzyme comprises an amylase.

[0012] Embodiment 6 is the composition of any one of the preceding embodiments, wherein the degradative enzymes comprise a lipase, a protease, and an amylase.

[0013] Embodiment 7 is the composition of any one of the preceding embodiments, wherein the degradative enzyme comprises a carbohydrase.

[0014] Embodiment 8 is the composition of any one of the preceding embodiments, wherein the degradative enzymes comprise a lipase, a protease, an amylase, and a carbohydrase.

[0015] Embodiment 9 is the composition of any one of the preceding embodiments, wherein the at least one degrading enzyme is present at about 1-10% v / v, about 1-4% v / v, about 3-6% v / v, about 3% v / v, or about 6% v / v.

[0016] Embodiment 10 is the composition of any one of the preceding embodiments, wherein each of the at least one degrading enzyme is present at about 1-4% v / v, about 1-10% v / v, about 3-6% v / v, or about 6% v / v.

[0017] Embodiment 11 is the composition of any one of embodiments 4 to 9, wherein the protease is present in a concentration of about 1 to 10% by volume.

[0018] Embodiment 12 is the composition of any one of embodiments 4 to 9, wherein the protease is present in a concentration of about 6% by volume.

[0019] Embodiment 13 is the composition of any one of embodiments 1-8, wherein the at least one degrading enzyme is present at about 1-10% w / v, about 1-4% w / v, about 3-6% w / v, about 3% w / v, or about 6% w / v.

[0020] Embodiment 14 is the composition of any one of embodiments 1-8 or 13, wherein each of the at least one degrading enzyme is present at about 1-4% w / v, about 1-10% w / v, about 3-6% w / v, or about 6% w / v.

[0021] Embodiment 15 is the composition of any one of embodiments 4-8 or 13, wherein the protease is present in a concentration of about 1-10% w / v.

[0022] Embodiment 16 is the composition of any one of embodiments 4-8 or 13, wherein the protease is present in a concentration of about 6% w / v.

[0023] Embodiment 17 is the composition of any one of embodiments 4 to 9 or 13, wherein the protease is a serine protease or a composition comprising multiple proteases.

[0024] Embodiment 18 is the composition of any one of embodiments 4-9 or 13, wherein the protease comprises a subtilisin, optionally wherein the subtilisin is a subtilisin or savinase derived from Bacillus lentus.

[0025] Embodiment 19 is the composition of any one of the preceding embodiments, wherein the detergent comprises an anionic detergent.

[0026] Embodiment 20 is the composition of any one of the preceding embodiments, wherein the cleaning agent is a composition comprising an alkyl sulfate.

[0027] Embodiment 21 is the composition of the immediately preceding embodiment, wherein the detergent is a composition comprising dodecyl sulfate.

[0028] Embodiment 22 is the composition of any one of the preceding embodiments, wherein the detergent is a composition comprising a sodium salt.

[0029] Embodiment 23 is the composition of any one of the preceding embodiments, wherein the cleaning agent is a composition comprising a lithium salt.

[0030] Embodiment 24 is the composition of the immediately preceding embodiment, wherein the lithium salt is lithium lauryl sulfate.

[0031] Embodiment 25 is the composition of any one of the preceding embodiments, wherein the detergent comprises a nonionic or zwitterionic detergent.

[0032] Embodiment 26 is the composition of any one of the preceding embodiments, further comprising a detergent booster, coupling agent, or complexing agent.

[0033] Embodiment 27 is the composition of any one of the preceding embodiments, wherein the detergent booster, coupling agent, or complexing agent comprises sodium xylene sulfonate, zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl or alkenyl succinic acid, soluble silicate, or layered silicate; optionally the layered silicate is Hoechst SKS-6.

[0034] Embodiment 28 is the composition of any one of the preceding embodiments, wherein the detergent is present in less than about 10% by weight of the composition, optionally, the detergent is present in about 0.05-10% by weight of the composition, or about 0.1-0.15% by weight of the composition.

[0035] Embodiment 29 is the composition of any one of embodiments 1-27, wherein the detergent is present at less than about 10% w / v, optionally wherein the detergent is present at about 0.05-10% w / v, or about 0.1-0.15% w / v.

[0036] Embodiment 30 is the composition of any one of the preceding embodiments, further comprising a bleaching system.

[0037] Embodiment 31 is the composition of the immediately preceding embodiment, wherein the bleaching system includes a source of H2O2.

[0038] Embodiment 32 is the composition of the immediately preceding embodiment, wherein the source of H2O2 comprises a perborate or percarbonate.

[0039] Embodiment 33 is the composition of embodiment 25 or 26, wherein the source of H2O2 is a composition comprising a peracid-forming bleach activator.

[0040] Embodiment 34 is the composition of the immediately preceding embodiment, wherein the peracid-forming bleach activator comprises tetraacetylethylenediamine or nonanoyloxybenzenesulfonate.

[0041] Embodiment 35 is a composition of any one of the preceding embodiments, further comprising a stabilizer.

[0042] Embodiment 36 is the composition of the immediately preceding embodiment, wherein the stabilizing agent comprises propylene glycol, a polyol, a sugar, a sugar alcohol, lactic acid, boric acid, or a boric acid derivative.

[0043] Embodiment 37 is the composition of the immediately preceding embodiment, wherein the boric acid derivative comprises a boric acid ester, an aromatic boric acid ester, a phenylboronic acid, optionally wherein the phenyl is a substituted phenyl, or 4-formylphenylboronic acid.

[0044] Embodiment 38 is the composition of any one of the preceding embodiments, further comprising one or more of a clay, a foam booster, a defoaming agent, an anti-corrosion agent, a soil suspension agent, a soil redeposition inhibitor, a fungicide, an anti-tarnish agent, or a hydrotrope, optionally wherein the hydrotrope comprises urea, tosylate, cumene sulfonate, or xylene sulfonate.

[0045] Embodiment 39 is the composition of any one of the preceding embodiments, further comprising a surfactant.

[0046] Embodiment 40 is the composition of the immediately preceding embodiment, wherein the surfactant is present in less than about 10% by weight of the composition, and optionally the surfactant is present in about 0.5-10% by weight of the composition or about 2-6% by weight of the composition.

[0047] Embodiment 41 is the composition of embodiment 39, wherein the surfactant is present at less than about 10% w / v, optionally wherein the surfactant is present at about 0.1% w / v to 0.3% w / v.

[0048] Embodiment 42 is the composition of any one of embodiments 39-41, wherein the surfactant comprises a partially fluorinated alkyl polyether, a C12-C14-secondary ethoxylated alcohol, an anionic low-foaming surfactant, or a combination thereof.

[0049] Embodiment 43 is the composition of any one of embodiments 1 to 40, comprising an enzymatic cleaner product component selected from Endozime AW Triple Plus with APA, Endozime Xtreme Power, Elementum, Elementum AW, and Endozime BioClean.

[0050] Embodiment 44 is the composition of embodiment 43, wherein the enzymatic detergent product component is a composition diluted in a solution comprising one or more of water; a phosphate buffer; an anionic detergent; and a divalent cation chelator.

[0051] Embodiment 45 is the composition of embodiment 44, wherein the enzyme cleaning product components are diluted to about 1% to 80% of their original concentration.

[0052] Embodiment 46 is the composition of embodiment 44, wherein the enzyme cleaning product components are diluted to about 40% to 75% of their original concentration, about 1% to 10% of their original concentration, about 3% to 6% of their original concentration, or about 6% of their original concentration.

[0053] Embodiment 47 is the composition of any one of the preceding embodiments, wherein the composition has a pH of 3-14.

[0054] Embodiment 48 is the composition of any one of the preceding embodiments, having a pH of 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, or 13-14.

[0055] Embodiment 49 is the composition of any one of the preceding embodiments, wherein the buffer is a phosphate buffer or an alkali salt thereof.

[0056] Embodiment 50 is the composition of any one of the preceding embodiments, wherein the buffer comprises sodium phosphate.

[0057] Embodiment 51 is the composition of any one of the preceding embodiments, wherein the buffer comprises HEPES.

[0058] Embodiment 52 is the composition of any one of the preceding embodiments, wherein the divalent cation chelator comprises a magnesium chelator and / or a calcium chelator.

[0059] Embodiment 53 is the composition of any one of the preceding embodiments, wherein the divalent cation chelator comprises Trilon M, trisodium citrate, EDTA, or EGTA.

[0060] Embodiment 54 is the composition of any one of the preceding embodiments, wherein the composition further comprises a preservative.

[0061] Embodiment 55 is the composition of the immediately preceding embodiment, wherein the preservative comprises a microbicide, bactericide, biocide, and / or fungicide.

[0062] Embodiment 56 is the composition of embodiment 54, wherein the preservative comprises a benzisothiazolinone (BIT).

[0063] Embodiment 57 is the composition of any one of the preceding embodiments, further comprising a biological sample.

[0064] Embodiment 58 is the composition of the immediately preceding embodiment, wherein the biological sample is a composition comprising at least one viscous polymer.

[0065] Embodiment 59 is the composition of the immediately preceding embodiment, wherein the at least one viscosity polymer comprises a polysaccharide.

[0066] Embodiment 60 is the composition of embodiment 58, wherein the at least one viscous polymer is a composition comprising an extracellular nucleic acid.

[0067] Embodiment 61 is the composition of any one of embodiments 57 to 60, wherein the biological sample is a lipid-containing composition.

[0068] Embodiment 62 is the composition of any one of embodiments 57 to 61, wherein the biological sample is a composition comprising a lipid phase or a lipid emulsion.

[0069] Embodiment 63 is a composition according to any one of embodiments 57 to 62, wherein the biological sample is a composition comprising protein, mucin, sputum, saliva, nasal secretion, mucoidal secretion, nasal mucus, pulmonary secretion, urine, blood, plasma, or serum.

[0070] Embodiment 64 is the composition of any one of embodiments 57 to 63, wherein the biological sample further comprises one or more pharmaceutical molecules.

[0071] Embodiment 65 is the composition of any one of embodiments 57 to 64, wherein the biological sample is of plant or animal origin.

[0072] Embodiment 66 is the composition of any one of embodiments 57 to 65, wherein the biological sample comprises particulate matter.

[0073] Embodiment 67 is the composition of any one of embodiments 57 to 66, wherein the biological sample is a suspension.

[0074] Embodiment 68 is the composition of any one of embodiments 57 to 67, wherein the biological sample is of mammalian origin.

[0075] Embodiment 69 is the composition of any one of embodiments 57 to 68, wherein the biological sample is of human origin.

[0076] Embodiment 70 is the composition of any one of embodiments 1 to 69, further comprising a target sample.

[0077] Embodiment 71 is the composition of embodiment 70, wherein the target sample is a composition comprising nucleic acid.

[0078] Embodiment 72 is the composition of embodiment 71, wherein the nucleic acid is RNA.

[0079] Embodiment 73 is the composition of embodiment 71, wherein the nucleic acid is DNA.

[0080] Embodiment 74 is the composition of any one of embodiments 57 to 69, wherein the biological sample is a composition containing or suspected of containing a respiratory pathogen.

[0081] Embodiment 75 is the composition of embodiment 74, wherein the respiratory pathogen is a virus.

[0082] Embodiment 76 is the composition of embodiment 74, wherein the respiratory pathogen is a coronavirus, parainfluenza virus (HPIV), influenza virus, adenovirus, human metapneumovirus (HMPV), rhinovirus, enterovirus, or respiratory syncytial virus.

[0083] Embodiment 77 is the composition of embodiment 74, wherein the respiratory pathogen is a bacterium.

[0084] Embodiment 78 is the composition of embodiment 75, wherein the bacteria is Bordetella pertussis, Chlamydia pneumoniae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, or methicillin-resistant Staphylococcus aureus (MRSA).

[0085] Embodiment 79 is the composition of embodiment 74, wherein the respiratory pathogen is a fungus.

[0086] Embodiment 80 is the composition of embodiment 79, wherein the fungus is Aspergillus fumigatus, Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, Rhizopus species, Mucor species, or Pneumocystis jirovecii.

[0087] Embodiment 81 is a composition described in any one of embodiments 57 to 80, wherein the sample type is any type of lung sample, optionally selected from sputum, nasal mucous, nasal secretion, oropharyngeal swab sample, bronchoalveolar lavage fluid, bronchial washing, and nasopharyngeal swab sample.

[0088] Embodiment 82 is a method for processing a biological sample, the method comprising a step of contacting the biological sample with the composition described in any one of embodiments 1 to 81.

[0089] Embodiment 83 is a method for inactivating an infectious pathogen, the method comprising contacting the biological sample with a composition described in any one of embodiments 1 to 82.

[0090] Embodiment 84 is the method of embodiment 83, wherein the biological sample comprises at least one viscous polymer.

[0091] Embodiment 85 is the method of embodiment 83, wherein the at least one viscosity polymer comprises a polysaccharide.

[0092] Embodiment 86 is the method of embodiment 83, wherein the at least one viscous polymer comprises an extracellular nucleic acid.

[0093] Embodiment 87 is the method of any one of embodiments 82 to 88, wherein the biological sample comprises lipids.

[0094] Embodiment 88 is the method of any one of embodiments 82 to 87, wherein the lipid comprises a lipid phase or a lipid emulsion.

[0095] Embodiment 89 is a method according to any one of embodiments 82 to 88, wherein the biological sample comprises protein, mucin, sputum, saliva, nasal secretion, mucoidal secretion, nasal mucus, pulmonary secretion, urine, blood, plasma, or serum.

[0096] Embodiment 90 is the method of any one of embodiments 82 to 89, wherein the biological sample is of plant or animal origin.

[0097] Embodiment 91 is the method according to any one of embodiments 82 to 90, wherein the biological sample is of mammalian origin.

[0098] Embodiment 92 is the method according to any one of embodiments 82 to 91, wherein the biological sample is of human origin.

[0099] Embodiment 93 is the method according to any one of embodiments 82 to 92, further comprising a target sample.

[0100] Embodiment 94 is the method of embodiment 93, wherein the target sample comprises nucleic acid.

[0101] Embodiment 95 is the method of embodiment 94, wherein the nucleic acid is RNA.

[0102] Embodiment 96 is the method of embodiment 94, wherein the nucleic acid is DNA.

[0103] Embodiment 97 is the method of any one of embodiments 82 to 96, wherein the biological sample is a suspension.

[0104] Embodiment 98 is the method of any one of embodiments 82 to 97, wherein the biological sample comprises particulate matter.

[0105] Embodiment 99 is the method of any one of embodiments 82 to 98, wherein the biological sample contains or is suspected of containing a respiratory pathogen.

[0106] Embodiment 100 is the method of embodiment 99, wherein the respiratory pathogen is a virus.

[0107] Embodiment 101 is the method of embodiment 100, wherein the virus is a coronavirus, parainfluenza virus (HPIV), influenza virus, adenovirus, human metapneumovirus (HMPV), rhinovirus, enterovirus, or respiratory syncytial virus.

[0108] Embodiment 102 is the method of embodiment 99, wherein the respiratory pathogen is a bacterium.

