WO2018038549A1 - Method for concentrating microorganism or extracting nucleic acid using dtbp - Google Patents

Method for concentrating microorganism or extracting nucleic acid using 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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Prior art keywords
nucleic acid
triethoxysilane
dtbp
propyl
microorganism
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PCT/KR2017/009254
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French (fr)
Korean (ko)
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신용
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울산대학교 산학협력단
재단법인 아산사회복지재단
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Priority to JP2019509478A priority Critical patent/JP6793248B2/en
Priority to US16/326,376 priority patent/US11584924B2/en
Priority to CN201780055666.9A priority patent/CN109996875B/en
Priority to EP17843969.1A priority patent/EP3505628B1/en
Priority claimed from KR1020170107007A external-priority patent/KR102026683B1/en
Publication of WO2018038549A1 publication Critical patent/WO2018038549A1/en

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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

The present invention relates to a method for concentrating microorganisms and extracting nucleic acid using DTBP. A technology capable of concentrating microorganisms using DTBP has been developed, and also a method capable of extracting nucleic acid directly from the concentrated microorganisms has been developed. As the technologies of concentrating microorganisms and extracting nucleic acid can be implemented in one tube or chip, it is expected to significantly reduce contamination from the outside, costs, time, complexities, and the like.

Description

DTBP를 이용한 미생물 농축 또는 핵산 추출 방법Microbial Concentration or Nucleic Acid Extraction Method Using DTBP
본 발명은 3,3'-디티오비스프로피온이미데이트를 이용한 미생물 농축 또는 핵산 추출 방법에 대한 것이다.The present invention relates to a method for concentrating microorganisms or extracting nucleic acids using 3,3'-dithiobispropionimate.
핵산은 질병 상태를 확인하기 위한 중요한 분석 수단이며, DNA 생체표지자(biomarker) 예를 들어, 단일염기다형성(SNP), 돌연변이 또는 DNA 메틸화는 연구자가 암의 원인을 찾도록 돕고 질병의 초기 단계 동안 질병의 상태를 진단하고 관찰하는 것은 물론 예후와 감시에 대한 큰 기회를 제공하는 데 중요한 실마리를 제공한다. 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.
DNA와 같은 핵산은 단백질과 같은 다른 성분에 비해 매우 낮은 생리적 농도로 존재하기 때문에(예를 들어, 전혈 마이크로리터당 수십 나노그람의 DNA 대 수십 마이크로그람의 단백질), 임상 시료로부터 DNA를 효과적으로 추출하고 예비 농축하는 것은 증폭 및 검출과 같은 이후의 공정에 매우 중요하다. 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.
기존의 미생물 검출을 위한 방법들은 환자 샘플 1∼2 ㎖에서 전체 용액을 다 이용하지 못하고, 그 중 일부만을 이용해서 핵산을 추출하고 이를 이용한 검출을 진행해 왔다. 많은 양의 미생물의 경우에는 큰 문제가 없지만, 적은 양의 미생물의 경우, 정확한 검출이 되지 못해서 추가적 감염병에 대한 조절에 문제가 생기는데, 1∼2 ㎖ 샘플에서 최대한 모든 미생물을 이용하기 위한 농축 방법에 대한 연구가 필요하다.Existing methods for detecting microorganisms have not used the entire solution in 1-2 ml of patient samples, and only a part of them has been used to extract nucleic acids and perform detection using the same. For large amounts of microorganisms, this is not a big problem, but for small amounts of microorganisms, it is not possible to detect them correctly, which leads to problems in controlling additional infectious diseases. Research is needed.
또한, 최근 들어 생명공학을 비롯한 진단의학, 약물의학, 대사의학 등 다양한 분야에서 고순도로 정제된 핵산의 사용량이 늘어남에 따라 다양한 생물시료로부터 보다 신속하고 순수하게 핵산을 분리하고자 하는 노력이 계속되고 있다.In recent years, as the use of highly purified nucleic acids has increased in various fields such as biotechnology, diagnostic medicine, pharmacy medicine, and metabolic medicine, efforts have been continuously made to separate nucleic acids from various biological samples more quickly and purely. .
그러나 현재까지 핵산의 분리 방법에 있어 가장 크게 발전한 부분은 유전체 DNA, 플라스미드 DNA, 메신저 RNA, 단백질, 세포 잔해 입자등 세포 용해 용액내에 포함된 여러 종류의 물질들로부터 특이적으로 핵산만을 흡착시키는 담체에 관한 기술 등 거의 모든 연구의 초점은 핵산을 흡착시키는 물질에 관한 연구와 개발에 집중되어 있는 한계가 있었다.However, the most advanced part of the nucleic acid separation method to date is that 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.
