WO2020256438A1 - Procédé de concentration d'un sujet et d'extraction d'acide nucléique à l'aide d'acide bis(sulfosuccinimidyl) subérique ou d'un dérivé de ce dernier - Google Patents

Procédé de concentration d'un sujet et d'extraction d'acide nucléique à l'aide d'acide bis(sulfosuccinimidyl) subérique ou d'un dérivé de ce dernier Download PDF

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WO2020256438A1
WO2020256438A1 PCT/KR2020/007911 KR2020007911W WO2020256438A1 WO 2020256438 A1 WO2020256438 A1 WO 2020256438A1 KR 2020007911 W KR2020007911 W KR 2020007911W WO 2020256438 A1 WO2020256438 A1 WO 2020256438A1
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triethoxysilane
integer
propyl
bis
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신용
진충은
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Infusion Tech
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Infusion Tech
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    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
    • 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
    • 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
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/46—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom

Definitions

  • the present invention relates to a method for enriching a subject and extracting nucleic acids using bissulfosuccinimidyl suberic acid or a derivative thereof.
  • Nucleic acid is an important analysis tool for identifying disease states, and DNA biomarkers such as single nucleotide polymorphism (SNP), mutations or DNA methylation allow researchers to find the cause of cancer. It provides important clues in helping to help and providing great opportunities for prognosis and surveillance as well as diagnosing and observing the condition of the disease during the early stages of the disease.
  • SNP single nucleotide polymorphism
  • DNA can be effectively extracted from clinical samples. Pre-concentration is very important for subsequent processes such as amplification and detection.
  • nucleic acids have been on carriers that specifically adsorb only nucleic acids from various types of substances contained in the cell lysis solution such as genomic DNA, plasmid DNA, messenger RNA, proteins, and cell debris particles.
  • the focus of almost all studies, such as related technology, has a limitation that is focused on research and development on substances that adsorb nucleic acids.
  • An object of the present invention is a composition for concentrating a subject comprising bissulfosuccinimidyl suberic acid or a derivative thereof, a method for concentrating a subject using the same, and a kit; It is to provide a composition for concentration and nucleic acid extraction of a subject comprising bissulfosuccinimidyl suberic acid or a derivative thereof, a method for enriching and extracting nucleic acids using the same, and a kit.
  • the present invention provides a composition for concentration of a subject comprising a compound represented by the following formula (1).
  • the present invention provides a kit for concentrating a subject comprising the composition.
  • the present invention is a first step of modifying by introducing an amine group to the object; And a second step of contacting a sample containing the object on the modified object and a compound represented by the following formula (1).
  • the present invention provides a composition for enriching a subject and extracting nucleic acids comprising a compound represented by the following formula (1).
  • the present invention provides a kit for concentration and nucleic acid extraction of a subject comprising the composition.
  • the present invention is a first step of modifying by introducing an amine group to the object; A second step of concentrating the subject by contacting a sample containing the subject on the modified subject with a compound represented by the following formula (1); A third step of isolating nucleic acids from the concentrated subject; A fourth step of forming a complex between the isolated nucleic acid and the compound; And a fifth step of extracting nucleic acids by treating the object with the complex formed with an elution buffer to extract the nucleic acid from the concentrated object.
  • X is (CH 2 ) n , CH 2 -(CH 2 -O-CH 2 ) m -CH 2 , (CH 2 ) p -SS-(CH 2 ) q or Is,
  • n is an integer of 3 to 6
  • n is an integer from 5 to 9
  • p or q are each an integer of 1 to 3
  • s is an integer from 1 to 5
  • R or R' is each hydrogen or a sulfonyl salt.
  • the method for concentrating a subject (cell or pathogen) and extracting nucleic acids using bissulfosuccinimidyl suberic acid or a derivative thereof can quickly extract a small amount of a subject contained in a sample without the use of special equipment. It can be used as a field diagnosis method, and it is possible to concentrate and extract nucleic acids simultaneously from one tube or chip, so it is more efficient than the conventional method, saves time and cost, and is easy to use. There is an advantage.
