WO2024006548A1 - Nucleic acid preparation - Google Patents

Nucleic acid preparation Download PDF

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Publication number
WO2024006548A1
WO2024006548A1 PCT/US2023/026778 US2023026778W WO2024006548A1 WO 2024006548 A1 WO2024006548 A1 WO 2024006548A1 US 2023026778 W US2023026778 W US 2023026778W WO 2024006548 A1 WO2024006548 A1 WO 2024006548A1
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Prior art keywords
range
composition
sample
concentration
nucleic acid
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PCT/US2023/026778
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French (fr)
Inventor
Mary Wilson
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Sherlock Biosciences, Inc.
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Publication of WO2024006548A1 publication Critical patent/WO2024006548A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D1/00—Detergent compositions based essentially on surface-active compounds; Use of these compounds as a detergent
    • C11D1/66—Non-ionic compounds
    • C11D1/75—Amino oxides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D1/00—Detergent compositions based essentially on surface-active compounds; Use of these compounds as a detergent
    • C11D1/86—Mixtures of anionic, cationic, and non-ionic compounds

Definitions

  • the present disclosure describes technologies (e.g., compositions, mixtures, methods, and/or systems, etc.) for preparing of lysates.
  • the present disclosure provides technologies for nucleic acid preparations.
  • the present disclosure provides technologies for preparation of lysates from cells (e.g., eukaryotic, prokaryotic, etc.).
  • the present disclosure provides technologies for preparation of lysates from samples.
  • the present disclosure provides technologies for preparation of lysates from infectious agents.
  • the present disclosure provides technologies for preparation of lysates of viral particles (e.g., enveloped viruses, non-enveloped viruses).
  • the present disclosure demonstrates that provided technologies achieve nucleic acid preparations amenable to sensitive detection technologies, including specifically technologies that detect (e.g., via processes that may include direct binding to, such as by hybridization and/or protein binding) particular target sequence(s) that may be present in nucleic acid(s) of the preparation.
  • the present disclosure demonstrates surprising effectiveness of certain detergents (e.g., certain zwitterionic detergents) for use in lysing cells and/or viral particles and/or releasing nucleic acids from cells and/or viral particles, so that a nucleic acid preparation as described herein is obtained.
  • certain detergents e.g., certain zwitterionic detergents
  • Advantages of certain embodiments of provided technologies may include, among other things, that a useful nucleic acid preparation is provided without use of one or more traditional processing steps - such as purification, isolation or extraction steps that are commonly required or utilized to remove detergents.
  • the present disclosure identifies the source of a problem with many traditional sample (including e.g., a cell and/or an infectious agent such as a bacterium or a virus) lysis and/or nucleic acid isolation technologies, for example in the reliance of such technologies on reagents and/or steps that can prolong or complicate provision of a useful nucleic acid preparation, including specifically of a nucleic acid preparation amenable to analysis (e.g., directly amenable to analysis) as described herein.
  • kits for lysis of viral particles may include rapid lysis of viral particles, such as envelope particles (e.g., ranging from 1 second to 120 seconds, such as 30 seconds).
  • envelope particles e.g., ranging from 1 second to 120 seconds, such as 30 seconds.
  • technologies are compatible with a variety of samples, wherein the sample is or comprises saliva and/or nasal swab eluates.
  • the present disclosure provides a surprising insight that levels of viral lysis comparable to those achieved by heat treatment (e.g., as described for example in Wateson et al Release of Intracellular Protein By Thermolysis. Ellis Horwood; London, UK, 1987 pp 105-109, incorporated herein by reference) can be achieved at ambient temperature through use of particular reagents (e.g., zwitterionic detergents) as described herein.
  • particular reagents e.g., zwitterionic detergents
  • Figure 1 depicts an exemplary workflow for viral lysis at ambient temperature (e.g., at 22 °C), using e.g., 3 -Dodecylamido-N,N' -Dimethylpropyl Amine Oxide • 3-Laurylamido-N,N'-Dimethylpropyl Amine Oxide (LAP AO) and hydrochloric acid (HC1), or using N,N-Dimethyl-1-Dodecanamine-N-Oxide (LDAO), sodium decanoate (NaClO), and HC1.
  • LAP AO 3-Laurylamido-N,N'-Dimethylpropyl Amine Oxide
  • LDAO N,N-Dimethyl-1-Dodecanamine-N-Oxide
  • NaClO sodium decanoate
  • the workflow includes an incubation step of the sample with a lysis reagent, followed by lysis reaction and a neutralization step.
  • the workflow also depicts an optional nucleic acid
  • Figure 2 depicts effect(s) of pH on viral lysis efficiency at ambient temperature (22°C) using detergent compositions as described herein.
  • Figure 3 demonstrates reproducibility of viral lysis reactions as described herein (e.g., in Figures 1 and 2).
  • Figure 3 also demonstrates that LAP AO detergent concentrations between about 0.03% and 0.3% (e.g., specifically between 0.0375% and 0.3%, inclusive) achieved better cell lysis than did lower concentrations (e.g., 0.018% and below).
  • Figure 4 demonstrates reproducibility of viral lysis reactions as described herein (e.g., in Figures 1 and 2).
  • Figure 5 demonstrates that viral lysis reactions as described herein e.g., in the Figures) can be conducted in water (i.e., that provided lysis reaction mixtures can be effective in water and/or in buffered solutions).
  • Figure 6 demonstrates compatibility of viral lysis reach on(s) as described herein (e.g., in the Figures) with INSPECTRTM nucleic acid detection.
  • Figure 7 demonstrates viral lysis reactions, as described herein, comprising the presence of at least one detergent alone (LAP AO) or in combination with HC1 (LAP AO + HC1). Heat lysis is used as a positive control and the NEG (negative control) is performed without any detergent or HC1.
  • Ambient temperature is the temperature of surroundings.
  • ambient temperature is to be understood as the temperature of any object or environment surrounding an item. Measuring an ambient temperature can be accomplished by using a thermometer or sensor. The ambient temperature of an item is dependent on the temperature of the surrounding of the item.
  • the surroundings can have any temperature, such as a temperature below 95°C, such as below 90°C, such as below 85°C, such as below 80°C, such as below 75°C, such as below 70°C, such as below 65°C, such as below 60°C, such as below 55°C, such as below 50°C, such as below 45°C, such as below 40°C, such as below 35°C, such as below 30°C, such as below 25°C, such as below 24°C, such as below 23°C, such as below 22°C, such as below 21°C, such as below 20°C.
  • a temperature below 95°C such as below 90°C, such as below 85°C, such as below 80°C, such as below 75°C, such as below 70°C, such as below 65°C, such as below 60°C, such as below 55°C, such as below 50°C, such as below 45°C, such as below 40°C, such as below 35°C, such as below 30°C, such as below 25
  • Exemplary ambient temperature ranges include 5°C to 50°C, such as 10°C to 40°C, such as 15°C to 35°C, such as 20°C to 30°C, such as 20°C to 25°C, such as 20°C to 22°C.
  • Binding typically refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts - including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and/or in a biological system or cell).
  • biological sample typically refers to a sample obtained or derived from a biological source (e.g., a tissue or organism or cell culture) of interest, as described herein.
  • a source of interest is or comprises an organism, such as an animal or human.
  • a biological sample is or comprises biological tissue or fluid.
  • a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or broncheoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and/or excretions; and/or cells therefrom, etc.
  • a biological sample is or comprises viral particles.
  • biological samples comprising viral particles are obtained from an individual.
  • a biological sample is or comprises cells obtained from an individual.
  • a biological sample is or comprises cells comprising viral particles obtained from an individual.
  • a biological sample is or comprises viral particles or fragments thereof in a cell lysate matrix.
  • a biological sample is or comprises a nasal swab.
  • a biological sample is or comprises saliva
  • obtained cells are or include cells from an individual from whom the sample is obtained.
  • obtained viral particles are or include viral particles from an individual from whom the sample is obtained.
  • a sample is a “primary sample” obtained directly from a source of interest by any appropriate means.
  • a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc.
  • body fluid e.g., blood, lymph, feces etc.
  • sample refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semipermeable membrane.
  • Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and/or purification of certain components, etc.
  • cellular lysate refers to a fluid containing contents of one or more disrupted cells (i.e., cells whose membrane has been disrupted).
  • a cellular lysate includes both hydrophilic and hydrophobic cellular components.
  • a cellular lysate includes predominantly hydrophilic components; in some embodiments, a cellular lysate includes predominantly hydrophobic components.
  • a cellular lysate is a lysate of one or more cells selected from the group consisting of plant cells, microbial (e.g., bacterial or fungal) cells, animal cells (e.g., mammalian cells), human cells, and combinations thereof.
  • a cellular lysate is a lysate of one or more abnormal cells, such as cancer cells.
  • a cellular lysate is a crude lysate in that little or no purification is performed after disruption of the cells; in some embodiments, such a lysate is referred to as a “primary” lysate.
  • a cellular lysate comprises viral particles.
  • viral particles e.g., intact viral particle
  • a cellular lysate comprises spiked-in viral particles.
  • compositions or methods comprising one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method.
  • any composition or method described as “comprising” (or which "comprises") one or more named elements or steps also describes the corresponding, more limited composition or method “consisting essentially of' (or which "consists essentially of) the same named elements or steps, meaning that the composition or method includes the named essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the composition or method.
  • composition or method described herein as “comprising” or “consisting essentially of one or more named elements or steps also describes the corresponding, more limited, and closed- ended composition or method “consisting of (or “consists of) the named elements or steps to the exclusion of any other unnamed element or step.
  • known or disclosed equivalents of any named essential element or step may be substituted for that element or step.
  • determining involves manipulation of a physical sample.
  • determining involves consideration and/or manipulation of data or information, for example utilizing a computer or other processing unit adapted to perform a relevant analysis.
  • determining involves receiving relevant information and/or materials from a source.
  • determining involves comparing one or more features of a sample or entity to a comparable reference.
  • expression of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and/or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
  • Lysis reagent composition refers to a discrete physical entity that comprises one or more specified components described herein.
  • the lysis reagent composition comprises components capable of disrupting membranes, such as a cell membrane and/or a viral particles (e.g. a capsid and/or lipid bilayer).
  • the lysis reagent composition comprises a detergent (e.g, a zwitterionic detergent); an acid (e.g., HC1); and, optionally, a salt (e.g., sodium decanoate).
  • the term “lysis reaction mix” refers to a discrete physical entity that comprises one or more specified components described herein.
  • the lysis reaction mix comprises at least a sample.
  • the sample is preferably a biological sample comprising viral particles and/or cells.
  • the lysis reaction mix comprises a detergent (e.g, a zwitterionic detergent); an acid (e.g., HC1); a sample (e.g., a biological sample) and, optionally, a salt (e.g., sodium decanoate).
  • Neutralization refers to the stoppage of a reaction by addition of one or more neutralizing reagents.
  • a lysis reaction is neutralized by a neutralization buffer.
  • Nucleic acid refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain.
  • a nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage.
  • nucleic acid refers to an individual nucleic acid residue (e.g., a nucleotide and/or nucleoside); in some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising individual nucleic acid residues.
  • a "nucleic acid” is or comprises RNA; in some embodiments, a “nucleic acid” is or comprises DNA.
  • a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues.
  • a nucleic acid is, comprises, or consists of one or more nucleic acid analogs.
  • a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone.
  • a nucleic acid is, comprises, or consists of one or more "peptide nucleic acids", which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention.
  • a nucleic acid has one or more phosphorothioate and/or 5'-N-phosphoramidite linkages rather than phosphodiester bonds.
  • a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine).
  • adenosine thymidine, guanosine, cytidine
  • uridine deoxyadenosine
  • deoxythymidine deoxy guanosine
  • deoxycytidine deoxycytidine
  • a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5- fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5- methylcytidine, 2- aminoadenosine, 7-deazaad enosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)- methylguanine, 2-thiocytidine, methylated bases, intercal
  • a nucleic acid comprises one or more modified sugars (e.g., 2'- fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids.
  • a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein.
  • a nucleic acid includes one or more introns.
  • nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis.
  • a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long.
  • a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded.
  • a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a nucleic acid has enzymatic activity.
  • Polypeptide ' refers to any polymeric chain of amino acids.
  • a polypeptide has an amino acid sequence that occurs in nature.
  • a polypeptide has an amino acid sequence that does not occur in nature.
  • a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man.
  • a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both.
  • a polypeptide may comprise or consist of only natural amino acids or only nonnatural amino acids.
  • a polypeptide may comprise D- amino acids, L-amino acids, or both.
  • a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and/or may comprise a cyclic portion.
  • a polypeptide is not cyclic and/or does not comprise any cyclic portion.
  • a polypeptide is linear.
  • a polypeptide may be or comprise a stapled polypeptide.
  • the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides.
  • exemplary polypeptides within the class whose amino acid sequences and/or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family.
  • a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and/or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class).
  • a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%.
  • a conserved region that may in some embodiments be or comprise a characteristic sequence element
  • Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids.
  • a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide.
  • a useful polypeptide as may comprise or consist of a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and/or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide.
  • Protein refers to a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds). Proteins may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means.
  • Polypeptides may contain L-amino acids, Damino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc.
  • proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.
  • the term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids.
  • proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.
  • Reference' As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment.
  • sample typically refers to an aliquot of material obtained or derived from a source of interest, as described herein.
  • a source of interest is a biological or environmental source.
  • a source of interest may be or comprise a cell or an organism, such as a microbe, a plant, or an animal (e.g., a human).
  • a sample is or comprises viral particles (e.g., enveloped or non-enveloped viral particles).
  • a source of interest is or comprises biological tissue or fluid.
  • a biological tissue or fluid may be or comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secreations, vitreous humour, vomit, and/or combinations or component(s) thereof.
  • a biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and/or a transcellular fluid.
  • a biological fluid may be or comprise a plant exudate.
  • a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., brocheoalvealar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage).
  • a biological sample is or comprises cells obtained from an individual.
  • a sample is a “primary sample” obtained directly from a source of interest by any appropriate means.
  • the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane.
  • a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acid, isolation and/or purification of certain components, etc.
  • a sample may be a “crude” sample in that it has been subjected to relatively little processing and/or is complex in that it includes components of relatively varied chemical classes.
  • Specific binding refers to an ability to discriminate between possible binding partners in the environment in which binding is to occur.
  • a binding agent that interacts with one particular target when other potential targets are present is said to “bind specifically” to the target with which it interacts.
  • specific binding is assessed by detecting or determining degree of association between the binding agent and its partner; in some embodiments, specific binding is assessed by detecting or determining degree of dissociation of a binding agentpartner complex; in some embodiments, specific binding is assessed by detecting or determining ability of the binding agent to compete an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detections or determinations across a range of concentrations.
  • Specificity is a measure of the ability of a particular ligand to distinguish its binding partner from other potential binding partners.
  • Subject refers to an organism, for example, a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, a dog).
  • a human subject is an adult, adolescent, or pediatric subject.
  • a subject is suffering from a disease, disorder or condition, e.g., a disease, disorder or condition that can be treated as provided herein, e.g., a cancer or a tumor listed herein.
  • a subject is susceptible to a disease, disorder, or condition; in some embodiments, a susceptible subject is predisposed to and/or shows an increased risk (as compared to the average risk observed in a reference subject or population) of developing the disease, disorder or condition.
  • a subject displays one or more symptoms of a disease, disorder or condition.
  • a subject does not display a particular symptom (e.g., clinical manifestation of disease) or characteristic of a disease, disorder, or condition.
  • a subject does not display any symptom or characteristic of a disease, disorder, or condition.
  • a subject is a patient.
  • a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
  • the present disclosure provides technologies (e.g., compositions, mixtures, methods, and/or systems, etc.) for preparation of lysates.
  • the present disclosure provides technologies (e.g., compositions, mixtures, methods, and/or systems, etc.) for preparation of lysates from samples as described herein.
  • the present disclosure provides technologies (e.g., compositions, mixtures, methods, and/or systems, etc.) for preparation of lysates cells (e.g., eukaryotic, prokaryotic, etc.).
  • the present disclosure provides technologies (e.g., compositions, mixtures, methods, and/or systems, etc.) for preparation of lysates of infectious agents (e.g., microbes).
  • the present disclosure provides technologies (e.g., compositions, mixtures, methods, and/or systems, etc.) for preparation of lysates of viral particles (e.g., enveloped viruses, non-enveloped viruses).
  • the present disclosure provides technologies (e.g., compositions, mixtures, methods, and/or systems, etc) for preparation of cell lysates (e.g., particularly of cells comprising nucleic acids of interest - e.g., target nucleic acids).
  • the present disclosure provides lysis reagent compositions (e.g., comprising one or more detergents), as well as lysis reaction mixtures (e.g., comprising the lysis reagent and cells), lysed compositions (i.e., comprising cell lysates), and/or methods for preparing and/or for using any or all of the foregoing.
  • the present disclosure provides a surprising insight that levels of cell and/ or viral particle lysis comparable to those achieved by heat treatment (e.g., 20 minutes for temperatures above 60 °C, or 5 minutes for temperatures above 65°C, such as temperatures of or above 95 °C) can be achieved at ambient temperature through use of appropriate reagents.
  • provided lysis reagent compositions achieve lysis at ambient conditions to a level or degree that is not significantly increased by exposure of the relevant system(s) to heat treatment.
  • one or more reagents utilized in accordance with the present disclosure may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g., in concentrate or lyophilized form).
  • the lysis reagents composition comprises one or more reagents, detergents, surfactants, buffers, or salts as provided in table 1.
  • a lysis reagent composition comprises one or more components such as, for example, detergents, buffers, acids, and salts.
  • a lysis reagent composition may be or comprise a detergent, such as one or more detergent(s) or combinations of detergents.
  • Exemplary zwitterionic detergents for use in accordance with the present disclosure may be or comprise, for example, 3-Dodecylamido-N,N'-Dimethylpropyl Amine Oxide also known as (aka) 3-Laurylamido-N,N' -Dimethylpropyl Amine Oxide or LAP AO, Lauryl dimethylamine-N-Oxide aka LDAO, N,N-Dimethyl-1-Dodecanamine-N-Oxide or DDAO, 3-[(3-Cholamidopropyl)dimethylammonio]-l-propanesulfonate aka CHAPS, 3-([3- Cholamidopropyl]dimethylammonio)-2 -hydroxy-1 -propanesulfonate aka CHAPSO.
  • 3-Dodecylamido-N,N'-Dimethylpropyl Amine Oxide also known as (aka) 3-Laurylamido-N,N' -Dimethylpropy
  • a lysis reagent composition is or comprises a zwitterionic detergent and HC1.
  • a zwitterionic detergent is selected from the group consisting of LAP AO, LDAO, and DDAO.
  • a zwitterionic detergent is present in a lysis reagent composition at a concentration within a range of about 0.001% to about 10%, such as between about 0.01% and about 9%, such as between about 0.05% and about 8%, such as between about 0.1% and about 5%, such as between about 0.5% and about 4%, such as between about 1% and about 3%, such as between about 1.5% and about 2.5%, such as between about 1.75% and about 2.25%.
  • a zwitterionic detergent may be provided and/or utilized in a stock solution at a concentration that is higher (and typically by a multiple or other amount appropriate for use by dilution into a reaction) than that at which it is present in a lysis reagent composition and/or in a lysis reaction as described herein.
  • a percentage of zwitterionic detergent in a lysis reagent composition utilized in accordance with the present disclosure is not more than about 30%, not more than about 20%, not more than about 10%, not more than about 5%, not more than about 4%, not more than about 3%, not more than about 2%, not more than about 1%, etc
  • lysis reagent compositions according to the invention are or comprise LAP AO.
  • a zwitterionic detergent used herein is LAP AO.