이에, 보다 신속하고 순수하게 핵산을 분리하기 위하여 무엇보다 세포 잔해 입자와 단백질 변성 응집물, 기타 다양한 세포 분해 물질들로부터 신속하게 원하는 핵산만을 분리할 수 있는 기술의 개발이 절실한 실정이다.Accordingly, in order to separate nucleic acids more quickly and purely, the development of a technology capable of rapidly separating only desired nucleic acids from cell debris particles, protein denatured aggregates, and various other cell lysates is urgently needed.
본 발명의 목적은 3,3'-디티오비스프로피온이미데이트(Dimethyl 3,3'-dithiobispropionimidate; 이하 'DTBP')를 유효성분으로 포함하는 미생물 농축용 조성물, 이를 이용한 미생물 농축 방법 및 키트; DTBP를 유효성분으로 포함하는 핵산 추출용 조성물, 이를 이용한 핵산 추출 방법 및 키트; 및 DTBP를 유효성분으로 포함하는 미생물 농축 및 핵산 추출용 조성물, 이를 이용한 미생물 농축과 동시에 상기 농축된 미생물로부터 핵산을 추출하는 방법 및 키트를 제공하는데 있다.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; And a composition for concentrating microorganisms and extracting nucleic acids comprising DTBP as an active ingredient, a method and kit for extracting nucleic acids from the concentrated microorganisms simultaneously with the concentration of microorganisms using the same.
상기 목적을 달성하기 위하여, 본 발명은 DTBP를 유효성분으로 포함하는 미생물 농축용 조성물을 제공한다.In order to achieve the above object, the present invention provides a composition for concentrating microorganisms comprising DTBP as an active ingredient.
또한, 본 발명은 상기 조성물을 포함하는 미생물 농축 키트를 제공한다.The present invention also provides a microbial enrichment kit comprising the composition.
또한, 본 발명은 상기 DTBP에 미생물이 함유된 시료를 접촉시키는 단계를 포함하는 미생물 농축 방법을 제공한다.The present invention also provides a method for concentrating microorganisms comprising contacting a sample containing microorganisms with the DTBP.
또한, 본 발명은 DTBP를 유효성분으로 포함하는 핵산 추출용 조성물을 제공한다.The present invention also provides a nucleic acid extracting composition comprising DTBP as an active ingredient.
또한, 본 발명은 상기 조성물을 포함하는 핵산 추출 키트를 제공한다.The present invention also provides a nucleic acid extraction kit comprising the composition.
또한, 본 발명은 대상물에 아민기를 도입하여 개질하는 단계(제1단계); 상기 개질된 대상물 상에 핵산 시료와 DTBP를 주입하고 핵산과 상기 DTBP 간의 복합체를 형성시키는 단계(제2단계); 및 상기 복합체가 형성된 대상물에 용출 완충액을 처리하여 핵산을 추출하는 단계(제3단계)를 포함하는 핵산 추출방법을 제공한다.In addition, 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).
또한, 본 발명은 DTBP를 유효성분으로 포함하는 미생물 농축 및 핵산 추출용 조성물을 제공한다.The present invention also provides a composition for microorganism concentration and nucleic acid extraction comprising DTBP as an active ingredient.
또한, 본 발명은 상기 조성물을 포함하는 미생물 농축 및 핵산 추출 키트를 제공한다.The present invention also provides a microbial enrichment and nucleic acid extraction kit comprising the composition.
또한, 본 발명은 대상물에 아민기를 도입하여 개질하는 단계(제1단계); 상기 개질된 대상물 상에 미생물이 함유된 시료와 DTBP를 접촉시켜 미생물을 농축시키는 단계(제2단계); 상기 농축된 미생물로부터 핵산을 분리하는 단계(제3단계); 상기 분리된 핵산과 상기 DTBP 간의 복합체를 형성시키는 단계(제4단계); 및 상기 복합체가 형성된 대상물에 용출 완충액을 처리하여 핵산을 추출하는 단계(제5단계)를 포함하는 미생물 농축과 동시에 상기 농축된 미생물로부터 핵산을 추출하는 방법을 제공한다.In addition, 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.
본 발명은 DTBP를 이용한 미생물 농축 또는 핵산 추출 방법에 관한 것으로서, 본 발명자들은 DTBP를 이용해서 미생물 농축이 가능한 기술을 개발하였으며, 또한 농축된 미생물에서 바로 핵산 추출할 수 있는 방법을 개발하였다. 미생물 농축과 핵산 추출 기술을 하나의 튜브(tube) 또는 칩(chip)에서 가능하게 함으로써, 외부로 오는 오염이나, 비용, 시간, 복잡성 등을 확연히 줄일 것으로 예상된다.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. By enabling microbial enrichment and nucleic acid extraction techniques in a single tube or chip, it is expected to significantly reduce external contamination, cost, time and complexity.
