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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L9/00—Disinfection, sterilisation or deodorisation of air
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  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Disinfection or sterilisation of materials or objects, in general; Accessories therefor
  • A61L2/16—Disinfection or sterilisation of materials or objects, in general; Accessories therefor using chemical substances
  • A61L2/18—Liquid substances
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  • A61L2/00—Disinfection or sterilisation of materials or objects, in general; Accessories therefor
  • A61L2/16—Disinfection or sterilisation of materials or objects, in general; Accessories therefor using chemical substances
• • A—HUMAN NECESSITIES
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  • A61L9/00—Disinfection, sterilisation or deodorisation of air
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  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L9/00—Disinfection, sterilisation or deodorisation of air
  • A61L9/14—Disinfection, sterilisation or deodorisation of air using sprayed or atomised substances including air-liquid contact processes
  • A61L9/145—Disinfection, sterilisation or deodorisation of air using sprayed or atomised substances including air-liquid contact processes air-liquid contact processes, e.g. scrubbing
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
  • C08B37/0045—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Galacturonans, e.g. methyl ester of (alpha-1,4)-linked D-galacturonic acid units, i.e. pectin, or hydrolysis product of methyl ester of alpha-1,4-linked D-galacturonic acid units, i.e. pectinic acid; Derivatives thereof
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  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
  • C08L5/06—Pectin; Derivatives thereof
• • 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
  • C11D17/00—Detergent materials or soaps characterised by their shape or physical properties
  • C11D17/04—Detergent materials or soaps characterised by their shape or physical properties combined with or containing other objects
• • 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
  • C11D17/00—Detergent materials or soaps characterised by their shape or physical properties
  • C11D17/04—Detergent materials or soaps characterised by their shape or physical properties combined with or containing other objects
  • C11D17/049—Cleaning or scouring pads; Wipes
• • 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
  • C11D7/00—Compositions of detergents based essentially on non-surface-active compounds
  • C11D7/22—Organic compounds
  • C11D7/26—Organic compounds containing oxygen
  • C11D7/268—Carbohydrates or derivatives thereof
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  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D7/00—Compositions of detergents based essentially on non-surface-active compounds
  • C11D7/22—Organic compounds
  • C11D7/32—Organic compounds containing nitrogen
  • C11D7/329—Carbohydrate or derivatives thereof
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2101/00—Chemical composition of materials used in disinfecting, sterilising or deodorising
  • A61L2101/32—Organic compounds
  • A61L2101/46—Macromolecular compounds
  • A61L2101/50—Polysaccharides or derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2103/00—Materials or objects being the target of disinfection or sterilisation
  • A61L2103/05—Living organisms or biological materials
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2103/00—Materials or objects being the target of disinfection or sterilisation
  • A61L2103/15—Laboratory, medical or dentistry appliances, e.g. catheters or sharps
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2209/00—Aspects relating to disinfection, sterilisation or deodorisation of air
  • A61L2209/20—Method-related aspects
  • A61L2209/21—Use of chemical compounds for treating air or the like
• • 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
  • C11D2111/00—Cleaning compositions characterised by the objects to be cleaned; Cleaning compositions characterised by non-standard cleaning or washing processes
  • C11D2111/10—Objects to be cleaned
• • 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
  • C11D2111/00—Cleaning compositions characterised by the objects to be cleaned; Cleaning compositions characterised by non-standard cleaning or washing processes
  • C11D2111/10—Objects to be cleaned
  • C11D2111/14—Hard surfaces
  • C11D2111/20—Industrial or commercial equipment, e.g. reactors, tubes or engines

Definitions

• sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification.
• the name of the text file containing the sequence listing is 70l34_Seq_Final_20l9-08-l4.txt.
• the text file is 2.23 KB; was created on August 14, 2019; and is being submitted via EFS-Web with the filing of the specification.
• the invention relates to methods and cleaning compositions for reduction of nucleic acid contamination on surfaces, in air, and in solutions.
• nucleic acid amplification-based techniques such as polymerase chain reaction (PCR)
• PCR polymerase chain reaction
• the high sensitivity of these techniques makes them vulnerable to contamination.
• Nucleic acid cross-contamination in the laboratory presents a serious problem for highly sensitive amplification-based assays.
• the repeated amplification of the target sequence itself, which leads to accumulation of amplification products, or so called amplicons, in the laboratory environment, is of the largest sources of cross-contamination.
• Prevention of amplicon carryover and/or sterilization of the generated amplicons by destroying them or rendering them ineligible for amplification are critical in molecular biology and diagnostic applications.
• a method of reducing nucleic acid contamination on a surface comprising contacting a surface contaminated with a nucleic acid with a composition comprising a modified pectin, wherein the modified pectin comprises a plurality of amino groups.
