WO2018038549A1 - Procédé de concentration de micro-organismes ou d'extraction d'acide nucléique à l'aide de dtbp - Google Patents

Procédé de concentration de micro-organismes ou d'extraction d'acide nucléique à l'aide de dtbp Download PDF

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Publication number
WO2018038549A1
WO2018038549A1 PCT/KR2017/009254 KR2017009254W WO2018038549A1 WO 2018038549 A1 WO2018038549 A1 WO 2018038549A1 KR 2017009254 W KR2017009254 W KR 2017009254W WO 2018038549 A1 WO2018038549 A1 WO 2018038549A1
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nucleic acid
triethoxysilane
dtbp
propyl
microorganism
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PCT/KR2017/009254
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English (en)
Korean (ko)
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신용
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울산대학교 산학협력단
재단법인 아산사회복지재단
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Priority to US16/326,376 priority Critical patent/US11584924B2/en
Priority to EP17843969.1A priority patent/EP3505628B1/fr
Priority to CN201780055666.9A priority patent/CN109996875B/zh
Priority to JP2019509478A priority patent/JP6793248B2/ja
Priority claimed from KR1020170107007A external-priority patent/KR102026683B1/ko
Publication of WO2018038549A1 publication Critical patent/WO2018038549A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • 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
    • 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

Definitions

  • the present invention relates to a method for concentrating microorganisms or extracting nucleic acids using 3,3'-dithiobispropionimate.
  • Nucleic acids are an important analytical tool for identifying disease states, and DNA biomarkers, such as single nucleotide polymorphism (SNP), mutations or DNA methylation, help researchers find the cause of cancer and disease during the early stages of the disease. Diagnosing and observing the condition of the doctor also provides important clues in providing great opportunities for prognosis and monitoring.
  • SNP single nucleotide polymorphism
  • Nucleic acids such as DNA are present at very low physiological concentrations compared to other components such as proteins (eg, tens of nanograms of DNA per microliter of whole blood versus tens of micrograms of protein), effectively extracting and preliminary DNA from clinical samples. Concentration is very important for subsequent processes such as amplification and detection.
  • the carrier specifically absorbs the nucleic acid only from various kinds of substances contained in the cell lysis solution such as genomic DNA, plasmid DNA, messenger RNA, protein, and cell debris particles.
  • the focus of almost all research, including related technologies, has been limited to research and development of substances that adsorb nucleic acids.
  • An object of the present invention is a composition for concentrating microorganisms comprising 3,3'-dithiobispropionimidate (Dimethyl 3,3'-dithiobispropionimidate; hereinafter 'DTBP') as an active ingredient, a method and kit for concentrating microorganisms using the same;
  • a nucleic acid extracting composition comprising DTBP as an active ingredient, a nucleic acid extracting method and kit using the same;
  • the present invention provides a composition for concentrating microorganisms comprising DTBP as an active ingredient.
  • the present invention also provides a microbial enrichment kit comprising the composition.
  • the present invention also provides a method for concentrating microorganisms comprising contacting a sample containing microorganisms with the DTBP.
  • the present invention also provides a nucleic acid extracting composition comprising DTBP as an active ingredient.
  • the present invention also provides a nucleic acid extraction kit comprising the composition.
  • the present invention comprises the steps of modifying by introducing an amine group to the object (first step); Injecting a nucleic acid sample and DTBP onto the modified object and forming a complex between the nucleic acid and the DTBP (second step); And it provides a nucleic acid extraction method comprising the step of extracting the nucleic acid by processing the elution buffer to the object formed with the complex (third step).
  • the present invention also provides a composition for microorganism concentration and nucleic acid extraction comprising DTBP as an active ingredient.
  • the present invention also provides a microbial enrichment and nucleic acid extraction kit comprising the composition.