  • LAP AO has the molecular formula CI 7 H 36 N 2 O 2 and the molecular structure as set forth herein below:
  • a lysis reagent composition is or comprises LAP AO in a concentration suitable for achieving cellular lysis.
  • a concentration range of LAP AO as utilized herein is within 0.001% and 10%, such as 0.01% and 9%, such as 0.05% and 8%, such as 0.1% and 5%, such as 0.5% and 4%, such as 1% and 3%, such as 1.5% and 2.5%, such as 1.75% and 2.25%.
  • a percentage of LAP AO utilized in accordance with the present disclosure is at least 0.001%, such as at least 0.01%, such as at least 0.05%, such as at least 0.1%, such as at least 0.5%, such as at least 1%, such as at least 1.25%, such as at least 1.5%, such as at least 1.75%, such as at least 2%, such as at least 2.25%, such as at least 2.5%, such as at least 3%, such as at least 3.5%, such as at least 4.5%, such as at least 5%, such as at least 5.5%, such as at least 6%, such as at least 6.5%, such as at least 7%, such as at least 7.5%, such as at least 8%, such as at least 8.5%, such as at least 9%, such as at least 9.5%, such as at least 10%.
  • a percentage of LAP AO utilized in accordance with the present disclosure is not more than about 30%, not more than about 20%, not more than about 10%, not more than about 5%, not more than about 4%, not more than about 3%, not more than about 2%, not more than about 1%, etc..
  • a lysis reagent composition comprises LAP AO and one or more reagents, such as buffers, acids, and salts.
  • provided lysis reagent compositions are or comprise LDAO.
  • the zwitterionic detergent used herein is LDAO.
  • LDAO has the molecular formula C14H31NO and the molecular structure as shown herein below:
  • a lysis reagent composition is or comprises LDAO in a concentration suitable for achieving cellular lysis.
  • LDAO is utilized in accordance with the present disclosure (e.g., in a lysis reagent composition, a lysis reaction, and/or a stock composition) at a concentration within a range of about 0.001% to aboutlO%, such as between about 0.01% and about 9%, such as between about 0.05% and about 8%, such as between about 0.1% and about 5%, such as between about 0.5% and about 4%, such as between about 1% and about 3%, such as between about 1.5% and about 2.5%, such as between about 1.75% and 2.25%.
  • LDAO is utilized in accordance with the present disclosure at a concentration of at least 0.001%, such as at least 0.01%, such as at least 0.05%, such as at least 0.1%, such as at least 0.5%, such as at least 1%, such as at least 1.25%, such as at least 1.5%, such as at least 1.75%, such as at least 2%, such as at least 2.25%, such as at least 2.5%, such as at least 3%, such as at least 3.5%, such as at least 4.5%, such as at least 5%, such as at least 5.5%, such as at least 6%, such as at least 6.5%, such as at least 7%, such as at least 7.5%, such as at least 8%, such as at least 8.5%, such as at least 9%, such as at least 9.5%, such as at least 10%.
  • at least 0.001% such as at least 0.01%, such as at least 0.05%, such as at least 0.1%, such as at least 0.5%, such as at least 1%, such as at least 1.25%,
  • LDAO is utilized in accordance with the present disclosure (and particularly as utilized in a lysis reagent composition and/or a lysis reaction) at a concentration that is not more than about 30%, not more than about 20%, not more than about 10%, not more than about 5%, not more than about 4%, not more than about 3%, not more than about 2%, not more than about 1%, not more than 0.5%, not more than 0.05%, not more than 0.005%, etc.
  • a lysis reagent composition comprises LDAO and one or more reagents, such as buffers, acids, and salts.
  • the lysis reaction composition sodium decanoate, Capric acid sodium salt, aka Decanoic acid sodium salt, Sodium caprate, or NaClO.
  • NaClO has the molecular formula CH3(CH2)sCOONa and the molecular structure as shown herein below:
  • a lysis reagent composition comprising a zwitterionic detergent further comprises sodium decanoate.
  • a lysis reagent composition comprising LAP AO further comprises sodium decanoate.
  • a lysis reagent composition comprising LDAO further comprises sodium decanoate.
  • such lysis reaction compositions also comprise HC1.
  • NaClO is utilized in accordance with the present disclosure (e.g., in a lysis reagent composition, a lysis reaction and/or a stock solution) in an about within a range of about 0.02% and about 20%, such as between about 0.1% and about 10%, such as between about 0.5% and about 5%, such as between about 1% and about 2%.
  • a lysis reagent composition or component thereof, a detergent (or component thereof), a buffer (or component thereof), and/or a combination thereof is provided and/or utilized as a stock preparation (z.e., in which it is present at a concentration higher than that, and typically a multiple of that, at which it is used during the lysis reaction).
  • a lysis reagent composition is prepared and/or utilized as a stock preparation.
  • one or more components of a lysis reagent composition is prepared and/or utilized as a stock preparation.
  • a stock preparation includes each of its components (e.g., buffer, detergent, salt, acid and/or a combination of some or all of the foregoing) at a concentration that is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 or more times its working concentration in a lysis reaction.
  • components e.g., buffer, detergent, salt, acid and/or a combination of some or all of the foregoing
  • a lysis reagent composition or component thereof and/or a detergent and/or one or more other components of a lysis reaction are each combined with (e.g., added to) a sample (e.g., sequentially or simultaneously, e.g., by prior combination).
  • a lysis reagent composition is prepared by combining one or more components, or all components, with one another before combining the lysis reagent composition with a sample; alternatively, in some embodiments, one or more components of a lysis reagent composition is combined sequentially or simultaneously with a sample. pH
  • a lysis reagent composition as described herein e.g., comprising a zwitterionic detergent such as, for example, LAP AO
  • a lysis reaction has a pH between 0 and 7.
  • such a composition includes HC1.
  • a lysis reagent composition and/or a lysis reaction has a pH between 0 and 6, or between 0 and 5, or between 0 and 4 or between 0 and 3 or between 0 and 2 or between 0 and 1.
  • a relevant pH is at the most 3, such as at the most 2, such as at the most 1.5, such as at the most 1, such as at the most 0.8, such as at the most 0.6, such as at the most 0.4, such as at the most 0.2.
  • pH of a lysis reagent composition, and/or a lysis reaction, comprising a zwitterionic detergent (e.g., LDAO), NaClO, and HC1 is between 0 and 7 or between 0 and 6 or between 0 and 5 or between 0 and 4 or between 0 and 3 or between 0 and 2 or between 0 and 1.
  • pH of a lysis reagent composition, and/or a lysis reaction, comprising a zwitterionic detergent, NaClO, and HC1 is at the most 3, such as at the most 2, such as at the most 1.5, such as at the most 1, such as at the most 0.8, such as at the most 0.6, such as at the most 0.4, such as at the most 0.2.
  • viral particle and “virus” are used interchangeable herein.
  • Viruses are small intracellular parasites, which contain either a RNA or DNA genome.
  • the viral genome often with associated basic proteins, is packaged inside a symmetric protein capsid.
  • a nucleocapsid is the capsid and the nucleic acid of the virus (e.g., a non-enveloped virus).
  • a viral particle comprises a nucleocapsid.
  • a viral particle is a non-enveloped viral particle.
  • the nucleocapsid is surrounded by a lipid bilayer (e.g., derived from a modified host cell membrane and often studded with an outer layer of virus envelope glycoproteins).
  • a viral particle is an enveloped viral particle.
  • viral particle fragments comprises nucleotides (e.g., target nucleotides) surrounded by a viral capsid, but not a lipid bilayer.
  • viruses e.g. SARS- CoV; influenza; herpes, etc.
  • heat treatment includes, but is not limited to treating the virus at temperatures above 75 °C for 3 minutes for, at temperatures above 65 °C for 5 minutes, or at temperatures above 60 °C for 20 minutes.
  • the present disclosure provides exemplary compositions and methods for the preparation and/or detection of target nucleic acids in a sample.
  • the sample is or comprises a viral particle.
  • the sample is or comprises a viral particle.
  • a sample is an environmental sample.
  • a sample is a biological sample (e.g., a sample from an organism, and/or of a tissue or fluid thereof, e.g., comprising one or more cells or cell components and/or one or more viral particles).
  • a sample is a crude sample (e.g., a primary sample or a sample that has undergone minimal processing).
  • a primary sample is collected from a subject (e.g., a human or animal subject).
  • an animal subject may be a wild or undomesticated animal such as animals living in the sea or in a jungle.
  • an animal subject may be a domesticated animal, such as a farm animal or a pet.
  • an animal subject may be a cat, cow, dog, goat, horse, llama, pig, sheep, bird, etc.
  • an animal subject may be a rodent.
  • a subject may be a primate.
  • a subject may be a human.
  • a primary sample is obtained directly from a subject - e.g., from a fluid or tissue of the subject.
  • a sample is saliva, urine, perspiration, breath, blood, tissue, hair, skin, or any other excretion product or body fluid.
  • a sample is obtained from a subject by means of a swab, an aspirate, or a lavage.
  • a sample is obtained from a subject by means of a nasal swab, nasopharyngeal swab, oropharyngeal swab, nasal aspirate, sputum, bronchoalveolar lavage.
  • a biological sample is selected from nasal swab, nasopharyngeal swab, oropharyngeal swab, nasal aspirate, sputum, bronchoalveolar lavage, blood, serum, feces, and saliva samples.
  • a sample is or comprises a nasal swab.
  • a sample is or comprises saliva.
  • a swab used to collect a sample is placed promptly (e.g., immediately) into a sterile tube containing 2-3 ml of viral transport media (i.e. VTM, UTM, M4RT).
  • VTM viral transport media
  • a sample is a pooled sample - e.g., a sample obtained by combining samples from multiple sources (e.g., pooled saliva sample).
  • a sample (or a reaction in which such a sample is lysed) includes a spiked-in viral particles comprising nucleic acid target (e.g., which may be processed and/or detected as a “control”).
  • a spiked-in sample is generated by growing viral particles inside cells (e.g., mammalian cells). Cells are thereafter lysed, thereby leaving the viral particles intact in a matrix of cell lysate.
  • a sample is or comprises viral particles in a cell lysate matrix.
  • a sample is or comprises a viral particle thereof within a cell.
  • a sample (or reaction) into which a target nucleic acid (e.g., a control nucleic acid target) is spiked is a “mock” sample; in some such embodiments, the spiked-in nucleic acid is a target nucleic acid of interest, so that the mock sample acts as a positive control for detection of the target nucleic acid.
  • a target nucleic acid e.g., a control nucleic acid target
  • a sample (or reaction) into which a nucleic acid is spiked is a test sample, and the spiked-in nucleic acid is a control target (e.g., a target other than the target of interest) whose processing and/or detection acts as a control for successful nucleic acid processing or detection.
  • a control target e.g., a target other than the target of interest
  • a spiked-in target is or comprises an infectious agent (e.g., a bacterium or virus), such as an inactivated infectious agent.
  • an inactivated agent is or comprises a heat-treated agent, a chemically-treated agent, and/or a y-irradiated agent.
  • an inactivated agent is a y-irradiated virus.
  • a sample is prepared and/or utilized as a liquid sample.
  • a liquid sample is an aqueous sample.
  • a liquid sample is a buffered sample (e.g., includes a buffer system).
  • a liquid sample does not include a buffer.
  • a liquid sample is in water.
  • a sample may be processed by one or more steps prior to lysis as described herein.
  • a sample is a processed sample.
  • a sample is processed by addition of one or more reagents (e.g., to separate cells in a tissue), dilution, filtration, clarification, distillation, separation, isolation, freezing, drying, cryopreservation, etc).
  • a sample is processed by isolation of specific component(s) (e.g., viral particles and/or cells).
  • a sample is processed by a mechanical process (e.g., centrifugation).
  • the sample (e.g., comprising at least one viral particle or fragment thereof) is combined with a lysis reagent composition according to the present invention hereby generating a lysis reaction mix.
  • the present disclosure provides technologies in which a lysis reagent composition or component thereof, such as one or more detergents, acids, salts, and buffers is combined with e.g, added to) a sample (e.g., sequentially or simultaneously, e.g, by prior combination).
  • a lysis reagent composition or component thereof such as one or more detergents, acids, salts, and buffers is combined with e.g, added to) a sample (e.g., sequentially or simultaneously, e.g, by prior combination).
  • the present disclosure provides cell lysis reaction mixtures (e.g., eukaryotic cells, prokaryotic cells, etc.). In some embodiments, the present disclosure provides microbe lysis reaction mixtures. In some embodiments, the present disclosure provides viral particle lysis reaction mixtures (e.g., enveloped viruses, nonenveloped viruses).
  • lysis reaction mixtures comprise a lysis reagent composition and a sample as described herein.
  • lysis reaction mixtures comprise a detergent, an acid, a sample and optionally a salt.
  • a sample comprises cells or components thereof.
  • a lysis reagent composition comprising a zwitterionic detergent, and HC1 as provided herein is added to a sample.
  • the zwitterionic detergent is LAP AO.
  • the sample comprises cells.
  • the sample comprises cells comprising at least one target nucleic acid.
  • addition of the lysis reagent composition to the sample causes at least some of the cells in the sample to lyse and release their nucleic acid content into the sample.
  • a lysis reaction mix comprises LAP AO in a concentration suitable for achieving cellular lysis.
  • a concentration range of the LAP AO in the lysis reaction mix is within 0.001% and 10%, such as 0.01% and 1%, such as 0.05% and 0.5%, such as 0.075% and 0.2%, such as 0.09% and 0.11%. In some embodiments, these concentrations represent a dilution of a stock solution.
  • a percentage of the LAP AO in the lysis reaction mix is at least 0.001%, such as at least 0.005%, such as at least 0.01%, such as at least 0.02%, such as at least 0.03%, such as at least 0.04%, such as at least 0.05%, such as at least 0.06%, such as at least 0.07%, such as at least 0.08%, such as at least 0.09%, such as at least 0.1%.
  • a percentage of LAP AO in the lysis reaction mix is as at the most 3%, such as at the most 2.5%, such as at the most 2%, such as at the most 1.5%, such as at the most 1%, such as at the most 0.8%, such as at the most 0.5%, such as at the most 0.3%, such as at the most 0.2%, such as at the most 0.1%.
  • the pH of a lysis reaction mix comprising a zwitterionic detergent, and HC1 is at the most 3, such as at the most 2, such as at the most 1.5, such as at the most 1, such as at the most 0.8, such as at the most 0.6, such as at the most 0.4, such as at the most 0.2.
  • a lysis reagent composition comprising a zwitterionic detergent, NaClO, and HC1 as provided herein is added to a sample.
  • the zwitterionic detergent is LDAO.
  • a sample comprises cells.
  • a sample comprises cells comprising at least one target nucleic acid.
  • addition of a composition to a sample causes at least some of the cells in the sample to lyse and release their nucleic acid content into the sample.
  • a lysis reaction mix comprises LDAO in a concentration suitable for achieving cellular lysis.
  • a concentration range of LDAO in the lysis reaction mix is within 0.001% and 10%, such as 0.005% and 1%, such as 0.01% and 0.1%, such as 0.02% and 0.05%, such as 0.02% and 0.04%. In some embodiments, these concentrations represent a dilution of a stock solution.
  • a percentage of LDAO in a lysis reaction mix is at least 0.001%, such as at least 0.002%, such as at least 0.003%, such as at least 0.004%, such as at least 0.005%, such as at least 0.006%, such as at least 0.007%, such as at least 0.008%, such as at least 0.009%, such as at least 0.01%, such as at least 0.015%, such as at least 0.02%, such as at least 0.0225%, such as at least 0.025%, such as at least 0.05%, such as at least 0.1%.
  • a percentage of LDAO in the lysis reaction mix is at the most 2%, such as at the most 1.5%, such as at the most 1%, such as at the most 0.5%, such as at the most 0.4%, such as at the most 0.3%, such as at the most 0.2%, such as at the most 0.1%, such as at the most 0.075%, such as at the most 0.050%, such as at the most 0.0375%, such as at the most 0.03%.
  • a mixture of a lysis reagent composition (and optionally a detergent and/or buffer and/or one or more other components) and the sample is incubated at ambient temperature.
  • a lysis reaction mix comprising zwitterionic detergent is incubated at ambient temperature.
  • the lysis reaction mix comprising LAP AO is incubated at ambient temperature.
  • a lysis reaction mix comprising LDAO is incubated at ambient temperature.
  • a lysis reaction mix comprising zwitterionic detergent and a sample comprising a viral particle or fragment thereof is incubated at ambient temperature.
  • the ambient temperature is at the most 95°C, such as at the most 90°C, such as at the most 85°C, such as at the most 80°C, such as at the most 75°C, such as at the most 70°C, such as at the most 65°C, such as at the most 60°C, such as at the most 55°C, such as at the most 50°C, such as at the most 45°C, such as at the most 40°C, such as at the most 35°C, such as at the most 30°C, such as at the most 25°C, such as at the most 24°C, such as at the most 23 °C, such as at the most 22°C, such as at the most 21 °C, such as at the most 20°C.
  • Exemplary ambient temperature ranges include 10°C and 40°C, such as 15°C to 35°C, such as 20°C to 30°C, such as 20°C to 25°C, such as 20°C to 22°C.
  • a mixture of a lysis reagent composition and a sample is incubated for a predetermined period of time.
  • a lysis reaction mix comprising zwitterionic detergent is incubated for a predetermined period of time.
  • a lysis reaction mix comprising LAP AO is incubated for a predetermined period of time.
  • a lysis reaction mix comprising LDAO is incubated for a predetermined period of time.
  • Exemplary ranges for an incubation time include 0.1 second to 30 minutes, such as 1 second to 20 minutes, such as 2 seconds to 10 minutes, such as 5 seconds to 5 minutes, such as 10 seconds to 1 minute, such as 20 seconds to 40 seconds. pH
  • a pH range of a lysis reaction mix, and/or lysis reaction, comprising a zwitterionic detergent, and HC1 is within 0 and 7. In some embodiments, a pH range of a lysis reaction mix, and/or lysis reaction, comprising a zwitterionic detergent and HC1 is within 0 and 6. In some embodiments, a pH range of a lysis reaction mix, and/or lysis reaction, comprising a zwitterionic detergent, and HC1 is within 0 and 5. In some embodiments, a pH range of a lysis reaction mix, and/or lysis reaction, comprising a zwitterionic detergent, and HC1 is within 0 and 4.
  • a pH range of a lysis reaction mix, and/or lysis reaction, comprising a zwitterionic detergent, and HC1 is within 0 and 3. In some embodiments, a pH range of a lysis reaction mix comprising a zwitterionic detergent, and HC1 is within 0 and 2. In some embodiments, a pH range of a lysis reaction mix comprising a zwitterionic detergent, and HC1 is within 0 and 1. In some embodiments, a pH range of a lysis reaction mix comprising a zwitterionic detergent, and HC1 is within 1 and 5. In some embodiments, a pH range of a lysis reaction mix comprising a zwitterionic detergent, and HC1 is within 2 and 4. In some embodiments, a pH range of a lysis reaction mix comprising a zwitterionic detergent, and HC1 is within 2 and 3.
  • a pH of a lysis reaction mix comprising a zwitterionic detergent, and HC1 is at the most 6, such as at the most 5, such as at the most 4, such as at the most 3, such as at the most 2.5.
  • a pH range of a lysis reaction mix comprising a LAP AO, and HC1 is within 0 and 7. In some embodiments, a pH range of a lysis reaction mix comprising a LAP AO and HC1 is within 0 and 6. In some embodiments, a pH range of a lysis reaction mix comprising a LAP AO, and HC1 is within 0 and 5. In some embodiments, a pH range of a lysis reaction mix comprising a LAP AO, and HC1 is within 0 and 4. In some embodiments, a pH range of a lysis reaction mix comprising a LAP AO, and HC1 is within 0 and 3.