[0109] Embodiment 103 is the method of embodiment 102, wherein the bacterium is Bordetella pertussis, Chlamydia pneumoniae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, or methicillin-resistant Staphylococcus aureus (MRSA).

[0110] Embodiment 104 is a composition described in any one of embodiments 82 to 103, wherein the sample type is any type of lung sample, optionally selected from sputum, nasal mucous, nasal secretion, oropharyngeal swab sample, bronchoalveolar lavage fluid, bronchial washings, and nasopharyngeal swab sample.

[0111] Embodiment 105 is the method according to any one of embodiments 82 to 104, further comprising the step of amplifying the nucleic acid.

[0112] Embodiment 106 is a method according to any one of embodiments 82 to 105, wherein the biological sample contains a coated or enveloped virus, and the method further comprises a step of releasing nucleic acid from the coat or envelope.

[0113] Embodiment 107 is a method according to any one of embodiments 82 to 106, wherein the biological sample contains at least one target nucleic acid, and the method comprises capturing the target nucleic acid with at least one capture probe or isolating the target nucleic acid by precipitation or chromatography.

[0114] Embodiment 108 is a kit comprising the composition of any one of embodiments 1-56.

[0115] Embodiment 109 is the kit of embodiment 108, wherein the composition is contained in a tube.

[0116] Embodiment 110 is the kit of embodiment 108 or 109, wherein the composition is contained in a tube comprising a cap.

[0117] Embodiment 111 is the kit of any one of embodiments 108 to 110, wherein the kit further comprises a swab comprising a tip portion and a stem portion.

[0118] Embodiment 112 is the kit of embodiment 111, wherein the tip of the swab is a flocked tip.

[0119] Embodiment 113 is the kit of embodiment 111 or 112, wherein the stem portion of the swab is a flexible plastic stem.

[0120] Embodiment 114 is the kit according to any one of embodiments 111 to 113, wherein the swab is a nasopharyngeal swab.

[0121] Embodiment 115 is the kit of any one of embodiments 108 to 114, wherein the kit contains 1 mL to 10 mL of the composition.

[0122] Embodiment 116 is the kit of embodiment 115, wherein the composition comprises 2% to 4% (w / v) propylene glycol, 2% to 10% (w / v) of a degrading enzyme that is a protease, and about 1% to 4% of an alkyl sulfate detergent, preferably a dodecyl sulfate detergent.

[0123] Embodiment 117 is the kit of embodiment 115 or 116, wherein the composition comprises 2% to 3% (w / v) Good's buffer and 0.05% to 0.3% (w / v) of a surfactant.

[0124] Embodiment 118 is the kit of embodiment 117, wherein the Good's buffer is HEPES.

[0125] Embodiment 119 is the kit of any one of embodiments 108 to 118, wherein the kit further comprises instructions for use.

[0126] Embodiment 120 is the kit of embodiment 119, wherein the instructions for use are provided as a digital document.

[0127] Embodiment 121 is the kit of embodiment 119 or 120, wherein the instructions for use include instructions for collecting a biological sample.

[0128] Embodiment 122 is the kit of any of embodiments 119 to 121, wherein the instructions for use include instructions for collecting a nasopharyngeal sample.

[0129] Embodiment 123 is the use of a kit according to any one of embodiments 108 to 122 for the collection of a biological sample for a diagnostic test.

[0130] Embodiment 124 is the use according to embodiment 123, wherein the biological sample is collected for a nucleic acid diagnostic test.

[0131] Embodiment 125 is the use of embodiment 123 or 124, wherein the biological sample is collected for a diagnostic test to determine the presence or absence of a respiratory pathogen.

[0132] Embodiment 126 is the use of embodiment 125, wherein the respiratory pathogen is one or more of a coronavirus, parainfluenza virus (HPIV), influenza virus, adenovirus, human metapneumovirus (HMPV), rhinovirus, enterovirus, or respiratory syncytial virus.

[0133] Embodiment 127 is the use of embodiment 123 or 124, wherein the biological specimen is collected for a diagnostic test to determine the presence or absence of a bacterial pathogen.

[0134] Embodiment 128 is the use according to embodiments 123 to 127, wherein the biological sample comprises one or more of protein, mucin, sputum, saliva, nasal secretions, mucus-like secretions, nasal mucus, pulmonary secretions, urine, blood, plasma, or serum. DETAILED DESCRIPTION OF THE INVENTION

[0135] Detailed Description Before describing the present teachings in detail, it should be understood that the present disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "an oligomer" includes a plurality of oligomers, and the like. The conjunction "or" should be construed in its inclusive sense, i.e., equivalent to "and / or," unless such inclusive sense is unreasonable in the context. Measured and measurable values ​​are understood to be approximate, taking into account significant digits and errors associated with their measurement.

[0136] The use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," "includes," and "including" is not to be construed as limiting. It is to be understood that both the foregoing general and detailed descriptions are exemplary and explanatory of teachings only and are not limiting. To the extent that any material incorporated by reference does not contradict the explicit context of this disclosure, the explicit content thereof will control.

[0137] Unless specifically noted, embodiments herein that are described as "comprising" various components are also contemplated as "consisting of" or "consisting essentially of" the described components.

[0138] A. Overview Certain types of biological samples (e.g., those containing cells, tissues, cell debris, and / or extracellular biopolymers) can be difficult to analyze using nucleic acid amplification and / or detection procedures. As noted above, samples containing viscous or particulate matter (e.g., mucus; clots) can be particularly problematic in liquid handling steps. Thus, it is often desirable to process such samples prior to downstream analytical steps, e.g., by degrading and / or solubilizing non-nucleic acid sample components. As described in detail below, it has been found that certain compositions can facilitate efficient isolation of nucleic acids by reducing viscosity or otherwise making the sample more suitable for liquid handling procedures such as aspiration and dispensing. The degree of degradation and / or solubilization of non-nucleic acid components is not necessarily absolute, but may nevertheless be sufficient to result in improvements such as reduced clogging / clotting, reduced pressure levels, and more consistent results during liquid handling and downstream analytical steps (e.g., nucleic acid amplification or other forms of detection or measurement).

[0139] B. Definition A "biological sample" includes any specimen that may contain target material (e.g., nucleic acids, viruses, or other biomolecules) such as any tissue or material derived from or containing a living or dead organism, or that may contain target material derived therefrom (e.g., peripheral blood, plasma, serum, lymph nodes, digestive tissue, cerebrospinal fluid, proteins, mucin, sputum, mucus, mucus-like secretions, nasal mucus, pulmonary secretions, feces, urine, semen, vaginal secretions, saliva, biopsy material, other bodily fluids or materials, soil, sludge, or microbial growths (e.g., bacterial, archaeal, fungal, or protist colonies or biofilms)). The biological sample may be treated to physically or mechanically disrupt tissue or cellular structures, thus releasing subcellular components into solution or suspension, and / or may be combined with buffers or other components. Biological samples also include crude samples and processed samples (e.g., obtained from passing a sample over or through a filtration device, or after centrifugation, or by adsorption to a medium, matrix, or support) as discussed below.

[0140] A "crude sample" is a biological sample obtained directly from a living or dead organism, food, the environment, etc., as opposed to an extract, lysate, filtrate, or eluate. Simply dissolving, suspending, or otherwise mixing a crude material in or with a liquid does not transform it into a processed sample.

[0141] A liquid is "aqueous" if it is water, a mixture of water and a water-miscible liquid (where water is present in more than trace amounts), or a solution whose solvent is any of the above. In some embodiments, an aqueous liquid is at least 50% water by weight.

[0142] "Particulate matter" includes any material (excluding whole cells and cell debris) of sufficient solidity to partially or completely occlude the opening at the tip of a pipette or micropipette. Solutes dissolved in solution are not particulate.

[0143] A "suspension" is a liquid containing undissolved solid or particulate matter.

[0144] "Aliquot" is used simply to refer to some or all of a sample. The first and second portions of an aliquot may or may not represent the entire aliquot.

[0145] A "viscous polymer" is a polymer dissolved in a sufficient amount to detectably increase the viscosity of a liquid upon dissolution. In aqueous or polar liquids, viscous polymers may contain multiple hydrogen bond donors and acceptors (e.g., hydroxyl, amine, oxo (carbonyl oxygen), etc.) such that the polymer molecules exert sufficient intermolecular attractive forces toward multiple other polymer molecules and solvent molecules, thereby increasing viscosity. Polysaccharides, polypeptides, nucleic acids, and various synthetic polymers can all be viscous polymers. Any suitable approach for measuring viscosity can be used. Exemplary instruments for determining viscosity include capillary viscometers, Zahn cups, oscillatory viscometers, and rotational viscometers.

[0146] "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 lipids or polypeptides) and may or may not be derivatized (e.g., phosphorylated, acetylated, alkylated, etc.). Polysaccharide chains may use glycosidic or other linkages (e.g., phosphodiester in certain polysaccharides such as poly-N-acetylmannosamine phosphate).

[0147] "Lipids" include fatty acids, triglycerides, phospholipids, sterols, steroids, waxes, and oils.

[0148] "Food" includes any substance intended or suitable for consumption, including solids, suspensions, emulsions, and gels (and thus includes gelatin, milk, ice cream, fruit smoothies, emulsified cheese dips, fruit purees, nut butters, processed and / or textured proteins, etc., as well as bread, fruit, vegetables, meats, etc., but does not include clear monodisperse solutions or water).

[0149] "Nucleic acid" refers to a multimeric compound containing two or more covalently linked nucleosides or nucleoside analogs with a nitrogenous heterocyclic base or base analog, wherein the nucleosides are linked together by phosphodiester 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" (LNAs), 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 43:1323-41, 2004). The nucleic acid "backbone" can be composed of various linkages, including one or more of a sugar-phosphodiester bond, a peptide-nucleic acid bond as in PNA, a phosphorothioate bond, a methylphosphonate bond, or a combination thereof. The sugar moiety of a nucleic acid can be either ribose or deoxyribose, or similar compounds with known substitutions, such as 2'-methoxy and 2'-halide substitutions (e.g., 2'-F). The nitrogenous bases can be conventional bases (A, G, C, T, U), their analogs (e.g., inosine, 5-methylisocytosine, isoguanine; see, e.g., *The Biochemistry of the Nucleic Acids* 5-36, Adams et al., eds., 11th ed., 1992; Abraham et al., 2007, BioTechniques 43: 617-24). They can also be derivatives of purine or pyrimidine bases (e.g., N 4The polynucleotides include -methyldeoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substitutions at the 5- or 6-position, purine bases with altered or replaced substituents at the 2-, 6-, and / or 8-positions, etc. (see generally U.S. Pat. Nos. 5,378,825, 6,949,367, and International Patent Application Publication No. WO 93 / 13121, each of which is incorporated herein by reference), and / or "abasic" residues (see, e.g., U.S. Pat. No. 5,585,481, incorporated herein by reference). The term "polynucleotide" as used herein refers to a nucleic acid chain. A "nucleotide" is a subunit of a nucleic acid having a phosphate group, a five-carbon sugar, and a nitrogenous base. The term also includes analogs of such subunits, such as a methoxy group at the 2' position of ribose (also referred to herein as "2'-O-Me" or "2'-methoxy").

[0150] As used herein, a "target" substance (e.g., a target nucleic acid; other embodiments of a target substance include cells, viruses, and other biomolecules) is a substance to be detected or quantified. The target nucleic acid may be DNA or RNA as described herein, and may be either single-stranded or double-stranded. The target nucleic acid may contain other sequences than the target sequence, which may not be detected or quantified.

[0151] The interchangeable terms "oligomer," "oligo," and "oligonucleotide" refer to nucleic acids comprising polymers generally having less than 1,000 nucleotide (nt) residues, ranging from about 5 nt residues at the lower end and about 500-900 nt residues at the upper end. In some embodiments, oligonucleotides range in size from about 12-15 nt at the lower end and about 50-600 nt at the upper end, while other embodiments range from about 15-20 nt at the lower end and about 22-100 nt at the upper end. Oligonucleotides can serve one or more of a variety of different functions, e.g., as primers and / or promoters, detection probes, capture oligomers, etc.

[0152] "Amplifying" or "amplification" refers to any known procedure for obtaining multiple copies of a target nucleic acid sequence, or its complement, or a fragment thereof. The multiple copies may be referred to as amplicons or amplification products, which may be double-stranded or single-stranded and may contain DNA, RNA, or both. Amplification of a "fragment" refers to the production of amplified nucleic acids containing less than the entire target nucleic acid or its complement, for example, by using amplification oligonucleotides that hybridize to and initiate polymerization at an internal position of the target nucleic acid. Known 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 (e.g., QB-replicase (see, e.g., U.S. Pat. No. 4,786,600, incorporated herein by reference)). PCR amplification uses DNA polymerase, a pair of primers, and thermal cycling to synthesize multiple copies of two complementary strands of dsDNA, or from cDNA (see, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,800,159, each incorporated herein by reference). (See, e.g., U.S. Patent Nos. 5,427,930 and 5,516,663, each of which is incorporated herein by reference). LCR amplification uses four or more different oligonucleotides to amplify a target and its complementary strand by employing multiple cycles of hybridization, ligation, and denaturation (see, e.g., U.S. Patent Nos. 5,427,930 and 5,516,663, each of which is incorporated herein by reference). SDA uses primers containing restriction sites for the restriction endonuclease and an endonuclease to nick one strand of a hemimodified DNA duplex containing the target sequence, whereby amplification occurs in a series of primer extension and strand displacement steps (see, e.g., U.S. Patent Nos. 5,422,252; 5,547,861; and 5,648,211, each of which is incorporated herein by reference).Helicase-dependent amplification uses a helicase to separate the two strands of a DNA duplex to generate a single-stranded template, to which a sequence-specific primer is subsequently hybridized and extended by a DNA polymerase to amplify the target sequence (see, e.g., U.S. Pat. No. 7,282,328, incorporated herein by reference). Amplification can be linear or exponential.

[0153] "Detection probe," "detection oligonucleotide," "probe oligomer," and "detection probe oligomer" are used interchangeably to refer to a nucleic acid oligomer that specifically hybridizes to a target sequence in a nucleic acid or amplified nucleic acid under conditions that promote hybridization to allow detection of the target sequence or amplified nucleic acid. Detection can be either direct (e.g., the probe is hybridized directly to its target sequence) or indirect (e.g., the probe is linked to its target via an intermediate molecular structure). Examples include invasive probes and primary probes (discussed below). Detection probes can be DNA, RNA, analogs thereof, or combinations thereof (e.g., DNA / RNA chimeras), and they can be labeled or unlabeled. The "target sequence" of a detection probe generally refers to a smaller nucleic acid sequence region within a larger nucleic acid sequence that specifically hybridizes to at least a portion of the probe oligomer through base pairing. Detection probes can contain target-specific sequences and other sequences that contribute to the three-dimensional conformation of the probe (see, e.g., U.S. Patent Nos. 5,118,801; 5,312,728; 6,849,412; 6,835,542; 6,534,274; and 6,361,945; and U.S. Patent Application Publication No. 20060068417, each of which is incorporated herein by reference).