  • the bissulfosuccinimidyl suberic acid or its derivatives are highly water-soluble and have excellent storage properties, and their efficiency does not decrease even after storage, and the biomaterials extracted using them are usefully used for diagnosis and treatment of diseases. Can be.
  • 1 shows the principle of concentration and nucleic acid extraction of a subject using an NHS ester compound.
  • 2 to 4 show the concentration of the subject (cells and pathogens) and nucleic acid extraction efficiency using a sulfo-NHS ester/thin film device.
  • 5 and 6 show the nucleic acid extraction efficiency according to bis(sulfosuccinimidyl)suberic acid or a derivative thereof.
  • Figure 7 shows the pathogen concentration efficiency according to the NHS charge.
  • the inventors of the present invention have developed a method for concentrating a small amount of pathogens or cells contained in a sample and extracting nucleic acids from the concentrated pathogens or cells, and the method for enriching pathogens or cells and extracting nucleic acids of the present invention is
  • the present invention was completed by finding that it is possible to concentrate pathogens or cells and extract nucleic acids at the same time at a simpler and lower cost than that, and that on-site diagnostics can be performed without using large equipment.
  • the present invention provides a composition for concentrating a subject comprising a compound represented by the following formula (1).
  • X is (CH 2 ) n , CH 2 -(CH 2 -O-CH 2 ) m -CH 2 , (CH 2 ) p -SS-(CH 2 ) q or Is,
  • n is an integer of 3 to 6
  • n is an integer from 5 to 9
  • p or q are each an integer of 1 to 3
  • s is an integer from 1 to 5
  • R or R' is each hydrogen or a sulfonyl salt.
  • the sulfonyl salt is preferably in the form of a salt in which an alkali metal such as sodium, potassium, or lithium is bound to a sulfonyl group.
  • the subject may be a pathogen or a cell
  • the pathogen may be a virus, a bacterium, a fungus, a protozoan, a rickettsia, or a spirochete as a microorganism, but is not limited thereto.
  • the compound is bis(sulfosuccinimidyl)suberate; BS 3 ], bis(sulfosuccinimidyl)2,2,7,7-suberic acid-d 4 [bis(sulfosuccinimidyl)2,2,7,7-suberate-d 4 , BS 3 -d 4 ], Bis(sulfosuccinimidyl)glutaric acid-d 0 [bis(sulfosuccinimidyl)glutarate-d 0 ; BS 2 Gd 0 ], bis(sulfosuccinimidyl)2,2,7,7-glutaric acid-d 4 [bis(sulfosuccinimidyl)2,2,4,4-glutarate-d 4 ;BS 2 Gd 4 ] , Bis(NHS)PEG5 [bis(NHS)PEG 5 ; BS(PEG) 5 ], bis(NHS)PEG9 [BS(NHS)PEG
  • the present invention provides a kit for concentrating a subject comprising the composition.
  • the present invention is a first step of modifying by introducing an amine group to the object; And a second step of contacting a sample containing the object on the modified object and a compound represented by the following formula (1).
  • X is (CH 2 ) n , CH 2 -(CH 2 -O-CH 2 ) m -CH 2 , (CH 2 ) p -SS-(CH 2 ) q or Is,
  • n is an integer of 3 to 6
  • n is an integer from 5 to 9
  • p or q are each an integer of 1 to 3
  • s is an integer from 1 to 5
  • R or R' is each hydrogen or a sulfonyl salt.
  • the sulfonyl salt is preferably in the form of a salt in which an alkali metal such as sodium, potassium, or lithium is bound to a sulfonyl group.
  • the subject may be a pathogen or a cell
  • the pathogen may be a virus, a bacterium, a fungus, a protozoan, a rickettsia, or a spirochete as a microorganism, but is not limited thereto.
  • the object of the first step may be a solid material or a solid support, for example, a thin film device, a magnetic bead, a ring resonator, or a nanoparticle, but is not limited thereto. Is specified.
  • the object of the first step may be modified with a silane compound.
  • the silane compound may be a compound represented by the following formula (2), but is not limited thereto.
