US20140113294A1 - Direct nucleic acid amplification kit, reagent and method - Google Patents

Direct nucleic acid amplification kit, reagent and method Download PDF

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US20140113294A1
US20140113294A1 US13/799,800 US201313799800A US2014113294A1 US 20140113294 A1 US20140113294 A1 US 20140113294A1 US 201313799800 A US201313799800 A US 201313799800A US 2014113294 A1 US2014113294 A1 US 2014113294A1
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nucleic acid
cyclodextrin
dried reagent
reagent composition
amplification
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US13/799,800
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English (en)
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Jeffrey K. Horton
Peter J. Tatnell
Kathryn L. Lamerton
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GE Healthcare UK Ltd
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GE Healthcare UK Ltd
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Assigned to GE HEALTHCARE UK LIMITED reassignment GE HEALTHCARE UK LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HORTON, JEFFREY K., LAMERTON, KATHRYN L., TATNELL, PETER J.
Priority to CN202110196732.2A priority Critical patent/CN112899354A/zh
Priority to CN201380055445.3A priority patent/CN104736726A/zh
Priority to JP2015538433A priority patent/JP2015533507A/ja
Priority to IN2793DEN2015 priority patent/IN2015DN02793A/en
Priority to PCT/EP2013/072206 priority patent/WO2014064169A1/en
Priority to EP13779893.0A priority patent/EP2912191B1/en
Publication of US20140113294A1 publication Critical patent/US20140113294A1/en
Priority to US15/432,476 priority patent/US10907201B2/en
Priority to JP2018128740A priority patent/JP7009011B2/ja
Abandoned legal-status Critical Current

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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q2527/00—Reactions demanding special reaction conditions
    • C12Q2527/125—Specific component of sample, medium or buffer