  • the present invention comprises the steps of modifying by introducing an amine group to the object (first step); Concentrating the microorganism by contacting the sample containing the microorganism with DTBP on the modified object (second step); Separating nucleic acid from the concentrated microorganism (third step); Forming a complex between the separated nucleic acid and the DTBP (fourth step); And it provides a method for extracting the nucleic acid from the concentrated microorganism at the same time the concentration of the microorganism comprising the step of extracting the nucleic acid by the treatment of the elution buffer solution to the object formed complex.
  • the present invention relates to a method for concentrating a microorganism or extracting nucleic acids using DTBP, and the present inventors have developed a technology capable of concentrating microorganisms using DTBP, and also a method for extracting nucleic acids directly from concentrated microorganisms.
  • FIG. 1 is a schematic view of the present invention DTBP / Thin film Sample analysis.
  • FIG. 2 is an exploded view showing the configuration of a thin film device.
  • 3 is a diagram showing the DNA extraction efficiency according to the DTBP concentration change.
  • FIG. 4 is a diagram illustrating end-point PCR (a) and real-time PCR (b) of DNA extracted from a colon cancer cell line (HCT116 cell line; hereinafter 'HCT116').
  • FIG. 5 is a diagram illustrating end-point PCR (a) and real-time PCR (b) of DNA extracted from a breast cancer cell line (MCF cell line; hereinafter 'MCF').
  • FIG. 6 shows PCR results of DNA extracted from Brucella Ovis.
  • FIG. M DNA marker
  • Q DNA sample extracted by the existing Qiagen method
  • DTBP DNA sample extracted using the DTBP method of the present invention
  • N negative control (negative control).
  • FIG. 7 is a diagram illustrating a method of extracting RNA (a) and a method of simultaneously extracting RNA and cDNA (b).
  • Figure 9 shows the results of the nucleic acid extraction analysis at the same time the concentration of pathogens (E. coli) in one chip using DTBP.
  • the present invention provides a composition for concentrating microorganisms comprising DTBP as an active ingredient.
  • the microorganism may be any negatively charged microorganism such as bacteria, viruses or cells.
  • the present invention also provides a microbial enrichment kit comprising the composition.
  • the kit may further include a buffer solution for adjusting pH required for effective microbial concentration.
  • the present invention comprises the steps of modifying by introducing an amine group to the object (first step); And contacting a sample containing microorganisms with DTBP on the modified object (second step).
  • the object may be one modified with a silane compound on the surface.
  • the silane compound may be a compound represented by the following Chemical Formula 1, but is not limited thereto.
  • R 1 to R 3 may each be the same or different, either C1 to C4 alkyl or C1 to C4 alkoxy, 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 (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, (6-aminohexyl) triethoxysilane, (6-aminohexyl) triethoxysilane, 3-aminopropyl (diethoxy Methylsilane (3-aminopropyl (diethoxy) methylsilane;
  • silane compound is most suitably (3-aminopropyl) triethoxysilane (APTES) or 3-aminopropyl (diethoxy) methylsilane (APDMS) described in the examples of the present invention.
  • APTES (3-aminopropyl) triethoxysilane
  • APIDMS 3-aminopropyl (diethoxy) methylsilane
  • the sample containing the microorganism is feces, urine, tears, saliva, external secretions of the skin, external secretions of the respiratory tract, external secretions of the intestinal tract, external secretions of the digestive tract, plasma, Serum, blood, spinal fluid, lymph, body fluids and tissues, but are not limited thereto.
  • the present invention also provides a nucleic acid extracting composition comprising DTBP as an active ingredient.
  • the nucleic acid may be DNA or RNA, but is not limited thereto.
  • the present invention also provides a nucleic acid extraction kit comprising the composition.
  • the kit may further include a lysis buffer or a protease for lysing the cells in the sample to release the nucleic acid from inside the cell, and a buffer for pH adjustment required for effective nucleic acid extraction. It may additionally be included.