도 1은 본 발명인 DTBP/Thin film Sample 분석의 개략적 모식도를 나타낸 도면이다.1 is a schematic view of the present invention DTBP / Thin film Sample analysis.
도 2는 박막 장치의 구성을 나타낸 분해도이다.2 is an exploded view showing the configuration of a thin film device.
도 3은 DTBP 농도 변화에 따른 DNA 추출 효율을 나타낸 도면이다.3 is a diagram showing the DNA extraction efficiency according to the DTBP concentration change.
도 4는 대장암 세포주(HCT116 cell line; 이하 'HCT116')로부터 추출한 DNA의 End-point PCR(a) 및 Real-time PCR(b)을 나타낸 도면이다.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').
도 5는 유방암 세포주(MCF cell line; 이하 'MCF')로부터 추출한 DNA의 End-point PCR(a) 및 Real-time PCR(b)을 나타낸 도면이다.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').
도 6은 브루셀라 오비스(Brucella Ovis)로부터 추출한 DNA의 PCR 결과를 나타낸 도면이다. M: DNA 마커(marker), Q: 기존 Qiagen 방법으로 추출한 DNA 샘플, DTBP: 본 발명의 DTBP 방식을 이용하여 추출한 DNA 샘플, N: 음성 대조군(negative control). 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).
도 7은 RNA 추출 방법(a) 및 RNA와 cDNA의 동시 추출하는 방법(b)에 대해 나타낸 도면이다. (a) 동일한 샘플을 4회 반복하여 실험을 수행하였다. L: DNA 마커(marker), N: 음성 대조군(negative control). (b) 동일한 샘플을 2회 반복하여 실험을 수행하였다. L: DNA 마커(marker).7 is a diagram illustrating a method of extracting RNA (a) and a method of simultaneously extracting RNA and cDNA (b). (a) The experiment was performed by repeating the same sample four times. L: DNA marker, N: negative control. (b) The experiment was performed by repeating the same sample twice. L: DNA marker.
도 8은 DTBP를 이용한 병원균(대장균) 농축 분석 결과를 나타낸다.8 shows the results of pathogen (E. coli) concentration analysis using DTBP.
도 9는 DTBP를 이용하여 하나의 칩에서 병원균(대장균) 농축과 동시에 핵산 추출 분석을 진행한 결과를 나타낸다.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.
도 10은 DTBP를 이용한 병원균(PIV3-RNA 바이러스) 농축 분석 결과를 나타낸다. P: 양성 대조군(positive control), N: 음성 대조군(negative control), L: 50 bp DNA ladder, 1번 내지 4번 샘플: 기존 Qiagen 방법으로 추출한 DNA 샘플, 1-1번 내지 4-1번 샘플: DTBP 방식을 이용하여 농축하여 추출한 DNA 샘플. 1번 샘플의 경우, 기존 방법으로는 음성으로 진단되었으나, DTBP 농축한 1-1번 샘플에서는 양성으로 확인되었다. 2, 3번 샘플의 경우, 기존 방법 및 DTBP 농축한 샘플 모두 확실한 음성이며, 4번 샘플의 경우, 기존에 양성이던 샘플의 밴드 세기(intensity)를 DTBP 농축한 4-1번 샘플은 확연히 증가시킨 것을 확인하였다. 10 shows the results of pathogen (PIV3-RNA virus) enrichment analysis using DTBP. P: positive control, N: negative control, L: 50 bp DNA ladder, samples 1 to 4: DNA samples extracted by conventional Qiagen method, samples 1-1 to 4-1 : DNA sample concentrated and extracted using DTBP method. In case of sample 1, it was diagnosed as a negative method in the conventional method, but it was confirmed as positive in sample 1-1 with DTBP concentration. For samples 2 and 3, both the conventional method and the DTBP-concentrated samples were positive for samples, and for sample 4, the samples 4-1 with DTBP-concentrated samples were significantly increased. It was confirmed.
본 발명은 DTBP를 유효성분으로 포함하는 미생물 농축용 조성물을 제공한다.The present invention provides a composition for concentrating microorganisms comprising DTBP as an active ingredient.
바람직하게는 상기 미생물은 박테리아, 바이러스 또는 세포 등 음전하를 띄는 모든 미생물일 수 있다.Preferably the microorganism may be any negatively charged microorganism such as bacteria, viruses or cells.
또한, 본 발명은 상기 조성물을 포함하는 미생물 농축 키트를 제공한다. 상기 키트에는 효과적인 미생물 농축에 필요한 pH 조절 등을 위한 완충액 등이 추가적으로 포함될 수 있다.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.
또한, 본 발명은 대상물에 아민기를 도입하여 개질하는 단계(제1단계); 및 상기 개질된 대상물 상에 미생물이 함유된 시료와 DTBP를 접촉시키는 단계(제2단계)를 포함하는 미생물 농축 방법을 제공한다.In addition, 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).