Definitions

  • the present invention relates to the field of nucleic acid amplification, particularly to the use of a polymerase chain reaction to amplify nucleic acids.
  • the invention provides methods and kits which can be used to amplify nucleic acids by lyophilizing or freeze-drying nucleic acid amplification reagents for easy amplification of nucleic acid samples.
  • the invention has applications in the easy processing of nucleic acids and is particularly useful in genotyping, diagnostics and forensics.
  • PCR polymerase chain reaction
  • U.S. Pat. No. 5,705,345 (Lundin et al.) describes a method of nucleic acid preparation whereby the sample containing cells is lysed to release nucleic acid and the sample is treated with cyclodextrin to neutralize the extractant.
  • the advantage of this system is that conventional detergent removal requires a separation step; however the addition of cyclodextrin to neutralize the detergent removes the need for the separation step and thus reduces the risk of contamination.
  • WO 99/38962 (Health, Gentra Systems Inc.) describes a solid support with a bound lysis reagent.
  • the lysis reagent can comprise a detergent, a chelating agent, water and optionally an RNA digesting enzyme.
  • the method for PCR amplification requires further steps for purification of the nucleic acid for amplification analysis.
  • WO 91/02040 describes an invention using cyclodextrin-labelled primers in an amplification reaction mixture for qualitative and quantitative nucleic acid sequence analysis.
  • the benefits of this invention are a higher signal efficiency and versatility in label colours.
  • WO 95/32739 (Agrawal) describes an oligonucleotide non-covalently complexed with a cyclodextrin.
  • cyclodextrin with oligonucleotides was for the cellular uptake of oligonucleotides and not for the amplification of nucleotides in a PCR reaction.
  • WO 2010/066908 (Beckers et al.,) describes the use of cyclodextrins to improve the specificity, sensitivity and/or yield of PCR.
  • the method discloses an amplification reaction which is performed in a reaction mixture comprising at least one cyclodextrin and performing the amplification reaction on the reaction mixture. Again the advantage being a combination of cyclodextrin in the liquid amplification reaction mixture would reduce the risk of contamination by reducing the number of purification steps.
  • column-based nucleic acid purification is a typical solid phase extraction method to purify nucleic acids. This method relies on the nucleic acid binding through adsorption to silica or other supports depending on the pH and the salt content of the buffer.
  • suitable buffers include Tris-EDTA (TE) buffer or Phosphate buffer (used in DNA microarray experiments due to the reactive amines).
  • TE Tris-EDTA
  • Phosphate buffer used in DNA microarray experiments due to the reactive amines.
  • Nucleic acid purification on spin columns typically involves three time-consuming and complex steps/stages: the sample containing nucleic acid is added to the column and the nucleic acid binds due to the lower pH (relative to the silanol groups on the column) and salt concentration of the binding solution, which may contain buffer, a denaturing agent (such as guanidine hydrochloride), Triton X-100, isopropanol and a pH indicator; the column is washed with 5 mM KPO 4 pH 8.0 or similar, 80% EtOH); and the column is eluted with buffer or water.
  • a denaturing agent such as guanidine hydrochloride
  • Triton X-100 Triton X-100
  • isopropanol a pH indicator
  • the column is washed with 5 mM KPO 4 pH 8.0 or similar, 80% EtOH
  • the column is eluted with buffer or water.
  • chaotropic agents such that DNA binds to silica or glass particles or glass beads. This property was used to purify nucleic acid using glass powder or silica beads under alkaline conditions.
  • Typical chaotropic agents include guanidinium thiocyanate or guanidinium hydrochloride and recently glass beads have been substituted with glass containing minicolumns.
  • the best defense against PCR amplification failure in forensics applications is to combine sound sample handling and processing techniques with extraction systems proven to efficiently purify DNA.
  • PCR reagents are stored in glycerol solution which must be maintained at temperatures below room temperature.
  • Lyophilisation or freeze drying is a process widely used in the preparation of reagents for nucleic acid analysis and other biological processes because it allows for long term stability of otherwise labile biomolecules, and provides a convenient method of storage, shipping and reconstitution.
  • Current technology for producing dry biological reagent compositions involves procedures such as dry-blending, spray-drying, freeze-drying, fluidized bed drying, and/or cryogenic freezing. All of these procedures have limitations and drawbacks including consistency and reliability.
  • Spray-drying technology provides more homogeneous blends of chemicals because the reagents are first dissolved in solution. With spray-drying, however, it is difficult to dispense precise amounts of blended chemicals. To overcome this drawback, the resulting particles are usually reprocessed by agglomeration to obtain uniform particle sizes such as tablets. However, the agglomerated particles are generally less soluble than the original spray-dried particles or powders. Also, these procedures sometimes use fluorocarbon cryogenic solutions which can be hazardous to the environment.
  • Fluid bed technology (Rubina, 1999 , Pharmaceutical Technology, 23, 104-113) relies upon spraying a liquid reagent blend onto a particle and drying the liquid to obtain a particle coated with the blended reagents. Using this procedure, it is difficult to obtain uniformly sized particles and to produce a uniform coating.
  • freeze-drying Another method for stabilizing biologics is freeze-drying.
  • One drawback to the freeze-drying is the use of fluorocarbon refrigerants which are difficult to dispose of and the freeze-drying process may be imprecise and also difficult to regulate. Indeed, regular freeze drying of reagents may not provide an entire solution for particularly labile reagents used in molecular biology processes. Furthermore, degradation of the product during the freeze drying process is common and a freeze dried product is not always perfectly stable during storage. Process control is critical and can be difficult to regulate (Tang & Pakil, 2004, Design of Freeze-Drying Processes for Pharmaceuticals: Practical Advice; Pharmaceutical Research, 21, 191-200).
  • Another method of stabilizing biologics is by air-drying biological reagent compositions (Ratti, 2001, Hot Air and Freeze-Drying of High Value Foods: A Review. J. Food Engineering 49, 311-319).