• the modified pectin is an amidated pectin. In some embodiments, the modified pectin comprises one or more monomeric units represented by Formula:
• n 0, 1, 2, or 3;
• R 1 is H or C J -C3 alkyl
• X at each occurrence, is independently C 2 -C 4 alkylene or C 4 -C 6 heteroalkylene;
• Y is a C 2 -C 3 alkylene or C 4 -C 6 heteroalkylene
• R 2 and R 3 are independently H or C j -C 3 alkyl.
• the molecular weight of the amidated pectin is between about 0.5 kDa and about 500 kDa or between about 100 kDa and about 300 kDa.
• the amidated pectin is in solution at a concentration of about 0.001% to about 5%, about 0.01% to about 1%, or about 0.1% and about 0.5%. In some embodiments, the amidated pectin is in solution with a concentration of about 0.1 pg/mL to about 1,000 pg/mL, about 1 pg/mL to about 500 pg/mL, or about 1 pg/mL to about 100 pg/mL.
• the amidated pectin is amidated citrus pectin or amidated apple pectin.
• the composition is present on a swab, wipe, cloth, filter, pad, or sponge.
• the surface is a surface of an instrument or a laboratory bench surface.
• a method for reducing nucleic acid contamination in a solution comprising: contacting a solution contaminated with a nucleic acid with a solid support comprising a modified pectin covalently bound thereto, wherein the modified pectin comprises a plurality of amino groups.
• the modified pectin is an amidated pectin.
• the modified pectin e.g., amidated pectin, comprises one or more monomeric units represented by Formula:
• n 0, 1,2, or 3;
• R 1 is H or C j -C3 alkyl
• X at each occurrence, is independently C2-C4 alkylene or C4-C6 heteroalkylene
• Y is a C 2 -C 3 alkylene or C 4 -C 6 heteroalkylene
• R 2 and R 3 are independently H or C 3 -C3 alkyl.
• the solid support is a magnetic bead, glass bead, polystyrene bead, polystyrene filter, or glass filter. In some embodiments, the solid support is a swab, wipe, cloth, filter, or sponge.
• method of reducing aerosolized nucleic acid contamination in air comprising contacting air contaminated with aerosolized nucleic acid with a composition comprising a modified pectin, wherein the modified pectin comprises a plurality of amino groups.
• the modified pectin is an amidated pectin.
• the modified pectin e.g., amidated pectin, comprises one or more monomeric units represented by Formula:
• n 0, 1, 2, or 3;
• R 1 is H or C J -C3 alkyl
• X at each occurrence, is independently C 2 -C 4 alkylene or C 4 -C 6 heteroalkylene;
• Y is a C2-C3 alkylene or C 4 -Cg heteroalkylene
• R 2 and R 3 are independently H or C l -C 3 alkyl.
• the contacting comprises passing the contaminated air through an aqueous composition comprising the modified pectin, e.g. a solution or suspension of the amidated pectin in water. In some embodiments, the contacting comprises passing the contaminated air through a filter comprising the amidated pectin. In some embodiments, the amidated pectin is covalently bound to the filter.
• the nucleic acid is a product of nucleic acid amplification reaction.
• the amplification reaction is a polymerase chain reaction.
• the amidated pectin is a pectin comprising one or more monomeric units represented by formula:
• the amidated pectin comprises one or more monomeric units represented by formula:
• FIGURE 1 is an amplification plot of amplicon DNA post treatment with various amounts of an exemplary polyamine modified polysaccharide (spermine-pectin).
• FIGURE 2 demonstrates binding of contaminant DNA with an exemplary polyamine modified polysaccharide (spermine-pectin) followed by amplification of target DNA.
• spermine-pectin polyamine modified polysaccharide
• FIGURE 3 shows that when 10 million copies of dsDNA (126 bp) were treated with 1 pg of an exemplary spermine-modified polysaccharide, the amplification was completely inhibited (B). After more copies (1.9 x 10 10 ) were added to the spermine- pectin-DNA mixture, the amplification recovered (C) and is comparable to amplification of untreated DNA (A). This shows how the spermine-pectin conjugate can be used to bind minor DNA contaminates, and then allow amplification of target to take place.
• FIGETRE 4 compares averages of eight PCR runs of two sets of air samples collected over 24 h 3 cm above the surface treated with dry amplicon.
• Graph (A) is the amplicon on the surface without treatment and graph (B) is the dry surface with amplicon sprayed with 0.1% solution of an exemplary spermine-modified polysaccharide.
• the present invention provides a method of reducing nucleic acid contamination on a surface by contacting the surface to be decontaminated with a decontaminating agent comprising a modified pectin, e.g., an amidated pectin, comprising a plurality of amino groups.