  • a pH range of a lysis reaction mix comprising a LAP AO, and HC1 is within 0 and 2. In some embodiments, a pH range of a lysis reaction mix comprising a LAP AO, and HC1 is within 0 and 1. In some embodiments, a pH range of a lysis reaction mix comprising a LAP AO, and HC1 is within 1 and 5. In some embodiments, a pH range of a lysis reaction mix comprising a LAP AO, and HC1 is within 2 and 4. In some embodiments, a pH range of a lysis reaction mix comprising a LAP AO, and HC1 is within 2 and 3.
  • a pH of a lysis reaction mix comprising a LAPO, and HC1 is at the most 6, such as at the most 5, such as at the most 4, such as at the most 3, such as at the most 2.5.
  • a pH range of a lysis reaction mix comprising a LDAO, NaClO and HC1 is within 0 and 7. In some embodiments, the pH range of a lysis reaction mix comprising a LDAO, NaClO and HC1 is within 0 and 6. In some embodiments, a pH range of a lysis reaction mix comprising a LDAO, NaClO, and HC1 is within 0 and 5. In some embodiments, the pH range of a lysis reaction mix comprising a LDAO, NaClO, NaClO, and HC1 is within 0 and 4.
  • a pH range of a lysis reaction mix comprising a LDAO, NaClO, and HC1 is within 0 and 3. In some embodiments, a pH range of a lysis reaction mix comprising a LDAO, NaClO, and HC1 is within 0 and 2. In some embodiments, a pH range of a lysis reaction mix comprising a LDAO, NaClO, and HC1 is within 0 and 1. In some embodiments, a pH range of a lysis reaction mix comprising a LDAO, NaClO, and HC1 is within 1 and 5.
  • a pH range of a lysis reaction mix comprising a LDAO, NaClO, and HC1 is within 2 and 4. In some embodiments, a pH range of a lysis reaction mix comprising a LDAO, NaClO, and HC1 is within 2 and 3.
  • a pH of a lysis reaction mix comprising a LAPO, NaClO, and HC1 is at the most 6, such as at the most 5, such as at the most 4, such as at the most 3, such as at the most 2.5.
  • a lysis reaction is performed in the same buffer (or absence of buffer) in which the sample was prepared.
  • a different buffer or absence of buffer is utilized, for example, by dialyzing, diluting, resuspending, or otherwise adjusting a prepared sample.
  • lysis reactions can be performed in the presence of one or more buffers and, in some embodiments, lysis reactions can be performed in the absence of a buffer.
  • teachings of the present disclosure are not limited to a particular buffer system.
  • a buffer utilized in a lysis reaction may be or comprise, for example a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof.
  • the buffer is acidic.
  • a buffer has a pH from about 0 to about 7.
  • a lysis reaction is performed in a liquid system (e.g., in an aqueous system). In some embodiments, a lysis reaction is performed in a buffered system. In some embodiments, a lysis reaction is performed in a system that is not buffered. In some embodiments, a lysis reaction is performed in water.
  • a lysis reaction as described herein may be stopped, e.g., by neutralization.
  • stoppage is achieved by addition of one or more neutralizing reagents.
  • neutralizing reagents may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g., in concentrate or lyophilized form).
  • exemplary neutralizing reagents comprise buffers, and bases.
  • a buffer is not limited to a particular buffer; in some embodiments, a buffer can be any buffer, including but not limited to a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES, NaOH, acetate buffer, PBS, and combinations thereof.
  • a buffer is alkaline.
  • a buffer has a pH from about 7 to about 11.
  • a lysis reaction is neutralized by the neutralization buffer.
  • a pH of the buffer is such that upon addition to reaction, the pH of the buffer and reaction mixture is within the rage of pH 7 and 9, such as within the range of pH 8 to 8.5.
  • an exemplary buffer used herein is a Tris buffer.
  • a Tris buffer is provided as a concentrate, i.e. stock solution.
  • Exemplary concentrations of the Tris buffer comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 times to working concentration of the buffer.
  • the concentration of the Tris buffer concentrate is within the range of 1 mM and 2M.
  • the concentration of the Tris buffer concentrate is within the range of 10 mM and IM, such as 25 mM and 500 mM, such as 50 mM and 400 mM, such as 75 mM and 300 mM, such as 100 mM to 200 mM.
  • a pH of the Tris buffer concentrate is within the range of 7 and 11, such as within the range of 7 and 10, such as within the range of 8 and 11, such as within the range of 8 and 10, such as within the range of 7 and 9.
  • a Tris buffer is added to an acidic reaction mix. In some embodiments, a Tris buffer is added to a reaction mix comprising viral particles, and/or a cell lysate comprising viral particles. In some embodiments, addition of Tris buffer to the reaction mix stops the reaction (e.g., cell lysis reaction).
  • working concentration ranges of the Tris buffer when added to the reaction mix are within 1 mM and 2M.
  • the concentration of the Tris buffer concentrate is within the range of 10 mM and IM, such as 25 mM and 500 mM, such as 50 mM and 400 mM, such as 75 mM and 300 mM, such as 100 mM to 200 mM.
  • working pH ranges of the composition resulting from the addition of the Tris buffer to the reaction mix are about 7 to about 9, such as within the range of 7 and 10, such as within the range of 8 and 11, such as within the range of 8 and 10, such as within the range of 7 and 9.
  • a composition described herein comprising a Tris buffer and a reaction mix is incubated at ambient temperature.
  • ambient temperature ranges include 10°C to 40°C, such as 15°C to 35°C, such as 20°C to 25°C, such as 15°C to 30°C or 15°Cto 25°C.
  • a composition described herein comprising a Tris buffer and a reaction mix is incubated for a predetermined period of time.
  • Exemplary ranges for an incubation time include 0.1 second to 35 minutes, such as 1 second to 20 minutes, such as 2 seconds to 10 minutes, such as 5 seconds to 5 minutes, such as 10 seconds to 1 minute, such as 20 seconds to 40 seconds.
  • nucleic acids prepared according to the present invention are not processed further and/or not used for any downstream reaction. In some embodiments, nucleic acids prepared according to the present invention are processed further and/or are used in downstream reactions.
  • no further nucleic acid isolation is performed prior to analysis of the sample, e.g., to detect nucleic acid(s) (e.g., target nucleic acid(s)) therein.
  • a sample comprising released nucleic acid is serially diluted after viral particle lysis and prior to analysis of the sample, e.g., to detect nucleic acid(s) (e.g., target nucleic acid(s)) therein.
  • a sample comprising released nucleic acid is stored. The sample with the prepared nucleic acids comprising the target nucleic acids may be stored in a freezer or a refrigerator.
  • a downstream reaction e.g., a nucleic acid processing and/or detection reaction
  • a downstream reaction is or comprises one or more of amplification, cleavage, digestion, hybridization, replication, etc.
  • nucleic acid processing and/or detection reaction is or comprises one or more of SHERLOCK, INSPECTRTM’ DETECTR, etc.
  • compositions, mixtures, methods and kits according to the present disclosure can be used in a number of applications, such as therapeutic and/or non-therapeutic applications.
  • methods of preparing nucleic acids from a sample and detecting a target nucleic acid in a sample are useful in therapeutic and/or non-therapeutic applications.
  • compositions, mixtures, methods, kits and uses described herein may be used for non-therapeutic purposes, such as diagnostic purposes.
  • compositions, mixtures, methods, kits and uses described herein may be used for therapeutic purposes.
  • the target is an infectious agent.
  • the present invention provides technologies (e.g., compositions, mixtures, methods, and/or kits, etc) for preparation of cell lysates comprising nucleic acids of interest - e.g., target nucleic acids.
  • the present invention provides technologies (e.g., compositions, mixtures, methods, and/or kits, etc) for preparation of viral particle lysates comprising nucleic acids of interest - e.g., target nucleic acids.
  • the disclosure provides a method of preparing nucleic acids from a sample, the method comprising: i) obtaining the sample comprising the nucleic acids from a subject; ii) contacting the sample with a composition comprising: a) LAP AO; and b) HC1; and iii) incubating the mixture of the sample and the composition of step ii), wherein the step of incubating comprises incubating at a temperature within the range of 10°C and 40°C.
  • the concentration of LAP AO is within the range of 0.01% and 10%, wherein the concentration of the HC1 is within the range of 4 mM and 4M, and wherein the pH of the composition is within the range of 0 and 6
  • the disclosure provides a method of preparing nucleic acids from a sample, the method comprising: i) obtaining the sample comprising the nucleic acids from a subject; ii) contacting the sample with a buffered preparation comprising: a) LDAO; b) sodium decanoate; and c) HC1; and iii) incubating the mixture of the sample and the buffered preparation of step ii), wherein the step of incubating comprises incubating at a temperature within the range of 10°C and 40°C.
  • the concentration of LDAO is within the range 0.02% and 4%, wherein the concentration of the sodium decanoate is within the range of 0.02% and 20%, wherein the concentration of the HC1 is within the range of 4 mM and 4M, and wherein the pH of the composition is within the range of 0 and 6
  • a sample may be any sample, such as an example described under the section “sample” and/or may be obtained by any suitable means.
  • a nucleic acid preparation developed by treatment of a sample with a lysis reagent composition as described herein is subjected to one or more downstream processing and/or detection processes, for example to achieve: detection of a target (e.g., a target nucleic acid sequence) present in the sample.
  • detection may accomplish or represent diagnosis, e.g., of a disease, disorder, condition (or stage thereof), or state (e.g., of presence of an infectious agent, or of a mutation or modification event or state, or developmental event or state).
  • provided technologies are useful for assessment of an environmental sample, such as a food sample (fresh fruits or vegetables, meats), a beverage sample, a paper surface, a fabric surface, a metal surface, a wood surface, a plastic surface, a soil sample, a freshwater sample, a wastewater sample, a saline water sample, exposure to atmospheric air or other gas sample, or a combination thereof.
  • an environmental sample such as a food sample (fresh fruits or vegetables, meats), a beverage sample, a paper surface, a fabric surface, a metal surface, a wood surface, a plastic surface, a soil sample, a freshwater sample, a wastewater sample, a saline water sample, exposure to atmospheric air or other gas sample, or a combination thereof.
  • household/commercial/industrial surfaces made of any materials including, but not limited to, metal, wood, plastic, rubber, or the like, may be swabbed and tested for contaminants.
  • soil samples may be tested for the presence of viral particles or fragments thereof, pathogenic bacteria or parasites, or other microbes, both for environmental purposes and/or for human, animal, or plant disease testing.
  • Water samples such as freshwater samples, wastewater samples, or saline water samples can be evaluated for cleanliness and safety, and/or potability, for example to detect the presence of, for example, viral particles, and/or Cryptosporidium parvum, Giardia lamblia, and/or other microbial contamination.
  • an environmental sample be a crude sample and/or the one or more target molecules may not be purified or amplified from the sample prior to application of provided technologies.
  • Identification of microbes may be useful and/or needed for any number of applications, and thus any type of sample from any source deemed appropriate by one of skill in the art may be used in accordance with the invention.
  • the technologies of the present inventions are useful in genotyping.
  • kit for performing the present methods may comprise a composition or a component thereof as described herein.
  • kits for performing methods of preparing nucleic acids from a sample.
  • a kit of parts comprises a lysis reagent composition according to the present invention, and/or one or more components thereof.
  • a kit of parts may comprise a neutralization buffer, a sample collection device, and/or instructions for use.
  • one or more components included in a provided kit is a stock solution as described herein.
  • the kit of parts comprises a positive control.
  • a positive control includes an inactivated envelope virus, optionally at a known concentration or a control nucleic acid, optionally at a known concentration.
  • kits of parts comprises a lysis reagent composition according to the present invention.
  • a kit of parts may also comprise a neutralization buffer, a nucleic acid sensor system, a sample collection device, and/or instructions for use.
  • a kit of parts may comprise a nucleic acid sensor system useful for detecting a target nucleic acid.
  • nucleic acid sensor systems such as an INSPECTRTM nucleic acid detection system, SHERLOCK nucleic acid detection system, etc.
  • a kit of parts also comprises a control nucleic acid, such as may be spiked into a sample as described herein.
  • the present Example illustrates effective viral particle lysis achieved at ambient temperature through use of technologies provided herein.
  • Figure 1 depicts exemplary lysis and nucleic acid processing steps performed in accordance with the present disclosure.
  • a “mock sample” (containing pooled human saliva plus an inactivated intact virus in a matrix of cell lysate) was first treated with a “lysis reagent composition” and then, without traditional processing or “clean-up” steps, it was treated with a “ligation solution”.
  • the cell lysate (matrix) is treated as background.
  • the mock sample comprises pooled saliva, BEI y-irradiated SARS-CoV-2 virus, RNAsin, and EDTA.
  • the starting pH of the pooled saliva was 8.4.
  • the BEI y- irradiated virus concentration in the sample was 10,000 cp/ul.
  • the RNAsin concentration in the sample was 1 U/pl (N2511, Promega).
  • the lysis reagent composition was added to the mock sample and the combination was incubated at ambient temperature, which in this case was 22°C, for a short period of time (specifically, 30 seconds in the reactions depicted in Figure 1).
  • the lysis reaction was stopped by adding a neutralization buffer (20x stock solution is 100 mM Tris, pH 9.0).
  • the lysis reaction was performed under different pH conditions, including pH 2.5, pH 4.5, pH 6, pH 6.5, pH 7.5, pH 8 and, pH 10. To achieve the desired pH, the reaction was adjusted with HC1.
  • Viral particle lysis/nucleic acid preparations were assessed by measuring Ct value of the samples. Poor to no viral particle lysis was indicated by high Ct values, while optimum viral particle lysis was indicated by lower Ct values.
  • No lysis comprises the mock sample (pooled saliva, BEI y-irradiated SARS- CoV-2 virus, RNAsin, and EDTA) without addition of the lysis reagent composition, including any detergents and HC1.
  • the samples were maintained at ambient temperature.
  • “No lysis” control samples were neither exposed to detergent(s), HC1 nor heat;
  • HC1 comprises the mock sample (pooled saliva, BEI y-irradiated SARS-CoV-2 virus, RNAsin, and EDTA) without addition of any detergents but it was exposed to HC1. The samples were maintained at ambient temperature. Thus, the “HQ” control samples were neither exposed to detergent(s) nor heat.
  • NTC No template control
  • a lysis reagent composition as described herein achieved effective lysis comparable to that achieved with the positive control under heating conditions, at least at pH conditions below about 6.0, and particularly below about 4.5.
  • Figure 2 shows results observed when lysis reactions as were performed under different pH conditions. In particular, Figure 2 shows that lower Ct values were achieved with lower pH values.
  • the present Example documents particular effectiveness (i.e., better viral particle lysis) of provided lysis reactions at low pH. Greatest levels of lysis were observed at lowest pH (2.5) tested. However, the limit may not have been reached in these studies.
  • a lysis reagent composition such as a zwitterionic detergent (e.g., LAP AO or LDAO) can achieve effective viral particle lysis in a biological sample (e.g., a crude sample, and in particular a saliva sample) at low pH (e.g., below 6.5, and preferably below 4.5 including specifically below about pH 3.0), even at ambient (e.g., about 22°C) temperatures.
  • a biological sample e.g., a crude sample, and in particular a saliva sample
  • low pH e.g., below 6.5, and preferably below 4.5 including specifically below about pH 3.0
  • ambient e.g., about 22°C
  • the achieved lysis can be comparable to heat lysis (e.g., that observed with incubated at 95°C for 5 min).
  • the present Example surprisingly demonstrates that provided effective lysis conditions can be rapidly and gently neutralized by simple addition of Tris, to generate a lysed composition amenable to further nucleic acid processing or manipulation.
  • the present disclosure provides an insight that use of certain zwitterionic detergents (e.g., LAP AO and LDAO) as described herein can achieve a “Goldilocks” effect of sufficient lysis without undesirable inhibition of downstream processing or reaction steps.
  • nucleic acids prepared by lysis as described herein does not require purification or extraction steps typically required or utilized to remove detergents. Rather, enzymes or other agents (c.g, ligase as depicted in Figure 1, alternatively or additionally, one or more cleavage systems such as CRISPR/Cas, TALEN, Zinc Finger, Restriction Enzyme, etc., and/or one or more hybridization reagents such as oligonucleotides - e.g., probes or primers, etc.) can be added directly to the lysis reaction (e.g, simultaneously or sequentially with any neutralization buffer).
  • enzymes or other agents c.g, ligase as depicted in Figure 1, alternatively or additionally, one or more cleavage systems such as CRISPR/Cas, TALEN, Zinc Finger, Restriction Enzyme, etc., and/or one or more hybridization reagents such as oligonucleotides - e.g., probes or
  • the present disclosure provides lysis technologies that are remarkably compatible with nucleic acid processing and/among other things, in some embodiments may permit so-called “one pot” assessments.
  • Example 2 Reproducibility of viral particle lysis and impact of detergent concentration
  • the present Example documents reproducibility of viral particle lysis using zwitterionic detergent lysis as described herein.
  • the present Example further documents effect(s) of detergent (in this case LAP AO) concentration on degree of lysis.
  • Figures 3 and 4 document reproducibility of viral particle lysis achieved by provided technologies, across the four replicates and across detergent concentration.
  • Figure 3 furthermore demonstrates that LAP AO detergent concentrations between about 0.03% and about 0.3% (e.g., specifically between 0.0375% and 0.3%, inclusive) achieved better lysis than the lower LAP AO detergent concentrations ((e.g., 0.018% and below).
  • Figure 3 also documents that the combination of a detergent, such as LAP AO detergent concentrations between about 0.03% and about 0.3% (e.g., specifically between 0.0375% and 0.3%, inclusive), and HC1 (i.e., pH) produces viral particle lysis that is more effective than HC1 alone.
  • a detergent such as LAP AO detergent concentrations between about 0.03% and about 0.3% (e.g., specifically between 0.0375% and 0.3%, inclusive)
  • HC1 i.e., pH
  • the present Example documents surprising versatility of provided lysis technologies, including that they can achieve effective lysis in a context other than saliva, and specifically in water.
  • Example 3 is performed using the same conditions as for Examples 1 and 2, i.e. wherein the lysis is performed at 95°C for the heat lysis and 30 seconds exposure to lysis reagent at ambient temperature, in this case 22°C.
  • the present Example demonstrates that presence of saliva is not required to achieve lysis and, moreover, demonstrates effectiveness of provided technologies even in water or other aqueous systems.
  • Figure 5 presents results achieved when different concentrations of zwitterionic detergents, as indicated, were utilized to lyse nasal swabs eluted in water.
  • both LAP AO and LDAO/NaClO achieved significant lysis, including lysis comparable to or better than that achieved with heat lysis, at various concentrations. Lysis was measured at pH 2.4.
  • a lysis reagent composition such as a zwitterionic detergent (e.g., LAP AO or LDAO) can achieve effective viral particle lysis in a water sample.
  • a zwitterionic detergent e.g., LAP AO or LDAO
  • Example 4 Compatibility of provided lysis technologies with downstream nucleic acid processing and/or analysis
  • the present Example documents one particularly surprising and useful feature of technologies provided herein - that they achieve preparation of nucleic acids (e.g., by release from lysed viral particles) under conditions that are amenable so downstream nucleic acid processing and/or analysis, for example without extensive isolation and/or “cleaning” steps.
  • Figure 6 documents end-to-end compatibility of provided technologies with an INSPECTRTM nucleic acid detection system.
  • a method as described herein can be used to prepare nucleic acids for use in detection of a target nucleic acid, e.g., with an INSPECTRTM nucleic acid detection system as described in applications W02020037038 and PCT/US2021/046438, each of which are incorporated herein by reference.