  • Some problems with air drying processes are that the dried product is not in a readily dispensable form.
  • the biological reagents must be stable at or above the temperature of the drying process and it is a difficult process to control accurately.
  • One type of carrier or filler which has been used to stabilize biological reagents are glass-forming filler materials (U.S. Pat. No. 5,565,318).
  • the biological reagent solutions are incorporated into the glass-forming filler materials (which are water soluble or a water-swellable substance). They are then dried to produce a glassy composition which immobilizes and stabilizes the biological reagent (U.S. Pat. No. 5,593,824).
  • Carbohydrates such as glucose, sucrose, maltose or maltotriose are an important group of glass-forming substances.
  • Other polyhydroxy compounds can be used such as carbohydrate derivatives like sorbitol and chemically modified carbohydrates.
  • Another important class of glass-forming substances are synthetic polymers such as polyvinyl pyrrolidone, polyacrylamide, or polyethyleneimine.
  • glass-forming substances include sugar copolymers such as FicollTM (U.S. Pat. No. 3,300,474).
  • Ficoll is a neutral, highly branched, high-mass, hydrophilic polysaccharide which dissolves readily in aqueous solutions. Ficoll radii range from 2-7 nm. It is prepared by reaction of the polysaccharide with epichlorohydrin.
  • Ficoll has molecular weights of between 5,000 to 1,000,000 and contains sucrose residues linked through ether bridges to bifunctional groups. Such groups may be an alkylene of 2, 3 or more carbon atoms but not normally more than 10 carbon atoms. The bifunctional groups serve to connect sugar residues together.
  • These polymers may, for example, be made by the reaction of sugar with a halohydrin or bis-epoxy compound.
  • a glass is typically defined as an undercooled liquid with a very high viscosity,
  • the present invention provides methods and kits which can be used to amplify nucleic acids by incorporating all the required PCR reagents into a lyophilized format for easy amplification of nucleic acid samples.
  • a dried reagent composition for nucleic acid amplification comprising, a sequestering reagent, a polymerase and a deoxyribonucleotide triphosphate (dNTP).
  • dNTP deoxyribonucleotide triphosphate
  • the nucleic acid is selected from the group consisting of DNA, RNA and oligonucleotide.
  • nucleic acid is used herein synonymously with the term “nucleotides” and includes DNA, such as plasmid DNA and genomic DNA; RNA, such as mRNA, tRNA, sRNA and RNAi; and protein nucleic acid, PNA.
  • the sequestering agent is a cyclodextrin.
  • the cyclodextrin may be selected from a group consisting of ⁇ -cyclodextrin, ⁇ -cyclodextrin, ⁇ -cyclodextrin and derivatives thereof. Cyclodextrin could consist of a group consisting of 6-O- ⁇ -D-Maltosyl- ⁇ cyclodextrin, hydroxyethyl- ⁇ -cyclodextrin, hydroxypropyl- ⁇ -cyclodextrin and 2-hydroxypropyl- ⁇ -cyclodextrin.
  • the sequestrant is preferably ⁇ -cyclodextrin.
  • the sequestering reagent is not a chelating agent.
  • a chelating agent is a chemical compound that combines with a metal to form a chelate, often used to trap heavy metal ions (Colins English Dictionary, ⁇ HarperCollins Publishers 2003).
  • a lysis reagent is sodium dodecyl suphate; sodium is a metal ion however according to Ramamurthy Palepu and Vincent C. Reinsborough (Can J. Chem Vol 66, 325-328, 1988) it is the hydrophobic tail that interacts with the cyclodextrin not the hydrophilic head.
  • the dried reagent composition comprises at least one primer.
  • the dried reagent composition additionally comprises an excipient mix.
  • excipient mix is used herein to denote additives or ingredients used to make up a preparation or mixture and for example may comprise of PCR buffer, Ficoll 70, Ficoll 400, Melezitose, Trehalose, stabilising proteins and nuclease free water.
  • PCR buffer is used herein to denote a buffer necessary to create optimal conditions for activity of a DNA polymerase and for example may comprise of Tris-HCl, KCl, MgCl 2 , gelatin and nuclease free water.
  • the dried reagent composition additionally comprises an exchange buffer.
  • exchange buffer is used herein to denote a buffer used for the removal of small ionic solutes, whereby one buffer is removed and replaced with another alternative buffer and for example may comprise of Tris/HCl, CaCl 2 , a detergent, RE960, MgCl 2 , KCl and nuclease free water.
  • the dried reagent composition additionally comprises bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the polymerase is an OmniKlen Taq (OKT) Polymerase.
  • the polymerase may be selected from the group consisting of T4 DNA Polymerase, Pol I and Klenow Fragment, T4 DNA Polymerase, Modified Bacteriophage T7 DNA Polymerase, Terminal Deoxynucleotide Transferase, Bst Polymerase, Taq Polymerase, Tth polymerase, Pow Polymerase, Vent Polymerase, Pab Pol I DNA Polymerase, Thermus thermophiles, Carboxydothermus hydrogenoformans, SP6 and SP7 RNA polymerase.
  • the preferred embodiment is a dried reagent composition
  • ⁇ -cyclodextrin comprising ⁇ -cyclodextrin, at least one primer, a polymerase, dNTP, BSA, an excipient mix and an exchange buffer.
  • a method for producing a dried reagent composition for nucleic acid amplification comprising the steps:
  • the mixture additionally comprises at least one primer.
  • the mixture additionally comprises an excipient mix.
  • the mixture additionally comprises an exchange buffer.
  • the drying step is achieved by lyophilizing the mixture.
  • amplification of nucleic acid comprising the steps:
  • the amplification method is a polymerase chain reaction.
  • the amplification method comprises reverse transcription polymerase chain reaction or isothermal amplification.
  • the solution is formed by adding water to the nucleic acid.
  • the nucleic acid is immobilized on a solid support.
  • the solid support is a cellulose based matrix.
  • the solid matrix is selected from the group consisting of glass, glass fiber, glass microfiber, silica, silica gel, silica oxide, cellulose, nitrocellulose, carboxymethylcellulose, polyester, polyamide, carbohydrate polymers, polypropylene, polytetraflurorethylene, polyvinylidinefluoride, wool and porous ceramics.
  • the solid matrix may comprise a glass or silica-based solid phase medium, a plastics-based solid phase medium or a cellulose-based solid phase medium.
  • the solid support is preferably a cellulose-based matrix. Examples of cellulose-based matrices include FTATM (data file 51668), 903 neonatal cards and 31-ETF cards available from GE Healthcare.