• a decontaminating agent comprising a modified pectin, e.g., an amidated pectin, comprising a plurality of amino groups.
• the amidated pectin comprises groups derived from polyamines.
• the amidated pectin is a pectin amidated with a polyamine.
• amidated pectin compounds disclosed herein are believed to facilitate flocculation of nucleic acids, the process by which individual molecules of nucleic acids aggregate or precipitate into small particles when bound to the modified, e.g., amidated pectins, thus rendering the nucleic acids unsuitable for amplification.
• the decontaminating agents can be used in the form of a solution, a suspension, or can be bound to a solid support, such as a wipe or a sponge.
• decontamination or "reducing nucleic acid contamination” means altering a nucleic acid in a way that makes it no longer capable of or less capable of acting as a template in an amplification reaction compared to a non-altered nucleic acid. Decontamination generally renders the nucleic acids incapable or less capable of interfering with other amplification reactions. In some embodiments, decontamination also means rendering the surfaces to be decontaminated substantially free of nucleic acid contaminants. “Substantially free of nucleic acid contaminants,” as used herein, is used to mean that the contaminating nucleic acid is unamplifiable and/or present at a concentration that cannot be detected by amplification-based nucleic acid detection methods.
• agent and “reagent” can be used interchangeably when referred to the decontaminating compositions, unless indicated otherwise.
• reducing contamination refers to reducing the ability of or preventing the nucleic acid from binding to another nucleic acid, protein, or other biological substance.
• Reducing nucleic acid contamination or nucleic acid decontamination also refers to preventing or making the nucleic acids less capable of serving as a substrate for an enzyme. Reducing nucleic acid contamination or nucleic acid decontamination, as used herein, does not refer to any particular mechanism by which the reduction in contamination, or decontamination, occurs.
• the decontaminating agents disclosed herein comprise a modified polysaccharide.
• the polysaccharides are pectins.
• Pectins are naturally occurring complex polysaccharides typically found in plant cell walls.
• Pectins comprise an alpha 1-4 linked polygalacturonic acid backbone intervened by rhamnose residues and modified with neutral sugar side chains and non-sugar components such as acetyl, methyl, and ferulic acid groups.
• the galacturonic acid residues in pectin are partly esterified and present as the methyl esters. The degree of esterification is defined as the percentage of carboxyl groups esterified.
• HM pectins with a degree of esterification are classified as high methyl ester (“HM”) pectins or high ester pectins, and pectins with a degree of esterification lower than 50% are referred to as low methyl ester (“LM”) pectins or low ester pectins.
• LM low methyl ester
• pectin found in fruits and vegetables are HM pectins.
• amidated pectin refers to any naturally occurring pectin that has been structurally modified, e.g., by chemical, physical, or biological (including enzymatic) means, or by some combination thereof, wherein some of the ester or acid groups have been converted to amide groups.
• Amidated pectins can be prepared by contacting unmodified pectin with a solution of a suitable amine thereby converting the ester groups of the unmodified pectin to amides.
• unmodified pectin or hydrolyzed pectin, including partially hydrolyzed pectin can be reacted with an amine in the presence of a suitable coupling agent to form amidated pectin.
• suitable coupling agents include carbodiimide coupling agents such as EDC and EDCI, phosphonium and imonium type reagents such as BOP, PyBOP, PyBrOP, TBTU, HBTU, HATU, COMU, and TFFH.
• a modified, e.g., amidated pectin can be obtained by any of the methods described herein.
• Particularly useful starting materials for synthesis of modified pectins include fruit pectins, for example, apple and citrus pectins.
• the precursor (unmodified) pectins have relative molecular weights between about 5 kDa and about 1,100 kDa, between about 10 kDa and about 500 kDa, between about 10 kDa and about 300 kDa, between about 20 kDa and about 200 kDa, or between about 20 kDa and about 100 kDa.
• the polysaccharide agents have relative molecular weights between about 120 kDa and about 300 kDa, between about l50kDa and about 300 kDa, or between about 120 kDa and about 175 kDa.
• the relative molecular weights of the amidated pectins can be determined by size exclusion chromatography using a molecular weight standard, such as Pullulan series standards, as a reference.
• the decontaminating reagents disclosed herein comprise an amidated pectin comprising one or more monomeric units substituted with at least one amino group.
• the amidated pectins comprise one or more monomeric units having the structure of Formula I:
• n 0, 1,2, or 3;
• R 1 is H or C j -C3 alkyl
• X at each occurrence, is independently C2-C4 alkylene or C4-C6 heteroalkylene
• Y is a C 2 -C 3 alkylene or C 4 -C 6 heteroalkylene
• R 2 and R 3 are independently H or C j -C 3 alkyl.