  • nucleic acids prepared using the indicated lysis conditions i.e., no detergent, 0.025% LDAO/ 0.06% NaClO, 0.0375% LAPAO-HC1, or 0.3% LAPAO-HC1
  • concentrations of the nucleic acids prepared using the indicated lysis conditions i.e., no detergent, 0.025% LDAO/ 0.06% NaClO, 0.0375% LAPAO-HC1, or 0.3% LAPAO-HC1
  • LDAO/NaClO-HCl 0.0375% LAPAO-HC1, and 0.3% LAPAO-HC1 shown, the target nucleic acid (RNA) was detected, and the polypeptide or reporter protein generated was detected at an intensity comparable to that of the “no detergent” condition.
  • the provided technologies achieved target detection with remarkable sensitivity. This finding confirmed that provided lysis technologies did not impact the sensitivity of the target detection. Furthermore, the intensity of the NTC bands did not increase as compared to the “no detergent” condition, indicating that provided lysis technologies generated a nucleic acid preparation in which target sequence(s) are detected with high specificity.
  • a provided nucleic acid preparation does not undergo further processing technologies before nucleic acid processing and/or analysis is performed.
  • a provided nucleic acid preparation is amenable to direct nucleic acid processing and/or analysis with one or more of a variety of technologies (e.g., analytic technologies such as detection technologies).
  • technologies e.g., analytic technologies such as detection technologies.
  • reagents useful or required to perform step(s) of such nucleic acid processing and/or detection technologies can be added directly to a viral particle lysis reaction (e.g., simultaneously or sequentially with any neutralization buffer) described herein.
  • the reagents can be added directly to the same reaction vessel, so that the present disclosure provides technologies for so-called “one pot” assessments (e.g., one-pot lysis and nucleic acid assessment - e.g., processing and/or detection).
  • a nucleic acid assessment involves and/or utilizes one or more of amplification, cleavage, digestion, hybridization, ligation, replication, reverse transcription, etc., and/or one or more of, for example a detection system, such as, but not limited to SHERLOCK, INSPECTRTM, a combinations thereof, etc.
  • a detection system such as, but not limited to SHERLOCK, INSPECTRTM, a combinations thereof, etc.
  • Ligation In some embodiments, methods as described herein can be used to prepare nucleic acids for use in e.g., ligation.
  • reagents for nucleic acid ligation may comprise one or more ligases, buffers, and probes; in some embodiments one or more of such reagents can be added to a provided nucleic acid preparation, for example simultaneously or sequentially with a neutralization buffer.
  • a ligation step is shown in Figure 1.
  • the ligase buffer (ligase, ET SSB, probes) is added simultaneously with the neutralization buffer (Tris pH 9.0) to the lysis reaction mixture for 35 min at 22°C.
  • reagents for nucleic acid amplification comprise one or more primer sets, a polymerase, a reverse transcriptase, and nucleotides can be added to a provided nucleic acid preparation, for example simultaneously or sequentially with a neutralization buffer.
  • Replication In some embodiments, methods as described herein can be used to prepare nucleic acids for use in e.g., replication.
  • reagent(s) for nucleic acid replication such as one or more primer sets, a polymerase, buffer(s), and/or nucleotides can be added to the prepared nucleic acids simultaneously or sequentially with any neutralization buffer.
  • nucleic acid hybridization In some embodiments, methods as described herein can be used to prepare nucleic acids for use in (e.g., for analysis and/or characterization, e.g., detection, by) e.g., nucleic acid hybridization.
  • SHERLOCK In some embodiments, methods as described herein can be used to prepare nucleic acids for use in detection of a target nucleic acid, e.g., with an SHERLOCK-type nucleic acid detection system as described in applications WO2017/218573, W02018/107129, WO2019/011022, W02019051318, W02019/104058, WO2020191376 (which describes a combination with an INSPECTR-type system) etc., each of which are incorporated herein by reference.
  • detection of target sequences within provided nucleic acid preparation(s) utilize one or more CRISPR/Cas enzymes. In some embodiments, such detection may involve use of one or more guide polynucleotides that recognize and bind a target sequence of interest.
  • Example 6 Synergistic effect
  • the present Example documents that the effect of a detergent (e.g., LAP AO) in combination with HC1 compared to a detergent alone (e.g., LAP AO) on viral particle lysis is more effective that HC1 alone.
  • a detergent e.g., LAP AO
  • Figure 7 shows that treating the sample with a detergent alone (e.g., LAP AO) in the absence of HC1 produces viral particle lysis that is less effective than the positive control (Heat lysis).
  • a detergent e.g., LAP AO
  • HC1 e.g., HC1

Abstract

The present disclosure provides technologies relating to cell and/or viral particle lysis and/or nucleic acid preparation.

Description

NUCLEIC ACID PREPARATION
BACKGROUND
[001] Nucleic acid analysis is playing an increasingly important role in public health.
SUMMARY
[002] The present disclosure describes technologies (e.g., compositions, mixtures, methods, and/or systems, etc.) for preparing of lysates. In some embodiments, the present disclosure provides technologies for nucleic acid preparations. In some embodiments, the present disclosure provides technologies for preparation of lysates from cells (e.g., eukaryotic, prokaryotic, etc.). In some embodiments, the present disclosure provides technologies for preparation of lysates from samples. In some embodiments, the present disclosure provides technologies for preparation of lysates from infectious agents. In some embodiments, the present disclosure provides technologies for preparation of lysates of viral particles (e.g., enveloped viruses, non-enveloped viruses). The present disclosure demonstrates that provided technologies achieve nucleic acid preparations amenable to sensitive detection technologies, including specifically technologies that detect (e.g., via processes that may include direct binding to, such as by hybridization and/or protein binding) particular target sequence(s) that may be present in nucleic acid(s) of the preparation.
[003] The present disclosure demonstrates surprising effectiveness of certain detergents (e.g., certain zwitterionic detergents) for use in lysing cells and/or viral particles and/or releasing nucleic acids from cells and/or viral particles, so that a nucleic acid preparation as described herein is obtained.
[004] Advantages of certain embodiments of provided technologies may include, among other things, that a useful nucleic acid preparation is provided without use of one or more traditional processing steps - such as purification, isolation or extraction steps that are commonly required or utilized to remove detergents. The present disclosure identifies the source of a problem with many traditional sample (including e.g., a cell and/or an infectious agent such as a bacterium or a virus) lysis and/or nucleic acid isolation technologies, for example in the reliance of such technologies on reagents and/or steps that can prolong or complicate provision of a useful nucleic acid preparation, including specifically of a nucleic acid preparation amenable to analysis (e.g., directly amenable to analysis) as described herein. Other advantages of provided technologies may include rapid lysis of viral particles, such as envelope particles (e.g., ranging from 1 second to 120 seconds, such as 30 seconds). Technologies are compatible with a variety of samples, wherein the sample is or comprises saliva and/or nasal swab eluates.
[005] Among other things, the present disclosure provides a surprising insight that levels of viral lysis comparable to those achieved by heat treatment (e.g., as described for example in Wateson et al Release of Intracellular Protein By Thermolysis. Ellis Horwood; London, UK, 1987 pp 105-109, incorporated herein by reference) can be achieved at ambient temperature through use of particular reagents (e.g., zwitterionic detergents) as described herein.
BRIEF DESCRIPTION OF THE DRAWING
[006] Figure 1 depicts an exemplary workflow for viral lysis at ambient temperature (e.g., at 22 °C), using e.g., 3 -Dodecylamido-N,N' -Dimethylpropyl Amine Oxide • 3-Laurylamido-N,N'-Dimethylpropyl Amine Oxide (LAP AO) and hydrochloric acid (HC1), or using N,N-Dimethyl-1-Dodecanamine-N-Oxide (LDAO), sodium decanoate (NaClO), and HC1. The workflow includes an incubation step of the sample with a lysis reagent, followed by lysis reaction and a neutralization step. The workflow also depicts an optional nucleic acid ligation step.
[007] Figure 2 depicts effect(s) of pH on viral lysis efficiency at ambient temperature (22°C) using detergent compositions as described herein.
[008] Figure 3 demonstrates reproducibility of viral lysis reactions as described herein (e.g., in Figures 1 and 2). Figure 3 also demonstrates that LAP AO detergent concentrations between about 0.03% and 0.3% (e.g., specifically between 0.0375% and 0.3%, inclusive) achieved better cell lysis than did lower concentrations (e.g., 0.018% and below). [009] Figure 4 demonstrates reproducibility of viral lysis reactions as described herein (e.g., in Figures 1 and 2).
[0010] Figure 5 demonstrates that viral lysis reactions as described herein e.g., in the Figures) can be conducted in water (i.e., that provided lysis reaction mixtures can be effective in water and/or in buffered solutions).
[0011] Figure 6 demonstrates compatibility of viral lysis reach on(s) as described herein (e.g., in the Figures) with INSPECTR™ nucleic acid detection.
[0012] Figure 7 demonstrates viral lysis reactions, as described herein, comprising the presence of at least one detergent alone (LAP AO) or in combination with HC1 (LAP AO + HC1). Heat lysis is used as a positive control and the NEG (negative control) is performed without any detergent or HC1.
DEFINITIONS
[0013] Ambient temperature: As used herein, the term “ambient temperature” is the temperature of surroundings. In general, the term ambient temperature is to be understood as the temperature of any object or environment surrounding an item. Measuring an ambient temperature can be accomplished by using a thermometer or sensor. The ambient temperature of an item is dependent on the temperature of the surrounding of the item. The surroundings can have any temperature, such as a temperature below 95°C, such as below 90°C, such as below 85°C, such as below 80°C, such as below 75°C, such as below 70°C, such as below 65°C, such as below 60°C, such as below 55°C, such as below 50°C, such as below 45°C, such as below 40°C, such as below 35°C, such as below 30°C, such as below 25°C, such as below 24°C, such as below 23°C, such as below 22°C, such as below 21°C, such as below 20°C. Exemplary ambient temperature ranges include 5°C to 50°C, such as 10°C to 40°C, such as 15°C to 35°C, such as 20°C to 30°C, such as 20°C to 25°C, such as 20°C to 22°C.
[0014] About'. The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.
[0015] Approximately. As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
[0016] Binding'. It will be understood that the term “binding”, as used herein, typically refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts - including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and/or in a biological system or cell).
[0017] Biological Sample. As used herein, the term “biological sample” typically refers to a sample obtained or derived from a biological source (e.g., a tissue or organism or cell culture) of interest, as described herein. In some embodiments, a source of interest is or comprises an organism, such as an animal or human. In some embodiments, a biological sample is or comprises biological tissue or fluid. In some embodiments, a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or broncheoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and/or excretions; and/or cells therefrom, etc. In some embodiments, a biological sample is or comprises viral particles. In some embodiments, biological samples comprising viral particles are obtained from an individual. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, a biological sample is or comprises cells comprising viral particles obtained from an individual. In some embodiments, a biological sample is or comprises viral particles or fragments thereof in a cell lysate matrix. In some embodiments, a biological sample is or comprises a nasal swab. In some embodiments, a biological sample is or comprises saliva In some embodiments, obtained cells are or include cells from an individual from whom the sample is obtained. In some embodiments, obtained viral particles are or include viral particles from an individual from whom the sample is obtained. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semipermeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and/or purification of certain components, etc.
[0018] Cellular lysate'. As used herein, the term “cellular lysate” or “cell lysate” refers to a fluid containing contents of one or more disrupted cells (i.e., cells whose membrane has been disrupted). In some embodiments, a cellular lysate includes both hydrophilic and hydrophobic cellular components. In some embodiments, a cellular lysate includes predominantly hydrophilic components; in some embodiments, a cellular lysate includes predominantly hydrophobic components. In some embodiments, a cellular lysate is a lysate of one or more cells selected from the group consisting of plant cells, microbial (e.g., bacterial or fungal) cells, animal cells (e.g., mammalian cells), human cells, and combinations thereof. In some embodiments, a cellular lysate is a lysate of one or more abnormal cells, such as cancer cells. In some embodiments, a cellular lysate is a crude lysate in that little or no purification is performed after disruption of the cells; in some embodiments, such a lysate is referred to as a “primary” lysate. In some embodiments, one or more isolation or purification steps is performed on a primary lysate; however, the term “lysate” refers to a preparation that includes multiple cellular components and not to pure preparations of any individual component. In some embodiments, a cellular lysate comprises viral particles. In some embodiments, viral particles (e.g., intact viral particle) are in a matrix of cell lysate. In some embodiments, a cellular lysate comprises spiked-in viral particles.
[0019] Composition'. Those skilled in the art will appreciate that the term
“composition”, as used herein, may be used to refer to a discrete physical entity that comprises one or more specified components. In general, unless otherwise specified, a composition may be of any form - e.g., gas, gel, liquid, solid, etc.
[0020] Comprising'. A composition or method described herein as
"comprising" one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method. To avoid prolixity, it is also understood that any composition or method described as "comprising" (or which "comprises") one or more named elements or steps also describes the corresponding, more limited composition or method "consisting essentially of' (or which "consists essentially of) the same named elements or steps, meaning that the composition or method includes the named essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the composition or method. It is also understood that any composition or method described herein as "comprising" or "consisting essentially of one or more named elements or steps also describes the corresponding, more limited, and closed- ended composition or method "consisting of (or "consists of) the named elements or steps to the exclusion of any other unnamed element or step. In any composition or method disclosed herein, known or disclosed equivalents of any named essential element or step may be substituted for that element or step.
[0021] Determine'. Many methodologies described herein include a step of
“determining”. Those of ordinary skill in the art, reading the present specification, will appreciate that such “determining” can utilize or be accomplished through use of any of a variety of techniques available to those skilled in the art, including for example specific techniques explicitly referred to herein. In some embodiments, determining involves manipulation of a physical sample. In some embodiments, determining involves consideration and/or manipulation of data or information, for example utilizing a computer or other processing unit adapted to perform a relevant analysis. In some embodiments, determining involves receiving relevant information and/or materials from a source. In some embodiments, determining involves comparing one or more features of a sample or entity to a comparable reference.
[0022] Expression'. As used herein, “expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and/or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
[0023] Lysis reagent composition: The term “lysis reagent composition”, as used herein, refers to a discrete physical entity that comprises one or more specified components described herein. The lysis reagent composition comprises components capable of disrupting membranes, such as a cell membrane and/or a viral particles (e.g. a capsid and/or lipid bilayer). For example, in some embodiments, the lysis reagent composition comprises a detergent (e.g, a zwitterionic detergent); an acid (e.g., HC1); and, optionally, a salt (e.g., sodium decanoate).
[0024] Lysis reaction mix'. As used herein, the term “lysis reaction mix” refers to a discrete physical entity that comprises one or more specified components described herein. In some embodiments, the lysis reaction mix comprises at least a sample. The sample is preferably a biological sample comprising viral particles and/or cells. For example, in some embodiments, the lysis reaction mix comprises a detergent (e.g, a zwitterionic detergent); an acid (e.g., HC1); a sample (e.g., a biological sample) and, optionally, a salt (e.g., sodium decanoate).
[0025] Neutralization: As used herein, the term “neutralization” refers to the stoppage of a reaction by addition of one or more neutralizing reagents. In some embodiments, a lysis reaction is neutralized by a neutralization buffer.
[0026] Nucleic acid. As used herein, in its broadest sense, refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments, "nucleic acid" refers to an individual nucleic acid residue (e.g., a nucleotide and/or nucleoside); in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, a "nucleic acid" is or comprises RNA; in some embodiments, a "nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid is, comprises, or consists of one or more "peptide nucleic acids", which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. Alternatively or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and/or 5'-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine). In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5- fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5- methylcytidine, 2- aminoadenosine, 7-deazaad enosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)- methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a nucleic acid comprises one or more modified sugars (e.g., 2'- fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a nucleic acid includes one or more introns. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a nucleic acid has enzymatic activity.
[0027] Polypeptide '. As used herein refers to any polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only nonnatural amino acids. In some embodiments, a polypeptide may comprise D- amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and/or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and/or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and/or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and/or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and/or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide. In some embodiments, a useful polypeptide as may comprise or consist of a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and/or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide.
[0028] Protein'. As used herein, the term “protein” refers to a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds). Proteins may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. Polypeptides may contain L-amino acids, Damino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.
[0029] Reference'. As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control. Sample: As used herein, the term “sample” typically refers to an aliquot of material obtained or derived from a source of interest, as described herein. In some embodiments, a source of interest is a biological or environmental source. In some embodiments, a source of interest may be or comprise a cell or an organism, such as a microbe, a plant, or an animal (e.g., a human). In some embodiments, a sample is or comprises viral particles (e.g., enveloped or non-enveloped viral particles). In some embodiments, a source of interest is or comprises biological tissue or fluid. In some embodiments, a biological tissue or fluid may be or comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secreations, vitreous humour, vomit, and/or combinations or component(s) thereof. In some embodiments, a biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, and/or a transcellular fluid. In some embodiments, a biological fluid may be or comprise a plant exudate. In some embodiments, a biological tissue or sample may be obtained, for example, by aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing or lavage (e.g., brocheoalvealar, ductal, nasal, ocular, oral, uterine, vaginal, or other washing or lavage). In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques such as amplification or reverse transcription of nucleic acid, isolation and/or purification of certain components, etc. In some embodiments, a sample may be a “crude” sample in that it has been subjected to relatively little processing and/or is complex in that it includes components of relatively varied chemical classes.
[0030] Specific binding: As used herein, the term “specific binding” refers to an ability to discriminate between possible binding partners in the environment in which binding is to occur. A binding agent that interacts with one particular target when other potential targets are present is said to “bind specifically” to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining degree of association between the binding agent and its partner; in some embodiments, specific binding is assessed by detecting or determining degree of dissociation of a binding agentpartner complex; in some embodiments, specific binding is assessed by detecting or determining ability of the binding agent to compete an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detections or determinations across a range of concentrations.
[0031] Specificity: As is known in the art, “specificity” is a measure of the ability of a particular ligand to distinguish its binding partner from other potential binding partners.
[0032] Subject. As used herein, the term “subject” refers to an organism, for example, a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, a dog). In some embodiments, a human subject is an adult, adolescent, or pediatric subject. In some embodiments, a subject is suffering from a disease, disorder or condition, e.g., a disease, disorder or condition that can be treated as provided herein, e.g., a cancer or a tumor listed herein. In some embodiments, a subject is susceptible to a disease, disorder, or condition; in some embodiments, a susceptible subject is predisposed to and/or shows an increased risk (as compared to the average risk observed in a reference subject or population) of developing the disease, disorder or condition. In some embodiments, a subject displays one or more symptoms of a disease, disorder or condition. In some embodiments, a subject does not display a particular symptom (e.g., clinical manifestation of disease) or characteristic of a disease, disorder, or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
Lysis Reactions
[0033] In some embodiments, the present disclosure provides technologies (e.g., compositions, mixtures, methods, and/or systems, etc.) for preparation of lysates. In some embodiments, the present disclosure provides technologies (e.g., compositions, mixtures, methods, and/or systems, etc.) for preparation of lysates from samples as described herein. In some embodiments, the present disclosure provides technologies (e.g., compositions, mixtures, methods, and/or systems, etc.) for preparation of lysates cells (e.g., eukaryotic, prokaryotic, etc.). In some embodiments, the present disclosure provides technologies (e.g., compositions, mixtures, methods, and/or systems, etc.) for preparation of lysates of infectious agents (e.g., microbes). In some embodiments, the present disclosure provides technologies (e.g., compositions, mixtures, methods, and/or systems, etc.) for preparation of lysates of viral particles (e.g., enveloped viruses, non-enveloped viruses).
[0034] In some embodiments, the present disclosure provides technologies (e.g., compositions, mixtures, methods, and/or systems, etc) for preparation of cell lysates (e.g., particularly of cells comprising nucleic acids of interest - e.g., target nucleic acids). Among other things, the present disclosure provides lysis reagent compositions (e.g., comprising one or more detergents), as well as lysis reaction mixtures (e.g., comprising the lysis reagent and cells), lysed compositions (i.e., comprising cell lysates), and/or methods for preparing and/or for using any or all of the foregoing.