  • the cellulose based matrix is in the form of a pre punched disc.
  • the cellulose based matrix is in the form of an FTA pre punched disc.
  • the lysis reagent is embedded onto said solid matrix.
  • the lysis reagent comprises an anionic surfactant or detergent.
  • Sodium dodecyl sulphate (SDS) is an example of an anionic surfactant frequently used to lyse biological cells.
  • the amplification is carried out in a single reaction vessel such as a test tube or the well of a multi-well plate.
  • the method of the invention can be used either in a single reaction well or a high-throughput 96-well format in combination with automated sample processing as described by Baron et al., (2011, Forensics Science International: Genetics Supplement Series, 93, e560-e561).
  • This approach would involve a minimal number of steps and increase sample throughput.
  • the risk of operator-induced error, such as cross-contamination is also reduced since this procedure requires fewer manipulations compared to protocols associated with currently used, more labor intensive kits (e.g. QIAmp DNA blood mini kit, Qiagen).
  • the risk of sample mix-up is also reduced since the procedure requires few manipulations.
  • the method is readily transferable to a multi-well format for high-throughput screening.
  • the present invention can thus improve sample processing for carrying out PCR reactions to aid genetic interrogations.
  • the invention can be conducted in a 96 well/high throughput format to facilitate sample handling and thus eliminate batch processing of samples.
  • the advantage of dried or lyophilized formulations of the polymerase chain reaction reagents is that they can be easily solubilized by the addition of water, thus saving operator time and facilitating operator usage.
  • the dried reagent mixture can be pre-dispensed into the reaction vessel, such as the well of a multi-well plate.
  • the preformulated, predispensed, ambient-temperature-stable beads or cakes allow amplification reactions to be carried out within a single well or reaction vessel and ensure greater reproducibility between reactions, minimize pipetting steps, and reduce the potential for pipetting errors and contamination.
  • a detection system comprising the steps:
  • the detection system is a PCR imaging system.
  • the detection system is a fluorescence or luminescent based system.
  • nucleic acid may be viral, prokaryotic or eukaryotic in origin.
  • the nucleic acid sample is present in a cellular sample.
  • the cellular sample may originate from a mammal, bird, fish or plant or a cell culture thereof.
  • the cellular sample is mammalian in origin, most preferably human in origin.
  • the sample containing the nucleic acid may be derived from any source. This includes, for example, physiological/pathological body fluids (e.g.
  • the method is for use as a tool selected from the group consisting of a molecular diagnostics tool, a human identification tool, a forensics tool, STR profiling tool and DNA profiling.
  • a kit for amplifying nucleic acid comprising the dried reagent composition hereinbefore described and instructions for use thereof.
  • FIG. 1 shows the results from PCR amplification of unwashed blood-spotted FTA paper with nucleic acid amplification reagent cakes with or without ⁇ -cyclodextrin.
  • FIG. 2 shows the results from PCR amplification of unwashed blood-spotted FTA paper with nucleic acid amplification reagent cakes with ⁇ -cyclodextrin.
  • FIG. 3 shows results from PCR amplification of titrated OmniKlen Taq (OKT) polymerase, in nucleic acid amplification reagent cakes, demonstrating the best concentration to freeze dry the nucleic acid amplification formulation.
  • OKT OmniKlen Taq
  • FTA papers for storing nucleic acid were obtained from GE Healthcare UK Limited; Normal human blood (Tissue Solutions Ltd); Genomic DNA (Promega product code G152A); 1 kb DNA ladder (Promega product code G571A); Harris Uni-core punch, 1.2 mm (Sigma, Catalogue number Z708860-25ea, lot 3110); OmniKlentaq Polymerase (Mo Bio Inc, catalogue code 1225-250); Deoxyribonucleotide triphosphate (dNTP) (Life Tech);
  • Samples were combined with the lyophilised nucleic acid amplification composition in a 96 well plate.
  • Blood-spotted FTA was added to a well with a nucleic acid amplification reagent cake that contained cyclodextrin or did not contain cyclodextrin.
  • Standards and samples were added to the appropriate wells.
  • the plates were centrifuged at 1000 rpm for 1 minute and sealed. PCR was carried out on an MJ Research PTC-200 Thermo Cycler following the manufacturer's user instructions.
  • the thermal cycling conditions were: 95° C. for 5 min, 95° C. for 30 sec, 55/65° C. for 1 min, 72° C. for 2 min followed by 35 cycles of: 95° C. for 30 sec, 55/65° C. for 1 min, 72° C. 2 min, followed by 72° C. for 10 mins.
  • the standard well of the 96 well PCR plate was loaded with 5 ⁇ l of the 1 Kb DNA ladder with 1 ⁇ l of 6 ⁇ loading buffer.
  • FIG. 1 shows PCR amplification of unwashed blood-spotted FTA with the lyophilized nucleic acid amplification composition with or without ⁇ -cyclodextrin: Lane M: 1 kb Ladder; Lane 1-4: FTA punch spotted with whole blood (1.2 mm) with a lyophilized nucleic acid amplification composition without cyclodextrin; Lane 5-8: FTA punch spotted with whole blood (1.2 mm) with the lyophilized nucleic acid amplification composition containing cyclodextrin.
  • FIG. 2 shows PCR amplification of unwashed blood-spotted FTA with the lyophilized nucleic acid amplification composition with or without ⁇ -cyclodextrin: Lane M: 1 kb Ladder; Lane 1-2: FTA punch spotted with whole blood (1.2 mm) with the lyophilized nucleic acid amplification composition containing cyclodextrin; Lane 3-4: FTA punch (1.2 mm) with the lyophilized nucleic acid amplification composition containing cyclodextrin; Lane 5-6: FTA punch spotted with whole blood (1.2 mm) with the lyophilized nucleic acid amplification composition without cyclodextrin.
  • FIG. 3 shows PCR amplification of unwashed blood-spotted FTA with the lyophilized nucleic acid amplification composition without ⁇ -cyclodextrin and with varying concentrations of OKT Taq polymerase: Lane 1-8 contains 6U OKT, Lane 1-3 FTA punch spotted with whole blood, Lane 4-6 genomic DNA, Lane 7-8 no DNA template; Lane 9-16 contains 8U OKT, Lane 9-11 FTA punch spotted with whole blood, Lane 12-14 genomic DNA, Lane 15-16 no DNA template; Lane 17-24 contains 10U OKT, Lane 17-19 FTA punch spotted with whole blood, Lane 20-22 genomic DNA, Lane 23-24 no DNA template; Lane 25-32 contains 12U OKT, Lane 25-27 FTA punch spotted with whole blood, Lane 28-30 genomic DNA, Lane 31-32 no DNA template.