• amidated pectins comprise one or more monomeric units represented by Formula II:
• n 0, 1, 2, or 3;
• n is independently 2, 3, or 4;
• p 2, 3, or 4;
• R 4 is H or C j -C3 alkyl
• R 5 and R 6 are independently H or C 1-C3 alkyl.
• the amidated pectin comprises one or more monomeric units comprising a primary amino group.
• the amidated pectin is amidated with a polyamine.
• a polyamine is a compound comprising two or more amino groups.
• Polyamines that can be used for modification of pectins of the solid supports disclosed herein include both synthetic polyamines and naturally occurring polyamines, e.g., spermidine, spermine, putrescine.
• the polyamine is selected from spermine, spermidine, cadaverine, ethylenediamine, and putrescine.
• the polyamine is spermine or spermidine.
• the amidated pectin comprises one or more units having the structure of Formula III or Formula IV, including their isomers and tautomers:
• the amidated pectin comprises one or more monomeric units represented by formula:
• the modified polysaccharide is a pectin that was obtained by periodate oxidation of an unmodified pectin followed by reductive amination, e.g., reductive amination with a polyamine.
• reductive amination e.g., reductive amination with a polyamine.
• alkyl As used herein, the terms “alkyl,” “alkenyl,” and “alkynyl” include straight-chain, branched-chain, and cyclic monovalent hydrocarbyl radicals, and combinations thereof, which contain only C and H when they are unsubstituted. Examples include methyl, ethyl, isobutyl, cyclohexyl, cyclopentylethyl, 2-propenyl, 3-butynyl, and the like.
• the total number of carbon atoms in each such group is sometimes described herein, e.g., when the group can contain up to ten carbon atoms, it can be represented as 1-10C, Cl- C10, CJ-CJO, C j.jO ’ or C1-10.
• heteroalky nyl as used herein, mean the corresponding hydrocarbons wherein one or more chain carbon atoms have been replaced by a heteroatom.
• exemplary heteroatoms include N, O, S, and P.
• the numbers describing the group though still written as e.g. C3-C10, represent the sum of the number of carbon atoms in the cycle or chain plus the number of such heteroatoms that are included as replacements for carbon atoms in the cycle or chain being described.
• a single group can include more than one type of multiple bond, or more than one multiple bond; such groups are included within the definition of the term "alkenyl” when they contain at least one carbon-carbon double bond, and are included within the term “alkynyl” when they contain at least one carbon-carbon triple bond.
• Alkyl, alkenyl, and alkynyl groups can be optionally substituted to the extent that such substitution makes sense chemically.
• Alkyl, alkenyl, and alkynyl groups can also be substituted by C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl or C5-C10 heteroaryl, each of which can be substituted by the substituents that are appropriate for the particular group.
• alkyl as used herein includes cycloalkyl and cycloalkylalkyl groups
• cycloalkyl is used herein to describe a carbocyclic non-aromatic group that is connected via a ring carbon atom
• cycloalkylalkyl is used to describe a carbocyclic non-aromatic group that is connected to the molecule through an alkyl linker.
• heterocyclyl is used to identify a non-aromatic cyclic group that contains at least one heteroatom as a ring member and that is connected to the molecule via a ring atom, which may be C or N; and “heterocyclylalkyl” can be used to describe such a group that is connected to another molecule through an alkylene linker. As used herein, these terms also include rings that contain a double bond or two, as long as the ring is not aromatic.
• Aromaatic or aryl substituent or moiety refers to a monocyclic or fused bicyclic moiety having the well-known characteristics of aromaticity; examples of aryls include phenyl and naphthyl.
• heteroaryl refers to such monocyclic or fused bicyclic ring systems which contain as ring members one or more heteroatoms. Suitable heteroatoms include N, O, and S, inclusion of which permits aromaticity in 5-membered rings as well as 6-membered rings.
• Typical heteroaromatic systems include monocyclic C5-C6 aromatic groups such as pyridyl, pyrimidyl, pyrazinyl, thienyl, furanyl, pyrrolyl, pyrazolyl, thiazolyl, oxazolyl, and imidazolyl, and fused bicyclic moieties formed by fusing one of these monocyclic groups with a phenyl ring or with any of the heteroaromatic monocyclic groups to form a C8-C10 bicyclic group such as indolyl, benzimidazolyl, indazolyl, benzotriazolyl, isoquinolyl, quinolyl, benzothiazolyl, benzofuranyl, pyrazolopyridyl, quinazolinyl, quinoxalinyl, cinnolinyl, and the like.