본 발명에 있어서, 상기 대상물은 표면에 실란 화합물로 개질된 것일 수 있다. 바람직하게는 상기 실란 화합물은 하기 화학식 1로 표시되는 화합물일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the object may be one modified with a silane compound on the surface. Preferably, the silane compound may be a compound represented by the following Chemical Formula 1, but is not limited thereto.
[화학식 1][Formula 1]
Figure PCTKR2017009254-appb-I000001
Figure PCTKR2017009254-appb-I000001
상기 식에서, R1 내지 R3는 각각 같거나 다를 수 있으며, C1 내지 C4의 알킬 또는 C1 내지 C4의 알콕시 중 어느 하나이고, R4는 아미노(C1 내지 C10)알킬, 3-(2-아미노(C1 내지 C4)알킬아미노)(C1 내지 C4)알킬 또는 3-[2-(2-아미노(C1 내지 C4)알킬아미노)(C1 내지 C4)알킬아미노](C1 내지 C4)알킬 중 어느 하나임.Wherein 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.
보다 바람직하게는 상기 실란 화합물은 (3-아미노프로필)트리에톡시실란((3-aminopropyl)triethoxysilane; APTES), (3-아미노프로필)트리메톡시실란((3-aminopropyl)trimethoxysilane), (1-아미노메틸)트리에톡시실란((1-aminomethyl)triethoxysilane), (2-아미노에틸)트리에톡시실란((2-aminoethyl)triethoxysilane), (4-아미노부틸)트리에톡시실란((4-aminobutyl)triethoxysilane), (5-아미노펜틸)트리에톡시실란((5-aminopentyl)triethoxysilane), (6-아미노헥실)트리에톡시실란((6-aminohexyl)triethoxysilane), 3-아미노프로필(디에톡시)메틸실란(3-aminopropyl(diethoxy)methylsilane; APDMS), N-[3-(트리메톡시실릴)프로필]에틸렌디아민(N-[3-(trimethoxysilyl)propyl]ethylenediamine), [3-(2-아미노에틸아미노)프로필]트리메톡시실란([3-(2-aminoethylamino)propyl]trimethoxysilane; AEAPTMS) 또는 3-[(트리메톡시실릴)프로필]디에틸렌트리아민(3-[(trimethoxysilyl)propyl]diethylenetriamine; TMPTA)일 수 있으나, 이에 제한되는 것은 아니다. 보다 더 바람직하게는 상기 실란 화합물은 본 발명의 실시예에서 기재한 (3-아미노프로필)트리에톡시실란(APTES) 또는 3-아미노프로필(디에톡시)메틸실란(APDMS)이 가장 적합하다.More preferably 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; APDMS), N- [3- (trimethoxysilyl) propyl] ethylenediamine (N- [3- (trimethoxysilyl) propyl] ethylenediamine), [3- (2- Aminoethylamino) propyl] trimethoxysilane ([3- (2-aminoethylamino) propyl] trimethoxysilane; AEAPTMS) or 3-[(trimethoxysilyl) propyl] diethylenetriamine (3-[(trimethoxysilyl) propyl] diethy lenetriamine; TMPTA), but is not limited thereto. Even more preferably the silane compound is most suitably (3-aminopropyl) triethoxysilane (APTES) or 3-aminopropyl (diethoxy) methylsilane (APDMS) described in the examples of the present invention.
바람직하게는, 상기 미생물이 함유된 시료는 미생물에 감염된 것으로 의심되는 객체의 분변, 소변, 눈물, 타액, 피부의 외부 분비물, 호흡관의 외부 분비물, 장관의 외부 분비물, 소화관의 외부 분비물, 혈장, 혈청, 혈액, 척수액, 림프액, 체액 및 조직일 수 있으나, 이에 제한되는 것은 아니다.Preferably, 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.
또한, 본 발명은 DTBP를 유효성분으로 포함하는 핵산 추출용 조성물을 제공한다. 바람직하게는, 상기 핵산은 DNA 또는 RNA일 수 있으나, 이에 제한되는 것은 아니다.The present invention also provides a nucleic acid extracting composition comprising DTBP as an active ingredient. Preferably, the nucleic acid may be DNA or RNA, but is not limited thereto.
또한, 본 발명은 상기 조성물을 포함하는 핵산 추출 키트를 제공한다. 상기 키트에는 시료 내 세포를 용해시켜 세포 내부로부터 핵산을 방출시키기 위한, 용해 완충액(lysis buffer) 또는 프로테아제(protease) 등이 추가로 포함될 수 있으며, 효과적인 핵산 추출에 필요한 pH 조절 등을 위한 완충액 등이 추가적으로 포함될 수 있다.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.