[0035] Among other things, the present disclosure provides a surprising insight that levels of cell and/ or viral particle lysis comparable to those achieved by heat treatment (e.g., 20 minutes for temperatures above 60 °C, or 5 minutes for temperatures above 65°C, such as temperatures of or above 95 °C) can be achieved at ambient temperature through use of appropriate reagents. In some embodiments, provided lysis reagent compositions achieve lysis at ambient conditions to a level or degree that is not significantly increased by exposure of the relevant system(s) to heat treatment.
[0036] In some embodiments, one or more reagents utilized in accordance with the present disclosure may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g., in concentrate or lyophilized form).
[0037] In some embodiments, the lysis reagents composition comprises one or more reagents, detergents, surfactants, buffers, or salts as provided in table 1.
Table 1 :
Figure imgf000015_0001
Figure imgf000016_0001
Detergents
[0038] In some embodiments, a lysis reagent composition comprises one or more components such as, for example, detergents, buffers, acids, and salts. In some embodiments, a lysis reagent composition may be or comprise a detergent, such as one or more detergent(s) or combinations of detergents.
[0039] In some embodiments, a detergent can be selected from the group consisting of nonionic, anionic, cationic, and zwitterionic detergents and combinations thereof. In some embodiments, exemplary detergents used herein are zwitterionic detergents. In some embodiments, a lysis reagent composition comprises at least one zwitterionic detergent. In some embodiments, a lysis reagent composition comprises a detergent component that consists of a zwitterionic detergent; in some such embodiments, a lysis reaction does not include a detergent component that is not a zwitterionic detergent.
[0040] Zwitterionic detergents (“zwittergents”) have characteristics of both ionic and non-ionic types. Like non-ionic detergents, the zwittergents are characterized by lack of (significant) net charge, lack of conductivity and electrophoretic mobility, and/or absence of (significant) binding toion-exchange resins. On the other hand, like ionic detergents, they can be efficient at breaking protein-protein interactions. Steroid-based zwittergents, are less denaturing than linear-chain zwitterionic detergents (e.g., dodecyldimethyldiamine oxide). In some embodiments, zwitterionic detergents utilized in accordance with the present disclosure do not possess a net charge, but maintain ability to break protein-protein interactions.
[0041] Exemplary zwitterionic detergents for use in accordance with the present disclosure may be or comprise, for example, 3-Dodecylamido-N,N'-Dimethylpropyl Amine Oxide also known as (aka) 3-Laurylamido-N,N' -Dimethylpropyl Amine Oxide or LAP AO, Lauryl dimethylamine-N-Oxide aka LDAO, N,N-Dimethyl-1-Dodecanamine-N-Oxide or DDAO, 3-[(3-Cholamidopropyl)dimethylammonio]-l-propanesulfonate aka CHAPS, 3-([3- Cholamidopropyl]dimethylammonio)-2 -hydroxy-1 -propanesulfonate aka CHAPSO. [0042] In some embodiments, a lysis reagent composition is or comprises a zwitterionic detergent and HC1. In some embodiments, a zwitterionic detergent is selected from the group consisting of LAP AO, LDAO, and DDAO.
[0043] In some embodiments, a zwitterionic detergent is present in a lysis reagent composition at a concentration within a range of about 0.001% to about 10%, such as between about 0.01% and about 9%, such as between about 0.05% and about 8%, such as between about 0.1% and about 5%, such as between about 0.5% and about 4%, such as between about 1% and about 3%, such as between about 1.5% and about 2.5%, such as between about 1.75% and about 2.25%.
[0044] Those skilled in the art will appreciate that, in some embodiments, a zwitterionic detergent may be provided and/or utilized in a stock solution at a concentration that is higher (and typically by a multiple or other amount appropriate for use by dilution into a reaction) than that at which it is present in a lysis reagent composition and/or in a lysis reaction as described herein.
[0045] In some embodiments, a percentage of zwitterionic detergent in a lysis reagent composition is at least 0.001%, such as at least 0.01%, such as at least 0.05%, such as at least 0.1%, such as at least 0.5%, such as at least 1%, such as at least 1.25%, such as at least 1.5%, such as at least 1.75%, such as at least 2%, such as at least 2.25%, such as at least 2.5%, such as at least 3%, such as at least 3.5%, such as at least 4.5%, such as at least 5%, such as at least 5.5%, such as at least 6%, such as at least 6.5%, such as at least 7%, such as at least 7.5%, such as at least 8%, such as at least 8.5%, such as at least 9%, such as at least 9.5%, such as at least 10%.
[0046] In some embodiments, a percentage of zwitterionic detergent in a lysis reagent composition utilized in accordance with the present disclosure (and particularly as utilized in a lysis reagent composition and/or a lysis reaction) is not more than about 30%, not more than about 20%, not more than about 10%, not more than about 5%, not more than about 4%, not more than about 3%, not more than about 2%, not more than about 1%, etc
[0047] In some embodiments, a zwitterionic detergent is utilized at a concentration within a range of about 0.01% and about 10%, wherein HC1 is present within the range of about 4 mM and about 4M, and wherein pH is within the range of 0 and 6. [0048] In some embodiments, a lysis reagent composition is or comprises a zwitterionic detergent and one or more reagents, such as buffers, acids, and salts, such as NaClO.
[0049] In some embodiments, lysis reagent compositions according to the invention are or comprise LAP AO. In some embodiments, a zwitterionic detergent used herein is LAP AO. LAP AO has the molecular formula CI7H36N2O2 and the molecular structure as set forth herein below:
Figure imgf000018_0001
[0050] In some embodiments, a lysis reagent composition is or comprises LAP AO in a concentration suitable for achieving cellular lysis.
[0051] In some embodiments, a concentration range of LAP AO as utilized herein (e.g., in a lysis reagent composition, a lysis reaction, or a stock solution) is within 0.001% and 10%, such as 0.01% and 9%, such as 0.05% and 8%, such as 0.1% and 5%, such as 0.5% and 4%, such as 1% and 3%, such as 1.5% and 2.5%, such as 1.75% and 2.25%.
[0052] In some embodiments, a percentage of LAP AO utilized in accordance with the present disclosure is at least 0.001%, such as at least 0.01%, such as at least 0.05%, such as at least 0.1%, such as at least 0.5%, such as at least 1%, such as at least 1.25%, such as at least 1.5%, such as at least 1.75%, such as at least 2%, such as at least 2.25%, such as at least 2.5%, such as at least 3%, such as at least 3.5%, such as at least 4.5%, such as at least 5%, such as at least 5.5%, such as at least 6%, such as at least 6.5%, such as at least 7%, such as at least 7.5%, such as at least 8%, such as at least 8.5%, such as at least 9%, such as at least 9.5%, such as at least 10%.
[0053] In some embodiments, a percentage of LAP AO utilized in accordance with the present disclosure (and particularly as utilized in a lysis reagent composition and/or a lysis reaction) is not more than about 30%, not more than about 20%, not more than about 10%, not more than about 5%, not more than about 4%, not more than about 3%, not more than about 2%, not more than about 1%, etc.. [0054] In some embodiments, a lysis reagent composition comprises LAP AO and one or more reagents, such as buffers, acids, and salts.
[0055] In some embodiments, provided lysis reagent compositions are or comprise LDAO. In some embodiments, the zwitterionic detergent used herein is LDAO. LDAO has the molecular formula C14H31NO and the molecular structure as shown herein below:
Figure imgf000019_0001
[0056] In some embodiments, a lysis reagent composition is or comprises LDAO in a concentration suitable for achieving cellular lysis.
[0057] In some embodiments, LDAO is utilized in accordance with the present disclosure (e.g., in a lysis reagent composition, a lysis reaction, and/or a stock composition) at a concentration within a range of about 0.001% to aboutlO%, such as between about 0.01% and about 9%, such as between about 0.05% and about 8%, such as between about 0.1% and about 5%, such as between about 0.5% and about 4%, such as between about 1% and about 3%, such as between about 1.5% and about 2.5%, such as between about 1.75% and 2.25%. In some embodiments, LDAO is utilized in accordance with the present disclosure at a concentration of at least 0.001%, such as at least 0.01%, such as at least 0.05%, such as at least 0.1%, such as at least 0.5%, such as at least 1%, such as at least 1.25%, such as at least 1.5%, such as at least 1.75%, such as at least 2%, such as at least 2.25%, such as at least 2.5%, such as at least 3%, such as at least 3.5%, such as at least 4.5%, such as at least 5%, such as at least 5.5%, such as at least 6%, such as at least 6.5%, such as at least 7%, such as at least 7.5%, such as at least 8%, such as at least 8.5%, such as at least 9%, such as at least 9.5%, such as at least 10%.
[0058] In some embodiments, LDAO is utilized in accordance with the present disclosure (and particularly as utilized in a lysis reagent composition and/or a lysis reaction) at a concentration that is not more than about 30%, not more than about 20%, not more than about 10%, not more than about 5%, not more than about 4%, not more than about 3%, not more than about 2%, not more than about 1%, not more than 0.5%, not more than 0.05%, not more than 0.005%, etc. [0059] In some embodiments, a lysis reagent composition comprises LDAO and one or more reagents, such as buffers, acids, and salts.
Acids (HC1)
[0060] In some embodiments, exemplary lysis reagent compositions comprise one or more acids. In some embodiments, lysis reagent compositions comprising a zwitterionic detergent further comprise one or more acids. In some embodiments, lysis reagent compositions comprising LAPOA and/or LDAO further comprise one or more acids. In some embodiments, an acid used in accordance with the present disclosure may be selected from hydrochloric acid (HC1), sulfuric acid, nitric acid, acetic acid, carbonic acid, citric acid, acetylsalicylic acid, etc., and combinations thereof..
[0061] In some embodiments, HC1 is utilized. In some embodiments, HC1 is utilized (e.g., in a lysis reagent composition, a lysis reaction, lysis reaction mix, and/or a stock solution) at a concentration within the range of about 4 mM and about 4 M. In some embodiments, HC1 is utilized at a concentration within a range of about 5 mM to about 4 M, or within a range of about 6 mM to about 4 M, , or within a range of about 7 mM to about 4 M, or within a range of about 8 mM to about 4 M, or within a range of about 9 mM to about 4 M, or within a range of about 10 mM to about 4 M, or within a range of about 11 mM to about 4 M, or within a range of about 12 mM to about 4 M, or within a range of about 13 mM to about 4 M, or within a range of about 14 mM to about 4 M, or within a range of about 15 mM to about 4 M, or within a range of about 16 mM to about 4 M, or within a range of about 17 mM to about 4 M, or within a range of about 18 mM to about 4 M, or within a range of about 19 mM to about 4 M, or within a range of about 20 mM to about 4 M, or within a range of about 25 mM to about 4 M, or within a range of about 30 mM to about 4 M, or within a range of about 35 mM to about 4 M, or within a range of about 40 mM to about 4 M, or within a range of about 45 mM to about 4 M, or within a range of about 50 mM to about 4 M, or within a range of about 60 mM to about 4 M, or within a range of about 70 mM to about 4 M, or within a range of about 80 mM to about 4 M, or within a range of about 90 mM to about 4 M, or within a range of about 100 mM to about 4 M, or within a range of about 200 mM to about 4 M, or within a range of about 300 mM to about 4 M, or within a range of about 400 mM to about 4 M, or within a range of about 500 mM to about 4 M, or within a range of about 600 mM to about 4 M, or within a range of about 700 mM to about 4 M, or within a range of about 800 mM to about 4 M, or within a range of about 900 mM to about 4 M, or within a range of about 1 M to about 4 M, or within a range of about 2 M to about 4 M, or within a range of about 3 M to about 4 M, or within a range of about 10 mM to about 1 M, or within a range of about 20 mM to about 900 mM, or within a range of about 50 mM to about 800 mM, or within a range of about 100 mM to about 700 mM, or within a range of about 200 mM to about 600 mM, or within a range of about 300 mM to about 500 mM, or within a range of about 350 mM to about 450 mM.
Sodium decanoate (NaClO)
[0062] In some embodiments, the lysis reaction composition sodium decanoate, Capric acid sodium salt, aka Decanoic acid sodium salt, Sodium caprate, or NaClO. NaClO has the molecular formula CH3(CH2)sCOONa and the molecular structure as shown herein below:
Figure imgf000021_0001
[0063] In some embodiments, a lysis reagent composition comprising a zwitterionic detergent further comprises sodium decanoate. In some embodiments, a lysis reagent composition comprising LAP AO further comprises sodium decanoate. In some embodiments, a lysis reagent composition comprising LDAO further comprises sodium decanoate. In some embodiments, such lysis reaction compositions also comprise HC1.
[0064] In some embodiments, NaClO is utilized in accordance with the present disclosure (e.g., in a lysis reagent composition, a lysis reaction and/or a stock solution) in an about within a range of about 0.02% and about 20%, such as between about 0.1% and about 10%, such as between about 0.5% and about 5%, such as between about 1% and about 2%.
Stock concentrations
[0065] In some embodiments, a lysis reagent composition or component thereof, a detergent (or component thereof), a buffer (or component thereof), and/or a combination thereof is provided and/or utilized as a stock preparation (z.e., in which it is present at a concentration higher than that, and typically a multiple of that, at which it is used during the lysis reaction). [0066] In some embodiments, a lysis reagent composition is prepared and/or utilized as a stock preparation. In some embodiments, one or more components of a lysis reagent composition is prepared and/or utilized as a stock preparation.
[0067] In some embodiments, a stock preparation includes each of its components (e.g., buffer, detergent, salt, acid and/or a combination of some or all of the foregoing) at a concentration that is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 or more times its working concentration in a lysis reaction.
[0068] In some embodiments, a lysis reagent composition or component thereof and/or a detergent and/or one or more other components of a lysis reaction are each combined with (e.g., added to) a sample (e.g., sequentially or simultaneously, e.g., by prior combination).
[0069] In one embodiment, a lysis reagent composition is prepared by combining one or more components, or all components, with one another before combining the lysis reagent composition with a sample; alternatively, in some embodiments, one or more components of a lysis reagent composition is combined sequentially or simultaneously with a sample. pH
[0070] In some embodiments, a lysis reagent composition as described herein (e.g., comprising a zwitterionic detergent such as, for example, LAP AO), and/or a lysis reaction, has a pH between 0 and 7. In some embodiments, such a composition includes HC1.
[0071] In some embodiments, a lysis reagent composition and/or a lysis reaction has a pH between 0 and 6, or between 0 and 5, or between 0 and 4 or between 0 and 3 or between 0 and 2 or between 0 and 1.
[0072] In some embodiments, a relevant pH is at the most 3, such as at the most 2, such as at the most 1.5, such as at the most 1, such as at the most 0.8, such as at the most 0.6, such as at the most 0.4, such as at the most 0.2.
[0073] In some embodiments, pH of a lysis reagent composition, and/or a lysis reaction, comprising a zwitterionic detergent (e.g., LDAO), NaClO, and HC1 is between 0 and 7 or between 0 and 6 or between 0 and 5 or between 0 and 4 or between 0 and 3 or between 0 and 2 or between 0 and 1. In some embodiments, pH of a lysis reagent composition, and/or a lysis reaction, comprising a zwitterionic detergent, NaClO, and HC1 is at the most 3, such as at the most 2, such as at the most 1.5, such as at the most 1, such as at the most 0.8, such as at the most 0.6, such as at the most 0.4, such as at the most 0.2.
Viral particle
[0074] The terms “viral particle” and “virus” are used interchangeable herein. Viruses are small intracellular parasites, which contain either a RNA or DNA genome. The viral genome, often with associated basic proteins, is packaged inside a symmetric protein capsid. A nucleocapsid is the capsid and the nucleic acid of the virus (e.g., a non-enveloped virus). In some embodiments, a viral particle comprises a nucleocapsid. In some embodiments, a viral particle is a non-enveloped viral particle.
[0075] In enveloped viruses, the nucleocapsid is surrounded by a lipid bilayer (e.g., derived from a modified host cell membrane and often studded with an outer layer of virus envelope glycoproteins). In some embodiments, a viral particle is an enveloped viral particle. In some embodiments, viral particle fragments comprises nucleotides (e.g., target nucleotides) surrounded by a viral capsid, but not a lipid bilayer.
[0076] It is understood in the art that viruses (e.g. SARS- CoV; influenza; herpes, etc.) can be lysed by heat treatment. In some embodiments, heat treatment includes, but is not limited to treating the virus at temperatures above 75 °C for 3 minutes for, at temperatures above 65 °C for 5 minutes, or at temperatures above 60 °C for 20 minutes.
Samples
[0077] In some embodiments, the present disclosure provides exemplary compositions and methods for the preparation and/or detection of target nucleic acids in a sample. In some embodiments, the sample is or comprises a viral particle. In some embodiments, the sample is or comprises a viral particle. In some embodiments, a sample is an environmental sample. In some embodiments, a sample is a biological sample (e.g., a sample from an organism, and/or of a tissue or fluid thereof, e.g., comprising one or more cells or cell components and/or one or more viral particles). In some embodiments, a sample is a crude sample (e.g., a primary sample or a sample that has undergone minimal processing). [0078] In some embodiments, a primary sample is collected from a subject (e.g., a human or animal subject). In some embodiments, an animal subject may be a wild or undomesticated animal such as animals living in the sea or in a jungle. In some embodiments, an animal subject may be a domesticated animal, such as a farm animal or a pet. In some embodiments, an animal subject may be a cat, cow, dog, goat, horse, llama, pig, sheep, bird, etc. In some embodiments, an animal subject may be a rodent. In some embodiments, a subject may be a primate. In some embodiments, a subject may be a human.
[0079] In some embodiments, a primary sample is obtained directly from a subject - e.g., from a fluid or tissue of the subject. In some embodiments, a sample is saliva, urine, perspiration, breath, blood, tissue, hair, skin, or any other excretion product or body fluid. In some embodiments, a sample is obtained from a subject by means of a swab, an aspirate, or a lavage. In some embodiments, a sample is obtained from a subject by means of a nasal swab, nasopharyngeal swab, oropharyngeal swab, nasal aspirate, sputum, bronchoalveolar lavage. In some embodiments, a biological sample is selected from nasal swab, nasopharyngeal swab, oropharyngeal swab, nasal aspirate, sputum, bronchoalveolar lavage, blood, serum, feces, and saliva samples. In some embodiments, a sample is or comprises a nasal swab. In some embodiments, a sample is or comprises saliva.
[0080] In some embodiments, a swab used to collect a sample is placed promptly (e.g., immediately) into a sterile tube containing 2-3 ml of viral transport media (i.e. VTM, UTM, M4RT).
[0081] In some embodiments, a sample is a pooled sample - e.g., a sample obtained by combining samples from multiple sources (e.g., pooled saliva sample).
[0082] In some embodiments, a sample (or a reaction in which such a sample is lysed) includes a spiked-in viral particles comprising nucleic acid target (e.g., which may be processed and/or detected as a “control”). In some embodiments, a spiked-in sample is generated by growing viral particles inside cells (e.g., mammalian cells). Cells are thereafter lysed, thereby leaving the viral particles intact in a matrix of cell lysate. In some embodiments, a sample is or comprises viral particles in a cell lysate matrix. In some embodiments, a sample is or comprises a viral particle thereof within a cell. In some embodiments, a sample (or reaction) into which a target nucleic acid (e.g., a control nucleic acid target) is spiked is a “mock” sample; in some such embodiments, the spiked-in nucleic acid is a target nucleic acid of interest, so that the mock sample acts as a positive control for detection of the target nucleic acid.
[0083] In some embodiments, a sample (or reaction) into which a nucleic acid is spiked is a test sample, and the spiked-in nucleic acid is a control target (e.g., a target other than the target of interest) whose processing and/or detection acts as a control for successful nucleic acid processing or detection.