  • the present invention comprises the steps of modifying by introducing an amine group to the object (first step); Injecting a nucleic acid sample and DTBP onto the modified object and forming a complex between the nucleic acid and the DTBP (second step); And it provides a nucleic acid extraction method comprising the step of extracting the nucleic acid by processing the elution buffer to the object formed with the complex (third step).
  • the object may be one modified with a silane compound on the surface.
  • the silane compound may be a compound represented by the following Chemical Formula 1, but is not limited thereto.
  • R 1 to R 3 may each be the same or different, either C1 to C4 alkyl or C1 to C4 alkoxy, 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.
  • silane compound is most suitably (3-aminopropyl) triethoxysilane (APTES) or 3-aminopropyl (diethoxy) methylsilane (APDMS) described in the examples of the present invention.
  • APTES (3-aminopropyl) triethoxysilane
  • APIDMS 3-aminopropyl (diethoxy) methylsilane
  • the present invention also provides a composition for microorganism concentration and nucleic acid extraction comprising DTBP as an active ingredient.
  • the microorganism may be any negatively charged microorganism such as bacteria, viruses or cells.
  • the nucleic acid may be DNA or RNA, but is not limited thereto.
  • the present invention also provides a microbial enrichment and nucleic acid extraction kit comprising the composition.
  • the kit may further include a buffer for effective microbial concentration and pH adjustment necessary for nucleic acid extraction, etc., and a lysis buffer or protease for lysing the cells in the sample to release the nucleic acid from inside the cell. And the like may further be included.
  • the present invention comprises the steps of modifying by introducing an amine group to the object (first step); Concentrating the microorganism by contacting the sample containing the microorganism with DTBP on the modified object (second step); Separating nucleic acid from the concentrated microorganism (third step); Forming a complex between the separated nucleic acid and the DTBP (fourth step); And it provides a method for extracting the nucleic acid from the concentrated microorganism at the same time the concentration of the microorganism comprising the step of extracting the nucleic acid by the treatment of the elution buffer solution to the object formed complex.
  • the object may be one modified with a silane compound on the surface.
  • the silane compound may be a compound represented by the following Chemical Formula 1, but is not limited thereto.
  • R 1 to R 3 may each be the same or different, either C1 to C4 alkyl or C1 to C4 alkoxy, 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.
  • silane compound is most suitably (3-aminopropyl) triethoxysilane (APTES) or 3-aminopropyl (diethoxy) methylsilane (APDMS) described in the examples of the present invention.
  • APTES (3-aminopropyl) triethoxysilane
  • APIDMS 3-aminopropyl (diethoxy) methylsilane
  • the sample containing the microorganism is feces, urine, tears, saliva, external secretions of the skin, external secretions of the respiratory tract, external secretions of the intestinal tract, external secretions of the digestive tract, plasma, Serum, blood, spinal fluid, lymph, body fluids and tissues, but are not limited thereto.
  • the "object” may be any one of a thin film device, a magnetic bead, a ring resonator, and a nanoparticle, but is not limited thereto. More preferably, the object is an upper thin film formed in each of the inlet and outlet holes described in the embodiment of the present invention; A lower thin film spaced apart from the upper thin film; The inlet end and the outlet end are formed so that the microchannels communicating in correspondence with the inlet and outlet holes of the upper thin film are formed inside, and the injection passage communicating with the inlet of the microchannel is adjacent to the inlet end. A micro channel chamber disposed between the upper thin film and the lower thin film; And sealing means for sealing each side of the upper thin film and the lower thin film to seal the micro channel chamber.
  • the nucleic acid analysis according to the present invention DTBP / Thin film Sample analysis, which is a nucleic acid analysis using DTBP in a thin film device, includes three steps of sample elution / culture, washing and elution, and is performed without centrifugation.
  • the thin film device is modified via 3-aminopropyltriethoxysilane (APTES) as the silane compound, which converts the hydrophobic thin film device into hydrophilic.
  • APTES 3-aminopropyltriethoxysilane
  • the nucleic acid sample, the elution buffer, and the DTBP solution are injected onto the modified thin film device.