또한, 본 발명은 대상물에 아민기를 도입하여 개질하는 단계(제1단계); 상기 개질된 대상물 상에 핵산 시료와 DTBP를 주입하고 핵산과 상기 DTBP 간의 복합체를 형성시키는 단계(제2단계); 및 상기 복합체가 형성된 대상물에 용출 완충액을 처리하여 핵산을 추출하는 단계(제3단계)를 포함하는 핵산 추출방법을 제공한다.In addition, 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).
바람직하게는 상기 대상물은 표면에 실란 화합물로 개질된 것일 수 있다. 보다 바람직하게는 상기 실란 화합물은 하기 화학식 1로 표시되는 화합물일 수 있으나, 이에 제한되는 것은 아니다.Preferably the object may be one modified with a silane compound on the surface. More preferably, the silane compound may be a compound represented by the following Chemical Formula 1, but is not limited thereto.
[화학식 1][Formula 1]
Figure PCTKR2017009254-appb-I000002
Figure PCTKR2017009254-appb-I000002
상기 식에서, R1 내지 R3는 각각 같거나 다를 수 있으며, C1 내지 C4의 알킬 또는 C1 내지 C4의 알콕시 중 어느 하나이고, R4는 아미노(C1 내지 C10)알킬, 3-(2-아미노(C1 내지 C4)알킬아미노)(C1 내지 C4)알킬 또는 3-[2-(2-아미노(C1 내지 C4)알킬아미노)(C1 내지 C4)알킬아미노](C1 내지 C4)알킬 중 어느 하나임.Wherein 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.
보다 더 바람직하게는 상기 실란 화합물은 본 발명의 실시예에서 기재한 (3-아미노프로필)트리에톡시실란(APTES) 또는 3-아미노프로필(디에톡시)메틸실란(APDMS)이 가장 적합하다.Even more preferably the silane compound is most suitably (3-aminopropyl) triethoxysilane (APTES) or 3-aminopropyl (diethoxy) methylsilane (APDMS) described in the examples of the present invention.
또한, 본 발명은 DTBP를 유효성분으로 포함하는 미생물 농축 및 핵산 추출용 조성물을 제공한다.The present invention also provides a composition for microorganism concentration and nucleic acid extraction comprising DTBP as an active ingredient.
바람직하게는 상기 미생물은 박테리아, 바이러스 또는 세포 등 음전하를 띄는 모든 미생물일 수 있다.Preferably the microorganism may be any negatively charged microorganism such as bacteria, viruses or cells.
바람직하게는, 상기 핵산은 DNA 또는 RNA일 수 있으나, 이에 제한되는 것은 아니다.Preferably, the nucleic acid may be DNA or RNA, but is not limited thereto.
또한, 본 발명은 상기 조성물을 포함하는 미생물 농축 및 핵산 추출 키트를 제공한다. 상기 키트에는 효과적인 미생물 농축 및 핵산 추출에 필요한 pH 조절 등을 위한 완충액 등이 추가적으로 포함될 수 있으며, 시료 내 세포를 용해시켜 세포 내부로부터 핵산을 방출시키기 위한, 용해 완충액(lysis buffer) 또는 프로테아제(protease) 등이 추가로 포함될 수 있다.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.
또한, 본 발명은 대상물에 아민기를 도입하여 개질하는 단계(제1단계); 상기 개질된 대상물 상에 미생물이 함유된 시료와 DTBP를 접촉시켜 미생물을 농축시키는 단계(제2단계); 상기 농축된 미생물로부터 핵산을 분리하는 단계(제3단계); 상기 분리된 핵산과 상기 DTBP 간의 복합체를 형성시키는 단계(제4단계); 및 상기 복합체가 형성된 대상물에 용출 완충액을 처리하여 핵산을 추출하는 단계(제5단계)를 포함하는 미생물 농축과 동시에 상기 농축된 미생물로부터 핵산을 추출하는 방법을 제공한다.In addition, 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.
바람직하게는 상기 대상물은 표면에 실란 화합물로 개질된 것일 수 있다. 보다 바람직하게는 상기 실란 화합물은 하기 화학식 1로 표시되는 화합물일 수 있으나, 이에 제한되는 것은 아니다.Preferably the object may be one modified with a silane compound on the surface. More preferably, the silane compound may be a compound represented by the following Chemical Formula 1, but is not limited thereto.
[화학식 1][Formula 1]
Figure PCTKR2017009254-appb-I000003
Figure PCTKR2017009254-appb-I000003
상기 식에서, R1 내지 R3는 각각 같거나 다를 수 있으며, C1 내지 C4의 알킬 또는 C1 내지 C4의 알콕시 중 어느 하나이고, R4는 아미노(C1 내지 C10)알킬, 3-(2-아미노(C1 내지 C4)알킬아미노)(C1 내지 C4)알킬 또는 3-[2-(2-아미노(C1 내지 C4)알킬아미노)(C1 내지 C4)알킬아미노](C1 내지 C4)알킬 중 어느 하나임.Wherein 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.