[0084] In some embodiments, a spiked-in target is or comprises an infectious agent (e.g., a bacterium or virus), such as an inactivated infectious agent. In some embodiments, an inactivated agent is or comprises a heat-treated agent, a chemically-treated agent, and/or a y-irradiated agent. In some particular embodiments, an inactivated agent is a y-irradiated virus.
[0085] In some embodiments, a sample is prepared and/or utilized as a liquid sample. In some embodiments, a liquid sample is an aqueous sample. In some embodiments, a liquid sample is a buffered sample (e.g., includes a buffer system). In some embodiments, a liquid sample does not include a buffer. In some embodiments, a liquid sample is in water.
[0086] In some embodiments, a sample may be processed by one or more steps prior to lysis as described herein. Thus, in some embodiments, a sample is a processed sample. For example, in some embodiments, a sample is processed by addition of one or more reagents (e.g., to separate cells in a tissue), dilution, filtration, clarification, distillation, separation, isolation, freezing, drying, cryopreservation, etc). In some embodiments, a sample is processed by isolation of specific component(s) (e.g., viral particles and/or cells). In some embodiments, a sample is processed by a mechanical process (e.g., centrifugation).
[0087] In some embodiments, the sample (e.g., comprising at least one viral particle or fragment thereof) is combined with a lysis reagent composition according to the present invention hereby generating a lysis reaction mix.
Lysis reaction mix
[0088] In some embodiments, the present disclosure provides technologies in which a lysis reagent composition or component thereof, such as one or more detergents, acids, salts, and buffers is combined with e.g, added to) a sample (e.g., sequentially or simultaneously, e.g, by prior combination).
[0089] In some embodiments, the present disclosure provides cell lysis reaction mixtures (e.g., eukaryotic cells, prokaryotic cells, etc.). In some embodiments, the present disclosure provides microbe lysis reaction mixtures. In some embodiments, the present disclosure provides viral particle lysis reaction mixtures (e.g., enveloped viruses, nonenveloped viruses).
[0090] In some embodiments, lysis reaction mixtures comprise a lysis reagent composition and a sample as described herein. In some embodiments, lysis reaction mixtures comprise a detergent, an acid, a sample and optionally a salt. In some embodiments, a sample comprises cells or components thereof.
[0091] In some embodiments, a lysis reagent composition comprising a zwitterionic detergent, and HC1 as provided herein is added to a sample. In some embodiments, the zwitterionic detergent is LAP AO. In some embodiments, the sample comprises cells. In some embodiments, the sample comprises cells comprising at least one target nucleic acid. In some embodiments, addition of the lysis reagent composition to the sample causes at least some of the cells in the sample to lyse and release their nucleic acid content into the sample.
[0092] In some embodiments, a lysis reaction mix comprises LAP AO in a concentration suitable for achieving cellular lysis. In some embodiments, a concentration range of the LAP AO in the lysis reaction mix is within 0.001% and 10%, such as 0.01% and 1%, such as 0.05% and 0.5%, such as 0.075% and 0.2%, such as 0.09% and 0.11%. In some embodiments, these concentrations represent a dilution of a stock solution.
[0093] In some embodiments, a percentage of the LAP AO in the lysis reaction mix is at least 0.001%, such as at least 0.005%, such as at least 0.01%, such as at least 0.02%, such as at least 0.03%, such as at least 0.04%, such as at least 0.05%, such as at least 0.06%, such as at least 0.07%, such as at least 0.08%, such as at least 0.09%, such as at least 0.1%.
[0094] In some embodiments, a percentage of LAP AO in the lysis reaction mix is as at the most 3%, such as at the most 2.5%, such as at the most 2%, such as at the most 1.5%, such as at the most 1%, such as at the most 0.8%, such as at the most 0.5%, such as at the most 0.3%, such as at the most 0.2%, such as at the most 0.1%. [0095] In some embodiments, the pH of a lysis reaction mix comprising a zwitterionic detergent, and HC1 is at the most 3, such as at the most 2, such as at the most 1.5, such as at the most 1, such as at the most 0.8, such as at the most 0.6, such as at the most 0.4, such as at the most 0.2.
[0096] In some embodiments, a lysis reagent composition comprising a zwitterionic detergent, NaClO, and HC1 as provided herein is added to a sample. In some embodiments, the zwitterionic detergent is LDAO. In some embodiments, a sample comprises cells. In some embodiments, a sample comprises cells comprising at least one target nucleic acid. In some embodiments, addition of a composition to a sample causes at least some of the cells in the sample to lyse and release their nucleic acid content into the sample.
[0097] In some embodiments, a lysis reaction mix comprises LDAO in a concentration suitable for achieving cellular lysis. In some embodiments, a concentration range of LDAO in the lysis reaction mix is within 0.001% and 10%, such as 0.005% and 1%, such as 0.01% and 0.1%, such as 0.02% and 0.05%, such as 0.02% and 0.04%. In some embodiments, these concentrations represent a dilution of a stock solution.
[0098] In some embodiments, a percentage of LDAO in a lysis reaction mix is at least 0.001%, such as at least 0.002%, such as at least 0.003%, such as at least 0.004%, such as at least 0.005%, such as at least 0.006%, such as at least 0.007%, such as at least 0.008%, such as at least 0.009%, such as at least 0.01%, such as at least 0.015%, such as at least 0.02%, such as at least 0.0225%, such as at least 0.025%, such as at least 0.05%, such as at least 0.1%.
[0099] In some embodiments, a percentage of LDAO in the lysis reaction mix is at the most 2%, such as at the most 1.5%, such as at the most 1%, such as at the most 0.5%, such as at the most 0.4%, such as at the most 0.3%, such as at the most 0.2%, such as at the most 0.1%, such as at the most 0.075%, such as at the most 0.050%, such as at the most 0.0375%, such as at the most 0.03%.
[00100] Those of ordinary skill in the art, reading the present disclosure and appreciating that ambient temperature lysis can be achieved as described herein, will appreciate that certain modifications to exemplified lysis reagent composition may also be useful and/or effective to achieve ambient temperature lysis (and/or lysis under conditions other than traditional heat treatment). For example, one of ordinary skill in the art, reading the present disclosure, will appreciate that, in some embodiments, one or more of the reagents provided in Table 1 or 2 may be tested or utilized together with or as an alternative to one or more components of a particular reagent system exemplified herein.
Temperature
[00101] In some embodiments, a mixture of a lysis reagent composition (and optionally a detergent and/or buffer and/or one or more other components) and the sample is incubated at ambient temperature. In some embodiments, a lysis reaction mix comprising zwitterionic detergent is incubated at ambient temperature. In some embodiments, the lysis reaction mix comprising LAP AO is incubated at ambient temperature. In some embodiments, a lysis reaction mix comprising LDAO is incubated at ambient temperature. In some embodiments, a lysis reaction mix comprising zwitterionic detergent and a sample comprising a viral particle or fragment thereof is incubated at ambient temperature. In some embodiments, the ambient temperature is at the most 95°C, such as at the most 90°C, such as at the most 85°C, such as at the most 80°C, such as at the most 75°C, such as at the most 70°C, such as at the most 65°C, such as at the most 60°C, such as at the most 55°C, such as at the most 50°C, such as at the most 45°C, such as at the most 40°C, such as at the most 35°C, such as at the most 30°C, such as at the most 25°C, such as at the most 24°C, such as at the most 23 °C, such as at the most 22°C, such as at the most 21 °C, such as at the most 20°C. Exemplary ambient temperature ranges include 10°C and 40°C, such as 15°C to 35°C, such as 20°C to 30°C, such as 20°C to 25°C, such as 20°C to 22°C.
Time
[00102] In some embodiments, a mixture of a lysis reagent composition and a sample is incubated for a predetermined period of time. In some embodiments, a lysis reaction mix comprising zwitterionic detergent is incubated for a predetermined period of time. In some embodiments, a lysis reaction mix comprising LAP AO is incubated for a predetermined period of time. In some embodiments, a lysis reaction mix comprising LDAO is incubated for a predetermined period of time.
[00103] Exemplary ranges for an incubation time include 0.1 second to 30 minutes, such as 1 second to 20 minutes, such as 2 seconds to 10 minutes, such as 5 seconds to 5 minutes, such as 10 seconds to 1 minute, such as 20 seconds to 40 seconds. pH
[00104] In some embodiments, a pH range of a lysis reaction mix, and/or lysis reaction, comprising a zwitterionic detergent, and HC1 is within 0 and 7. In some embodiments, a pH range of a lysis reaction mix, and/or lysis reaction, comprising a zwitterionic detergent and HC1 is within 0 and 6. In some embodiments, a pH range of a lysis reaction mix, and/or lysis reaction, comprising a zwitterionic detergent, and HC1 is within 0 and 5. In some embodiments, a pH range of a lysis reaction mix, and/or lysis reaction, comprising a zwitterionic detergent, and HC1 is within 0 and 4. In some embodiments, a pH range of a lysis reaction mix, and/or lysis reaction, comprising a zwitterionic detergent, and HC1 is within 0 and 3. In some embodiments, a pH range of a lysis reaction mix comprising a zwitterionic detergent, and HC1 is within 0 and 2. In some embodiments, a pH range of a lysis reaction mix comprising a zwitterionic detergent, and HC1 is within 0 and 1. In some embodiments, a pH range of a lysis reaction mix comprising a zwitterionic detergent, and HC1 is within 1 and 5. In some embodiments, a pH range of a lysis reaction mix comprising a zwitterionic detergent, and HC1 is within 2 and 4. In some embodiments, a pH range of a lysis reaction mix comprising a zwitterionic detergent, and HC1 is within 2 and 3.
[00105] In some embodiments, a pH of a lysis reaction mix comprising a zwitterionic detergent, and HC1 is at the most 6, such as at the most 5, such as at the most 4, such as at the most 3, such as at the most 2.5.
[00106] In some embodiments, a pH range of a lysis reaction mix comprising a LAP AO, and HC1 is within 0 and 7. In some embodiments, a pH range of a lysis reaction mix comprising a LAP AO and HC1 is within 0 and 6. In some embodiments, a pH range of a lysis reaction mix comprising a LAP AO, and HC1 is within 0 and 5. In some embodiments, a pH range of a lysis reaction mix comprising a LAP AO, and HC1 is within 0 and 4. In some embodiments, a pH range of a lysis reaction mix comprising a LAP AO, and HC1 is within 0 and 3. In some embodiments, a pH range of a lysis reaction mix comprising a LAP AO, and HC1 is within 0 and 2. In some embodiments, a pH range of a lysis reaction mix comprising a LAP AO, and HC1 is within 0 and 1. In some embodiments, a pH range of a lysis reaction mix comprising a LAP AO, and HC1 is within 1 and 5. In some embodiments, a pH range of a lysis reaction mix comprising a LAP AO, and HC1 is within 2 and 4. In some embodiments, a pH range of a lysis reaction mix comprising a LAP AO, and HC1 is within 2 and 3.
[00107] In some embodiments, a pH of a lysis reaction mix comprising a LAPO, and HC1 is at the most 6, such as at the most 5, such as at the most 4, such as at the most 3, such as at the most 2.5.
[00108] In some embodiments, a pH range of a lysis reaction mix comprising a LDAO, NaClO and HC1 is within 0 and 7. In some embodiments, the pH range of a lysis reaction mix comprising a LDAO, NaClO and HC1 is within 0 and 6. In some embodiments, a pH range of a lysis reaction mix comprising a LDAO, NaClO, and HC1 is within 0 and 5. In some embodiments, the pH range of a lysis reaction mix comprising a LDAO, NaClO, NaClO, and HC1 is within 0 and 4. In some embodiments, a pH range of a lysis reaction mix comprising a LDAO, NaClO, and HC1 is within 0 and 3. In some embodiments, a pH range of a lysis reaction mix comprising a LDAO, NaClO, and HC1 is within 0 and 2. In some embodiments, a pH range of a lysis reaction mix comprising a LDAO, NaClO, and HC1 is within 0 and 1. In some embodiments, a pH range of a lysis reaction mix comprising a LDAO, NaClO, and HC1 is within 1 and 5. In some embodiments, a pH range of a lysis reaction mix comprising a LDAO, NaClO, and HC1 is within 2 and 4. In some embodiments, a pH range of a lysis reaction mix comprising a LDAO, NaClO, and HC1 is within 2 and 3.
[00109] In some embodiments, a pH of a lysis reaction mix comprising a LAPO, NaClO, and HC1 is at the most 6, such as at the most 5, such as at the most 4, such as at the most 3, such as at the most 2.5.
Buffer
[00110] In some embodiments, a lysis reaction is performed in the same buffer (or absence of buffer) in which the sample was prepared. In some embodiments, a different buffer (or absence of buffer) is utilized, for example, by dialyzing, diluting, resuspending, or otherwise adjusting a prepared sample.
[00111] The present disclosure demonstrates, as documented here (see, Examples), that, in some embodiments, lysis reactions can be performed in the presence of one or more buffers and, in some embodiments, lysis reactions can be performed in the absence of a buffer. Thus, teachings of the present disclosure, in many embodiments, are not limited to a particular buffer system.
[00112] In some embodiments, a buffer utilized in a lysis reaction provided by the present disclosure may be or comprise, for example a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof. In some embodiments, the buffer is acidic. In some embodiments, a buffer has a pH from about 0 to about 7.
[00113] In some embodiments, a lysis reaction is performed in a liquid system (e.g., in an aqueous system). In some embodiments, a lysis reaction is performed in a buffered system. In some embodiments, a lysis reaction is performed in a system that is not buffered. In some embodiments, a lysis reaction is performed in water.
Neutralization
[00114] In some embodiments, a lysis reaction as described herein may be stopped, e.g., by neutralization.
[00115] In some embodiments, stoppage (e.g., neutralization) is achieved by addition of one or more neutralizing reagents. Those skilled in the art, reading the present disclosure, will appreciate that neutralizing reagents may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g., in concentrate or lyophilized form). In some embodiments, exemplary neutralizing reagents comprise buffers, and bases.
[00116] In some embodiments, a buffer is not limited to a particular buffer; in some embodiments, a buffer can be any buffer, including but not limited to a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES, NaOH, acetate buffer, PBS, and combinations thereof. In some embodiments, a buffer is alkaline. In some embodiments, a buffer has a pH from about 7 to about 11. In some embodiments, a lysis reaction is neutralized by the neutralization buffer. In some embodiments, a pH of the buffer is such that upon addition to reaction, the pH of the buffer and reaction mixture is within the rage of pH 7 and 9, such as within the range of pH 8 to 8.5. In some embodiments, an exemplary buffer used herein is a Tris buffer. In some embodiments, a Tris buffer is provided as a concentrate, i.e. stock solution. Exemplary concentrations of the Tris buffer comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 times to working concentration of the buffer. In some embodiments, the concentration of the Tris buffer concentrate is within the range of 1 mM and 2M. In some embodiments, the concentration of the Tris buffer concentrate is within the range of 10 mM and IM, such as 25 mM and 500 mM, such as 50 mM and 400 mM, such as 75 mM and 300 mM, such as 100 mM to 200 mM.
[00117] In some embodiments, a pH of the Tris buffer concentrate is within the range of 7 and 11, such as within the range of 7 and 10, such as within the range of 8 and 11, such as within the range of 8 and 10, such as within the range of 7 and 9.
[00118] In some embodiments, a Tris buffer is added to an acidic reaction mix. In some embodiments, a Tris buffer is added to a reaction mix comprising viral particles, and/or a cell lysate comprising viral particles. In some embodiments, addition of Tris buffer to the reaction mix stops the reaction (e.g., cell lysis reaction).
[00119] In some embodiments, working concentration ranges of the Tris buffer when added to the reaction mix are within 1 mM and 2M. In some embodiments, the concentration of the Tris buffer concentrate is within the range of 10 mM and IM, such as 25 mM and 500 mM, such as 50 mM and 400 mM, such as 75 mM and 300 mM, such as 100 mM to 200 mM.
[00120] In some embodiments, working pH ranges of the composition resulting from the addition of the Tris buffer to the reaction mix are about 7 to about 9, such as within the range of 7 and 10, such as within the range of 8 and 11, such as within the range of 8 and 10, such as within the range of 7 and 9.
[00121] In some embodiments, a composition described herein comprising a Tris buffer and a reaction mix is incubated at ambient temperature. Exemplary ambient temperature ranges include 10°C to 40°C, such as 15°C to 35°C, such as 20°C to 25°C, such as 15°C to 30°C or 15°Cto 25°C.
[00122] In some embodiments, a composition described herein comprising a Tris buffer and a reaction mix is incubated for a predetermined period of time. Exemplary ranges for an incubation time include 0.1 second to 35 minutes, such as 1 second to 20 minutes, such as 2 seconds to 10 minutes, such as 5 seconds to 5 minutes, such as 10 seconds to 1 minute, such as 20 seconds to 40 seconds.
[00123] In some embodiments, nucleic acids prepared according to the present invention are not processed further and/or not used for any downstream reaction. In some embodiments, nucleic acids prepared according to the present invention are processed further and/or are used in downstream reactions.
Downstream Reactions
[00124] In some embodiments, no further nucleic acid isolation is performed prior to analysis of the sample, e.g., to detect nucleic acid(s) (e.g., target nucleic acid(s)) therein. In some embodiments, a sample comprising released nucleic acid is serially diluted after viral particle lysis and prior to analysis of the sample, e.g., to detect nucleic acid(s) (e.g., target nucleic acid(s)) therein. In some embodiments, a sample comprising released nucleic acid is stored. The sample with the prepared nucleic acids comprising the target nucleic acids may be stored in a freezer or a refrigerator.
[00125] In some embodiments, a downstream reaction (e.g., a nucleic acid processing and/or detection reaction) is or comprises one or more of amplification, cleavage, digestion, hybridization, replication, etc.
[00126] In some embodiments, (e.g., a nucleic acid processing and/or detection reaction) is or comprises one or more of SHERLOCK, INSPECTR™’ DETECTR, etc.
Uses and methods
[00127] Compositions, mixtures, methods and kits according to the present disclosure can be used in a number of applications, such as therapeutic and/or non-therapeutic applications. In some embodiments, methods of preparing nucleic acids from a sample and detecting a target nucleic acid in a sample are useful in therapeutic and/or non-therapeutic applications. In some embodiments, compositions, mixtures, methods, kits and uses described herein may be used for non-therapeutic purposes, such as diagnostic purposes. In some embodiments, compositions, mixtures, methods, kits and uses described herein may be used for therapeutic purposes. In some embodiments, the target is an infectious agent. [00128] In some embodiments, the present invention provides technologies (e.g., compositions, mixtures, methods, and/or kits, etc) for preparation of cell lysates comprising nucleic acids of interest - e.g., target nucleic acids. In some embodiments, the present invention provides technologies (e.g., compositions, mixtures, methods, and/or kits, etc) for preparation of viral particle lysates comprising nucleic acids of interest - e.g., target nucleic acids.
[00129] In some embodiments, the disclosure provides a method of preparing nucleic acids from a sample, the method comprising: i) obtaining the sample comprising the nucleic acids from a subject; ii) contacting the sample with a composition comprising: a) LAP AO; and b) HC1; and iii) incubating the mixture of the sample and the composition of step ii), wherein the step of incubating comprises incubating at a temperature within the range of 10°C and 40°C.
[00130] In some embodiments, the concentration of LAP AO is within the range of 0.01% and 10%, wherein the concentration of the HC1 is within the range of 4 mM and 4M, and wherein the pH of the composition is within the range of 0 and 6
[00131] In some embodiments, the disclosure provides a method of preparing nucleic acids from a sample, the method comprising: i) obtaining the sample comprising the nucleic acids from a subject; ii) contacting the sample with a buffered preparation comprising: a) LDAO; b) sodium decanoate; and c) HC1; and iii) incubating the mixture of the sample and the buffered preparation of step ii), wherein the step of incubating comprises incubating at a temperature within the range of 10°C and 40°C.