  • the cross-linking mechanism between the amino acid of the nucleic acid and the DTBP by interaction with the bifunctional amine reactor of the DTBP may be used to form a complex between the nucleic acid and the DTBP and extract DNA from the sample.
  • Example 1 thin film device fabrication and pretreatment
  • the thin film device of the present invention was manufactured using a laser cutting machine (Universal Laser Systems, Scottsdale, USA) (see Fig. 2 (a)).
  • the thin film device comprises an upper thin film and a lower thin film, and a microfluidic chamber inserted between the upper thin film and the lower thin film, the microfluidic chamber being connected to each other by flow paths in the chamber to extract DNA from a nucleic acid source. It consists of slot-type microwells.
  • the microfluid chamber design was cut by a laser cutting machine on a 300 ⁇ m thick double-sided tape (100 ⁇ m thick polyester film sandwiched between 100 ⁇ m thick double-sided tape) to manufacture a microfluidic chamber. It was. Thin films (top and bottom) were cut to the same dimensions as the microfluidic chamber using a laser cutting machine.
  • Inlets and outlets which are through holes, were manufactured in the upper thin film.
  • the laser cutting thin films top and bottom were adhered to the surfaces of the upper and lower portions of the laser cutting microfluidic chamber, respectively, using a permanent adhesive.
  • the height of the microfluidic chamber was about 300 ⁇ m and the total volume was 300 ⁇ l (300 ⁇ l amount, 8.4 cm ⁇ 3.7 cm).
  • a tubing adapter for injecting a nucleic acid source was prepared by attaching a cast acrylic sheet (MARGA CIPTA, Indonesia) having a thickness of 3 mm to one side of a double-sided tape, and cutting and drilling with a laser cutting machine.
  • the manufactured tubing adapter was attached to the inlet and the outlet of the microfluidic chamber, respectively.
  • the pre-cut Tygon tubing (AAC02548; Cole-Parmer, Vernon Hills, USA) was then placed in the hole of the adapter and sealed with epoxy.
  • the thin film device manufactured as described above has the advantage of being capable of processing various nucleic acid samples (100 ⁇ l, 300 ⁇ l, and 500 ⁇ l).
  • the inside of the thin film device was treated with oxygen plasma for 10 minutes, and the plasma treated thin film device was treated with 2% 3-aminopropyltriethoxysilane (APTES) for 10 to 60 minutes at 65 ° C. D) was immersed in an aqueous solution containing), and then washed thoroughly with deionized water. After cleaning, the thin film device was quickly dried under a stream of nitrogen to modify the thin film device with amine to cure the thin film device.
  • APTES 3-aminopropyltriethoxysilane
  • the water contact angle of the amine-modified thin film device using the Drop Shape Analyzer showed that the hydrophilicity of the thin film device changed considerably with temperature and incubation time. After silanization of the thin film device with APTES for 10 minutes at 65 ° C., the thin film surface hydrophilicity was increased (about 30-40 ° C.).
  • DTBP dimethyl 3,3'-dithiobispropionimate
  • an optimized assay solution was first prepared to extract DNA using a thin film device (300 ⁇ l amount, 8.4 cm ⁇ 3.7 cm) modified with amine.
  • the optimized assay solution was prepared by mixing elution buffer containing 100 mM tris-hydrochloric acid (pH 8.0), 10 mM EDTA, 1% SDS, 10% Triton X-100 with DTBP (100 mg / ml)
  • elution buffer containing 100 mM tris-hydrochloric acid (pH 8.0), 10 mM EDTA, 1% SDS, 10% Triton X-100 with DTBP (100 mg / ml)
  • 100 ⁇ l of each sample derived from cells, bacteria, blood or urine was mixed with 200 ⁇ l of the assay solution.
  • the mixed mixture of the mixed nucleic acid assay sample and the assay solution is introduced into the upper substrate inlet of the thin film device modified with amine, and the mixed solution is moved into the microfluidic chamber to bind two amine groups and DNA of DTBP.