보다 더 바람직하게는 상기 실란 화합물은 본 발명의 실시예에서 기재한 (3-아미노프로필)트리에톡시실란(APTES) 또는 3-아미노프로필(디에톡시)메틸실란(APDMS)이 가장 적합하다.Even more preferably the silane compound is most suitably (3-aminopropyl) triethoxysilane (APTES) or 3-aminopropyl (diethoxy) methylsilane (APDMS) described in the examples of the present invention.
바람직하게는, 상기 미생물이 함유된 시료는 미생물에 감염된 것으로 의심되는 객체의 분변, 소변, 눈물, 타액, 피부의 외부 분비물, 호흡관의 외부 분비물, 장관의 외부 분비물, 소화관의 외부 분비물, 혈장, 혈청, 혈액, 척수액, 림프액, 체액 및 조직일 수 있으나, 이에 제한되는 것은 아니다.Preferably, 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.
본 발명에 있어서, "대상물"은 박막장치, 자성 비드(magnetic bead), 링 공진기(ring resonator) 또는 나노입자(nanoparticle) 중 어느 하나일 수 있으나, 이에 제한되는 것은 아니다. 보다 바람직하게는 상기 대상물은 본 발명의 실시예서 기재한 입구홀과 출구홀이 각각 관통 형성된 상부박막; 상기 상부박막으로부터 이격 배치된 하부박막; 그 입구단 및 출구단이 상기 상부박막의 입구홀 및 출구홀에 각각 대응하여 연통하는 마이크로 채널이 내측에 패턴 형성되고, 상기 마이크로 채널의 입구부와 연통하는 주입로가 상기 입구단에 인접하도록 형성되며, 상기 상부박막과 하부박막 사이에 배치되는 마이크로 채널챔버; 및 상기 상부박막 및 하부박막의 각 측부를 밀봉하여 상기 마이크로 채널챔버를 밀봉시키는 밀봉수단을 포함하는 박막장치일 수 있다.In the present invention, 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.
이하, 하기 실시예에 의해 본 발명을 보다 상세하게 설명한다. 다만, 이러한 실시예에 의해 본 발명이 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.
도 1을 참조하면, 본 발명에 따른 핵산 분석은 박막 장치 내에서 DTBP를 이용한 핵산 분석인 DTBP/Thin film Sample 분석으로서, 시료 용출/배양, 세척 및 용출의 세 단계를 포함하고 있으며, 원심분리하지 않고 수행한다. 예를 들어, 실란 화합물로서 3-아미노프로필트리에톡시실란(APTES)을 통해 박막 장치를 개질하고, 이러한 개질로 인하여 소수성인 박막 장치를 친수성으로 전환시킨다.Referring to Figure 1, 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. For example, the thin film device is modified via 3-aminopropyltriethoxysilane (APTES) as the silane compound, which converts the hydrophobic thin film device into hydrophilic.
상기 개질된 박막 장치 상에 핵산시료, 및 용출 완충액과 DTBP 용액을 주입한다. 상기 핵산의 아미노기와 DTBP의 이-기능성 아민 반응기와의 상호작용에 의한 핵산과 DTBP 사이에 가교 메카니즘을 이용하여 핵산과 DTBP 간의 복합체를 형성시키고 시료에서 DNA를 추출할 수 있다.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.
이때 DTBP는 하기 화학식 2 구조를 가지며, 이-기능성 이미도에스터(imidoesters) 및 이황화(disulfide) 결합을 포함하고 있기 때문에, 상기 DTBP는 가역적인 가교 구조를 형성함으로써 세포, 단백질 및 핵산의 아미노 반응성 가교제로 사용되었다. DTBP와 단백질과의 상호작용이 아니라 DTBP와 핵산 사이의 빠르고 강한 상호결합을 통해 핵산 시료로부터 고효율의 핵산 추출이 가능하다.In this case, since the DTBP has a structure of Formula 2 and includes di-functional imidoesters and disulfide bonds, the DTBP forms a reversible crosslinking structure to form an amino reactive crosslinking agent for cells, proteins and nucleic acids. Was used. Rather than interacting with DTBP and proteins, fast and strong interactions between DTBP and nucleic acids allow for highly efficient nucleic acid extraction from nucleic acid samples.
[화학식 2][Formula 2]
Figure PCTKR2017009254-appb-I000004
Figure PCTKR2017009254-appb-I000004
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.