[00132] In some embodiments, the concentration of LDAO is within the range 0.02% and 4%, wherein the concentration of the sodium decanoate is within the range of 0.02% and 20%, wherein the concentration of the HC1 is within the range of 4 mM and 4M, and wherein the pH of the composition is within the range of 0 and 6
[00133] In some embodiments, a sample may be any sample, such as an example described under the section “sample” and/or may be obtained by any suitable means.
[00134] In some embodiments, a nucleic acid preparation developed by treatment of a sample with a lysis reagent composition as described herein (e.g., comprising a zwitterionic detergent and, in some embodiments, lacking any detergent that is not zwitterionic, is subjected to one or more downstream processing and/or detection processes, for example to achieve: detection of a target (e.g., a target nucleic acid sequence) present in the sample. In some embodiments, detection may accomplish or represent diagnosis, e.g., of a disease, disorder, condition (or stage thereof), or state (e.g., of presence of an infectious agent, or of a mutation or modification event or state, or developmental event or state).
[00135] Alternatively or additionally, in some embodiments, provided technologies are useful for assessment of an environmental sample, such as a food sample (fresh fruits or vegetables, meats), a beverage sample, a paper surface, a fabric surface, a metal surface, a wood surface, a plastic surface, a soil sample, a freshwater sample, a wastewater sample, a saline water sample, exposure to atmospheric air or other gas sample, or a combination thereof. For example, household/commercial/industrial surfaces made of any materials including, but not limited to, metal, wood, plastic, rubber, or the like, may be swabbed and tested for contaminants.
[00136] To give but a few examples, soil samples may be tested for the presence of viral particles or fragments thereof, pathogenic bacteria or parasites, or other microbes, both for environmental purposes and/or for human, animal, or plant disease testing. Water samples such as freshwater samples, wastewater samples, or saline water samples can be evaluated for cleanliness and safety, and/or potability, for example to detect the presence of, for example, viral particles, and/or Cryptosporidium parvum, Giardia lamblia, and/or other microbial contamination. [00137] In some particular embodiments, an environmental sample be a crude sample and/or the one or more target molecules may not be purified or amplified from the sample prior to application of provided technologies.
[00138] Identification of microbes may be useful and/or needed for any number of applications, and thus any type of sample from any source deemed appropriate by one of skill in the art may be used in accordance with the invention.
[00139] In some embodiments, the technologies of the present inventions are useful in genotyping.
Kit
[00140] Provided herein is a kit for performing the present methods. The kit of parts may comprise a composition or a component thereof as described herein.
[00141] In some embodiments, a kit is provided for performing methods of preparing nucleic acids from a sample. In some embodiments, a kit of parts comprises a lysis reagent composition according to the present invention, and/or one or more components thereof. In some embodiments, a kit of parts may comprise a neutralization buffer, a sample collection device, and/or instructions for use.
[00142] In some embodiments, one or more components included in a provided kit is a stock solution as described herein.
[00143] In some embodiments, the kit of parts comprises a positive control. A nonlimiting example of a positive control includes an inactivated envelope virus, optionally at a known concentration or a control nucleic acid, optionally at a known concentration.
[00144] In some embodiments, the kit is provided for performing methods of detecting a target nucleic acid in a sample. In some embodiments, a kit of parts comprises a lysis reagent composition according to the present invention. In some such embodiments, a kit of parts may also comprise a neutralization buffer, a nucleic acid sensor system, a sample collection device, and/or instructions for use. For example, in some particular embodiments, a kit of parts may comprise a nucleic acid sensor system useful for detecting a target nucleic acid. Non-limiting examples of nucleic acid sensor systems such as an INSPECTR™ nucleic acid detection system, SHERLOCK nucleic acid detection system, etc.
[00145] In some embodiments, a kit of parts also comprises a control nucleic acid, such as may be spiked into a sample as described herein.
EXAMPLES
Example 1. Ambient temperature viral particle lysis
[00146] The present Example illustrates effective viral particle lysis achieved at ambient temperature through use of technologies provided herein.
[00147] Figure 1 depicts exemplary lysis and nucleic acid processing steps performed in accordance with the present disclosure. As depicted, a “mock sample” (containing pooled human saliva plus an inactivated intact virus in a matrix of cell lysate) was first treated with a “lysis reagent composition” and then, without traditional processing or “clean-up” steps, it was treated with a “ligation solution”. The cell lysate (matrix) is treated as background.
[00148] The mock sample comprises pooled saliva, BEI y-irradiated SARS-CoV-2 virus, RNAsin, and EDTA. The starting pH of the pooled saliva was 8.4. The BEI y- irradiated virus concentration in the sample was 10,000 cp/ul. The RNAsin concentration in the sample was 1 U/pl (N2511, Promega).
[00149] Two different stock (20x) lysis reagents composition were prepared, each of which included a zwitterionic detergent. Specifically, an LAPAO/HC1 stock solution, and an LDAO/NaClO/HCl stock solution were prepared, as set forth in the following Table:
Table 2:
Figure imgf000038_0001
[00150] The lysis reagent composition was added to the mock sample and the combination was incubated at ambient temperature, which in this case was 22°C, for a short period of time (specifically, 30 seconds in the reactions depicted in Figure 1). The lysis reaction was stopped by adding a neutralization buffer (20x stock solution is 100 mM Tris, pH 9.0). The lysis reaction was performed under different pH conditions, including pH 2.5, pH 4.5, pH 6, pH 6.5, pH 7.5, pH 8 and, pH 10. To achieve the desired pH, the reaction was adjusted with HC1.
[00151] Viral particle lysis/nucleic acid preparations were assessed by measuring Ct value of the samples. Poor to no viral particle lysis was indicated by high Ct values, while optimum viral particle lysis was indicated by lower Ct values.
Positive control
[00152] As a positive control for lysis, an otherwise comparable mock sample comprising pooled saliva, BEI y-irradiated SARS-CoV-2 virus, RNAsin, and EDTA was exposed to high temperature (specifically, was incubated at 95°C for 5 minutes), without addition of the lysis reagent composition (for example LAPAO/HC1 stock solution, or an LDAO/NaClO/HCl solution stock) or other detergents.
Negative controls
[00153] Three different negative controls were prepared:
(i) “No lysis” comprises the mock sample (pooled saliva, BEI y-irradiated SARS- CoV-2 virus, RNAsin, and EDTA) without addition of the lysis reagent composition, including any detergents and HC1. The samples were maintained at ambient temperature. Thus, “No lysis” control samples were neither exposed to detergent(s), HC1 nor heat;
(ii) “HC1” comprises the mock sample (pooled saliva, BEI y-irradiated SARS-CoV-2 virus, RNAsin, and EDTA) without addition of any detergents but it was exposed to HC1. The samples were maintained at ambient temperature. Thus, the “HQ” control samples were neither exposed to detergent(s) nor heat.
(iii) “Neg” or No template control (NTC) comprises only water or a buffer.
[00154] As can be seen, for example, with reference to Figure 2, use of a lysis reagent composition as described herein achieved effective lysis comparable to that achieved with the positive control under heating conditions, at least at pH conditions below about 6.0, and particularly below about 4.5. Figure 2 shows results observed when lysis reactions as were performed under different pH conditions. In particular, Figure 2 shows that lower Ct values were achieved with lower pH values. Thus, the present Example documents particular effectiveness (i.e., better viral particle lysis) of provided lysis reactions at low pH. Greatest levels of lysis were observed at lowest pH (2.5) tested. However, the limit may not have been reached in these studies.
[00155] Among other things, the present Example surprisingly demonstrates that use of a lysis reagent composition such as a zwitterionic detergent (e.g., LAP AO or LDAO) can achieve effective viral particle lysis in a biological sample (e.g., a crude sample, and in particular a saliva sample) at low pH (e.g., below 6.5, and preferably below 4.5 including specifically below about pH 3.0), even at ambient (e.g., about 22°C) temperatures.
Moreover, the achieved lysis can be comparable to heat lysis (e.g., that observed with incubated at 95°C for 5 min).
[00156] Moreover, the present Example surprisingly demonstrates that provided effective lysis conditions can be rapidly and gently neutralized by simple addition of Tris, to generate a lysed composition amenable to further nucleic acid processing or manipulation. Among other things, the present disclosure provides an insight that use of certain zwitterionic detergents (e.g., LAP AO and LDAO) as described herein can achieve a “Goldilocks” effect of sufficient lysis without undesirable inhibition of downstream processing or reaction steps.
[00157] Thus, in many embodiments, subsequent manipulation and/or analysis of nucleic acids prepared by lysis as described herein does not require purification or extraction steps typically required or utilized to remove detergents. Rather, enzymes or other agents (c.g, ligase as depicted in Figure 1, alternatively or additionally, one or more cleavage systems such as CRISPR/Cas, TALEN, Zinc Finger, Restriction Enzyme, etc., and/or one or more hybridization reagents such as oligonucleotides - e.g., probes or primers, etc.) can be added directly to the lysis reaction (e.g, simultaneously or sequentially with any neutralization buffer).
[00158] Thus, the present disclosure provides lysis technologies that are remarkably compatible with nucleic acid processing and/among other things, in some embodiments may permit so-called “one pot” assessments.
Example 2. Reproducibility of viral particle lysis and impact of detergent concentration [00159] The present Example documents reproducibility of viral particle lysis using zwitterionic detergent lysis as described herein. The present Example further documents effect(s) of detergent (in this case LAP AO) concentration on degree of lysis.
[00160] For each detergent composition concentration, four replicates of detergent compositions were prepared using two different detergent dilutions. Positive and negative controls were generated as described in Example 1. For experimental samples, progressively increasing concentration of detergent in a saliva matrix was used to prepare the lysis reaction mix. Each detergent composition was mixed with a sample and incubated as described previously. The following final LAP AO concentrations were tested 0.009%, 0.018%, 0.0357%, 0.075%, 0.15%, 0.3% LAP AO at ambient temperature, which in this case was 22°C. Viral particle lysis was then assessed.
[00161] Figures 3 and 4 document reproducibility of viral particle lysis achieved by provided technologies, across the four replicates and across detergent concentration. Figure 3 furthermore demonstrates that LAP AO detergent concentrations between about 0.03% and about 0.3% (e.g., specifically between 0.0375% and 0.3%, inclusive) achieved better lysis than the lower LAP AO detergent concentrations ((e.g., 0.018% and below).
[00162] Figure 3 also documents that the combination of a detergent, such as LAP AO detergent concentrations between about 0.03% and about 0.3% (e.g., specifically between 0.0375% and 0.3%, inclusive), and HC1 (i.e., pH) produces viral particle lysis that is more effective than HC1 alone. As shown in Figure 3, addition of HC1 alone to the sample (HC1 column) produces viral particle lysis that is less effective than the positive control (heat lysis), and that is comparable to the “No lysis” control.
Example 3. Viral particle lysis in water
[00163] The present Example documents surprising versatility of provided lysis technologies, including that they can achieve effective lysis in a context other than saliva, and specifically in water.
[00164] Examples 1-2 herein documented effective lysis using provided technologies in a sample comprising saliva. Example 3 is performed using the same conditions as for Examples 1 and 2, i.e. wherein the lysis is performed at 95°C for the heat lysis and 30 seconds exposure to lysis reagent at ambient temperature, in this case 22°C. The present Example demonstrates that presence of saliva is not required to achieve lysis and, moreover, demonstrates effectiveness of provided technologies even in water or other aqueous systems.
[00165] Specifically, Figure 5 presents results achieved when different concentrations of zwitterionic detergents, as indicated, were utilized to lyse nasal swabs eluted in water. As can be seen, both LAP AO and LDAO/NaClO achieved significant lysis, including lysis comparable to or better than that achieved with heat lysis, at various concentrations. Lysis was measured at pH 2.4.
[00166] The present Example demonstrates that use of a lysis reagent composition such as a zwitterionic detergent (e.g., LAP AO or LDAO) can achieve effective viral particle lysis in a water sample.
Example 4. Compatibility of provided lysis technologies with downstream nucleic acid processing and/or analysis
[00167] The present Example documents one particularly surprising and useful feature of technologies provided herein - that they achieve preparation of nucleic acids (e.g., by release from lysed viral particles) under conditions that are amenable so downstream nucleic acid processing and/or analysis, for example without extensive isolation and/or “cleaning” steps.
[00168] For example, Figure 6 documents end-to-end compatibility of provided technologies with an INSPECTR™ nucleic acid detection system.
[00169] As shown in Figure 6, in some embodiments a method as described herein can be used to prepare nucleic acids for use in detection of a target nucleic acid, e.g., with an INSPECTR™ nucleic acid detection system as described in applications W02020037038 and PCT/US2021/046438, each of which are incorporated herein by reference.
[00170] Specifically, in the reactions presented in Figure 6, various concentrations of the nucleic acids prepared using the indicated lysis conditions (i.e., no detergent, 0.025% LDAO/ 0.06% NaClO, 0.0375% LAPAO-HC1, or 0.3% LAPAO-HC1), were used as targets in an INSPECTR™ reaction. No nucleic acid purification was performed post-lysis. Lateral flow was used as a readout of the reaction. The results showed that a negative control “NTC” band was detected for all controls. Further, for each of the three detergent compositions, LDAO/NaClO-HCl, 0.0375% LAPAO-HC1, and 0.3% LAPAO-HC1 shown, the target nucleic acid (RNA) was detected, and the polypeptide or reporter protein generated was detected at an intensity comparable to that of the “no detergent” condition.
[00171] The provided technologies achieved target detection with remarkable sensitivity. This finding confirmed that provided lysis technologies did not impact the sensitivity of the target detection. Furthermore, the intensity of the NTC bands did not increase as compared to the “no detergent” condition, indicating that provided lysis technologies generated a nucleic acid preparation in which target sequence(s) are detected with high specificity.
Example 5. Downstream processing
[00172] In some embodiments, a provided nucleic acid preparation does not undergo further processing technologies before nucleic acid processing and/or analysis is performed.
[00173] In some embodiments, a provided nucleic acid preparation is amenable to direct nucleic acid processing and/or analysis with one or more of a variety of technologies (e.g., analytic technologies such as detection technologies). Those skilled in the art are aware of a variety of nucleic acid processing and/or detection technologies and/or assays, and are familiar with reagents useful or required to perform step(s) of such nucleic acid processing and/or detection technologies. In some embodiments, one or more of such reagents can be added directly to a viral particle lysis reaction (e.g., simultaneously or sequentially with any neutralization buffer) described herein. In some embodiments, the reagents can be added directly to the same reaction vessel, so that the present disclosure provides technologies for so-called “one pot” assessments (e.g., one-pot lysis and nucleic acid assessment - e.g., processing and/or detection).
[00174] In some embodiments, a nucleic acid assessment (e.g., a nucleic acid processing and/or detection) involves and/or utilizes one or more of amplification, cleavage, digestion, hybridization, ligation, replication, reverse transcription, etc., and/or one or more of, for example a detection system, such as, but not limited to SHERLOCK, INSPECTR™, a combinations thereof, etc. [00175] Ligation: In some embodiments, methods as described herein can be used to prepare nucleic acids for use in e.g., ligation. In some embodiments, reagents for nucleic acid ligation may comprise one or more ligases, buffers, and probes; in some embodiments one or more of such reagents can be added to a provided nucleic acid preparation, for example simultaneously or sequentially with a neutralization buffer. A ligation step is shown in Figure 1. In Figure 1, the ligase buffer (ligase, ET SSB, probes) is added simultaneously with the neutralization buffer (Tris pH 9.0) to the lysis reaction mixture for 35 min at 22°C.
[00176] Amplification: In some embodiments, methods as described herein can be used to prepare nucleic acids for use in e.g., amplification, reagents for nucleic acid amplification comprise one or more primer sets, a polymerase, a reverse transcriptase, and nucleotides can be added to a provided nucleic acid preparation, for example simultaneously or sequentially with a neutralization buffer.
[00177] Replication: In some embodiments, methods as described herein can be used to prepare nucleic acids for use in e.g., replication. In some embodiments, reagent(s) for nucleic acid replication, such as one or more primer sets, a polymerase, buffer(s), and/or nucleotides can be added to the prepared nucleic acids simultaneously or sequentially with any neutralization buffer.
[00178] Nucleic acid hybridization: In some embodiments, methods as described herein can be used to prepare nucleic acids for use in (e.g., for analysis and/or characterization, e.g., detection, by) e.g., nucleic acid hybridization.
[00179] SHERLOCK: In some embodiments, methods as described herein can be used to prepare nucleic acids for use in detection of a target nucleic acid, e.g., with an SHERLOCK-type nucleic acid detection system as described in applications WO2017/218573, W02018/107129, WO2019/011022, W02019051318, W02019/104058, WO2020191376 (which describes a combination with an INSPECTR-type system) etc., each of which are incorporated herein by reference. In some embodiments, detection of target sequences within provided nucleic acid preparation(s) utilize one or more CRISPR/Cas enzymes. In some embodiments, such detection may involve use of one or more guide polynucleotides that recognize and bind a target sequence of interest. Example 6. Synergistic effect
[00180] The present Example documents that the effect of a detergent (e.g., LAP AO) in combination with HC1 compared to a detergent alone (e.g., LAP AO) on viral particle lysis is more effective that HC1 alone.
[00181] Figure 7 shows that treating the sample with a detergent alone (e.g., LAP AO) in the absence of HC1 produces viral particle lysis that is less effective than the positive control (Heat lysis). In Figure 7, it can also be seen that optimal viral particle lysis is achieved when the samples are exposed to both a detergent (e.g., LAP AO) and HC1.
[00182] A synergistic effect between detergent concentration and HC1 is observed (Figures 3 and 7), as HC1 alone does not have an effect on viral particle lysis (Figure 3) and a detergent alone (e.g., LAP AO) is less effective that the combination of a detergent and HC1 (Figure 7). Thus, a synergistic effect between the detergent and HC1 results in effective lysis comparable to that of a control (Heat lysis).

Claims

Claims We claim:
1. A composition comprising: i) zwitterionic detergent; and ii) HC1, wherein the concentration of the zwitterionic detergent is within the range of 0.01% and 10%, wherein the concentration of the HC1 is within the range of 4 mM and 4M, and wherein the pH of the composition is within the range of 0 and 6.
2 The composition of claim 1, wherein the composition further comprises sodium decanoate.
3. The composition of claim 1, wherein the zwitterionic detergent is selected from the group consisting of LAP AO, LDAO, and DDAO.
4. A lysis reaction mix comprising: i) sample; ii) zwitterionic detergent; and iii) HC1.
5. The reaction mix of claim 4, wherein the sample is a biological sample.
6. The reaction mix of claim 4, wherein the sample is or comprises a viral particle or fragments thereof.
7. The reaction mix of claim 4, wherein the sample comprises a viral particle within a cell, such as a human or mammalian cell.
8. The reaction mix of claim 5, wherein the biological sample comprises a viral particle or fragment thereof within a cell or cell component.
9. The reaction mix of claim 4, wherein the concentration of the zwitterionic detergent is within the range of 0.01% and 10%.
10. The reaction mix of claim 4, wherein the concentration of the concentration of the HC1 is within the range of 4 mM and 4M.
11. The reaction mix of claim 4, wherein the pH of the composition is within the range of 0 and 6.
12. The reaction mix of claim 4, wherein the composition further comprises NaClO.
13. The reaction mix of claim 4, wherein the zwitterionic detergent is selected from the group consisting of LAP AO, LDAO, and DDAO.
14. A composition comprising: i) LAP AO; and ii) HC1, wherein the concentration of the LAP AO is within the range of 0.01% and 10%, wherein the concentration of the HC1 is within the range of 4 mM and 4M, and wherein the pH of the composition is within the range of 0 and 6.