[0154] As used herein, "label" refers to a moiety or compound directly or indirectly attached to a probe that is detected or produces a detectable signal. Direct labeling can occur through a bond or interaction (including covalent or non-covalent bonds, e.g., hydrogen bonds, hydrophobic interactions, and ionic interactions) that links the label to the probe, or through the formation of a chelate or coordination complex. Indirect labeling can occur through the use of a bridging moiety or "linker" (e.g., a binding pair member, an antibody, or an additional oligomer) that can be labeled either directly or indirectly and amplify the detectable signal. Labels include any detectable moiety (e.g., a radionuclide, a ligand (e.g., biotin, avidin), an enzyme or enzyme substrate, a reactive group, or a chromophore (e.g., a dye, particle, or bead that imparts a detectable color), a light-emitting compound (e.g., a bioluminescent, phosphorescent, or chemiluminescent label), or a fluorophore. Labels may be detectable in homogeneous assays in which bound labeled probes in a mixture exhibit a detectable change (e.g., instability or different degradation properties) that differs from that of unbound labeled probes.

[0155] The terms "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 binding to a binding partner on the immobilized probe to capture the target nucleic acid to a support. One example of a capture oligomer contains two binding regions: a sequence binding region (e.g., a target-specific portion) and an immobilized probe binding region (usually on the same oligomer), although the two regions may be on two different oligomers linked together by one or more linkers. Another embodiment of a capture oligomer uses a target sequence binding region containing a random or nonrandom poly-GU, poly-GT, or poly-U sequence to nonspecifically bind to the target nucleic acid and link the capture oligomer to a probe immobilized on a support.

[0156] "Separating" or "purifying" means that one or more components of a sample are removed or separated from other sample components. Sample components include target materials, e.g., target nucleic acids, which are typically present in an aqueous solution phase, which may also include suspended matter, cell fragments, proteins, carbohydrates, lipids, and other nucleic acids. "Separating" or "purifying" does not imply any particular degree of purification. In some embodiments, 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.

[0157] C. Compositions for Sample Processing Compositions for sample processing may include degradative enzymes, detergents, and at least one additive (e.g., a buffer and / or a chelating agent). In some embodiments, the compositions may include two or more degradative enzymes.

[0158] 1. Degradative enzymes In some embodiments, degradative enzymes may be wild-type, chemically modified, or engineered. Enzymes may differ in some engineered manner from the related wild-type sequence by amino acid substitutions. For example, nucleic acid substitutions may result in enzyme variants that differ in their specific activity, thermostability, or pH optimum. In some embodiments, enzyme variants may comprise an amino acid sequence that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the wild-type enzyme.

[0159] In some embodiments, the enzyme may be amylase, arabinase, carbohydrase, cellulase, cutinase, esterase, galactanase, glucanase, lipase, mannanase, oxidase, pectinase, peroxidase, protease, or xylanase, or a combination thereof. In some embodiments, the composition may include a plurality (e.g., at least two, at least three, or at least four) of these exemplary enzymes. In some embodiments, the composition may include lipase, protease, and / or amylase. In some embodiments, the composition may include lipase, protease, carbohydrase, and / or amylase.

[0160] In some embodiments, the enzymes may include one or more of the enzymes present in enzyme-based cleaning products (e.g., Endozime AW Triple Plus with APA (Ruhof Corporation, catalog number 34521), Endozime Xtreme Power (Ruhof Corporation, catalog number 34530), Elementum (Ruhof Corporation, catalog number 54309), Elementum AW (Ruhof Corporation, catalog number 34511), Endozime BioClean (Ruhof Corporation, catalog number 34524), Pure Enzymatic Detergent (Boston Scientific, catalog number SEE-573-4), Revital-Ox Enzymatic Detergents (Steris Healthcare, catalog number 2D97AW), and Metrizyme (Metrix Research LLC, catalog number 10-4000)). In some embodiments, the enzymes include each of the enzymes present in enzyme-based cleaning products (e.g., Endozime AW Triple Plus with APA, Endozime Xtreme Power, Elementum, Elementum AW (Ruhof Corporation catalog number 54310), Endozime BioClean, Pure Enzymatic Detergent, Revital-Ox Enzymatic Detergents, and Metrizyme). In any of the foregoing embodiments, the components of the enzyme-based cleaning product are diluted in a solution comprising one or more of water; a phosphate buffer; an anionic detergent; and a divalent cation chelator.In some embodiments, the enzymes include each of the enzymes present in an enzyme-based cleaning product (e.g., Endozime AW Triple Plus with APA, Endozime Xtreme Power, Elementum, Elementum AW, Endozime BioClean, Pure Enzymatic Detergent, Revital-Ox Enzymatic Detergents, and Metrizyme). If desired, the components of the enzyme-based cleaning product can be diluted, for example, to about 1% to 80% of their original concentration. In some embodiments, the enzymes include each of the enzymes present in an enzyme-based cleaning product (e.g., Endozime AW Triple Plus with APA, Endozime Xtreme Power, Elementum, Elementum AW, Endozime BioClean, Pure Enzymatic Detergent, Revital-Ox Enzymatic Detergents, and Metrizyme), where the components of the enzyme-based cleaning product are diluted to about 40% to 75% of their original concentration. In some embodiments, the enzymes include each of the enzymes present in an enzyme-based cleaning product (e.g., Endozime AW Triple Plus with APA, Endozime Xtreme Power, Elementum, Elementum AW, Endozime BioClean, Pure Enzymatic Detergent, Revital-Ox Enzymatic Detergents, and Metrizyme), wherein the components of the enzyme-based cleaning product are diluted to about 50% to 65% of their original concentration.In some embodiments, the enzymes include each of the enzymes present in an enzyme-based cleaning product (e.g., Endozime AW Triple Plus with APA, Endozime Xtreme Power, Elementum, Elementum AW, Endozime BioClean, Pure Enzymatic Detergent, Revital-Ox Enzymatic Detergents, and Metrizyme), wherein the components of the enzyme-based cleaning product are diluted to about 1% to 10% of their original concentration, about 3% to 6% of their original concentration, or about 6% of their original concentration.

[0161] In some embodiments, the composition comprises about 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% The enzyme concentration may be about 1-4% w / v, about 3-6% w / v, or about 6% w / v. In some embodiments, the enzyme concentration is about 1-4% w / v. In some embodiments, the enzyme concentration is about 3% w / v. In some embodiments, the enzyme concentration is about 1-10% w / v, about 3-6% w / v, or about 6% w / v.

[0162] In some embodiments, the enzyme may be present at a concentration of less than about 20% by weight (i.e., w / w) of the composition, or 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%, 8.5-9.5%, 9-10%, 9.5-10.5% by weight of the composition. In some embodiments, the enzyme is present at about 1-4% by weight of the composition. In some embodiments, the enzyme is present at about 3% by weight of the composition.

[0163] In some embodiments, the composition comprises an enzyme (e.g., protease) concentration of about 0 to 16 Anson Units / g (AnsonU / g), or about 0.16 AnsonU / g, about 0.32 AnsonU / g, about 0.48 AnsonU / g, about 0.72 AnsonU / g, about 0.96 AnsonU / g, about 1.6 AnsonU / g, about 3.2 AnsonU / g, about 4.8 AnsonU / g, about 7.2 AnsonU / g, or about 9.6 AnsonU / g.

[0164] a) Lipase Suitable lipases include microbial lipases of bacterial or fungal origin, including wild-type, chemically modified, and protein-engineered variants.

[0165] Examples of bacteria from which lipases can be derived include those of the genera Brochothris, Lactobacillus, and Pseudomonas, including Brochothris thermosohata, Lactobacillus curvatus, Pseudomonas cepacian, Pseudomonas plantari, and Pseudomonas stutzeri.

[0166] Examples of fungi from which lipases can be derived include fungi of the genera Candida, Humicola, Rhizomucor, and Thermomyces. Examples of 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.

[0167] b) protease Suitable proteases include those of animal, plant, or microbial origin, including wild-type, chemically modified, and protein-engineered variants.

[0168] Examples of proteases include serine proteases, neutral proteases, threonine proteases, cysteine ​​proteases, aspartic acid proteases, glutamic acid proteases, metalloproteases, and alkaline proteases.

[0169] Serine proteases are enzymes that catalyze the hydrolysis of peptide bonds and contain an essential serine residue in the active site. In some embodiments, the protease is porcine trypsin, bovine trypsin, or Fusarium protease. In some embodiments, the protease is derived from Aspergillus, Rhizopus, Bacillus alcalophilus, B. cereus, B. vulgatus, B. myocide, or Nocardiopsis N. natto.

[0170] Neutral proteases are enzymes that catalyze the hydrolysis of peptide bonds in neutral, slightly acidic, or slightly alkaline environments. Neutral proteases have optimal proteolytic activity in the neutral pH range of about 6 to about 8 and can be derived from bacterial, fungal, yeast, plant, or animal sources. Examples of neutral proteases include aspartic acid proteases and metalloproteases. In some embodiments, the metalloprotease is Neutrase® (produced by submerged fermentation of certain strains of Bacillus subtilis).

[0171] Alkaline proteases are catalytically active at pHs ranging from neutral to alkaline. Examples of alkaline proteases include RP-II protease and subtilisin. In some embodiments, the protease is derived from Bacillus, Nocardiopsis spe, or Nocardiopsis dassonvillei. In some embodiments, the protease is subtilisin Novo, subtilis Carlsberg, subtilisin 309, subtilisin 147, or subtilisin 168. In some embodiments, the protease is wild-type RP-II protease or a variant thereof, or wild-type subtilisin or a variant thereof. In some embodiments, the subtilisin variants contain substitutions at one or more of the following positions relative to the 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. For an exemplary wild-type subtilisin sequence, see U.S. Patent No. 6,605,458 B1, Figure 1 (the BLSAVI sequence of U.S. Patent No. 6,605,458 B1, SEQ ID NO: 10). U.S. Patent No. 6,605,458 B1 is incorporated herein by reference.

[0172] In some embodiments, commercially available proteases may be used (e.g., Alcalase TM , Duralase TM , Esperase TM , FN2 TM , FN3 TM , Kannase TM , Maxacal TM , Maxapem TM , Maxatase TM , Primase TM , Properase TM , Purafect OxP TM , Purafect TM, and Savinase®). In some embodiments, the protease is Savinase®. In some embodiments, the protease is a subtilisin derived from Bacillus lentus (the species Bacillus lentus is also known as Lederbergia lentus). Savinase® is an example of a commercially available subtilisin derived from Bacillus lentus.

[0173] Suitable commercially available proteases produced by conventional fermentation can include, for example, Alcalase® (produced by submerged fermentation of certain strains of Bacillus licheniformis), Durazyme® (a protein-engineered variant of Savinase®), Esperase® (produced by submerged fermentation of alkaliphilic species of Bacillus), Rennilase® (produced by submerged fermentation of non-pathogenic strains of Mucor miehei), and Savinase® (produced by submerged fermentation of genetically modified strains of Bacillus).

[0174] In some embodiments, the protease is subtilisin A, Alcalase (登録商標) , Savinase (登録商標) , Esperase (登録商標) , Neutrase (登録商標) , rTrypsin (登録商標) , or any combination thereof.

[0175] In some embodiments, the composition comprises about 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% The protease concentration may be about 1% to 4% w / v. In some embodiments, the protease concentration is about 3% w / v. In some embodiments, the protease concentration is about 1% to 10% w / v, about 3% to 6% w / v, or about 6% w / v.

[0176] In some embodiments, the protease may be present at a concentration (i.e., w / w) of less than about 10% by weight of the composition. In some embodiments, the protease may be 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. In some embodiments, the protease may be present at about 1-4% by weight of the composition. In some embodiments, the protease may be present at about 3% by weight of the composition. In some embodiments, the protease may be present at about 1% to 10% by weight of the composition, about 3% to 6% by weight of the composition, or about 6% by weight of the composition.

[0177] In some embodiments, the composition comprises about 0-0.5% v / v, 0.25-0.75% v / v, 0.5-1.0% v / v, 0.75-1.25% v / v, 1.0-1.5% v / v, 1.25-1.75% v / v, 1.5-2.0% v / v, 1.75-2.25% v / v, 2-2.5% v / v, 2.25-2.75% v / v, 2.5-3.0% v / v, 2.75-3.25% v / v, 3-3.5% v / v, 3.25-3.75% v / v, 3.5-4.0% v / v, 3.75-4.25% v / v, 4.0-4.5% v / v, 4.25-4.75% v / v, 4.5-5.0% v / v, The protease concentration may be about 1% to 4% v / v. In some embodiments, the protease concentration is about 2.1% to 3% v / v. In some embodiments, the protease concentration is about 1% v / v, 2% v / v, 2.1% v / v, 2.3% v / v, 3% v / v, or 4% v / v.

[0178] i. Protease combinations Combinations of proteases in a composition may provide synergistic benefits. In some embodiments, a composition may include RP-II and a subtilisin of subtilase group I-S2 (Siezen et al., Protein Science, 6: 501-523 91997) or a highly alkaline subtilisin. Examples of I-S2 proteases include ESPERASE®, MAXACAL®, Savinase®, subtilisin 147, subtilisin 309, subtilisin PB92, and alkaline elastase YaB. In some embodiments, the I-S2 protease is a subtilisin derived from Bacillus lentus (the species Bacillus lentus is also known as Lederbergia lentus).

[0179] Combinations of proteases may provide advantages for use with detergents. In some embodiments, the composition comprises BLC and JA96 and variants thereof in combination with Savinase. TM and variants thereof (e.g., Duralase TM , FN2 TM , FN3 TM , Kannase TM , Maxacal TM , Maxapem TM , Maxatase TM , Properase TM , Purafect OxP TM , and Purafect TM may include:

[0180] c) amylase Suitable amylases (α and / or β) include those of bacterial or fungal origin, including wild-type, chemically modified, and protein-engineered variants. In some embodiments, the amylase is an α-amylase, which may be obtained from a particular strain of Bacillus, such as B. licheniformis. In some embodiments, the amylase is derived from Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus stearothermophilus, or Bacillus subtilis. In some embodiments, the amylase is derived from Aspergillus (e.g., Aspergillus niger or Aspergillus oryzae). In some embodiments, the amylase variants contain substitutions at one or more of the following positions relative to the 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., variants described in WO 94 / 02597, WO 94 / 18314, WO 96 / 23873, and WO 97 / 43424, which are incorporated herein by reference). An exemplary A. oryzae α-amylase sequence is found on page 7, lines 18-33 of WO 94 / 02597, which is incorporated herein by reference.

[0181] In some embodiments, commercially available amylases may be used (e.g., BAN TM , Bioamylase D(G), Duramyl TM , FungaMyl TM , Kam's Enzyme Plus, Kemzym TM AT 9000, Purafect TM OxAm, Purastar TM HPAmL, Purastar TM St, Rapidase TM TEX, StainzymeTM , and Termamyl®).

[0182] d) Carbohydrase Carbohydrases are enzymes that can degrade oligosaccharides or polysaccharides. In some embodiments, carbohydrases other than amylases are used (e.g., cellulases, maltases, galactosidases, lactases, pectinases, xylanases, etc.).