<< 실시예Example 1> 박막 장치 제작 및 전처리 1> thin film device fabrication and pretreatment
1. 박막 장치 제작1. Thin film device fabrication
레이저 컷팅 기기(Universal Laser Systems, Scottsdale, USA)를 이용하여 본 발명의 박막 장치를 제작하였다[도 2(a) 참조]. 먼저, 상기 박막 장치는 상부 박막과 하부 박막, 그리고 상부 박막과 하부 박막 사이에 삽입된 마이크로 유체 챔버로 구성되며, 상기 마이크로 유체 챔버는 핵산 공급원으로부터 DNA를 추출하기 위해 챔버 내의 유로에 의해 서로 연결된 다수의 슬롯-형 마이크로 웰로 이루어져 있다. The thin film device of the present invention was manufactured using a laser cutting machine (Universal Laser Systems, Scottsdale, USA) (see Fig. 2 (a)). First, 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.
상기 마이크로 유체 챔버를 제작하기 위해, 300 ㎛ 두께의 양면테이프(100 ㎛ 두께의 양면테이프 사이에 끼워진 100 ㎛ 두께의 폴리에스터 막)에 마이크로 유체 챔버 디자인을 레이저 컷팅 기기로 절단하여 마이크로 유체 챔버를 제작하였다. 박막(상부 및 하부)은 레이저 컷팅 기기를 이용하여 마이크로 유체 챔버와 동일한 치수로 절단하였다. In order to fabricate the microfluidic chamber, the microfluid chamber design was cut by a laser cutting machine on a 300 µm thick double-sided tape (100 µm thick polyester film sandwiched between 100 µm 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.
상기 상부 박막에 관통 구멍인 입구(inlet)와 출구(outlet)를 제작하였다. 레이저 컷팅 마이크로 유체 챔버의 상부 및 하부의 표면에 영구 접착제를 이용하여 각각 레이저 컷팅 박막(상부 및 하부)을 접착시켰다. 상기 마이크로 유체 챔버의 높이는 약 300 ㎛이며, 총 부피는 300 ㎕이었다(300 ㎕ 양, 8.4 cm × 3.7 cm). 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 μm and the total volume was 300 μl (300 μl amount, 8.4 cm × 3.7 cm).
핵산 공급원을 주입하기 위한 튜빙 어댑터는 3 mm 두께를 갖는 캐스트 아크릴 시트(MARGA CIPTA, Indonesia)를 양면테이프 한쪽 면에 부착하였고, 레이저 컷팅 기기로 절단 및 천공하여 제조하였다. 상기 제조된 튜빙 어댑터는 마이크로 유체 챔버의 유입구와 배출구에 각각 부착하였다. 이 후 미리 절단된 타이곤 튜빙(AAC02548; Cole-Parmer, Vernon Hills, USA)을 어댑터의 구멍에 위치시킨 후 에폭시를 사용하여 밀봉하였다. 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.
이렇게 제작된 박막 장치는 다양한 용량의 핵산 시료들(100 ㎕, 300 ㎕, 및 500 ㎕)을 처리할 수 있는 장점이 있다.The thin film device manufactured as described above has the advantage of being capable of processing various nucleic acid samples (100 μl, 300 μl, and 500 μl).
2. 박막 장치 전처리2. Thin film device pretreatment
상기 박막 장치를 이용한 DNA 분석을 위하여, 박막 장치 내부를 10분 동안 산소 플라즈마로 처리한 후 상기 플라즈마 처리된 박막 장치를 65℃에서 10 내지 60분 동안 2% 3-아미노프로필트리에톡시실란(APTES)을 함유한 수용액에 침지시킨 후 탈이온수로 완전히 세정하였다. 세정 후, 박막 장치를 경화(cure)시키기 위해, 상기 세정된 박막 장치를 신속하게 질소 기류 하에서 건조시켜 박막 장치를 아민으로 개질하였다.For DNA analysis using the thin film device, 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.
Drop Shape Analyzer(DSA100, KRUSS, Germany)을 이용한 아민-개질된 박막 장치의 물 접촉각 측정을 통해 온도 및 배양시간에 따라 박막 장치의 친수성이 상당히 변화하였음을 알 수 있었다. 65 ℃에서 10분 동안 상기 박막 장치를 APTES로 실란화(silanization)한 후, 상기 박막 표면 친수성은 증가하였다(약 30 내지 40℃). The water contact angle of the amine-modified thin film device using the Drop Shape Analyzer (DSA100, KRUSS, Germany) 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.).
<< 실시예Example 2>  2> DTBPDTBP /Thin film Sample 분석/ Thin film Sample analysis
본 발명에서는 앞서 제작된 박막 장치에 디메틸3,3'-디티오비스프로피온이미데이트(DTBP)를 적용한 핵산 분석법을 DTBP로 명명하였으며, 이하 실험에서 DTBP 분석을 수행하였다.In the present invention, a nucleic acid analysis method in which dimethyl 3,3'-dithiobispropionimate (DTBP) was applied to the previously prepared thin film device was named DTBP, and DTBP analysis was performed in the following experiment.