  • R 1 to R 3 may each be the same or different, C1 to C4 alkyl or C1 to C4 alkoxy, and R 4 is amino (C1 to C10) alkyl, 3-(2-amino( C1 to C4)alkylamino)(C1 to C4)alkyl or 3-[2-(2-amino(C1 to C4)alkylamino)(C1 to C4)alkylamino](C1 to C4)alkyl.
  • the silane compound is (3-aminopropyl) triethoxysilane ((3-aminopropyl) triethoxysilane; APTES), (3-aminopropyl) trimethoxysilane ((3-aminopropyl) trimethoxysilane), ( 1-aminomethyl) triethoxysilane, (2-aminoethyl) triethoxysilane, (4-aminobutyl) triethoxysilane ((4 -aminobutyl)triethoxysilane), (5-aminopentyl) triethoxysilane ((5-aminopentyl) triethoxysilane), (6-aminohexyl) triethoxysilane ((6-aminohexyl) triethoxysilane), 3-aminopropyl (die Oxy)methylsilane (3-aminopropyl(diethoxy)methyls
  • Samples containing the subject include feces, urine, tears, saliva, external secretions from the skin, external secretions from the respiratory tract, external secretions from the intestine, external secretions from the digestive tract, plasma, serum, blood, It should be noted that it may be any one selected from the group consisting of spinal fluid, lymph fluid, body fluid, and tissue, but is not limited thereto.
  • the present invention provides a composition for enriching a subject and extracting nucleic acids comprising a compound represented by the following formula (1).
  • X is (CH 2 ) n , CH 2 -(CH 2 -O-CH 2 ) m -CH 2 , (CH 2 ) p -SS-(CH 2 ) q or Is,
  • n is an integer of 3 to 6
  • n is an integer from 5 to 9
  • p or q are each an integer of 1 to 3
  • s is an integer from 1 to 5
  • R or R' is each hydrogen or a sulfonyl salt.
  • the sulfonyl salt is preferably in the form of a salt in which an alkali metal such as sodium, potassium, or lithium is bound to a sulfonyl group.
  • the subject may be a pathogen or a cell
  • the pathogen may be a virus, a bacterium, a fungus, a protozoan, a rickettsia, or a spirochete as a microorganism, but is not limited thereto.
  • the nucleic acid may be any one or more selected from the group consisting of DNA, cfDNA, ctDNA, RNA, mRNA, and cfRNA, but is not limited thereto.
  • the cfDNA refers to a circulating free nucleic acid that is circulating in the blood as cell free DNA.
  • the circulating free nucleic acid specifically includes circulating free DNA, circulating free RNA, and the like.
  • the circulating free nucleic acid generally exhibits a length of 1000 bp or less (DNA), 100 nt or less (RNA) in plasma or serum, but is not limited thereto.
  • the ctDNA refers to a nucleic acid that is isolated from cancer cells as circulating tumor DNA and exists in circulating blood. It has both tumor-specific mutations and epigenetic mutations and accounts for a very small amount of total circulating DNA in the blood. It should be noted that the size of circulating tumor DNA varies from 50 bp to 250 bp, but is not limited thereto.
  • the present invention provides a kit for concentration and nucleic acid extraction of a subject comprising the composition.
  • the kit may additionally include a buffer solution necessary for effective nucleic acid extraction.
  • the present invention is a first step of modifying by introducing an amine group to the object; A second step of concentrating the subject by contacting a sample containing the subject on the modified subject with a compound represented by the following formula (1); A third step of isolating nucleic acids from the concentrated subject; A fourth step of forming a complex between the isolated nucleic acid and the compound; And a fifth step of extracting nucleic acids by treating the object with the complex formed with an elution buffer to extract the nucleic acid from the concentrated object.
  • X is (CH 2 ) n , CH 2 -(CH 2 -O-CH 2 ) m -CH 2 , (CH 2 ) p -SS-(CH 2 ) q or Is,
  • n is an integer of 3 to 6
  • n is an integer from 5 to 9
  • p or q are each an integer of 1 to 3
  • s is an integer from 1 to 5
  • R or R' is each hydrogen or a sulfonyl salt.