[0183] 2. Cleaning agents Detergents can be used to lyse cells containing target substances and / or solubilize substances that are otherwise insoluble or poorly soluble in aqueous liquids. Target substances are discussed in Section B above. The detergent is suitable for cell lysis and can contain a concentration desired for the catalytic efficiency of the enzyme in the composition. The detergent can be used in concentrated or diluted form. Detergents can be anionic, nonionic, or zwitterionic. In some embodiments, the detergent comprises an alkyl sulfate (e.g., dodecyl sulfate (also known as lauryl sulfate)). In some embodiments, the detergent comprises a sodium salt and / or a lithium salt. While not wishing to be bound by any particular theory, the combination of an alkyl sulfate detergent with other components described herein (e.g., two or more degradative enzymes) can beneficially reduce problems associated with sample aspiration due to clots while maintaining nucleic acids in a form suitable for subsequent amplification and / or detection.

[0184] The detergent may contain nonionic, anionic, amphoteric, or cationic surfactants, or any combination thereof. Examples of nonionic surfactants include alkanolamides, amine oxides, block polymers, ethoxylated primary and secondary alcohols, ethoxylated alkylphenols, ethoxylated fatty esters, sorbitan derivatives, glycerol esters, propoxylated and ethoxylated fatty acids, alcohols, and alkylphenols, glycol esters, polymeric polysaccharides, sulfates and sulfonates of ethoxylated alkylphenols, and polymeric surfactants. Examples of anionic surfactants include ethoxylated amines or amides, sulfosuccinates and derivatives, sulfates of ethoxylated alcohols, sulfates, sulfonates, and sulfonic acid derivatives of alcohols, phosphate esters, and polymeric surfactants. Examples of amphoteric surfactants include betaine derivatives. Examples of cationic surfactants include amine surfactants.

[0185] In some embodiments, the detergent comprises a lithium salt, a lauryl sulfate, or lithium lauryl sulfate.

[0186] In some embodiments, the composition comprises about 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, 5.5~6.0% w / v, 5.75~6.25% w / v, 6~6.5% w / v, 6.25~6.75% w / v, 6.5~7.0% w / v, 6.75~7.25% w / v, 7.0~7.5% w / v, 7.25~7.75% w / v, 7.5~8.0% w / v, 7.75~8.25% w / v, 8.0~8.5% w / v, 8.25~8.75% w / v, 8.5~9.0% w / v, 8.75~9.25% w / v, 9.0~9.5% w / v, 9.25~9.75% The detergent may comprise a detergent concentration of 9.5-10.0% w / v, 9.75-10.25% w / v, 10.0-10.5% w / v, 10.25-10.75% w / v, 10.5-11.0% w / v, 10.75-11.25% w / v, 11.0-11.5% w / v, or higher. In some embodiments, the detergent concentration is 2.75-3.25% w / v. In any of the foregoing embodiments, the detergent may comprise a lithium salt. In any of the foregoing embodiments, the detergent may comprise a lauryl sulfate. In any of the foregoing embodiments, the detergent may comprise lithium lauryl sulfate.

[0187] In some embodiments, the detergent may be present at a concentration of less than about 10% by weight (i.e., w / w) of the composition. In some embodiments, the detergent is present at about 0.05-10%, 2-6%, 0.05-0.5%, 0.1-0.15%, 0.1-1.0%, 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. In any of the foregoing embodiments, the detergent may include a lithium salt. In any of the foregoing embodiments, the cleaning agent may include lauryl sulfate. In any of the foregoing embodiments, the cleaning agent may include lithium lauryl sulfate.

[0188] 3. Additives The compositions of the present disclosure may include additives, for example, at a concentration of about 0.1 wt% to about 2.0 wt% of the total weight of the composition. Examples of additives include bactericides, biocides, biopolymer degrading agents, bleaching agents, bleaching systems, buffers, chelating agents, enhancing agents, fungicides, polymers, reagents, salts, preservatives, and stabilizers.

[0189] a) Buffer A buffering agent can be used to maintain the pH of the composition within a desired range, which in some embodiments can be selected from the following pH ranges: 3-11, 3-14, 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, 8.5-9.5, 9-10, 9.5-10.5, 10-11, 10.5-11.5, 11-12, 11.5-12.5, 12-13, 12.5-13.5, and 13-14. Examples of buffering agents include Good's buffer; alkali salt, ammonia buffer; 3-(N-morpholino)propanesulfonic acid (MOPS); bis(tris(hydroxymethyl)methylamino)propane (BIS-TRIS); borate buffer; carbonate buffer; citrate buffer; hydroxyethylpiperazineethanesulfonic acid (HEPES); phosphate buffer (e.g., sodium phosphate); tartrate buffer; and tris(hydroxymethyl)aminomethane (TRIS) (e.g., TRIS-hydrochloride (TRIS-HCl) and TRIS-EDTA). In some embodiments, the buffering agent comprises sodium phosphate.In some embodiments, the composition comprises 0 to 0.2% w / v, 0.1 to 0.3% w / v, 0.2 to 0.4% w / v, 0.3 to 0.5% w / v, 0.4 to 0.6% w / v, 0.5 to 0.7% w / v, 0.6 to 0.8% w / v, 0.7 to 0.9% w / v, 0.8 to 1.0% w / v, 0.9 to 1.1% w / v, 1.0 to 1.2% w / v, 1.1 to 1.3% w / v, 1.2 to 1.4% w / v, 1.3 to 1.5% w / v, 1.4 to 1.6% w / v, 1.5 to 0.7% w / v, 1.6 to 1.8% w / v, 1.7 to 1.9% w / v, 1.8 to 2.0% w / v, 1.9 to 2.1% w / v. w / v, 2.0-2.2% w / v, 2.1-2.3% w / v, 2.2-2.4% w / v, 2.3-2.5% w / v, 2.4-2.6% w / v, 2.5-2.7% w / v, 2.6-2.8% w / v, 2.7-2.9% w / v, 2.8-3.0% w / v, 2.9-3.1% w / v, 3.1-3.3% w / v, 3.2-3.4% w / v, 3.3-3.5% w / v, 3.4-3.6% w / v, 3.5-3.7% w / v, 3.6-3.8% w / v, 3.7-3.9% w / v, 3.8-4.0% w / v, 3.9-4.1% w / v, or higher buffer concentrations. In some embodiments, the buffer concentration is 0.4 to 0.5% w / v, hi some embodiments, the buffer concentration is about 3% w / v.

[0190] b) Chelating agents Chelating agents can be used to inhibit divalent cation-dependent proteases. Examples of chelating agents include magnesium chelating agents and calcium chelating agents. In some embodiments, the chelating agent is trisodium salt of methylglycine diacetic acid (MGDA) (Trilon M), trisodium citrate, ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), or a salt thereof. In some embodiments, the chelating agent includes EDTA and EGTA.

[0191] In some embodiments, the composition comprises a chelating agent 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. In some embodiments, the composition comprises a chelating agent 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 higher. In some embodiments, the chelating agent concentration is 700-800 mg / L.

[0192] c) Salt Salts can be used to maintain the osmolarity of the composition. Exemplary salts that can be used include alkali salts, sodium salts, potassium salts, chloride salts, and acetate salts. In some embodiments, the composition has an osmolarity of 0-50 mOsm, 20-50 mOsm, 50-100 mOsm, 100-200 mOsm, 200-300 mOsm, 300-400 mOsm, 400-500 mOsm, 500-600 mOsm, 600-700 mOsm, 700-800 mOsm, or 800-1000 mOsm.

[0193] d) Detergent enhancers, coupling agents, or complexing agents In some embodiments, the composition may include a detergent booster, coupling agent, or complexing agent (e.g., sodium xylene sulfonate, zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl or alkenyl succinic acid, soluble or layered silicate (e.g., Hoechst's SKS-6), for example, in a concentration of less than or equal to about 65% by weight.

[0194] e) Bleaching system In some embodiments, the composition may include a bleaching system, which may include an HO source (e.g., perborate, percarbonate, or peracid-forming bleach activator). In some embodiments, the composition may include a peracid-forming bleach activator (e.g., tetraacetylethylenediamine or nonanoyloxybenzenesulfonate). In some embodiments, the bleaching system may include, for example, an amide, imide, or sulfone-type peroxyacid.

[0195] f) Stabilizers Stabilizers can be used to stabilize the enzymes in the composition or target substances (e.g., RNA or DNA). The stabilizer can be selected from boric acid, boric acid derivatives (e.g., aromatic boric acid esters) or phenylboronic acid derivatives (e.g., 4-formylphenylboronic acid), calcium ions, glycerol, lactic acid, magnesium ions, polyethylene glycol, propylene glycol, sodium borate, sugars, sugar alcohols, and appropriate enzymes. In some embodiments, the enzymes in the composition can be stabilized using, for example, propylene glycol, polyols (e.g., propylene glycol or glycerol), sugars or sugar alcohols, lactic acid, boric acid, or boric acid derivatives (e.g., aromatic boric acid esters), or phenylboronic acid derivatives (e.g., 4-formylphenylboronic acid), and the compositions can be formulated, for example, as described in WO 92 / 19709 and WO 92 / 19708.

[0196] In some embodiments, the stabilizer comprises one or more ribonuclease (RNase) inhibitors. RNase inhibitors can inhibit RNase A, RNase B, RNase C, and / or any enzyme with RNA hydrolysis activity. RNase inhibitors include small molecules, enzymes, or antibodies used to inhibit RNases from degrading RNA molecules. RNAse inhibitor enzymes and antibodies can be of eukaryotic origin. Exemplary RNase inhibitors include ribonucleoside-vanadyl complexes, guanadine thiocyanate, polyvinyl sulfonic acid, and human placenta RNase inhibitor. Commercially available RNase inhibitors that can be used include RNeasy, SUPERase-In, and others. TM , RNaseOUT TM , Ambion (登録商標) RNase inhibitor, Ambion (登録商標) RNAsecure TM , RNasin (登録商標) , RNasin (登録商標)Plus, RIBOPROTECT, RiboLock, and RiboSafe.

[0197] In some embodiments, the stabilizer comprises one or more deoxyribonuclease (DNase) inhibitors. DNase inhibitors can inhibit DNase I, DNase II, TREX1 (i.e., DNase III), and / or any enzyme with DNA hydrolysis activity. DNase inhibitors include small molecules, enzymes, or antibodies used to inhibit DNases from degrading DNA molecules. DNase inhibitors can be of eukaryotic or prokaryotic origin. Exemplary DNase inhibitors include DNase inhibitors from human, bovine, calf, rabbit, rat, Nicotiana tabacum, Streptomyces species, Adenovirus species, Micromonospora echinospora, and Escherichia coli. Exemplary small molecule DNAse inhibitors include anthracycline and aminoglycoside antibiotics.

[0198] g) Surfactants In some embodiments, the composition may include one or more surfactants, which may include nonionic, anionic, amphoteric, cationic, or a combination of surfactants. In some embodiments, the composition may include nonionic surfactants such as alkanolamides, amine oxides, block copolymers, ethoxylated primary and secondary alcohols, ethoxylated alkylphenols, ethoxylated fatty esters, sorbitan derivatives, glycerol esters, propoxylated and ethoxylated fatty acids, alcohols, and alkylphenols, glycol esters, polymeric polysaccharides, sulfates and sulfonates of ethoxylated alkylphenols, and polymeric surfactants. In some embodiments, the composition may include anionic surfactants such as ethoxylated amines or amides, sulfosuccinates and derivatives, sulfates of ethoxylated alcohols, sulfates of alcohols, sulfonates and sulfonic acid derivatives, phosphate esters, and polymeric surfactants. In some embodiments, the composition may include amphoteric surfactants (e.g., betaine derivatives). In some embodiments, the composition may include a cationic surfactant (e.g., an amine surfactant). In some embodiments, the composition may include a fluorinated surfactant (e.g., a partially or fully fluorinated alkyl polyether). In some embodiments, the composition may include one or more alcohols (e.g., a c12-c14-secondary ethoxylated alcohol (e.g., Tergitol)).

[0199] In some embodiments, the concentration of the surfactant can vary based on the enzymatic activity of the enzyme, the exposure time of the detergent to the biofilm, whether the detergent is in solid, liquid, spray, or gel form, the medical device exposed to the detergent, and the cleaning system utilized. The surfactant can be present in the composition at a concentration of less than about 10% (i.e., wt / wt or wt %) of the total weight of the composition. In some embodiments, the surfactant is present at about 0.05-10%, 2-6%, 0.05-0.5%, 0.1-0.15%, 0.1-1.0%, 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. In some embodiments, the concentration of surfactant can vary based on the enzymatic activity of the enzyme, the exposure time of the cleaning agent to the biofilm, whether the cleaning agent is in solid, liquid, spray, or gel form, the medical device exposed to the cleaning agent, and the cleaning system utilized.

[0200] h) Polymer In some embodiments, the composition may include 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 acid / acrylic acid copolymers, and lauryl methacrylate / acrylic acid copolymers.

[0201] i) Preservatives In some embodiments, the composition may contain a preservative. Any compound that can function as a microbicide, bactericide, biocide, or fungicide may be used as a preservative in the composition. Any compound that can maintain the stability of nucleic acids may also be used as a preservative in the composition. In some embodiments, the preservative is benzoisothiazolinone (1,2-benzisothiazol-3(2H)-one; BIT).

[0202] j) Other ingredients In some embodiments, the composition may also include other ingredients such as clay, foam boosters, suds suppressors, anti-corrosion agents, soil suspension agents, soil redeposition inhibitors, dyes, fungicides, hydrotropes, and tarnish inhibitors. In some embodiments, the hydrotropes may be selected from urea, tosylates, cumene sulfonates, and xylene sulfonates.

[0203] D. Biological Samples Biological samples may include bodily fluids obtained from a subject, which may contain target substances. Examples of biological samples and target substances are provided in Section B above.

[0204] In some embodiments, the biological sample is sputum, saliva, mucus-like secretions, nasal mucus, nasal secretions, pulmonary secretions, nasopharyngeal swab samples, oropharyngeal swab samples, bronchoalveolar lavage fluid, bronchial washings, urine, blood, plasma, or serum. In some embodiments, any type of lung sample is a suitable biological sample. In some embodiments, the target substance comprises nucleic acids and / or respiratory pathogens.

[0205] 1. Mucus-like secretions In some embodiments, the biological sample is a type of mucus-like secretion or mucus, which is an aqueous, viscous liquid containing mucin (i.e., a glycolic protein with a large water-holding capacity). Mucus-like secretions are produced by mucus cells, which may be organized into mucus glands and / or mucosa. Many types of mucus-like secretions can be analyzed for target nucleic acids and / or respiratory pathogens. Examples of mucus-like secretions include nasal secretions, pulmonary secretions, gastric mucus, and vaginal secretions.

[0206] 2. Pharmaceutical molecules In some embodiments, biological samples may contain one or more types of pharmaceutical molecules. For example, biological samples may be collected from subjects who have been treated with pharmaceutical molecules. In these cases, the biological samples may be tested to ensure that the pharmaceutical molecules do not interfere with sample storage, processing, or analysis.