  • DNA was modified by combining the modified amine group with DNA to form a complex.
  • the thin film device was placed in any one of a thermoelectric cooler (TEC) including an incubator or a controller (Alpha Omega Instruments) maintained at a constant temperature (56 ° C) for 20 minutes to sufficiently extract DNA from the nucleic acid analysis sample. .
  • DNA was extracted using elution buffer (10 mM sodium bicarbonate, pH 10.6). After measuring the amount and purity of the extracted DNA, the optical density ratio of the sample at 260 nm (DNA) and 280 nm (protein) was determined using Enspire Multimode Plate Reader (PerkinElmer). In order to compare the conventional DNA extraction and DTBP analysis of the present invention, it was analyzed using a QIAmp DNA mini kit according to a known method (Qiagen, Hilden, Germany).
  • the DNA binding efficiency was confirmed to be the highest when the DTBP concentration was 100 mg / ml.
  • Plastic of high glucose Dulbecco's modified eagle medium (DMEM, DMEM Life Technology) supplemented with 10% fetal calf serum (FCS) in a humidified incubator at 37 ° C in a 5% CO 2 atmosphere
  • FCS fetal calf serum
  • HCT116 and MCF were purchased from the US ATCC (https://www.atcc.org).
  • End-point PCR and real time PCR were performed to confirm the amount and purity of DNA. Forward and reverse primers of some genes (Actin) were synthesized to a normal length of about 24 base pairs. End-point PCR was performed at 95 ° C. for 15 minutes at the initial denaturation step; 45 cycles totaling 45 seconds at 95 ° C., 45 seconds at 59 ° C. (Actin), and 45 seconds at 72 ° C .; And 72 ° C., final extension for 10 minutes.
  • end-point PCR (FIGS. 4A and 5A) and real-time PCR (FIGS. 4B and 5B) are extracted using DTBP.
  • end-point PCR of DNA HCT116 was extracted about 1.3 times and MCF was about 2.6 times higher than that of Qiagen product, and real-time PCR result was not significantly different from Qiagen product. there was.
  • PCR-based DNA amplification was performed using DNA extracted using DTBP analysis. All primers are Escherichia coli, Mycobacterium abscessus, Mycobacterium gordonae and Salmonella Strains (Salmonella Typhimurium, Salmonella Typhimurium) Commercial primers from Salmonella Newport, and Salmonella Saintpaul were used. Commercial primers of the strains used in this example were purchased from the US IDT (https://www.idtdna.com).
  • elution buffer containing 100 mM tris-hydrochloric acid (pH 8.0), 10 mM EDTA, 1% SDS, 10% Triton X-100 and 20 mg / ml of lysozyme DTBP (50 mg / ml) Mixed with.
  • General PCR was performed to validate the DTBP method of the present invention.
  • E. coli XL1 blue strain purchased from Korea Microbiological Resource Center; https://kctc.kribb.re.kr
  • Echerichia coli, Mycobacterium abscessus, Mycobacterium gordonae and Salmonella strains cultured for use in DTBP assays and Qiagen kit assays Bacterial DNA was extracted from Salmonella Typhimurium, Salmonella Newport, Salmonella Saintpaul, and Brucella Ovis. The strain used in this example was purchased from the Korea Microbial Resources Center (https://kctc.kribb.re.kr).
  • PCR amplifications were visualized by gel electrophoresis to separate PCR products on 2% agarose gels containing ethidium bromide (EtBr) (Sigma-Aldrich). The gel was visualized using the Gel Doc System (Bio-Rad). Measurement of DNA concentration and purity was performed with a UV spectrometer (Perkin-Elmer).
  • the elution buffer containing proteinase K and DTBP and the bodily fluid sample were introduced into the previously prepared thin film device, and then moved to the microchannel chamber to form a complex between the DNA and the DTBP in the bodily fluid sample.