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Application Number Priority Date Filing Date Title
CN202110196732.2A CN112899354A (zh) 2012-10-24 2013-10-23 直接核酸扩增试剂盒、试剂和方法
CN201380055445.3A CN104736726A (zh) 2012-10-24 2013-10-23 直接核酸扩增试剂盒、试剂和方法
JP2015538433A JP2015533507A (ja) 2012-10-24 2013-10-23 直接核酸増幅キット、試薬及び方法
IN2793DEN2015 IN2015DN02793A (zh) 2012-10-24 2013-10-23
PCT/EP2013/072206 WO2014064169A1 (en) 2012-10-24 2013-10-23 Direct nucleic acid amplification kit, reagent and method
EP13779893.0A EP2912191B1 (en) 2012-10-24 2013-10-23 Direct nucleic acid amplification kit, reagent and method
US15/432,476 US10907201B2 (en) 2012-10-24 2017-02-14 Direct nucleic acid amplification kit, reagent and method
JP2018128740A JP7009011B2 (ja) 2012-10-24 2018-07-06 直接核酸増幅キット、試薬及び方法

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WO2016099999A1 (en) * 2014-12-18 2016-06-23 Ge Healthcare Uk Limited Analyte detection on a solid support by nucleic acid amplification coupled to an immunoassay
WO2016106113A1 (en) * 2014-12-23 2016-06-30 Ge Healthcare Uk Limited Methods and reagents for reverse-transcription polymerase chain reaction
WO2016106129A3 (en) * 2014-12-23 2016-12-08 Ge Healthcare Uk Limited Methods and reagents for reverse-transcription polymerase chain reaction
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US10590408B2 (en) 2016-02-18 2020-03-17 Ge Healthcare Uk Limited Method and composition for biomolecule stabilization
US10907201B2 (en) 2012-10-24 2021-02-02 Global Life Sciences Solutions Operations UK Ltd Direct nucleic acid amplification kit, reagent and method
US10968478B2 (en) 2014-12-23 2021-04-06 Global Life Sciences Solutions Operations UK Ltd Methods and reagents for reverse-transcription polymerase chain reaction
US11286526B2 (en) * 2017-05-19 2022-03-29 Gen-Probe Incorporated Dried compositions containing flap endonuclease
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EP4289507A3 (en) * 2017-07-10 2024-02-28 Gen-Probe Incorporated Analytical systems and methods for nucleic acid amplification using sample assigning parameters
US11987836B2 (en) 2014-09-30 2024-05-21 Global Life Sciences Solutions Usa Llc Method for nucleic acid analysis directly from an unpurified biological sample