• monocyclic C5-C6 aromatic groups such as pyridyl, pyrimidy
• any monocyclic or fused ring bicyclic system which has the characteristics of aromaticity in terms of electron distribution throughout the ring system is included in this definition. It also includes bicyclic groups where at least the ring which is directly attached to the remainder of the molecule has the characteristics of aromaticity.
• the ring systems contain 5-14 ring member atoms.
• monocyclic heteroaryls contain 5-6 ring members, and bicyclic heteroaryls contain 8-10 ring members.
• Aryl and heteroaryl moieties can be substituted with a variety of substituents including C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C5-C12 aryl, C1-C8 acyl, and heteroforms of these, each of which can itself be further substituted; other substituents for aryl and heteroaryl moieties include halogens (F, Cl, Br, I), OR, NR 2 , SR, S0 2 R, S0 2 NR 2 , NRS0 2 R, NRCONR 2 , NRC(0)OR, NRC(0)R, CN, C(0)OR, C(0)NR 2 , OC(0)R, C(0)R, and N0 2 , wherein each R is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl,
• an aryl or heteroaryl group can be further substituted with the groups described herein as suitable for each type of such substituents or for each component of the substituent.
• an arylalkyl substituent can be substituted on the aryl portion with substituents described herein as typical for aryl groups, and it can be further substituted on the alkyl portion with substituents described herein as typical or suitable for alkyl groups.
• Optionally substituted indicates that the particular group being described can have one or more hydrogen substituents replaced by a non-hydrogen substituent. In some optionally substituted groups or moieties, all hydrogen substituents are replaced by a non-hydrogen substituent (e.g., a polyfluorinated alkyl such as trifluoromethyl). If not otherwise specified, the total number of such substituents that can be present is equal to the number of H atoms present on the unsubstituted form of the group being described.
• a non-hydrogen substituent e.g., a polyfluorinated alkyl such as trifluoromethyl
• the group takes up two available valences, so the total number of substituents that may be included is reduced according to the number of available valences.
• the decontaminating reagent comprises a solution of an amidated pectin having one or more monomeric units represented by formula:
• n 0, 1, 2, or 3;
• n 2, 3, or 4;
• p 2, 3, or 4;
• R 1 , R 2 , and R 3 are independently H or -C3 alkyl.
• the amidated pectin comprises one or more primary amino groups. In some embodiments, the amidated pectin is amidated with a polyamine. In some embodiments, the polyamine is selected from spermine, spermidine, cadaverine, ethylenediamine, and putrescine.
• the amidated pectin comprises one or more units represented by formulas:
• the amidated pectin comprises one or more monomeric units represented by formula:
• the amidated pectins comprise a plurality of monomeric units represented by formulae shown above.
• the term "plurality" means more than one.
• a plurality of monomeric units means at least two monomeric units, at least three monomeric units, or at least monomeric units, and the like. If an embodiment of the present invention comprises more than one types of monomeric units, they may also be referred to as a first monomeric unit, a second monomeric unit, a third monomeric unit, etc.
• the method for reducing nucleic acid contamination on a surface comprising: contacting the surface to be decontaminated with a solution comprising a dissolved or suspended modified pectin comprising a plurality of amino groups, e.g., an amidated pectin.
• the modified pectin is an amidated pectin, wherein the amidated pectin comprises one or more monomeric units having the structure of Formulae I-V.
• the concentration of the amidated pectin in the decontaminating solution is between about 0.01% and about 5%, between about 0.01% and about 1%, between about 0.01% and about 0.1%, between about 0.
• concentration of the amidated pectin in the decontaminating solution is between about 0.1 pg/mL and about 10,000 pg/mL, between about 0. 1 pg/mL and about 5,000 pg/mL, between about 0.1 pg/mL and about 1,000 pg/mL, between about 0.1 pg/mL and about 100 pg/mL, between about 0.1 pg/mL and about 5 pg/mL.
• concentration of the amidated pectin in the decontaminating solution is between about 0.1 pg/mL and about 10,000 pg/mL, between about 0. 1 pg/mL and about 5,000 pg/mL, between about 0.1 pg/mL and about 1,000 pg/mL, between about 0.1 pg/mL and about 100 pg/mL, between about 0.1 pg/mL and about 5 pg/mL.
• a person skilled in the art can select the suitable amount and/or concentration of
• the decontaminating compositions disclosed herein for example, solutions or suspensions of amidated pectins, are stable upon storage at room temperature.
• the decontaminating compositions can further comprise additives such soaps, detergents, disinfecting agents, and/or other suitable chemicals.
• suitable disinfecting agents may include, for example, quaternary ammonium compounds, colloidal silver, acetic acid, hydrogen peroxide, or a combination thereof.
• the decontaminating solution is present on a swab, wipe, cloth, filter, or sponge.