15. The composition of claim 14, wherein the concentration of the LAP AO is within the range of 0.1% and 5%.
16. The composition of claim 14, wherein the concentration of the LAP AO is within the range of 1% and 3%.
17. The composition of claim 14, wherein the concentration of the LAP AO in the composition is 2%.
18. The composition of claim 14, wherein the concentration of the HC1 is within the range of 40 mM and 2M.
19. The composition of claim 14, wherein the concentration of the HC1 is within the range of 100 mM and IM.
20. The composition of claim 14, wherein the concentration of the HC1 is within the range of 200 mM and 600 mM.
21. The composition of claim 14, wherein the concentration of the HC1 in the composition is 400 mM.
22. The composition of claim Al 14 wherein the pH of the composition is within the range of 1 and 5.
23. The composition of claim 15, wherein the pH of the composition is within the range of 1 and 4.
24. The composition of claim 16, wherein the pH of the composition is within the range of 2 and 3.
25. The composition of claim 17, wherein the pH of the composition is 2.5.
26. A lysis reaction mix comprising: i) a sample; ii) LAP AO; and iii) HC1.
27. The reaction mix of claim 26, wherein the sample is a biological sample.
28. The reaction mix of claim 27, wherein the biological sample is or comprises a viral particle or fragments thereof.
29. The reaction mix of claim 28, wherein the biological sample further comprises cells or cell components selected from plant, microbial, animal, and human.
30. The reaction mix of claim 28, wherein the biological sample comprises a viral particle or fragment thereof within a cell or cell component.
31. The reaction mix of claim 27, wherein the biological sample is selected from nasal swab, nasopharyngeal swab, oropharyngeal swab, nasal aspirate, sputum, bronchoalveolar lavage, blood, serum, feces, and saliva samples.
32. The reaction mix of claim 27, wherein the biological sample is a crude sample.
33. The reaction mix of claim 32, wherein the crude sample is selected from amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humour, vomit, intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, a transcellular fluid and/or combinations or component(s) thereof.
34. The reaction mix of claim 26, wherein the sample is a processed sample.
35. The reaction mix of claim 29 or 30, wherein the cells are human cells.
36. The reaction mix of claim 26, wherein the concentration of the LAP AO is within the range of 0.01% and 10%.
37. The reaction mix of claim 26, wherein the concentration of the LAP AO is within the range of 0.001% and 1%.
38. The reaction mix of claim 37, wherein the concentration of the LAP AO is within the range of 0.01% and 0.5%.
39. The reaction mix of claim 38, wherein the concentration of the LAP AO is within the range of 0.09% and 0.2%.
40. The reaction mix of claim 39, wherein the concentration of the LAP AO in the composition is 0.1%.
41. The reaction mix of claim 26, wherein the concentration of the HC1 is within the range of 0.4 mM and 400 mM.
42. The reaction mix of claim 41, wherein the concentration of the HC1 is within the range of 1 mM and 100 mM.
43. The reaction mix of claim 42, wherein the concentration of the HC1 is within the range of 10 mM and 50 mM.
44. The reaction mix of claim 43, wherein the concentration of the HC1 is within the range of 15 mM and 25 mM.
45. The reaction mix of claim 44, wherein the concentration of the HC1 is 20 mM.
46. The reaction mix of claim 26, wherein the pH of the mix is within the range of 0 and 6.
47. A composition comprising: i) LDAO; ii) sodium decanoate; and iii) HC1.
48. The composition of claim 47, wherein the concentration of the LDAO is within the range of 0.02% and 4%.
49. The composition of claim 48, wherein the concentration of the LDAO is within the range of 0.09% and 2%.
50. The composition of claim 49, wherein the concentration of the LDAO is within the range of 0.2% and 1%.
51. The composition of claim 50, wherein the concentration of the LDAO is 0.5%.
52. The composition of claim 47, wherein the concentration of the sodium decanoate is within the range of 0.02% and 20%.
53. The composition of claim 52, wherein the concentration of the sodium decanoate is within the range of 0.2% and 10%.
54. The composition of claim 53, wherein the concentration of the sodium decanoate is within the range of 1% and 2%.
55. The composition of claim 54, wherein the concentration of sodium decanoate is 1.2%.
56. The composition of claim 26, wherein the concentration of the HC1 is within the range of 4 mM and 4M.
57. The composition of claim 56, wherein the concentration of the HC1 is within the range of 40 mM and 2M.
58. The composition of claim 57, wherein the concentration of the HC1 is within the range of 100 mM and IM.
59. The composition of claim 58, wherein the concentration of the HC1 is within the range of 200 mM and 600 mM.
60. The composition of claim 59, wherein the concentration of the HC1 in the composition is 400 mM.
61. The composition of claim 47, wherein the pH is within the range of 0 and 6.
62. The composition of claim 61, wherein the pH is within the range of 1 and 5.
63. The composition of claim 62, wherein the pH is within the range of 1 and 4.
64. The composition of claim 63, wherein the pH is within the range of 2 and 3.
65. The composition of claim 64, wherein the pH is 2.5.
66. A lysis reaction mix comprising: i) a sample; ii) LDAO; iil) sodium decanoate; and iv) HC1.
67. The reaction mix of claim 66, wherein the sample is a biological sample.
68. The reaction mix of claim 66, wherein the biological sample is or comprises a viral particle or fragment thereof.
69. The reaction mix of claim 68, wherein the biological sample further comprises cells or cell components selected from plant, microbial, animal, and human.
70. The reaction mix of claim 67, wherein the biological sample comprises a viral particle or fragment thereof within a cell or cell component.
71. The reaction mix of claim 67, wherein the biological sample is selected from nasal swab, nasopharyngeal swab, oropharyngeal swab, nasal aspirate, sputum, bronchoalveolar lavage, blood, serum, feces, and saliva samples.
72. The reaction mix of claim 67, wherein the biological sample is a crude sample.
73. The reaction mix of claim 67, wherein the biological sample include cells or cell components selected from plant, microbial, animal, and human.
74. The reaction mix of claim 72, wherein the crude sample is selected from amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humour, vomit, intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, a transcellular fluid and/or combinations or component(s) thereof.
75. The reaction mix of claim 66, wherein the sample is a processed sample.
76. The reaction mix of claim 69 or 70, wherein the cells are human cells.
77. The reaction mix of claim 66, wherein the concentration of the LDAO is within the range of 0.002% and 0.2%.
78. The reaction mix of claim 77, wherein the concentration of the LDAO is within the range of 0.01% and 0.1%.
79. The reaction mix of claim 78, wherein the concentration of the LDAO is within the range of 0.02% and 0.04%.
80. The reaction mix of claim 79, wherein the concentration of the LDAO is 0.025%.
81. The reaction mix of claim 66, wherein the concentration of the sodium decanoate is within the range of 0.001% and 1%.
82. The reaction mix of claim 81, wherein the concentration of the sodium decanoate is within the range of 0.005% and 0.5%.
83. The reaction mix of claim 82, wherein the concentration of the sodium decanoate is within the range of 0.01% and 0.1%.
84. The reaction mix of claim 83, wherein the concentration of sodium decanoate is 0.06%.
85. The reaction mix of claim 66, wherein the concentration of the HC1 is within the range of 0.4 mM and 400 mM.
86. The reaction mix of claim 85, wherein the concentration of the HC1 is within the range of 1 mM and 100 mM.
87. The reaction mix of claim 86, wherein the concentration of the HC1 is within the range of 10 mM and 50 mM.
88. The reaction mix of claim 87, wherein the concentration of the HC1 is within the range of 15 mM and 25 mM.
89. The reaction mix of claim 88, wherein the concentration of the HC1 is 20 mM.
90. A composition comprising: i) products of a lysis reaction; and ii) a neutralization buffer.
91. The composition of claim 90, wherein the composition comprises one or more nucleic acids, viral particle lysate components, reagents for nucleic acid amplification, enzymes, reagents for lysing cells, and reagents for neutralizing lysis reagents.
92. The composition of claim 91, wherein the one or more nucleic acids are DNA and RNA.
93. The composition of claim 91, wherein the viral particle lysate components comprise crude viral particle lysate.
94. The composition of claim 93, wherein the composition further comprises ET SSB and probes.
95. The composition of claim 91, wherein the reagents for nucleic acid amplification comprise one or more primer sets, a polymerase, a reverse transcriptase, and nucleotides.
96. The composition of claim 90, wherein the composition comprises ligases.
97. The composition of claim 90, wherein the neutralization buffer comprise Tris.
98. The composition of claim 97, wherein the concentration of the Tris is within the range of 0.5 mM and 100 mM.
99. The composition of claim 98, wherein the concentration of the Tris is within the range of 5 mM and 80 mM.
100. The composition of claim 99, wherein the concentration of the Tris is 20 mM.
101. The composition of claim 90, wherein the pH of the composition is within the range of 3 and 11.
102. The composition of claim 101, wherein the pH of the composition is within the range of 4 and 10.
103. The composition of claim 102, wherein the pH of the composition is within the range of 5 and 9.
104. The composition of claim 103, wherein the pH of the composition is within the range of 6 and 8.
105. The composition of claim 104, wherein the pH of the composition is 7.
106. A method of preparing nucleic acids from a sample, the method comprising: i) obtaining the sample comprising the nucleic acids from a subject; ii) contacting the sample with a composition comprising: a) LAP AO; and b) HC1, and iii) incubating the mixture of the sample and the composition of step ii), wherein the step of incubating comprises incubating at a temperature within the range of 10°C and 40°C.
107. The method of claim 106, wherein the concentration of the LAP AO is within the range of 0.01% and 10%.
108. The method of claim 106, wherein the concentration of the HC1 is within the range of 4 mM and 4M.
109. The method of claim 106, wherein the pH of the composition is within the range of 0 and 6.
110. The method of claim 106, wherein the sample is a biological sample.
111. The method of claim 106, wherein the sample is or comprises a viral particle or fragments thereof.
112. The method of claim 111, wherein the biological sample comprises cells or cell components selected from plants, microbes, animals, and humans.
113. The method of claim 111, wherein the biological sample comprises a viral particle or fragment thereof within a cell or cell component.
114. The method of claim 106, wherein the sample is selected from amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humour, vomit, intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, a transcellular fluid and/or combinations or component(s) thereof.
115. The method of claim 114, wherein the cells are human cells.
116. The method of claim 106, wherein the step of incubating comprises incubating at a temperature within the range of 15°C and 35°C.
117. The method of claim 116, wherein the step of incubating comprises incubating at a temperature within the range of 17°C and 30°C.
118. The method of claim 117, wherein the step of incubating comprises incubating at a temperature within the range of 20°C and 25°C.
119. The method of claim 118, wherein the step of incubating comprises incubating at 22°C.
120. The method of claim 106, wherein the step of incubating is performed for a period of time within a range of 0.1 second to 5 minutes.
121. The method of claim 120, wherein the step of incubating is performed for a period of time within a range of 1 second to 4 minutes.
122. The method of claim 121, wherein the step of incubating is performed for a period of time within a range of 10 seconds to 3 minutes.
123. The method of claim 122, wherein the step of incubating is performed for a period of time within a range of 20 seconds to 2 minutes.
124. The method of claim 106, wherein the step of incubating is performed for a period of time within a range of 25 seconds to 1 minute.
125. The method of claim 124, wherein the step of incubating is performed for 30 seconds.
126. The method of claim 106 further comprising a step of neutralizing, wherein the step of neutralizing comprises incubating the mixture of the sample and the buffered preparation of step iii) with a neutralization buffer.
127. The method of claim 126, wherein the neutralization buffer comprises Tris.
128. A method of preparing nucleic acids from a sample, the method comprising: i) obtaining the sample comprising the nucleic acids from a subject; ii) contacting the sample with a buffered preparation comprising: a) LDAO; b) sodium decanoate; and c) HC1, iii) incubating the mixture of the sample and the buffered preparation of step ii), wherein the step of incubating comprises incubating at a temperature within the range of 10°C and 40°C.
129. The method of claim 128, wherein the concentration of the LDAO is within the range of 0.02% and 4%.
130. The method of claim 128, wherein the concentration of the sodium decanoate is within the range of 0.02% and 20%.
131. The method of claim 128, wherein the concentration of the HC1 is within the range of 4 mM and 4M.
132. The method of claim 128, wherein the pH of the composition is within the range of 0 and 6.
133. The method of claim 128, wherein the sample is a biological sample.
134. The method of claim 128, wherein the sample is or comprises a viral particle or fragments thereof.
135. The method of claim 134, wherein the sample comprises cells or cell components selected from plants, microbes, animals, and humans.
136. The method of claim 134, wherein the sample comprises a viral particle or fragment thereof within a cell or cell component.
137. The method of claim 128, wherein the sample is selected from amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humour, vomit, intracellular fluid, an extracellular fluid, an intravascular fluid (blood plasma), an interstitial fluid, a lymphatic fluid, a transcellular fluid and/or combinations or component(s) thereof.
138. The method of claim G135 or 136 wherein the cells are human cells.
139. The method of claim 128, wherein the step of incubating comprises incubating at a temperature within the range of 15°C and 35°C.
140. The method of claim 139, wherein the step of incubating comprises incubating at a temperature within the range of 17°C and 30°C.
141. The method of claim 140, wherein the step of incubating comprises incubating at a temperature within the range of 20°C and 25°C.
142. The method of claim 141, wherein the step of incubating comprises incubating at 22°C.
143. The method of claim 128, wherein the step of incubating is performed for a period of time within a range of 0.1 second to 5 minutes.
144. The method of claim 143, wherein the step of incubating is performed for a period of time within a range of 1 second to 4 minutes.
145. The method of claim 144, wherein the step of incubating is performed for a period of time within a range of 10 seconds to 3 minutes.
146. The method of claim 145, wherein the step of incubating is performed for a period of time within a range of 20 seconds to 2 minutes.
147 The method of claim 146, wherein the step of incubating is performed for a period of time within a range of 25 seconds to 1 minute.
148. The method of claim 147, wherein the step of incubating is performed for 30 seconds.
149. The method of claim 128 further comprising a step of neutralizing, wherein the step of neutralizing comprises incubating the mixture of the sample and the buffered preparation of step iii) with a neutralization buffer.
150. The method of claim 149, wherein the neutralization buffer comprises Tris.
151. A method for detecting a target nucleic acid in a sample, the method comprising: i) providing a sample comprising the target nucleic acid; ii) contacting the sample comprising the target nucleic acid with the composition of any of claims 1-3, 14-25 or 47-65; iii) incubating the reaction product of step ii) at ambient temperature within a range of 5°C to 50°C for a period of time within a range of 0.1 second to 5 minutes; iv) contacting the reaction product of step iii) with a neutralization buffer, wherein the neutralization buffer comprises Tris; v) contacting the reaction product of step iv) with a nucleic acid sensor system comprising at least a first nucleic acid sensor part and a second nucleic acid sensor part.
152. The method of claim 151, wherein the nucleic acid sensor system comprising at least a first nucleic acid sensor part and a second nucleic acid sensor part, wherein: a) the first nucleic acid sensor part comprises:
1) a sequence that is, encodes, or templates coding of, at least one first reporting element component; and
2) a first target hybridization element; and b) the second sensor part comprises:
1) a sequence that is, encodes, or templates coding of, at least one second reporting element component; and
2) a second target hybridization element, wherein: the first and second target hybridization elements are related to one another in that, when the system is contacted with a sample comprising a target nucleic acid, hybridization of the target nucleic acid with both of the first and second target hybridization elements juxtaposes the first and second nucleic acid sensor parts with one another so that the juxtaposed parts are susceptible to linkage by one or more of: c) ligation to generate a ligation product; d) templated copying to generate a linked template product, vi) contacting the solution of step v) with nucleic acid amplification solution; vii) contacting the reaction product of step vi) with a cell-free expression system in the presence of a strand displacing DNA Polymerase and a primer, under conditions favorable to the production of a reporter protein; viii) contacting reaction product of step vii) with a reagent enabling the detection of the expression of the reporter protein; ix) measuring the expression of the reporter protein produced in step viii) to determine the presence and/or amount of the target nucleic acid in the sample.
153. A method for detecting a target nucleic acid in a sample, the method comprising: i) incubating the lysis reaction mix of claims 4-13, 26-46 or 66-89 at ambient temperature within a range of 5°C to 50°C for a period of time within a range of 0.1 second to 5 minutes; ii) contacting the reaction product of step i) with a neutralization buffer, wherein the neutralization buffer comprises Tris; iii) contacting the reaction mix of step ii) with a nucleic acid sensor system, comprising at least a first nucleic acid sensor part and a second nucleic acid sensor part.
154. The method of claim 153, wherein the nucleic acid sensor system comprising at least a first nucleic acid sensor part and a second nucleic acid sensor part, wherein: a) the first nucleic acid sensor part comprises:
1) a sequence that is, encodes, or templates coding of, at least one first reporting element component; and
2) a first target hybridization element; and b) the second sensor part comprises:
1) a sequence that is, encodes, or templates coding of, at least one second reporting element component; and
2) a second target hybridization element, wherein: the first and second target hybridization elements are related to one another in that, when the system is contacted with a sample comprising a target nucleic acid, hybridization of the target nucleic acid with both of the first and second target hybridization elements juxtaposes the first and second nucleic acid sensor parts with one another so that the juxtaposed parts are susceptible to linkage by one or more of: c) ligation to generate a ligation product; d) templated copying to generate a linked template product, iv) contacting the reaction product of step iii) with nucleic acid amplification solution; v) contacting the reaction product of step iv) with a cell-free expression system in the presence of a strand displacing DNA Polymerase and a primer, under conditions favorable to the production of a reporter protein; vi) contacting reaction product of step v) with a reagent enabling the detection of the expression of the reporter protein; vii) measuring the expression of the reporter protein produced in step vi) to determine the presence and/or amount of the target nucleic acid in the sample.
155. A method for detecting a target nucleic acid in a sample, the method comprising: SHERLOCK method.
156. A method for detecting a target nucleic acid in a sample, the method comprising: SHERLOCK/INSPECTR™ combination method such as is described, for example, in WO2020191376.
157. A kit compri sing i) a composition of any of the claims 1-3, 14-25 or 47-65; ii) a sample collection device; and iii) instructions for use.
158. The kit of claim 157, wherein the kit further comprises a neutralization buffer.
159. The kit of claim 157 or 158, wherein the kit further comprises a nucleic acid sensor system.
160; The kit of claim 157, wherein the kit further comprises a positive control.
161. The kit of claim 158, wherein the neutralization buffer comprises Tris.
162. The kit of claim 159, wherein the nucleic acid sensor system comprising at least a first nucleic acid sensor part and a second nucleic acid sensor part, wherein: a) the first nucleic acid sensor part comprises:
1) a sequence that is, encodes, or templates coding of, at least one first reporting element component; and
2) a first target hybridization element; and b) the second sensor part comprises:
1) a sequence that is, encodes, or templates coding of, at least one second reporting element component; and
2) a second target hybridization element, wherein: the first and second target hybridization elements are related to one another in that, when the system is contacted with a sample comprising a target nucleic acid, hybridization of the target nucleic acid with both of the first and second target hybridization elements juxtaposes the first and second nucleic acid sensor parts with one another so that the juxtaposed parts are susceptible to linkage by one or more of: c) ligation to generate a ligation product; d) templated copying to generate a linked template product, vi) contacting the solution of step v) with nucleic acid amplification solution; vii) contacting the reaction product of step vi) with a cell-free expression system in the presence of a strand displacing DNA Polymerase and a primer, under conditions favorable to the production of a reporter protein; viii) contacting reaction product of step vii) with a reagent enabling the detection of the expression of the reporter protein; ix) measuring the expression of the reporter protein produced in step viii) to determine the presence and/or amount of the target nucleic acid in the sample.
PCT/US2023/026778 2022-07-01 2023-06-30 Nucleic acid preparation WO2024006548A1 (en)

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