[0207] In some embodiments, the pharmaceutical molecule is a large molecule (e.g., an antibody, antibody fragment, antibody conjugate, or any protein that can have a therapeutic effect on the subject). In some embodiments, the pharmaceutical molecule is a small molecule that can regulate a biochemical process in the subject. In some embodiments, the pharmaceutical molecule is one that can be prescribed for a respiratory infection. In some embodiments, the pharmaceutical molecule is an antibiotic, antiviral, or antifungal. In some embodiments, the pharmaceutical molecule is one that can alleviate the symptoms of an infection (e.g., a respiratory infection).

[0208] E. Respiratory pathogens In some embodiments, the biological sample may contain target substances derived from respiratory pathogens. Any type of virus, fungus, and bacteria can infect the respiratory tract. Respiratory infections may share similar symptoms (e.g., bronchiolitis, chest pain, chills, cough, croup, fatigue, fever, headache, hypoxia, loss of appetite, muscle or body pain, pneumonia, sore throat, stuffy or runny nose, difficulty breathing, and / or whooping cough), but treatment may vary depending on the causative agent. Therefore, it may be useful to determine the respiratory pathogen behind the infection.

[0209] 1. Viruses In some embodiments, the respiratory pathogen is a virus, such as an influenza virus. Influenza viruses can cause seasonal influenza and seasonal epidemics of disease known as flu pandemics. Examples of influenza viruses include influenza A, influenza B, influenza C, and influenza D.

[0210] In some embodiments, the respiratory pathogen is a coronavirus. Coronaviruses can cause respiratory infections and have been the cause of 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).

[0211] In some embodiments, the respiratory pathogen is a paramyxovirus. Examples of paramyxoviruses include respiratory syncytial virus (RSV), including RSV subtypes A and B; human parainfluenza virus (HPIV), including HPIV subtypes 1-4; and human metapneumovirus (HMPV).

[0212] In some embodiments, the respiratory pathogen is an adenovirus. In addition to respiratory infections, adenoviruses can also cause diarrhea and conjunctivitis. Examples of adenoviruses include mammalian adenoviruses (mastadenoviruses) and avian adenoviruses (aviadenoviruses). Examples of adenoviruses that infect humans include adenovirus types 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).

[0213] In some embodiments, the respiratory pathogen is a rhinovirus. Examples of rhinoviruses include rhinovirus A, B, and C.

[0214] In some embodiments, the respiratory pathogen is an enterovirus. Examples of enteroviruses include EV-D68 and EV-71.

[0215] 2. Bacteria In some embodiments, the respiratory pathogen is a Bordetella species (eg, Bordetella pertussis, which causes whooping cough or pertussis, or Bordetella parapertussis).

[0216] In some embodiments, the respiratory pathogen is Staphylococcus aureus. In some embodiments, the S. aureus is methicillin-resistant S. aureus (MRSA).

[0217] In some embodiments, the respiratory pathogen is Streptococcus pneumoniae. Streptococcus pneumoniae infections can range from respiratory, ear, and sinus infections to pneumonia and bloodstream infections.

[0218] In some embodiments, the respiratory pathogen is a mycobacterium. This genus includes pathogens known to cause serious diseases in mammals, including tuberculosis and leprosy in humans. Examples of mycobacteria include Mycobacterium tuberculosis.

[0219] In some embodiments, the respiratory pathogen is Chlamydia, which is transmitted through small respiratory droplets. In some embodiments, the Chlamydia is Chlamydia pneumoniae.

[0220] In some embodiments, the respiratory pathogen is Mycoplasma pneumoniae, which can cause mild infections of the respiratory system and more serious lung infections requiring hospitalization.

[0221] 3.Fungi In some embodiments, the respiratory pathogen is an Aspergillus species (e.g., Aspergillus fumigatus). Infection with Aspergillus species can cause aspergillosis.

[0222] In some embodiments, the respiratory pathogen is a Blastomyces species (e.g., Blastomyces dermatitidis). Infection with Blastomyces dermatitidis can cause blastomycosis.

[0223] In some embodiments, the respiratory pathogen is a Coccidioides species (e.g., Coccidioides immitis). Infection with Coccidioides immitis can cause coccidioidomycosis (also known as valley fever).

[0224] In some embodiments, the respiratory pathogen is a Cryptococcus species (e.g., Cryptococcus neoformans). Infection with Cryptococcus neoformans can cause cryptococcosis.

[0225] In some embodiments, the respiratory pathogen is a Histoplasma species (e.g., Histoplasma capsulatum). Infection with Histoplasma capsulatum can cause histoplasmosis.

[0226] In some embodiments, the respiratory pathogen is a Rhizopus species or a Mucor species. Infection with a Rhizopus species or a Mucor species can cause mucormycosis.

[0227] In some embodiments, the respiratory pathogen is a Pneumocystis species (e.g., Pneumocystis jirovecii). Infection with Pneumocystis jirovecii can cause Pneumocystis pneumonia.

[0228] F. Gastrointestinal pathogens In some embodiments, the biological sample may contain target material derived from a gastrointestinal pathogen. Many types of viruses, parasites, and bacteria can cause gastrointestinal infections. Although gastrointestinal infections may share similar symptoms (e.g., nausea, vomiting, diarrhea, abdominal pain, fatigue, fever, headache, loss of appetite, and / or muscle or body pain), treatment may vary depending on the causative agent. Therefore, it may be useful to determine the gastrointestinal pathogen behind the infection.

[0229] 1. Bacteria In some embodiments, the gastrointestinal pathogen is a Yersinia species (e.g., Yersinia enterocolitica). Infection with Yersinia enterocolitica can cause yersiniosis.

[0230] In some embodiments, the gastrointestinal pathogen is a Vibrio species (e.g., Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae). Infection with Vibrio species can cause vibriosis.

[0231] In some embodiments, the gastrointestinal pathogen is an Escherichia species (eg, Escherichia coli O157, Escherichia coli Stx1, and Escherichia coli Stx2).

[0232] In some embodiments, the gastrointestinal pathogen is a Plesiomonas species (eg, Plesiomonas shigelloides).

[0233] In some embodiments, the gastrointestinal pathogen is a Campylobacter species (e.g., Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis). Infection with Campylobacter species can cause campylobacteriosis.

[0234] In some embodiments, the gastrointestinal pathogen is a Shigella species (e.g., Shigella boydii, Shigella dysenteriae, Shigella flexneri, and Shigella sonnei). Infection with Shigella species can cause shigellosis.

[0235] 2. Viruses In some embodiments, the gastrointestinal pathogen is a virus such as norovirus, rotavirus, astrovirus, adenovirus, and sapovirus.

[0236] 3. Parasites In some embodiments, the biological sample may contain target material derived from a parasite. Parasitic infections may share similar symptoms, but treatment may differ depending on the causative agent. Therefore, it may be useful to determine the parasite behind the infection.

[0237] In some embodiments, the parasite is a Giardia species (e.g., Giardia intestinalis). Infection with Giardia species can cause giardiasis.

[0238] In some embodiments, the parasite is a Cryptosporidium species (e.g., Cryptosporidium parvum). Infection with Cryptosporidium species can cause cryptosporidiosis.

[0239] In some embodiments, the parasite is an Entamoeba species (e.g., Entamoeba histolytica). Infection with Entamoeba hystolytica can cause amebiasis.

[0240] In some embodiments, the parasite is a Cyclospora species (e.g., Cyclospora cayetanensis). Infection with Cyclospora cayetanensis can cause cyclosporiasis.

[0241] In some embodiments, the bloodborne pathogen is a Babesia species (e.g., Babesia microti). Infection with Babesia species can cause babesiosis, which is spread by the bite of infected Ixodes scapularis ticks.

[0242] G. Bloodborne Pathogens In some embodiments, the biological sample may contain target material derived from a blood-borne pathogen. Blood-borne pathogens can be transmitted when any bodily fluid (e.g., blood, saliva, or mucous secretions) from an infected person enters another person's body through a needlestick, human bite, cut, abrasion, or through a mucous membrane. Many types of viruses, parasites, and bacteria can cause blood-borne infections.

[0243] 1. Parasites In some embodiments, the blood-borne pathogen is Plasmodium (e.g., Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi). Infection with Plasmodium can cause malaria.

[0244] 2. Viruses In some embodiments, the blood-borne pathogen is a hepatitis virus (e.g., hepatitis B virus (HBV) and hepatitis C virus (HCV)). In some embodiments, the blood-borne pathogen is human immunodeficiency virus (HIV).

[0245] H. Exemplary Methods 1. Contacting a biological sample with a swab, wherein an aliquot of the sample and the swab are associated; swab; sample In some embodiments, the methods disclosed herein use a swab associated with an aliquot of a biological sample. In some embodiments, a method according to the present disclosure may include contacting the biological sample with the swab, wherein the aliquot of sample and the swab are associated. The association between the sample aliquot and the swab should be at least partially temporary or reversible so that at least some analytes (e.g., cells or nucleic acids) can be released into the liquid. One skilled in the art can select an appropriate manner of contacting the sample with the swab to achieve appropriate association between the sample and the swab (e.g., dipping, wiping, poking, or rolling the swab into, on, or over the sample).

[0246] Any swab suitable for adsorbing and releasing biological materials can be used. In some embodiments, a swab is used that includes a stem having an absorbent and / or adsorbent material (e.g., a sponge or fiber, which can be, for example, cotton, alginate, silk, carbon fiber, or polymer fiber (e.g., rayon, polyester, or polyamide)) wrapped, glued, or attached to the end to form a tip, the absorbent or adsorbent material being suitable for adsorbing and / or absorbing the sample to be collected. In some embodiments, the tip has a thickness (measured perpendicular to the stem axis) of 0.5 to 3 mm. In some embodiments, the tip includes fibers having a linear density of 1 to 4 mg per 10 meters. In some embodiments, the swab is a nasal swab.

[0247] In some embodiments, a flocked swab is used, which includes an elongated support and a plurality of flocked fibers at the end of the support. An exemplary flocked swab is described in US 2006 / 0142668, which is incorporated herein by reference. The swab may have an elongated stem generally made of a plastic material (e.g., polystyrene).

[0248] Any biological sample that can be associated with a swab can be used in the methods according to the present disclosure. The biological sample used in the methods according to the present disclosure can be an emulsion, suspension, or solution (or a combination thereof) and can contain various types of impurities or contaminants (e.g., 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 materials derived from food, solid lipids, inorganic solids such as phosphates (e.g., calcium phosphate or iron phosphate), and insoluble fibrous materials (e.g., cellulose fibers). The sample can contain a lipid phase or lipid emulsion. The sample can contain cellular debris, which, depending on its size, solubility, and hydrophobicity, can exist at least partially as particulate matter, viscous polymers, lipid phase, emulsion, or a combination thereof. The sample may be crude or may have undergone some initial purification (e.g., filtration, extraction, or fractionation (e.g., chromatographic fractionation)). In some embodiments, the crude sample is diluted, suspended, or at least partially dissolved in an appropriate liquid (e.g., water or a solution, which may be buffered, saline, or isotonic with the sample).

[0249] Exemplary sample types that may contain particulate matter, viscous polymers, or emulsions are sputum, saliva, mucus-like secretions, mucus (e.g., nasal mucus, pulmonary secretions, blood, plasma, serum, urine, soil, sludge, feces, or microbial growth). Sludge and soil include natural and artificial versions thereof (e.g., mud, potting mix, chemical waste, etc.). Microbial growth includes bacteria, archaea, protists, or fungal growths (e.g., colonies, biofilms, mycelium, etc.). In some embodiments, the sample is a food sample.

[0250] In some embodiments, the sample contains or is suspected of containing a pathogen, virus, fungus, or bacterium. In some embodiments, the pathogen is a respiratory pathogen. Examples of respiratory pathogens are discussed above in Section E.

[0251] Any volume of material sufficient to yield a second portion suitable for the intended downstream use may optionally be associated with the swab. In some embodiments, a sample volume of 20 to 550 microliters, e.g., 20 to 50 μl, 50 to 100 μl, 100 to 150 μl, 150 to 200 μl, 200 to 250 μl, 250 to 300 μl, 300 to 350 μl, 350 to 400 μl, 450 to 500 μl, or 500 to 550 μl, or 20 to 75 μl, 75 to 125 μl, 125 to 175 μl, 175 to 225 μl, 225 to 275 μl, 275 to 325 μl, 325 to 375 μl, 375 to 425 μl, 425 to 475 μl, 475 to 525 μl, or 525 to 575 μl, is associated with the swab.

[0252] 2. Compositions for stabilizing nucleic acids In some embodiments, the compositions of the methods disclosed herein are useful for stabilizing nucleic acids in biological samples so that the nucleic acids can be protected or prevented from degradation. In some embodiments, the compositions stabilize DNA. In some embodiments, the compositions stabilize RNA. The compositions can be used according to the methods disclosed herein at any temperature. In some embodiments, the compositions are mixed with the biological sample at room temperature.

[0253] In some embodiments, the composition is used as a diluent when mixed with a biological sample. While the composition can be mixed with any biological sample, in some embodiments, the biological sample is whole blood or urine.

[0254] In some embodiments, the composition is used as a matrix when mixed with a biological sample. For example, the composition can be used to prepare RNA transcripts for testing in an RNA panel.

[0255] In some embodiments, the composition can be used as a storage medium for a biological sample or for nucleic acids associated with the biological sample.

[0256] 3. Compositions for inactivating infectious pathogens In some embodiments, the compositions of the methods disclosed herein are useful for inactivating infectious pathogens in biological samples so that the pathogens are less likely or can no longer cause disease. In some embodiments, the compositions destroy the pathogen's ability to replicate. In some embodiments, the compositions destroy the pathogen's ability to infect a host. In some embodiments, the compositions can cause the pathogen to lyse.

[0257] 4. Further processing and / or analysis of sample material containing cells, nucleic acids, or polypeptides; capture, amplification, and detection of targets; target bacterial or pathogen sequences In some embodiments, viruses or cells (e.g., bacteria) released into the liquid are disrupted or lysed, for example, by heating, sonication, vortexing, osmolysis, proteolysis, exposure to alkali and / or detergents, or a combination thereof. This can occur in the liquid either immediately upon transfer of the swab to the liquid (e.g., if the liquid is a lysis solution), or later, for example, after release of a portion of the aliquot into the liquid, in the case of further manipulation by adding reagents or using an instrument. In some embodiments, nucleic acids are isolated from the lysate, for example, by chromatography or precipitation, or by binding to a binding agent (e.g., at least one capture oligomer).

[0258] In some embodiments, the nucleic acid released from the swab into carrier liquid, or from the cell or virus released and lysed as discussed above, is further isolated or purified.For example, further isolation or purification can be carried out by using chromatography or precipitation, or by binding nucleic acid to a binding agent (for example, at least one capture oligomer).For example, it can be beneficial to further isolate or purify the nucleic acid intended for downstream analysis approach (for example, amplification or sequencing) (otherwise, this approach may be totally or partially inhibited due to impurities).