• the surface to be decontaminated is a surface of an instrument or a laboratory bench surface. In other embodiments, the solution is used to decontaminate laboratory pipettes.
• the methods of decontamination disclosed herein further comprise rinsing or wiping the surface to be decontaminated with water or another solution that does not comprise an amidated pectin.
• the methods further comprise contacting the surface or solution to be decontaminated from nucleic acid contaminants with a solution comprising a metal ion that can form a hydrogel with pectin thereby precipitating, trapping, complexing, or otherwise render insoluble the nucleic acid or the complex of the nucleic acid with the amidated pectins of the decontaminating solutions disclosed herein.
• Suitable metal ions include Ca 2+ and Mg 2+ ions. Any metal that can crosslink a pectin or an amidated pectin is suitable to be included in the decontaminating compositions disclosed herein.
• compositions comprising an amidated pectin covalently bound to a solid support, wherein the amidated pectin comprises a plurality of monomeric residues having the structure of Formulae I-V shown above.
• solid support refers to any substrate including paramagnetic particles, gels, controlled pore glass, magnetic beads, microspheres, nanospheres, capillaries, filter membranes, columns, cloths, wipes, paper, flat supports, multi-well plates, porous membranes, porous monoliths, wafers, combs, or any combination thereof.
• Solid supports can comprise any suitable material, including but not limited to glass, silica, titanium oxide, iron oxide, ethylenic backbone polymers, polypropylene, polyethylene, polystyrene, ceramic, cellulose, nitrocellulose, and divinylbenzene.
• Covalent attachment of modified pectins such as amidated pectins to solid supports can be achieved in any suitable manner by reacting the pectin with a solid support that comprises amine-reactive groups, for example, an epoxide, aldehyde, ketone, or activated ester.
• a solid support that comprises amine-reactive groups, for example, an epoxide, aldehyde, ketone, or activated ester.
• the amidated pectins of the disclosure are covalently attached to the solid supports via an amide bond, e.g., an amide bond formed between a carboxy group of the solid support and an amino group of the amidated pectin. Formation of the amide bond can be carried out by any suitable methods. For example, amidated pectin comprising one or more primary amino groups can be reacted with a substrate comprising one or more carboxylic acid groups in the presence of a suitable coupling agent.
• Non-limiting examples of suitable coupling agents include carbodiimide coupling agents such as DCC and EDCI, phosphonium and imonium type reagents such as BOP, PyBOP, PyBrOP, TBTU, HBTU, HATU, COMU, and TFFH.
• the carboxylic acid group of the solid substrate can be converted to an activated ester and then subsequently reacted with an amino group of the ami dated pectin.
• the solid supports comprise a compound of any one of Formulae I-V covalently attached to the solid support.
• the amidated pectins are incorporated into a cloth, sponge, pad, or wipe by impregnating or coating said cloth, sponge, pad, or wipe with the amidated pectin.
• examples include, but are not limited to cotton swabs, woven fiber pads, or wipes typically used for laboratory purposes, such as KimWipesTM, manufactured by Kimberly-Clark Corporation.
• a method of reducing aerosolized nucleic acid contamination in air comprising contacting air contaminated with aerosolized nucleic acid with a composition comprising a modified pectin, e.g., an amidated pectin comprising one or more monomeric units of any one of Formulae I-V or combinations thereof.
• contacting the air contaminated with aerosolized nucleic acid comprises passing the air through a solution or a suspension of the amidated pectin.
• contacting the air contaminated with aerosolized nucleic acid comprises passing the air through a filter comprising the amidated pectin.
• the amidated pectin is covalently bound to the filter.
• contacting the air contaminated with aerosolized nucleic acid comprises passing the air above a surface comprising the amidated pectin covalently bound thereto.
• the amidated pectin is a pectin amidated with spermine, e.g., by NHS/EDC coupling, or by sequential oxidation of a pectin with periodate and reductive amination with an amine, e.g., spermine, and sodium borohydride.
• Nucleic acid contaminants also can be present in the form of aerosols or upon dust particles.
• air filters comprising an amidated pectin of the invention can be useful for the removal of such contaminants.
• such filters can be prepared, for example, by dipping the filter material into a solution containing the amidated pectin of the invention and then drying the filter.
• the filter material can be covalently coated with the amidated pectins.
• the solution comprising the contaminant can be treated with a composition comprising an amidated pectin of the invention, for example, a solid support comprising an amidated pectin covalently attached to the solid support.
• a composition comprising an amidated pectin of the invention for example, a solid support comprising an amidated pectin covalently attached to the solid support.
• such treatment includes passing the solution to be decontaminated through a filter, a monolith, a membrane, or the like.