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Publication number Priority date Publication date Assignee Title
CN108291249A (zh) * 2015-11-18 2018-07-17 国立研究开发法人海洋研究开发机构 靶核酸的定量方法和用于该方法的试剂盒
CN106755414B (zh) * 2016-12-23 2020-09-01 宁波海尔施基因科技有限公司 一种检测dna遗传标记的方法
US20230242898A1 (en) * 2020-03-11 2023-08-03 Kao Corporation Method for preparing rna derived from skin surface lipids
CN111593112A (zh) * 2020-05-12 2020-08-28 深圳市星蝶科技有限公司 一种用于检测β-地中海贫血的PCR试剂及试剂盒
AU2022239609A1 (en) * 2021-03-19 2023-10-05 Becton, Dickinson And Company Isothermal amplification of pathogens

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5705345A (en) * 1991-01-10 1998-01-06 Amersham International Plc Methods and kits for preparing nucleic acids using cyclodextrin
US5972386A (en) * 1995-12-19 1999-10-26 Flinders Technologies Pty, Ltd. Dry solid medium for storage and analysis of genetic material
US20060160078A1 (en) * 2002-07-12 2006-07-20 Cardy Donald L N Lateral flow assay device and method
US7462475B2 (en) * 2004-05-20 2008-12-09 Dna Poleymerase Technology, Inc. Use of whole blood in PCR reactions

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5593824A (en) * 1994-09-02 1997-01-14 Pharmacia Biotech, Inc. Biological reagent spheres
US5565318A (en) * 1994-09-02 1996-10-15 Pharmacia Biotech, Inc. Room temperature stable reagent semi-spheres
US7122304B2 (en) * 1997-05-12 2006-10-17 Whatman, Inc. Methods for the storage and synthesis of nucleic acids using a solid support
WO1999038962A2 (en) * 1998-02-02 1999-08-05 Gentra Systems, Inc. Compositions and methods for using a lysing matrix for isolating dna
CN101516337A (zh) * 2006-09-18 2009-08-26 通用电气医疗集团生物科学公司 用玻璃覆盖的生物学试剂的制备
US8900525B2 (en) * 2006-09-18 2014-12-02 Ge Healthcare Bio-Sciences Corp. Preparation of glassified biological reagents
US8153401B2 (en) 2007-05-17 2012-04-10 Applied Biosystems, Llc Method for direct amplification from crude nucleic acid samples
SI2294225T1 (sl) 2008-06-30 2015-04-30 Life Technologies Corporation Postopek za neposredno amplifikacijo iz vzorcev surove nukleinske kisline
CN102317472A (zh) * 2008-12-12 2012-01-11 欧洲基因技术股份有限公司 使用环糊精改进核酸扩增反应的特异性、灵敏度和产率
CN102018672B (zh) * 2010-11-29 2013-09-18 广州朗圣药业有限公司 一种水溶性药物的脂质体冻干组合物及其制备方法
GB201219137D0 (en) 2012-10-24 2012-12-05 Ge Healthcare Uk Ltd Direct nucleic acid amplification kit, reagent and method
FR2998845B1 (fr) * 2012-12-04 2016-04-01 Autoliv Dev Generateur de gaz