  • the sulfonyl salt is preferably in the form of a salt in which an alkali metal such as sodium, potassium, or lithium is bound to a sulfonyl group.
  • the subject may be a pathogen or a cell
  • the pathogen may be a virus, a bacterium, a fungus, a protozoan, a rickettsia, or a spirochete as a microorganism, but is not limited thereto.
  • the object of the first step may be any one of a thin film device, a magnetic bead, a ring resonator, or a nanoparticle, but is not limited thereto.
  • the object of the first step may be modified with a silane compound.
  • the silane compound may be a compound represented by the following formula (2), but is not limited thereto.
  • R 1 to R 3 may each be the same or different, C1 to C4 alkyl or C1 to C4 alkoxy, and R 4 is amino (C1 to C10) alkyl, 3-(2-amino( C1 to C4)alkylamino)(C1 to C4)alkyl or 3-[2-(2-amino(C1 to C4)alkylamino)(C1 to C4)alkylamino](C1 to C4)alkyl.
  • the silane compound is (3-aminopropyl) triethoxysilane ((3-aminopropyl) triethoxysilane; APTES), (3-aminopropyl) trimethoxysilane ((3-aminopropyl) trimethoxysilane), (1 -Aminomethyl) triethoxysilane ((1-aminomethyl) triethoxysilane), (2-aminoethyl) triethoxysilane ((2-aminoethyl) triethoxysilane), (4-aminobutyl) triethoxysilane ((4- aminobutyl)triethoxysilane), (5-aminopentyl)triethoxysilane ((5-aminopentyl)triethoxysilane), (6-aminohexyl) triethoxysilane ((6-aminohexyl) triethoxysilane).
  • Samples containing the subject include feces, urine, tears, saliva, external secretions from the skin, external secretions from the respiratory tract, external secretions from the intestine, external secretions from the digestive tract, plasma, serum, blood, It should be noted that it may be any one selected from the group consisting of spinal fluid, lymph fluid, body fluid, and tissue, but is not limited thereto.
  • Example 1 thin film device fabrication and pretreatment
  • a low-cost thin film device for use as a microfluidic device was fabricated using a CO 2 laser cutter (VLS3.50 (610 x 305 mm); Universal Laser Systems, Scottsdale, AZ).
  • the thin film device is composed of an upper thin film and a lower thin film, and a microfluidic chamber interposed between the upper and lower thin films, and the thin film device is used for concentration of an object (cell, pathogen, etc.). It consists of a single microchannel microfluidic chamber combined with a hydroxysuccinimide (sulfo-NHS ester) compound.
  • the microfluidic chamber of the device is based on a closed device in order to prevent contamination caused by the open device.
  • the reaction sample remains in the microfluidic chamber of the sealed device to reduce contamination. Repeated rapid expansion and contraction in the flow cross-sectional area can create microvortices in liquid sample injection.
  • microfluidic chip was designed using AutoCAD (Autodesk, Inc, San Rafael, CA), and printed with a laser cutting machine used to fabricate a prototyping device having advantages of low cost, simplicity and speed.
  • a 3D disposable chip consisting of three layers, using a laser cutting machine (a 10.6 ⁇ CO 2 laser source with a power range of 10 W to 50 W), a 300 ⁇ m thick double-sided tape ( Adhesive 300LSE-9495LE, 3M, USA) and a 100 ⁇ m thick thin film (Kemafoil hydrophilic film, HNW-100, COVEME, Italy) as an outer layer.
  • the outer layer was attached to the permanent adhesive surfaces on the top and bottom of the inner layer to create a 3D disposable chip for processing large volumes of samples.
  • the height of the microfluidic chamber was about 600 ⁇ m, and the total volume was set to 700 ⁇ l.
  • the tubing adapter was prepared by attaching a cast acrylic sheet (MAGA CIPTA, Indonesia) having a thickness of 3 mm to one side of a double-sided tape, and cutting and perforating it with a laser cutting machine.
  • the prepared adapter was attached to the inlet and outlet of the 3D disposable chip, respectively.
  • pre-cut Tygon tubing (AAC02548; Cole-Parmer, Vernon Hills, IL) was placed in the adapter hole and then sealed using a thermally stable epoxy at 120°C.