• the solution to be decontaminated is contacted with a solid support, for example, a magnetic bead.
• the nucleic acid contaminant is an amplicon.
• compositions comprising the amidated pectins are used to remove amplicon contamination and/or to prevent said contamination of molecular diagnostics laboratory surfaces, instrumentation, and equipment. The decontamination of such surfaces, instrumentation, or equipment can be carried out in any suitable manner, for example, using any of the methods described above.
• the decontaminating solutions disclosed herein are added to an amplification reaction after completion of the amplification reaction and detection of the product of the amplification, thereby rendering the product of the amplification, e.g., an amplicon, substantially unamplifiable.
• the decontaminating solution is added to the amplification reaction post-amplification in an automated cartridge.
• the decontaminating agents disclosed herein have certain advantages because, unlike bleach or other oxidants that can react with certain components of molecular diagnostics tests with a release of toxic byproducts, the decontaminating agents do not react with such components.
• the decontaminating performance of the compositions disclosed herein can be tested using any suitable method, e.g., a wipe test.
• any methods of efficacy assessment of decontamination reagents disclosed in the literature can be used (see, e.g., Fischer M. et al, Efficacy Assessment of Nucleic Acid Decontamination Reagents Used in Molecular Diagnostics Laboratories, PLOS One , July 13, 2016).
• EDC N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride
• NHS N-hydroxysuccinimide
• the solution was then poured into rapidly stirring MeOH (2 L) and stirred for 20 min.
• the solids were collected by filtering the solution through a medium fritted glass funnel, and then washed with MeOH two times.
• the solids were dried under vacuum for 40 h at 50 °C.
• the solids were ground to a fine powder using an electric coffee grinder and suspended in 500 mL of an acid wash solution (55% isopropyl alcohol, 34.5% water, and 10.5% concentrated hydrochloric acid) and stirred for 4.5 h.
• the solution was filtered off, and the solids were washed additionally twice with acid wash solution and then dried under vacuum overnight at 50 °C.
• Synthesis of spermine conjugate Compound 2 by oxidative cleavage of pectin followed by reductive amination In this example, a general procedure is provided for the modification of polysaccharide polymers with various polyamines through oxidation followed by reductive amination.
• Solutions (1%, w/w) of spermine-pectin conjugates Compound 1 and Compound 2 were prepared by dissolving 1.0 g of the lyophilized spermine-pectin conjugates Compound 1 or Compound 2in 100 mL of Milli-Q filtered water and removing any insoluble particles by using centrifuge.
• the 0.1%, 0.01%, 0.001% and 0.0001% dilutions in water were prepared from 1% solution.
• Apple pectin (2.45 g) was slowly added to 250 mL of rapidly stirred Milli-Q filtered water. The solution was stirred until the pectin was thoroughly wetted (1.5 h). 2.5 M NaOH ( ⁇ 5 mL) was added until the pH of the solution was 12. The solution was stirred for 30 min, and then 1 N HC1 ( ⁇ 7 mL) was added until the pH 9. N-(3- dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (2.45 g, 12.8 mmol) and N- hydroxysuccinimide (1.45 g, 12.6 mmol) were added to the solution, and the mixture was stirred at RT for 1.25 h.
• Carboxyl-polystyrene Particles 5.11 pm (Spherotec, Germany, CP-50-10)
• Polystyrene beads ( ⁇ 5 micron, 2 mL of 5% wt suspension) modified with carboxyl groups (Spherotec, CP-50-10) were diluted with DI water (4 mL) and sonicated for 15 min.
• DI water 4 mL
• EDC.HC1 and 40mg of NHS were added to the bead suspension.
• the suspension was stirred for 24 h for activation, briefly centrifuged at 4000rpm for 5 min and the supernatant decanted. Beads were resuspended in 5ml DI water, and to this was added a 1% solution of amine polymer (5 mL).
• Amine pectin polymer solution was prepared by stirring amine pectin polymer in DI water for 18 h, and then centrifugation at 9000 rpm for 30 min to remove any dissolved material. Suspension was stirred for 18 h, then centrifuged at 9000rpm for 30 min, diluted with 45 mL of water and rinsed in the same manner. The process was repeated with 0.1 M NaOH (lx), 0.1 M HC1 (lx), and DI water (2x). Beads were resuspended in 5ml DI H20, sonicated for 30 min, and concentration measured by weighing the amount of beads in a 150 ul aliquot via Speedvac.
• Example 3 Decontamination of nucleic acids
• PCR reaction solution 25 mM KC1, 50 mM MgCl 2 , 4.2 mM HEPES, 0.1% Tween, 0.2 mM dNTP's, 1.5 mU/uL of hot start Taq enzyme, 400 nM probe and 400 nM primers.