  • Example 2 DNA was extracted in the same manner as.
  • the elution buffer and the bodily fluid sample were introduced into two different inlets at a flow rate of 1.5 ml / hr for 10 minutes using a syringe pump (KD Scientific, MA), and the cartridge was extracted for 20 minutes for extraction and purification of DNA.
  • RNA was extracted using DTBP analysis in the same manner as in Example 2, and RNA extraction was confirmed by amplification of 18S rRNA after cDNA synthesis from the outside as shown in FIG.
  • RNA extraction and cDNA synthesis could be performed on a single thin film apparatus through DTBP analysis as shown in FIG. 7 (b), and it was confirmed that this method was well developed through amplification of 18S rRNA later.
  • the previously produced thin film device was surface treated with 3-aminopropyl (diethoxy) methylsilane (APDMS) at 65 ° C. for 1 hour.
  • ADMS 3-aminopropyl (diethoxy) methylsilane
  • 1 to 2 ml of the pathogen sample was injected with DTBP (100 mg / ml) and concentrated at room temperature at a flow rate of 100 ⁇ l / min.
  • the concentrated sample was suspended in 100 ⁇ l.
  • E. coli was used as a pathogen sample. Meanwhile, DNA was extracted and compared with the Qiagen DNA Kit.
  • each sample was taken 1 ml and extracted with Qiagen kit to test PIV3-RNA virus detection (lanes 1, 2, 3, 4 of Figure 10) and concentrated in DTBP according to the present invention after extraction PIV3-RNA When virus detection was tested (lanes 1-1, 2-1, 3-1, 4-1 in Figure 10) were compared.

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Abstract

La présente invention concerne un procédé de concentration de micro-organismes et d'extraction d'acide nucléique à l'aide de DTBP. Il a été développé une technologie permettant de concentrer des micro-organismes à l'aide de la DTBP, ainsi qu'un procédé permettant d'extraire directement l'acide nucléique des micro-organismes concentrés. Étant donné que les technologies de concentration de micro-organismes et d'extraction d'acide nucléique peuvent être mises en oeuvre dans un tube ou une puce, on peut s'attendre à une réduction significative de la contamination provenant de l'extérieur, des coûts, du temps, de complexités et similaire.
PCT/KR2017/009254 2016-08-24 2017-08-24 Procédé de concentration de micro-organismes ou d'extraction d'acide nucléique à l'aide de dtbp WO2018038549A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US16/326,376 US11584924B2 (en) 2016-08-24 2017-08-24 Method for concentrating microorganism or extracting nucleic acid using DTBP
EP17843969.1A EP3505628B1 (fr) 2016-08-24 2017-08-24 Procédé de concentration de micro-organismes ou d'extraction d'acide nucléique à l'aide de dtbp
CN201780055666.9A CN109996875B (zh) 2016-08-24 2017-08-24 使用dtbp浓缩微生物或提取核酸的方法
JP2019509478A JP6793248B2 (ja) 2016-08-24 2017-08-24 Ｄｔｂｐを利用した微生物濃縮または核酸抽出方法

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KR10-2016-0107827 2016-08-24
KR20160107827 2016-08-24
KR1020170107007A KR102026683B1 (ko) 2016-08-24 2017-08-24 Dtbp를 이용한 미생물 농축 또는 핵산 추출 방법
KR10-2017-0107007 2017-08-24

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KR20130128348A (ko) * 2012-05-16 2013-11-26 중앙대학교 산학협력단 비피막성 바이러스의 분리, 검출 방법 및 이와 박테리아의 분리, 측정 방법 및 장치
KR20140134710A (ko) * 2012-03-16 2014-11-24 메르크 파텐트 게엠베하 표적화 아미노산 지질
KR20150096444A (ko) * 2012-12-13 2015-08-24 에이전시 포 사이언스, 테크놀로지 앤드 리서치 고형상 장치 상에서 핵산을 분리 및 분석하는 무표지 방법

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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