[0259] If at least one capture oligomer is used, it can be sequence-specific (i.e., it includes a target-hybridizing region configured to specifically hybridize with a site in the target nucleic acid to form a duplex). One example of a capture oligomer includes two binding regions: a sequence-binding region (e.g., a 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 linked together by one or more linkers. Alternatively, capture oligomers with randomized, repetitive, or non-specific sequences for hybridization to the target can be used (e.g., poly-(k) and poly-(r) capture oligomers and combinations thereof, as described, for example, in US 2013 / 0209992 and US 2020 / 0165599, which are incorporated herein by reference). In some embodiments, the capture oligomer uses a target sequence binding region comprising a random or non-random poly-GU, poly-GT, or poly-U sequence to nonspecifically bind to the target nucleic acid. In some embodiments, the capture oligomer uses a target sequence binding region comprising a random or non-random poly-GA sequence to nonspecifically bind to the target nucleic acid. In some embodiments, a combination of capture oligomers is used, where a first capture oligomer comprises a random or non-random poly-GA sequence and a second capture oligomer comprises a random or non-random poly-GU or poly-GT sequence. The capture oligomer may further comprise a sequence or moiety that binds to a probe immobilized on a support.

[0260] In some embodiments, the method further comprises determining the presence or absence of at least one macromolecule in the sample. In some embodiments, the nucleic acid isolated from the lysate can be subjected to one or more analytical procedures, such as detecting the presence or absence of one or more target sequences (e.g., using one or more probes (e.g., probe oligomers) for the target region), which can be followed by amplification of the target sequence or performed directly on the isolated nucleic acid (as in Southern blot, slot blot, or dot blot); sequencing; electrophoresis; molecular cloning; or microarray analysis. In some embodiments, the amplification reaction is carried out using an amplification method involving isothermal amplification or temperature cycling (e.g., polymerase chain reaction (PCR) and transcription-mediated amplification (TMA)), or any other type of amplification, including those discussed above.

[0261] In some embodiments, a probe oligomer or FRET cassette containing a label is used. Particularly suitable labels include compounds that emit detectable light signals (e.g., fluorophores or luminescent (e.g., chemiluminescent) compounds that can be detected in a homogeneous mixture). More than one kind of label and more than one type of label can be present on a specific probe, and detection can rely on using a mixture of probes, each of which is labeled with a compound that produces a detectable signal (see, for example, U.S. Patent Nos. 6,180,340 and 6,350,579, each of which is incorporated herein by reference). Labels can be attached to probes by various means, including covalent linkage, chelation, and ionic interaction, but in some embodiments, labels are covalently attached. For example, in some embodiments, the detection probe has an attached fluorescent or chemiluminescent label (such as, for example, 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 of which is incorporated herein by reference). In a typical variation, the label is attached to the probe via a non-nucleotide linker (see, e.g., U.S. Pat. Nos. 5,585,481; 5,656,744; and 5,639,604, each of which is incorporated herein by reference). In some embodiments, the probe oligomer is labeled with an interactive pair of detectable labels. Examples of detectable labels that are members of an interactive pair of labels include those that interact with each other via FRET or non-FRET energy transfer mechanisms. Fluorescence resonance energy transfer (FRET) involves the non-radiative transfer of an energy quantum from an absorption site to its utilization site in a molecule or molecular system by resonant interaction between chromophores over distances significantly greater than interatomic distances, without conversion to thermal energy and without kinetic collision of the donor and acceptor.The "donor" is the moiety that initially absorbs energy, and the "acceptor" is the moiety to which the energy is subsequently transferred. In addition to FRET, there are at least three other "non-FRET" energy transfer processes by which excitation energy can be transferred from a donor to an acceptor molecule. In some embodiments, the probe oligomer is a probe primer and can be used for nucleic acid amplification.

[0262] In some embodiments, at least one probe system is used, and the oligomers of the above-mentioned system function together to facilitate the detection of target sequence.For example, in INVADER or INVADER PLUS assay, invasive oligomer (which can also function as amplification oligomer, for example, primer, but not necessarily), primary probe and FRET cassette can be used.INVADER and INVADER PLUS assay are discussed in detail in, for example, US 2018 / 0163259.

[0263] Detection probe oligomers according to the present disclosure may contain sequences that do not hybridize to the target. In some applications, probes that exhibit at least some degree of self-complementarity are desirable to facilitate detection of probe:amplicon duplexes in a test sample without first requiring removal of unhybridized probes prior to detection. Specific embodiments of such detection probes include, for example, probes that form conformations (e.g., commonly referred to as hairpin conformations) that are maintained by intramolecular hybridization. Particularly suitable hairpin probes include "molecular torches" (see, e.g., U.S. Pat. Nos. 6,849,412; 6,835,542; 6,534,274; and 6,361,945, each of which is incorporated herein by reference) and "molecular beacons" (see, e.g., U.S. Pat. Nos. 5,118,801; 5,312,728; and 5,925,517, each of which is incorporated herein by reference). In yet other embodiments, the detection probe is a linear oligomer that does not substantially form a conformation held by intramolecular bonds.

[0264] In some embodiments, an amplification and detection procedure is performed (e.g., an INVADER or INVADER PLUS assay, a TaqMan assay, or a TMA reaction (e.g., using at least one probe oligomer or FRET cassette comprising a fluorophore and a FRET partner and exhibiting hybridization- and / or degradation-dependent fluorescence) with probe hybridization and detection). In some embodiments, 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, for example, based on a standard curve and / or one or more calibration standards.

[0265] Embodiments of fluorophores include those that absorb light in the range of about 495-650 nm and emit light in the range of about 520-670 nm, including FAM, TM , TET TM ,CAL FLUOR TM (Orange or Red), and QUASAR TM Fluorophores can be used in combination with quencher molecules that absorb light when in close proximity to the fluorophore to reduce background fluorescence. Such quenchers are well known in the art and include, for example, BLACK HOLE QUENCHER (BLACK HOLE QUENCHER) compounds. TM (or BHQ TM ) or TAMRA TM Compounds include:

[0266] Examples of interacting donor / acceptor label pairs that can be used in connection with the present disclosure, without attempting to distinguish FRET from non-FRET pairs, include fluorescein / tetramethylrhodamine, IAEDANS / fluororescein, EDANS / DABCYL, coumarin / DABCYL, fluorescein / fluorescein, BODIPY FL / BODIPY FL, fluorescein / DABCYL, Lucifer Yellow / DABCYL, BODIPY / DABCYL, eosin / DABCYL, erythrosine / DABCYL, tetramethylrhodamine / DABCYL, Texas Red / DABCYL, CY5 / BH1, CY5 / BH2, CY3 / BH1, CY3 / BH2, and fluorescein / QSY7 dye. Those skilled in the art will understand that when the donor and acceptor dyes are different, energy transfer can be detected by the appearance of sensitized fluorescence of the acceptor or by quenching of donor fluorescence. Non-fluorescent acceptors (e.g., DABCYL and QSY7 dyes) advantageously eliminate the potential problem of background fluorescence resulting from direct (i.e., unsensitized) acceptor excitation. Exemplary fluorophore moieties that can be used as one member of a donor-acceptor pair include fluorescein, ROX, and CY dyes (e.g., CY5). Exemplary quencher moieties that can be used as the other member of a donor-acceptor pair include DABCYL and BLACK HOLE QUENCHER moieties (which are commercially available from Biosearch Technologies, Inc., (Novato, Calif.)).

[0267] In some embodiments, amplification and detection procedures (e.g., quantitative PCR (qPCR), reverse transcriptase PCR (RT-PCR), or nucleic acid sequencing) are performed using non-specific DNA binding dyes. Examples of non-sequence-specific DNA binding dyes include SYBR (登録商標)Examples of non-sequence-specific DNA binding dyes used in sequencing reactions include dye-labeled terminators (e.g., d-rhodamine ddNTPs, BigDye 1, d-dimers, and d-dimers). (登録商標) ddNTPs, and MagaDye TM Examples of non-sequence-specific dyes used in microarrays include infrared (IR) dyes, near-infrared (NIR) dyes, rhodamine, cyanines (e.g., Cy3, Cy5, and Cy7), fluorescein isothiocyanates (e.g., FITC, Alexa 532, JOE, Oregon Green), and BODIPY dyes (e.g., BODIPY 576 / 589 and BODIPY 630 / 650).

[0268] If the sample contains or is suspected of containing a pathogen, the method may include determining the presence or absence of at least one nucleic acid characteristic of the pathogen.

[0269] In some embodiments, the polypeptides isolated from the lysate can be subjected to one or more analytical procedures, such as detecting the presence or absence of one or more targets, or other forms of characterization, which may include one or more of antibody or aptamer binding assays (e.g., ELISA or Western blot); electrophoresis; amino acid sequencing; or mass spectrometry. In some embodiments, at least one polypeptide is quantified, for example, based on a standard curve and / or one or more calibration standards.

[0270] In some embodiments, the cells released from the carrier liquid are fixed, e.g., with an aldehyde or other cross-linking agent. In some embodiments, the cells are stained, e.g., with a fluorescent dye or chromophore, which is optionally linked to a binding agent, e.g., a nucleic acid probe (e.g., a FISH probe), an antibody, or an aptamer. In some embodiments, the cells are subjected to flow cytometry, microscopy, or other optical analysis.

[0271] 5. Cell Culture and Pathogen Inactivation Testing In some embodiments, a formulation is tested for its ability to inactivate one or more pathogens. In some embodiments, the pathogen is a bacterial pathogen (e.g., those disclosed herein, including Staphylococcus aureus and Bordetella pertussis). In some embodiments, the bacterial pathogen is evaluated for colony formation on an agar plate. In some embodiments, the bacterial pathogen is evaluated for hemolytic activity. After the bacterial pathogen is treated with a formulation of interest, it is applied to a blood agar plate and evaluated for lysis of red blood cells. In some embodiments, the bacterial pathogen is applied at different concentrations or in a series of dilutions. The blood agar plate may be incubated (1) at a range of temperatures (including 20°C, 24°C, 30°C, and 37°C) and (2) for a period of time (15 minutes, 1 hour, 2 hours, 4 hours, 24 hours, overnight, or longer).

[0272] In some embodiments, the pathogen is a viral pathogen (e.g., those disclosed herein, including coronavirus 229E and adenovirus serotype 2). In some embodiments, the viral pathogen is assessed for plaque formation. After the viral pathogen is treated with a formulation of interest, the viral pathogen is applied to a cell or tissue type and assessed for cytopathic effect (CPE), e.g., formation of viral plaques. In some embodiments, the viral pathogen is applied at different concentrations or in a series of dilutions.

[0273] In embodiments in which a cell or tissue type is used, the cell or tissue type is grown to approximately 80-90% confluence before the viral pathogen is applied to the cell or tissue type. Any suitable cell or tissue type may be used, and each cell or tissue type is cultured according to materials and methods known to those skilled in the art. In some embodiments, the cell or tissue type is of mammalian or human origin. In some embodiments, the cell or tissue type is MRC-5.

[0274] I. Kit Also provided herein is a kit comprising any one of the compositions described herein. In some embodiments, the composition is contained in a tube, for example, a tube including a cap.

[0275] In some embodiments, the kit further includes a swab comprising a tip portion and a stem portion. In some embodiments, the tip portion of the swab is a flocked tip. In some embodiments, the stem portion of the swab is a flexible plastic stem. In some embodiments, the swab is a nasopharyngeal swab.

[0276] In some embodiments, the kit contains 1 mL to 10 mL of any one of the compositions described herein. In some embodiments, the composition contains 2% to 4% (w / v) propylene glycol, 2% to 10% (w / v) of a degrading enzyme that is a protease, and about 1% to 4% of an alkyl sulfate detergent (preferably a dodecyl sulfate detergent). In some embodiments, the composition contains 2% to 3% (w / v) of Good's buffer and 0.05% to 0.3% (w / v) of a surfactant. Good's buffer refers to any of the series of 20 dipolar (zwitterionic) buffers developed by Dr. Norman Good (see Good et al., Biochemistry 5(2):467-477 (1966), which is incorporated herein by reference), such as HEPES, BES, bicine, CAPS, CHES, MES, MOPS, Tricine, etc., as well as six later developed zwitterionic buffers, AMPSO, CAPSO, HEPPSO, MOPSO, and POPSO. In some embodiments, the Good's buffer is HEPES.

[0277] In some embodiments, the kit further comprises instructions for use. In some embodiments, the instructions are provided as a digital document. In some embodiments, the instructions comprise instructions for biological sample collection. In some embodiments, the instructions comprise instructions for nasopharyngeal sample collection.

[0278] Also provided herein is the use of any one of the kits described herein for collecting a biological sample for a diagnostic test. In some embodiments, the biological sample is collected for a nucleic acid diagnostic test. In some embodiments, the biological sample includes one or more of protein, mucin, sputum, saliva, nasal discharge, mucus-like secretions, nasal mucus, pulmonary secretions, urine, blood, plasma, or serum. In some embodiments, the biological specimen is collected for a diagnostic test to determine the presence or absence of a bacterial pathogen. In some embodiments, the biological sample is collected for a diagnostic test to determine the presence or absence of a respiratory pathogen. In some embodiments, the respiratory pathogen is one or more of coronavirus, parainfluenza virus (HPIV), influenza virus, adenovirus, human metapneumovirus (HMPV), rhinovirus, enterovirus, or respiratory syncytial virus. [Example]

[0279] Example 1: Effect of enzyme-containing detergents and sample transport media on sample viscosity and target nucleic acid amplification and detection This example compares the effects of treating samples with three conditions on reducing sample viscosity (i.e., breaking up sample clumps) and preserving RNA stability for subsequent amplification and detection: (1) commercially available Endozime XP (Ruhof Corporation, catalog number 34530) diluted to 50%, (2) commercially available Endozime AW Triple Plus with APA (unscented) (Endo; Ruhof Corporation, catalog number 34521), and (3) sample transport medium (STM). The STM recipe is as follows: 2.07 g / L monosodium phosphate monohydrate; 30 g / L lithium lauryl sulfate (LLS); 0.372 g / L disodium EDTA dihydrate; 0.38 g / L EGTA free acid; and 2.13 g / L disodium hydrogen phosphate.

[0280] 100 mL of each sample condition was added to FluA / B / RSV in vitro transcripts (IVT) as shown in Table 1. [Table 1]

[0281] Clinical specimens from 30 healthy volunteers were collected directly into the specimen condition using nasal swabs (i.e., the collected samples in Table 4). 30 clinical specimens were collected for each specimen condition. The centrifuged samples were loaded into the Panther Fusion instrument for nucleic acid extraction, amplification, and detection. Target nucleic acid extraction, amplification, and detection were performed using the Panther Fusion Flu A / B / RSV assay, generally according to the manufacturer's instructions (Hologic, Inc., catalog number PRD-04328).

[0282] The performance of the aspiration and dispensing pipettes was monitored for each sample during transfer to the Panther Fusion extraction assembly. Samples that caused pipetting failure were detected by real-time pressure monitoring, and further processing of those samples was stopped. The data are shown in Table 2, where "invalid rate" indicates the percentage of samples that caused pipetting failure. [Table 2]

[0283] Samples in 100% Endozime AW Triple Plus with APA (unscented) and 50% Endozime XP in water did not have high viscosity and / or clots and therefore were transferred to the Panther Fusion extraction assembly without blockage.