• Probe 5'-CGACGGCAACG(T-Dabcyl)CAGGAGGAACTACGA-3' SEQ ID NO: 3 modified with FAM (5') and BHQ (3').
• Double stranded DNA (126 bp), from a salmon genomic sequence, was purchased from Integrated DNA Technologies to be a model of an amplicon product from PCR.
• Amplicon template sequence (l26-mer):
• FKJFJRE 1 shows the PCR curves where 2.5 ng of template dsDNA (126 bp) was treated with spermine conjugated pectin. Each curve represents an experiment where 0.5 to 6.0 ng of spermine-pectin was added to 2.5 ng of dsDNA. The first curve on the left side has a Ct of 12.2 which represents the no spermine-pectin control. As more pectin is added to the separate reactions, the Cf s become longer, until at 6 ug of spermine-pectin, no Ct is observed. This is the concentration where all of the template is bound by the spermine-pectin conjugate, and the template cannot be amplified in the PCR reaction.
• Two separate vials were prepared each containing 6 pg of the spermine-pectin conjugate in 60 pL of water.
• 4 pg of hgDNA in 20 pL of water was added and allowed to sit at RT for 1 h.
• 2.5 ng of the salmon dsDNA (126 bp) model amplicon in 100 uL was then added to both vials.
• a 5 pL aliquot was removed from each vial to use in PCR as described below.
• the salmon dsDNA (126 bp) model amplicon was diluted to a concentration of 10 million copies per 1 mL of water. 1 mL of the DNA solution was added to each of three separate vials. One vial was used as the no spermine-pectin control sample. To the other two vials, 1 pg of the spermine-pectin conjugate in 10 pL of water was added. And to only one of these vials, an additional 2.5 ng of the salmon dsDNA (126 bp) model amplicon was added in 100 pL of water. After sitting for 1 h, 5 pL was used from each vial for the PCR reaction as described below.
• FIGURE 3 shows that when 10 million copies of dsDNA (126 bp) were treated with 1 ug of the spermine-pectin conjugate, the amplification was completely inhibited (B). After more copies (1.9 x 10 10 ) were added to the spermine-pectin-DNA mixture, the amplification recovered (C), comparable to untreated DNA amplification (A). This shows how the spermine-pectin conjugate can be used to bind minor DNA contaminates, and then allow amplification of target to take place.
• PCR with the salmon DNA target was performed by the method described above without using the probe. After 40 PCR cycles, the content of the PCR tube was diluted 1000 times giving "amplicon solution" with approximately 400B copies of DNA/mL. The ability to amplify and the number of copies/mL were confirmed by parallel comparison in PCR with standard 10K copies of the salmon DNA target. The amplicon solution was used in the following experiments. Preparation of dry amplicon on a surface
• a 31x23 cm polypropylene plate was roughened using P150 (very fine) sandpaper.
• the surface was cleaned with water, tested for wettability and dried.
• the surface was marked with a grid of 7.5 cm xl.8 cm spaciously separated segments for testing various treatments and running controls.
• 10 mL of amplicon solution was deposited onto the dry surface and spread consistently throughout the surface using a cotton swab.
• the plate was left on a horizontal surface and allowed to dry for 4 hr.
• the surface containing the dried amplicon was subjected to the following decontaminating and control experiments.
• Aerosol was collected above the surface of roughened 31x23 cm polypropylene surface containing dried analyte using a 7 cm diameter polypropylene funnel installed 3 cm above the surface.
• the funnel was connected by flexible tubing to a polypropylene impinger containing 30 mL of water.
• the outlet of the impinger was connected to a suction pump set at a 5 L/min rate.
• Collection of airborne particles was performed over 24 hr and a sample of the solution was submitted for PCR analysis. Untreated surface amplicon produced PCR signal at Ct 35.
• the surface that was treated by spraying of 0.1% Compound 1 solution and then dried overnight showed no detected amplicon (Figure 4).
• Aerosol was collected above the surface of roughened 31x23 cm polypropylene surface containing dried amplicon using a 7 cm diameter polypropylene funnel installed 3 cm above the surface.
• the funnel was connected by flexible tubing to a polypropylene impinger A containing 30 mL of water.
• Another impinger B with 30 mL of water was connected in series with impinger A.
• the outlet of the impinger B was connected to a suction pump set at a 5 L/min rate. Collection of airborne particles in impinger A and then subsequently in impinger B was performed over 24 hr and samples of the solutions from A and B were submitted to PCR analysis. Aerosol from surface amplicon produced PCR signal at Ct 35 from A and 42 from B. This experiment demonstrates that water was not effective scrubbing substance: amplicon still escape from impinger A resulting in detection of amplicon in impinger B.

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