  • a plastic cartridge was manufactured using a laser cutting machine.
  • the plastic cartridge (top and bottom part) serves to hold the 3D disposable chip during analysis; 105 mm long, 60 mm wide, 10 mm high.
  • the layout of each plastic component was designed using AutoCAD.
  • the structure was patterned on a transparent acrylonitrile butadiene styrene (ABS) sheet using a milling machine. After mounting the chip on the lower plastic part, the device was constructed by assembling it with the upper plastic part using 4 wrench bolts.
  • ABS transparent acrylonitrile butadiene styrene
  • a surface modification protocol was performed to use a sulfo-NHS ester compound as a thin film device for concentration of subjects (cells, pathogens, etc.) and a non-chaotropic reagent.
  • the inner surface was first treated with oxygen plasma (Covance Model, Femtoscience) for 10 minutes to change the characteristics of the inner surface from hydrophobic to hydrophilic, and the plasma treatment
  • the resulting thin film device was immersed in an aqueous 2% 3-aminopropyl diethoxymethylsilane (APDMS, Sigma-Aldrich) solution at 65° C. for 60 minutes, and then thoroughly washed with deionized water. After washing, in order to cure the thin film device, the cleaned thin film device was quickly dried under a nitrogen stream to modify the thin film device with an amine.
  • ADMS 3-aminopropyl diethoxymethylsilane
  • the hydrophilicity of the thin film device was significantly changed depending on the temperature and incubation time. After silanizing the thin film device at 65° C. for 60 minutes with APDMS, it was confirmed that the hydrophilicity of the surface of the thin film was increased (about 30 to 40° C.).
  • the device can be stored at room temperature until use.
  • the subject (cells, pathogens, etc.) was concentrated and the analysis conditions and reactions for extracting nucleic acids were optimized.
  • the sulfo-NHS ester compound can form a complex with an object on the surface of a thin film and capture nucleic acids.
  • the sulfo-NHS ester compound used in the experiment was bis(sulfosuccinimidyl)suberic acid [bis(sulfosuccinimidyl)suberate; BS 3 ], bis(sulfosuccinimidyl)2,2,7,7-suberic acid-d 4 [bis(sulfosuccinimidyl)2,2,7,7-suberate-d 4 , BS 3 -d 4 ], Bis(sulfosuccinimidyl)glutaric acid-d 0 [bis(sulfosuccinimidyl)glutarate-d 0 ; BS 2 Gd 0 ], bis(sulfosuccinimidyl)2,2,7,7-glutaric acid-d 4 [bis(sulfosuccinimidyl)2,2,4,4-glutarate-d 4 ;BS 2 Gd 4 ] , Bis(NHS)PEG5 [bis(
  • HCT116 cell line (ATCC_CCL-247) (1 x 10 5 cells ml -1 ) were added to each sulfo-NHS ester solution (100 mg ml -1 ) 100 ⁇ l, protease K (proteinase K) 10 ⁇ l, mixed with 100 ⁇ l of self lysis buffer (100 mM Tris-HCl (pH 8.0), 10 mM ethylenediaminetetraacetic acid, 1% sodium lauryl sulfate, 10% Triton X-100) The following pump (KD Scientific, MA) was injected into the device at a rate of 100 ⁇ l/min.
  • RNA To capture nucleic acid from the sample, DNA, 56°C for 10 minutes or room temperature for 10 minutes, RNA, The device was left at room temperature for 20 minutes. After washing the device with PBS to remove debris from the sample, concentrated by sulfo-NHS ester solution using an elution buffer (10 mM sodium bicarbonate, pH ⁇ 10.6, flow rate: 50 ⁇ l/min). Nucleic acid was extracted within minutes.
  • nucleic acids were extracted with a QIAamp® DNA mini kit or a QIAamp® viral RNA mini kit (Qiagen, Germany), and experiments were performed according to the protocol provided by the manufacturer.
  • the amount and purity of the extracted nucleic acid were analyzed by measuring the ratio of the optical density of the sample at 260 nm and 280 nm using Nano Drop (Thermo Fisher Scientific, USA).