[0284] Nine samples in the STM had high viscosity and / or clots that interfered with aspiration and dispensing, as indicated by real-time pressure monitoring. These samples were processed for reloading onto the assembly. In one sample, the blockage was resolved on the second loading attempt. In five other samples, the blockage was resolved after 15 seconds of human intervention by vortexing, and the sample was successfully loaded onto the assembly on the third attempt. Three samples did not resolve clots despite undergoing all of the above-described attempts. The results indicate that the use of a multi-enzyme detergent can reduce sample viscosity and improve sample loading, while the use of an STM did not.

[0285] Amplification and detection reactions were then performed on the samples. Reactions were performed approximately 12 and 16 hours after adding IVT to the samples. As shown in Table 4, amplification and detection of target nucleic acids was poor for samples in 50% Endozime XP or 100% Endozime AW Triple Plus with APA (unscented) (Endo). Of the samples in AC or Endo that tested positive for Flu A, RSV, or Flu B, the mean Ct did not show comparable Ct or analyte detection when sample matrix was introduced into the medium compared to STM.

[0286] In contrast, target nucleic acids were successfully amplified and detected in all samples in STM (Table 3). These results were consistent with the results of the no-clot positive control reactions (Table 4), where target nucleic acids for influenza A (Flu A), influenza B virus (Flu B), and respiratory syncytial virus A / B (RSV) were detected in all reactions performed approximately 0, 12, and 16 hours after addition of IVT. [Table 3] [Table 4]

[0287] The above data indicate that while the presence of an enzyme-containing detergent may reduce sample viscosity and aid sample processing, it did not effectively stabilize the target nucleic acid.

[0288] Example 2: Aspiration of viscous samples and / or samples with inhomogeneous content after collection onto several sample transport media The objective of this example was to reduce aspiration failures associated with specimens containing mucus-like or other viscous or inhomogeneous materials. Clinical specimens from 30 healthy volunteers were collected using nasal swabs. After collection, the swabs were individually placed in 1 ml of STM. The STM recipe was as described in Example 1 above. Each collected clinical specimen in specimen condition was vortexed and then centrifuged at 200 RCF for 1 minute. Each ml of STM contained approximately 6.9 copies / μL IVT for each of influenza A, influenza B, and respiratory syncytial virus target nucleic acids. The collected specimens were tested for the presence of blood and ranked as -blood, +blood, or ++blood depending on whether the specimen contained no blood, traces 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 (multi-enzyme detergent). The resulting sample transport media contained 0% Endozime AW Triple Plus with APA (ie, STM only), 50% Endozime AW Triple Plus with APA, or 65% Endozime AW Triple Plus with APA (Table 5). [Table 5]

[0289] Thirty clinical samples were prepared as described in Example 1, with each clinical specimen divided into each of three specimen conditions. Real-time pressure monitoring data was collected as described in Example 1.

[0290] In the 30 samples collected into the STM, there were six clots associated with pipetting failure. The 30 samples collected in 50% Endozime AW Triple Plus with APA produced one clot associated with pipetting failure. The 30 samples collected into 65% Endozime AW Triple Plus with APA produced no clots, and aspiration of all 30 samples was successful. These data indicate that the addition of a multi-enzyme detergent to the STM reduced viscosity and inhomogeneous content in the samples and reduced the frequency of pipetting failure.

[0291] Example 3: Amplification and detection of viscous samples and / or samples with non-homogeneous contents after collection onto several sample transport media Whole blood was spiked into the clinical specimens from Example 1. The specimens were then amplified and analyzed as described in Example 1 to determine the presence or absence of spiked IVT in each specimen condition. Extraction, amplification, and detection were performed using the Panther Fusion Flu A / B / RSV Assay generally as described by the manufacturer.

[0292] The results of this example are shown in Tables 6-8. The conditions used and the presence / absence of blood in the samples are indicated. The results show that the target nucleic acid was amplifiable and detectable in all three conditions tested (as shown in Table 5). The results also show approximately equal counts (Ct), but lower standard deviations for specimens processed in 50% Endozime AW Triple Plus with APA (50%) and 65% Endozime AW Triple Plus with APA (65%) compared to those processed in STM. [Table 6] [Table 7] [Table 8]

[0293] Example 4: Evaluation of assay performance in the presence of multi-enzyme detergents An additional set of assays was performed using the conditions of STM, 50% Endozime AW Triple Plus with APA (50%), and 65% Endozime AW Triple Plus with APA (65%), as described above in Example 2 and Table 6, except that patient specimens (volunteer nasal swabs) were not included in the samples. Approximately 6.9 copies / mL IVT for each of influenza A (Flu A), influenza B (Flu B), and respiratory syncytial virus (RSV) target nucleic acids were spiked into each sample. Target nucleic acid extraction, amplification, and detection were performed on a Panther Fusion system using the Panther Fusion Flu A / B / RSV assay according to the manufacturer's instructions. As shown in Table 9, the results show equal or lower Ct for target nucleic acids spiked into 50% or 65% Endozime AW Triple Plus with APA compared to those spiked into 0% Endozime AW Triple Plus with APA (i.e., STM in Table 9). This indicates that the use of a multi-enzyme detergent (eg, Endozime AW Triple Plus with APA) in combination with STM did not negatively impact assay performance. [Table 9]

[0294] Example 5: Enhanced STM (E-STM) for improved sample viscosity Based on the formulations and observations discussed in the preceding examples, new formulations were designed and tested for improvements in the following areas: (1) ability to reduce coagulation in biological samples, (2) stabilizing nucleic acids when specimens are collected and stored in the formulation, and (3) inactivation of pathogens, (4) formation of a homogenous solution during storage at various temperatures; and (5) stability at room temperature for at least one year.

[0295] This example discusses changes in formulation and how those changes affect sample viscosity. One set of assays was performed as described in Example 1 above using STM, 65% Endozime XP, 65% Endozime BioClean in STM, and 65% Elementum AW. The STM formulation was as described in Example 1 above. Data from the assays was as shown in Table 10 below. The presence of Endozime BioClean and Elementum AW reduced clot error compared to the STM control, without sample manipulation such as vortexing. [Table 10]

[0296] Example 6: E-STM exhibits improved nucleic acid preservation This example discusses formulation changes and how those changes affect nucleic acid stability in samples. Flu A, RSV, and Flu B IVT were spiked into one of 100% STM, 100% Endozime BioClean, 100% Endozime XP, 100% Elementum, 65% Endozime BioClean, 65% Endozime XP, or 65% Elementum AW. Formulations with 65% Endozime BioClean, 65% Endozime XP, or 65% Elementum AW were diluted from 100% to 65% using (1) 2 parts Endozime BioClean, Endozime XP, or Elementum AW, respectively, and (2) 1 part STM. Because STM contained 3% (w / v) LLS, E-STM formulations with 65% Endozime BioClean, 65% Endozime XP, or 65% Elementum AW each contained a final concentration of 1% (w / v) LLS. The stability of nucleic acids in these formulations was compared. Experiments were performed as described in Example 1 above.

[0297] As shown in Table 11 below, 100% Endozime BioClean, 100% Endozime XP, and 100% Elementum AW formulations did not sufficiently stabilize nucleic acids in the presence of patient swabs. However, as shown in Table 12 below, STM formulations supplemented with 65% Endozime BioClean or 65% Endozime XP stabilized nucleic acids in the presence of patient swabs. It was noted that the STM formulation supplemented with 65% Endozime BioClean containing 1% (w / v) LLS reduced CLT errors (Example 5), indicating stabilization of nucleic acids in the sample and facilitating amplification and detection. [Table 11-1] [Table 11-2] [Table 12-1] [Table 12-2]

[0298] Example 7: Lithium Lauryl Sulfate (LLS) for Recovery of Viral Nucleic Acids Formulations containing lithium lauryl sulfate (LLS) were tested for the recovery of human immunodeficiency virus (HIV) nucleic acid (NA) from dried blood spots (DBS) and cytomegalovirus (CMV) NA from whole blood diluents (WBD).

[0299] Various percentages of LLS in STM and blood processing medium (BPM; whole blood diluent containing 6% LLS) were tested. To prepare HIV-1-positive dried blood spots, Whatman 903 Specimen Collection Cards were loaded with 70 μL of HIV-1-positive blood (5 × 10 4Whole blood spiked with HIV-1 to a concentration of 100 copies / mL was spotted onto the blood and allowed to air dry for 24 hours. The dried blood spots were then excised and transferred to STM reagents containing one of several different LLS concentrations (0 wt%, 1 wt%, 3 wt%, 5 wt%, or 7 wt%; i.e., w / v (0-100 g / L)). These STM reagents were otherwise substantially identical to the STM formulations disclosed in Example 1 (above). After a 30-minute incubation, the STMs were transferred to separate tubes and tested in five replicates using the Aptima HIV-1 Quant Dx Assay (Hologic, Inc., catalog number PRD-03565) according to the manufacturer's instructions. The results from this test are as follows (mean log copies / mL recovery, standard deviation) and are shown in Table 13 (Experiment 1). Based on these data, STM containing 3% to 7% LLS (w / v) all showed robust amplification and detection results (approximately 3.5 to 3.7 log copies / mL recovery), while 0% and 1% showed lower recovery. In a separate experiment, 10% LLS (w / v) also showed lower recovery (not shown).

[0300] In further testing, HIV-1 was recovered from DBS in STM containing 0%, 1%, 1.5%, 2%, 2.5%, or 3% LLS (w / v). Dried blood spot samples were prepared and recovered as described above. After 30 minutes of incubation in an STM formulation substantially identical to that in Example 1, except that LLS was present in the STM at 0 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, or 30 g / L, the samples were tested for the presence of HIV-1 nucleic acid using the Aptima HIV-1 assay (catalog number PRD-03565). The results of this test are shown in Table 13 (Experiment 2) and demonstrate a decrease in assay performance below 1.5% LLS. Therefore, preferred LLS concentrations are above 1.5% (w / v). These data indicate that sample collection media formulations containing approximately 1.5% to approximately 7% LLS provide robust nucleic acid and recovery. The highest concentrations were obtained at about 2% to about 5% LLS. [Table 13]

[0301] Example 8: Protease stability in E-STM at room temperature This example discusses changes in formulation and how those changes affect formulation stability at various temperatures, which in turn affects protease stability at those temperatures. The protease present in the formulation in the example is subtilisin. Three formulations were stored for two weeks at room temperature, 4°C, or -20°C, and aspirate / dispense experiments were performed as described in Example 1. The formulations were STM, 65% Endozime BioClean in STM, and 100% Endozime BioClean supplemented with LLS to a final concentration of 3% (w / v). Samples in STM did not resolve clots as expected, but the 65% Endozime BioClean formulation lost its ability to resolve clots (Table 14). The 100% Endozime BioClean formulation with 3% (w / w) LLS, in contrast, retained its ability to resolve clots. [Table 14]

[0302] Therefore, the 65% EndoZime BioClean E-STM was deemed to lack sufficient protease stability to prevent clotting after two weeks at room temperature. Because STM contains EDTA and EGTA, it was noted that the chelating agents EDTA and EGTA were present in the 65% EndoZime BioClean E-STM. The lack of protease stability was attributed to inhibition of proteases in EndoZime BioClean by EDTA and EGTA. The 100% EndoZime BioClean formulation with 3% (w / w) LLS (which did not clot) did not contain chelating agents.

[0303] Aspiration / dispense experiments confirmed that the presence of active protease plays an important role in dissolving sample clots. The protease is believed to solubilize proteins in mucus-like respiratory samples, thereby reducing aspiration pressure and reducing clots. As shown in Table 15, the presence of 3% v / v protease dissolves clots in the samples. Furthermore, aspiration and dispensing pressures using the E-STM were only slightly lower than those using the STM, despite dissolving more clots than the STM. [Table 15]

[0304] Based on the observations noted in Examples 5-7, the formulations shown in Table 16 were considered for future testing. [Table 16]

[0305] Further aspiration / dispensing experiments showed that higher concentrations of protease were more effective in dissolving clots (Table 17). [Table 17]

[0306] Example 9: Guard band test for proteases in E-STM The above examples demonstrate that formulations containing protease resolve clots better than formulations without protease. Guard band studies were performed to determine the acceptable range of protease % in E-STM formulations without negatively impacting aspiration and dispensing pressures. 1%, 2%, 3%, 4%, 4.5%, or 6% (w / v) protease (Savinase (登録商標)E-STM formulations containing 12T, Strem Chemicals, Inc., Newburyport, MA, catalog number 06-3137, 16 Anson units / g) were tested and compared for their performance in dissolving mucus-like material in samples. All formulations tested in this example contained 3% (w / v) each of HEPES, propylene glycol, sodium xylene sulfonate, and lithium lauryl sulfate; 0.14% (w / v) partially fluorinated alkyl polyether; 0.1% (w / v) c12-c14-secondary ethoxylated alcohol; and 0.28% (w / v) anionic low-foaming surfactant and 1,2-benzisothiazol-3(2H)-one. The above formulations further contained protease at the following concentrations (all w / v): 0% (Formulation 3G0P), 1.2% (Formulation 3G1P), 2.1% (Formulation 3G2P); 3% (Formulation 3G3P); 3.9% (Formulation 3G4P); 4.5% (Formulation 3G5P); and 6% (Formulation 3G6P). A commercially available specimen transport medium was also included in this example (Hologic, Inc., catalog number PRD-04423). Nasal swab samples were collected from healthy donors, and the swabs were transferred to 2.9 mL of either the test formulation or the commercially available STM. Twenty replicates of each condition were prepared. Aspiration / dispensing experiments were performed as described in Example 1. The aspiration / dispensing data (Table 18) show that formulations containing 1.2% to 6% (w / v) protease removed mucus from all 20 nasal specimens, while the commercial STM and protease-free formulations removed mucus from 50% to 60% of the specimens (10 tested per condition). This example demonstrates that formulations containing protease more efficiently eliminate clotting errors and improve specimen pipetting errors. [Table 18]

[0307] Further testing was performed to determine whether formulations with higher concentrations of protease improved aspiration quality over formulations with lower concentrations of protease. Aspiration quality was assessed for nasal swab samples collected in one of several formulations. Aspiration quality was assessed by measuring the flow rate of the collected sample / formulation through a disposable tip. In this study, formulations 3G3P, 3G6P, and a formulation with 10% (w / v) protease were compared for aspiration quality. A formulation containing 10% protease was prepared as described immediately above, except that 10% (w / v) protease was included in the formulation (3G10P). Nasal swab samples were collected from healthy donors, and the swabs were transferred to 2.9 mL of one of the test formulations. The 3G3P condition was prepared in 285 replicates, while the 3G6P and 3G10P conditions were each prepared in 230 replicates. Aspiration pressure data for the samples showed 1.3% clots for the 3G3P condition, 0.4% clots for the 3G6P condition, and 1.3% clots for the 3G10P condition. In this study, the 3G3P condition was determined to have higher viscosity than either the 3G6P or 3G10P conditions, with these latter two conditions having better aspiration quality than the 3G3P condition. The data from this study demonstrate improved aspiration pressure profiles for formulations containing higher concentrations of protease, although there was no difference in quality above 6% (w / v) protease in these formulations.