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5705345A (en) * 1991-01-10 1998-01-06 Amersham International Plc Methods and kits for preparing nucleic acids using cyclodextrin
US5972386A (en) * 1995-12-19 1999-10-26 Flinders Technologies Pty, Ltd. Dry solid medium for storage and analysis of genetic material
US20060160078A1 (en) * 2002-07-12 2006-07-20 Cardy Donald L N Lateral flow assay device and method
US7462475B2 (en) * 2004-05-20 2008-12-09 Dna Poleymerase Technology, Inc. Use of whole blood in PCR reactions

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Kermekchiev et al. (2009) Nucl. Acids res. Vol. 37 no 5 e40 doi.1093/nar/gkn1055 *

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10907201B2 (en) 2012-10-24 2021-02-02 Global Life Sciences Solutions Operations UK Ltd Direct nucleic acid amplification kit, reagent and method
US20140322761A1 (en) * 2013-03-29 2014-10-30 Sony Corporation Method of preparing sample for nucleic acid amplification reaction, nucleic acid amplification method, and reagent and microchip for solid phase nucleic acid amplification reaction
US11987836B2 (en) 2014-09-30 2024-05-21 Global Life Sciences Solutions Usa Llc Method for nucleic acid analysis directly from an unpurified biological sample
WO2016099999A1 (en) * 2014-12-18 2016-06-23 Ge Healthcare Uk Limited Analyte detection on a solid support by nucleic acid amplification coupled to an immunoassay
US11008604B2 (en) 2014-12-18 2021-05-18 Global Life Sciences Solutions Operations UK Ltd Analyte detection on a solid support by nucleic acid amplification coupled to an immunoassay
CN107208136A (zh) * 2014-12-18 2017-09-26 通用电气医疗集团英国有限公司 通过与免疫测定法偶联的核酸扩增的固体支持物上的分析物检测
JP2018506020A (ja) * 2014-12-18 2018-03-01 ジーイー・ヘルスケア・ユーケイ・リミテッド 核酸増幅と組合せたイムノアッセイによる固体支持体上の被検体検出
US10968478B2 (en) 2014-12-23 2021-04-06 Global Life Sciences Solutions Operations UK Ltd Methods and reagents for reverse-transcription polymerase chain reaction
US10822645B1 (en) 2014-12-23 2020-11-03 Global Life Sciences Solutions Operations UK Ltd Methods and reagents for reverse-transcription polymerase chain reaction
US10858694B2 (en) 2014-12-23 2020-12-08 Global Life Sciences Solutions Operations UK Ltd Methods and reagents for reverse-transcription polymerase chain reaction
WO2016106129A3 (en) * 2014-12-23 2016-12-08 Ge Healthcare Uk Limited Methods and reagents for reverse-transcription polymerase chain reaction
WO2016106113A1 (en) * 2014-12-23 2016-06-30 Ge Healthcare Uk Limited Methods and reagents for reverse-transcription polymerase chain reaction
US10907148B2 (en) * 2015-08-28 2021-02-02 Global Life Sciences Solutions Operations UK Ltd Method and kit for analyte detection
WO2017036808A1 (en) * 2015-08-28 2017-03-09 Ge Healthcare Uk Limited A method and kit for analyte detection
US10590408B2 (en) 2016-02-18 2020-03-17 Ge Healthcare Uk Limited Method and composition for biomolecule stabilization
US11286526B2 (en) * 2017-05-19 2022-03-29 Gen-Probe Incorporated Dried compositions containing flap endonuclease
US11952630B2 (en) * 2017-05-19 2024-04-09 Gen-Probe Incorporated Dried compositions containing flap endonuclease
EP4289507A3 (en) * 2017-07-10 2024-02-28 Gen-Probe Incorporated Analytical systems and methods for nucleic acid amplification using sample assigning parameters
CN114317700A (zh) * 2021-12-28 2022-04-12 无锡科智达科技有限公司 荧光pcr试剂冻干保护剂、冻干方法及应用

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