  • HCT116 and E. coli nucleic acids extracted using the thin film apparatus or Qiagen kit of the present invention were amplified by real-time PCR. Briefly, 5 ⁇ l of DNA, 10 ⁇ l of 2X Brilliant III SYBR Green QPCR master mix, 25 pmol of each primer, and deionized water were added to adjust the total volume to 20 ⁇ l. PCR conditions were the initial denaturation step of 10 minutes at 95 °C; 95°C for 10 seconds, 58°C (Escherichia coli rodA gene) or 60°C (HCT116 ⁇ -actin gene) for 20 seconds, 72°C for 20 seconds, 40 cycles; It consists of a 30 second cooling step at 40°C.
  • RNA 5 ⁇ l, 2X Brilliant III SYBR Green QRT-PCR master mix 10 ⁇ l, each primer 25 pmol, RT mix, DTT, and RNase-Free water were added to adjust the total volume to 20 ⁇ l.
  • PCR conditions are the initial denaturation step of 20 minutes at 50 °C; 95°C for 10 minutes; 95°C for 10 seconds, 60°C (18s rRNA of HCT116) for 20 seconds, 72°C for 20 seconds, 40 cycles of stretching steps; It consists of a 30 second cooling step at 40°C.
  • the PCR product was isolated on a 2% agarose gel containing ethidium bromide (EtBr) using electrophoresis, and the gel was visualized using a GelDoc system (Clinx Science Instruments, Shanghi, China).
  • the HCT116 cell line and the sulfo-NHS ester solution were injected into the device, and real-time PCR was performed using the nucleic acid obtained therefrom.
  • RNA was extracted from HCT116 cell line with similar efficiency to the existing Qiagen kit method.
  • E. coli and various concentrations (10, 25, 50, 100 mg/mL) of sulfo-NHS ester solution were injected into the apparatus, and obtained from Real-time PCR was performed using nucleic acids.
  • DTSSP has a disulfide bond to a spacer arm, and a 50 mM TCEP solution reacting with a reducing agent instead of an elution buffer using sodium bicarbonate. (pH 7.0) was used.
  • Reducing agents such as TCEP create a cleavage site at the disulfide binding site and induce the bond to be broken, and the use of reducing agents such as TCEP and DTT solves problems caused by detection inhibitors that occur when elution buffers using pH are used. Can be solved.
  • BS(PEG) 5 ) was injected into the device, and the expression level of rodA was compared using the nucleic acid obtained therefrom.

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Abstract

La présente invention concerne un procédé de concentration d'un sujet et d'extraction d'un acide nucléique à l'aide d'acide bis(sulfosuccinimidyl) subérique ou d'un dérivé de ce dernier. Le procédé de concentration d'un sujet (cellule ou pathogène) et l'extraction d'un acide nucléique à l'aide d'un acide bis(sulfosuccinimidyl)) subérique ou un dérivé de ce dernier selon la présente invention peut extraire rapidement une petite quantité du sujet contenu dans un échantillon sans utiliser d'équipement spécial, et peut ainsi être utilisé en tant que procédé de diagnostic sur site. La présente invention peut concentrer simultanément un sujet et extraire un acide nucléique d'un seul tube ou puce, et présente ainsi l'avantage d'être plus efficace, de permettre des économies de temps et de coûts, et d'être plus commode à utiliser que les procédés classiques. De plus, l'acide bis(sulfosuccinimidyl) subérique ou son dérivé présente d'excellentes propriétés de stockage du fait qu'il est hautement soluble dans l'eau, et ne présente pas de diminution de son efficacité même lorsqu'il est utilisé après avoir été stocké. De plus, des biomatériaux extraits à partir de ces derniers peuvent être avantageusement utilisés pour le diagnostic et le traitement de maladies.
PCT/KR2020/007911 2019-06-19 2020-06-18 Procédé de concentration d'un sujet et d'extraction d'acide nucléique à l'aide d'acide bis(sulfosuccinimidyl) subérique ou d'un dérivé de ce dernier Ceased WO2020256438A1 (fr)

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