[0308] Example 10: Guardband testing for lithium lauryl sulfate (LLS) in E-STM Lithium lauryl sulfate (LLS) in commercially available analyte transport media stabilizes nucleic acids for subsequent processing. Guardband studies were conducted to determine the acceptable range of LLS percentages for enhancing analyte transport media formulations without negatively impacting performance. E-STM formulations containing 1%, 3%, or 4% (w / v) LLS were tested and compared. Several formulations were prepared as generally described in Example 9, without HEPES but with the addition of trisodium citrate dihydrate and protease (3% (w / v)), and with an LLS concentration of 1% w / v LLS (Formulation 3C1L), 3% w / v LLS (Formulation 3C3L), or 4% w / v LLS (Formulation 3C4L). Additional formulations were prepared as generally described in Example 9 with the addition of 6% (w / v) protease, and the LLS concentrations were one of 2% w / v LLS (formulation 3G6P2L), 3% w / v LLS (formulation 3G6P3L), or 4% w / v LLS (formulation 3G6P4L). Commercially available specimen transport media (Hologic, Inc.) was also included.

[0309] Testing of formulations 3C1L, 3C3L, and 3C4L was performed on day 0 and after 6 days of storage at 30°C. For these tests, nasal swabs were collected from 15 healthy volunteers for each formulation condition. Each nasal swab was collected into 2.9 mL of formulation condition. For testing with the Panther Fusion Bordetella Assay (Bordetella), Panther Fusion Adenovirus / Metapneumovirus / Rhinovirus Assay (AMR), and Panther Fusion Flu A / B / RSV Assay, the formulations were spiked with virus and bacteria to concentrations equal to 5 times the reported detection limits for these assays (Hologic, Inc., catalog numbers PRD-04868, PRD-04330, and PRD-04328, respectively). These spiked formulations were each divided into two incubation conditions: a 0-day incubation condition and a 6-day incubation condition at 30°C. After 0 or 6 days of incubation, the spiked formulations were loaded into a Hologic Panther instrument without further mixing and tested according to the respective assay protocol.

[0310] As shown in Tables 19-21 below, no differences in Ct values ​​were noted for the two time points in the detection of Bordetella (Table 19) or the six viral targets (Tables 20 and 21). Thus, the viral and bacterial nucleic acids were stable in each of the formulations tested during 6 days of storage at 30°C. [Table 19] [Table 20] [Table 21]

[0311] Testing with formulations 3G6P2L, 3G6P3L, and 3G6P4L was performed on nasal swabs collected from healthy volunteers. Thirty volunteers each provided four nasal swab samples, which were collected into 2.9 mL of 3G6P2L, 3G6P3L, 3G6P4L, or commercial STM to provide 30 replicates of each condition. Samples in media were shaken for 15 minutes. Twenty-five replicates of each condition were spiked with SARS-CoV-2 virus for testing in the Aptima SARS-CoV-2 assay at 3x the LoD of the virus according to the package insert (Hologic, Inc., catalog number PRD-06419). Five of the replicates served as negative controls. Samples were loaded into a Panther System (Hologic, Inc.) and assayed according to the manufacturer's instructions. The STM control condition produced one clot (1 / 25). Without being bound by any theory, it is believed that the mixing process liquefied any mucus-like material that may have been present in these STM conditions, giving them an improved clotting profile. All conditions showed 25 / 25 positive results for the added condition and 0 / 5 positive results for the negative condition. All tested conditions showed comparable mean cycle time values.

[0312] Nucleic acids were stabilized for at least 6 days in these sample transport formulations containing 1% to 6% LLS.

[0313] Example 11: Pathogen Inactivation The purpose of this example was to test several formulations for their ability to inactivate bacterial pathogens. Staphylococcus aureus and Bordetella pertussis stock suspensions were combined with one of the formulations or phosphate-buffered saline, serially diluted, and then plated on blood agar plates and evaluated for colony formation. The stock Staphylococcus aureus had a concentration of 2.6E9 CFU / mL, and the stock Bordetella pertussis suspension had a concentration of 4.7E9 CFU / mL. Seven different test formulations were prepared generally as described above (Formulations 1A, 3A, 3C, 3D, 3E, 3G, and 3I). The formulations contained the following: 1% (w / v) (Formulation 1A) or 3% (w / v) lithium lauryl sulfate, 0% or 0.1% (w / v) trisodium salt of methylglycine diacetic acid (Trilon M, Na3MGDA, BASF) (Formulation 3E), 0% or 3% (w / v) HEPES free acid (Formulations 3G and 3I), 2% (w / v) (Formulations 1A, 3A, and 3I) or 3% (w / v) each of propylene glycol and sodium xylene sulfonate, 0.1% (w / v) each of partially fluorinated alkyl polyether and c12-14 secondary ethoxylated alcohol, 0.2% (w / v) (Formulations 1A, 3A, and 3I) or 0.3% (w / v) each of anionic low-foaming surfactant and 1,2-benzisothiazol-3(2H)-one, 2% (w / v). Trisodium citrate (Formulations 1A and 3A) or 3% (w / v) trisodium citrate (Formulation 3C), and 2% (w / v) protease or 3% (w / v) protease (Formulations 3C, 3D, 3E, and 3G) were compared for their ability to inactivate these pathogens with commercially available specimen transport media (Hologic, Inc., San Diego, CA, catalog number PRD-04423).

[0314] Biological control sample dilutions were prepared by combining 50 μL of stock organism in 950 μL of PBS to give a 1E-6 dilution. Buffer control dilutions were prepared by creating an initial solution containing 50 μL of PBS in 950 μL of formulation, and then subsequently creating a 50:950 μL dilution in PBS to give a 1E-5 dilution. Test conditions were prepared separately for each organism by combining 50 μL of stock organism in 950 μL of formulation. Each test condition was then divided into four 250 μL aliquots, and the aliquots were incubated for 15 minutes, 1 hour, 2 hours, or 4 hours. At each incubation time point, the corresponding test condition was serially diluted to a 1E-6 dilution of the stock. Dilutions 1E-4 to 1E-6 for the organisms and 1E-5 to 1E-6 for the controls were plated in duplicate per dilution onto blood agar plates (Hardy Diagnostics, Santa Maria, CA, catalog number A10, and BD Bioscience, Franklin Lakes, NJ, catalog number BBL297876) in 100 μL. The plates were incubated at 37°C for 24 hours. After 24 hours of incubation, the number of colonies on each of the test condition plates and the organism control plates was counted. The control dilution series indicates the preferred dilution of the stock for growing these organisms. The buffer control series indicates that the buffer does not prevent the organisms from growing on the plates at any given dilution. The test condition series indicates whether the formulation inactivates the organisms.

[0315] The CFU counts on each plate were back-calculated to the stock suspension (undiluted) to determine CFU / mL. The back-calculated CFU / mL was compared to the original stock suspension to determine the fold reduction in pathogens under the test conditions. The results are shown in Table 22 below. [Table 22] *Conditions showing two results were tested in duplicate.

[0316] In Table 22, higher values ​​indicate greater effectiveness in target inactivation. Formulations containing 3% (w / v) LLS were more effective at inactivating target pathogens than formulations containing less than 3% (w / v) LLS. Formulation 3C inactivated test pathogens as effectively as commercial sample transport media. Based on the data from these studies, sample transport media with LLS concentrations greater than 1% (w / v) are preferred for efficient bacterial pathogen inactivation.

[0317] Further experiments were conducted on the inactivation of viral pathogens. The test formulation used in this experiment was based on one of the better-performing formulations from the bacterial inactivation experiment. The test formulation contained 3% (w / v) each of lithium lauryl sulfate, trisodium citrate, propylene glycol, and sodium xylene sulfonate; 0.1% (w / v) of a partially fluorinated alkyl polyether and a c12-c14 ethoxylated secondary alcohol; and 0.3% (w / v) of an anionic low-foaming surfactant and a benzisothiazolone. The protease component was omitted from the test formulation because protease activity against tissue culture media would result in inaccurate evaluation of the formulation. The test formulation was compared against a commercially available specimen transport medium (Hologic, Inc., San Diego, CA, catalog number PRD-04423). The test formulation and STM were combined with stock titers of coronavirus 229E and adenovirus serotype 2, respectively, and incubated for 1 hour at 37°C. After incubation, the test and STM conditions were diluted to a predetermined concentration shown to infect tissue culture cells, and each dilution was passed through a detergent removal column (Pierce Biotech, Rockform, IL, catalog number 87776) according to the manufacturer's instructions. The detergent-free dilutions were added (100 μL) to separate cultures of MRC-5 cells (ATCC, Manassas, VA, catalog number CCL-171) grown to 80%-90% confluence. A positive control condition was prepared using stock virus diluted in cell growth medium and added to the MRC-5 tissue cultures. A no-virus control was prepared to determine whether the formulation dilution condition negatively affected the tissue cultures. Tissue cultures for all conditions were then incubated at 37°C for 2 hours to allow infection of the cells with any virus from the test condition, the commercial STM condition, or the positive control condition. The medium was then changed to standard cell growth medium and the cultures were further incubated.Each tissue culture was then examined under a microscope to determine the presence and extent of cytopathic effect in the culture, and the ability of the test formulation to inactivate the virus compared to commercial STM was then determined. Positive control cultures exhibited cytopathic effects representative of each virus at each dilution. The results show that the test formulations inactivated the virus as effectively as commercial STM.

[0318] All formulations tested in this example were effective in inactivating various pathogens, with formulations containing greater than 2% (w / v) protease being more effective than those containing 2% (w / v) protease.

[0319] Example 12: Sedimentation Test Precipitate formation was observed for formulation 3C (described above) when stored at 4°C. At least one hour of incubation at room temperature was required to return the precipitant to solution, an additional step before the formulation could be used. Seven formulations (including formulation 3C) were prepared and evaluated for precipitant formation. The formulations included formulations 3C, 3D, 3E, 3G, and 3I (described in Example 11 above) and 3J. Formulation 3J contained 2% (w / v) propylene glycol and sodium xylene sulfonate, 3% (w / v) protease and lithium lauryl sulfonate, 0.2% (w / v) BIT preservative and anionic low-foaming surfactant, and 0.1% (w / v) partially fluorinated alkyl polyether and c12-c14 secondary ethoxylated alcohol. Each formulation was divided into two storage conditions: -20°C storage and 4°C storage. None of Formulations 3D, 3E, 3G, 3I, or 3J formed a precipitate when stored at 4° C. However, Formulations 3D and 3E formed a precipitate when thawed from −20° C.

[0320] Formulations containing 3% (w / v) trisodium citrate formed a precipitate when stored at 4° C. Removal of that reagent from the formulations resulted in some formulations that did not form a precipitate at 4° C. However, some of these formulations formed a precipitate when thawed from −20° C. Addition of HEPES free acid and / or reduction in the concentration of other formulation components resulted in formulations that did not form a precipitate when thawed from −20° C.

Claims

1. 1. A composition for use in collecting a biological sample for a diagnostic test comprising at least one protease and an anionic detergent comprising an alkyl sulfate, and optionally a buffer and / or a divalent cation chelator, the protease is present at about 3% w / v to about 6% w / v; the anionic detergent is present at about 2% w / v to about 5% w / v; the biological sample is a lung sample selected from the group consisting of sputum, nasal mucosa, nasal secretion, oropharyngeal swab sample, bronchoalveolar lavage fluid, bronchial washings, and nasopharyngeal swab sample; composition.

2. The composition described in claim 1, wherein the protease is present at about 3% w / v or about 6% w / v.

3. The composition described in claim 1 or 2, wherein the protease is a serine protease.

4. The composition described in claim 3, wherein the serine protease is subtilisin.

5. The composition of any one of claims 1 to 4, wherein the anionic detergent comprises a sodium or lithium salt.

6. The composition of claim 1, wherein the alkyl sulfate is dodecyl sulfate.

7. The composition of claim 6, wherein the anionic detergent comprises lithium lauryl sulfate.

8. 8. The composition of claim 1, further comprising a detergent booster, coupling agent, or complexing agent; optionally the detergent booster, coupling agent, or complexing agent comprises sodium xylene sulfonate, a zeolite, a diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, an alkyl or alkenyl succinic acid, a soluble silicate, or a layered silicate; and further optionally the layered silicate is Hoechst SKS-6.

9. 9. The composition of any one of claims 1 to 8, wherein the composition further comprises a bleaching system, optionally comprising a source of HO; and further optionally comprising a source of HO comprising a perborate, a percarbonate, a peracid forming bleach activator, or a combination thereof.

10. 10. The composition of any one of claims 1 to 9, wherein the composition further comprises a stabilizing agent; optionally the stabilizing agent comprises propylene glycol, a polyol, a sugar, a sugar alcohol, lactic acid, boric acid, or a boric acid derivative; and optionally the boric acid derivative comprises a borate ester, an aromatic borate ester, a phenylboronic acid (optionally the phenyl is a substituted phenyl), or 4-formylphenylboronic acid.

11. 11. The composition of any one of claims 1 to 10, wherein the composition further comprises one or more of a clay, a foam booster, a defoaming factor, an anti-corrosion agent, a soil suspension agent, a soil redeposition inhibitor, a fungicide, an anti-tarnish agent, or a hydrotrope, optionally wherein the hydrotrope comprises urea, a tosylate, a cumene sulfonate, or a xylene sulfonate.

12. The composition of any one of claims 1 to 11, wherein the composition further comprises a surfactant; optionally the surfactant is present at less than about 10% by weight of the composition.

13. The composition according to any one of claims 1 to 12, wherein the composition has a pH of from 3 to 14.

14. The composition of any one of claims 1 to 12, wherein the composition has a pH of 6 to 7, 7 to 8, 8 to 9, 9 to 10, or 10 to 11.

15. The composition of any one of claims 1 to 14, wherein the buffer is a phosphate buffer, sodium phosphate, HEPES, or an alkali salt thereof.

16. 16. The composition of any one of claims 1 to 15, wherein the divalent cation chelator comprises a magnesium and / or calcium chelator, optionally wherein the magnesium and / or calcium chelator comprises Trilon M, trisodium citrate, EDTA, or EGTA.

17. 17. The composition of any one of claims 1 to 16, wherein the composition further comprises a preservative; the preservative comprises a microbicide, a bactericide, a biocide, a fungicide, and / or a benzoisothiazolinone (BIT).

18. A composition described in any one of claims 1 to 17, wherein the biological sample contains or is suspected of containing extracellular nucleic acid of a respiratory pathogen.

19. 19. A method of processing a biological sample, the biological sample being a lung sample selected from the group consisting of sputum, nasal mucosa, nasal secretion, oropharyngeal swab sample, bronchoalveolar lavage fluid, bronchial washings, and nasopharyngeal swab sample, the method comprising contacting the biological sample with a composition according to any one of claims 1 to 18.

20. A method for inactivating an infectious pathogen in a biological sample, wherein the biological sample is a lung sample selected from the group consisting of sputum, nasal mucosa, nasal secretion, oropharyngeal swab sample, bronchoalveolar lavage fluid, bronchial washings, and nasopharyngeal swab sample, the method comprising the step of contacting the biological sample with a composition described in any one of claims 1 to 18.