즉, 앞서 아민으로 개질된 박막 장치(300 ㎕ 양, 8.4 cm × 3.7 cm)를 이용하여 DNA를 추출하기 위해 먼저 최적화된 분석 용액을 준비하였다. 상기 최적화된 분석 용액은 100 mM 트리스-염산 (pH 8.0)을 함유하는 용출 완충액, 10 mM의 EDTA, 1% SDS, 10%의 트리톤 X-100을 DTBP (100 mg/㎖)와 혼합하여 준비한 후, 핵산 분석 시료로서 세포, 박테리아, 혈액 또는 소변 유래 각 시료 100 ㎕를 상기 분석 용액 200 ㎕와 혼합하였다.That is, an optimized assay solution was first prepared to extract DNA using a thin film device (300 μl 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) As a nucleic acid analysis sample, 100 μl of each sample derived from cells, bacteria, blood or urine was mixed with 200 μl of the assay solution.
상기 혼합된 핵산 분석 시료와 분석 용액이 혼합된 혼합액을 아민으로 개질된 박막 장치의 상부 기판 입구로 유입시키고, 마이크로 유체 챔버 내로 상기 혼합액이 이동하면서 DTBP의 2개의 아민기와 DNA가 결합하며 또한 박막 장치 내 개질된 아민기와 DNA가 결합하여 복합체를 형성하여 DNA를 분리할 수 있었다. 이때, 박막 장치는 핵산 분석 시료로부터 DNA를 충분히 추출하기 위하여 20분 동안 항온(56℃)을 유지한 배양기 또는 컨트롤러(Alpha Omega Instruments)를 포함한 열전냉각기(thermoelectric cooler; TEC) 중 어느 하나에 놓아 두었다. 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. At this time, 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. .
DTBP-DNA 복합체 중 이물질을 제거하기 위해 PBS 완충액으로 세척한 후, 용출 완충액(10 mM의 중탄산나트륨, pH 10.6)을 사용하여 DNA를 추출하였다. 추출된 DNA의 양과 순도를 측정한 후 Enspire Multimode Plate Reader(PerkinElmer)를 이용하여 260 nm (DNA) 및 280 nm (단백질)에서의 시료의 광학 밀도 비율을 결정하였다. 종래 DNA 추출법과 본 발명의 DTBP 분석을 비교하기 위해, 알려진 방법(Qiagen, Hilden, Germany)에 따라 QIAmp DNA mini kit를 사용하여 분석하였다.After washing with PBS buffer to remove foreign substances in DTBP-DNA complex, 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).
도 3과 같이, DTBP 농도별로 결합력을 확인한 결과, DTBP 농도가 100 mg/㎖일 때 DNA 결합 효율이 가장 높은 것으로 확인되었다.As shown in FIG. 3, as a result of confirming the binding force for each DTBP concentration, the DNA binding efficiency was confirmed to be the highest when the DTBP concentration was 100 mg / ml.
<< 실시예Example 3>  3> DTBPDTBP 분석을 이용한  Analysis 진핵세로포부터의From eukaryotic cells DNA 추출 DNA extraction
5% CO2 분위기, 37℃ 습한 배양기에서 10 % 태아우아혈청(fetal calf serum; 이하 'FCS')을 보충한 고 글루코오스 둘베코 변형 이글 배지(Dulbecco's modified eagle medium; DMEM, DMEM Life Technology)의 플라스틱 배양 플레이트에서 두개의 진핵 세포인 HCT116 및 MCF를 각각 배양한 후, 실시예 1과 동일한 방법으로 진핵세로포부터 DNA를 추출하되, 게놈성 DNA를 추출하기 위하여 단백질 분해효소인 프로테이나제 K를 처리하였다. 그리고, 비교를 위하여, QIAmp DNA 미니 키트를 이용하여 진핵세포로부터 DNA를 추출하였다. 본 실시예에서 사용한 HCT116 및 MCF는 미국 ATCC(https://www.atcc.org)에서 구입하여 사용하였다.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 After culturing two eukaryotic cells, HCT116 and MCF, in a culture plate, DNA was extracted from eukaryotic cells in the same manner as in Example 1, but proteinase K, a proteinase, was extracted to extract genomic DNA Treated. And, for comparison, DNA was extracted from eukaryotic cells using a QIAmp DNA mini kit. HCT116 and MCF used in this example were purchased from the US ATCC (https://www.atcc.org).
DNA의 양과 순도를 확인하기 위해 End-point PCR 및 실시간 PCR(real time-PCR)을 수행하였다. 일부 유전자(Actin)의 정방향 및 역방향 프라이머를 약 24 염기쌍의 정상 길이로 합성하였다. End-point PCR은 15분 동안 95℃에서 초기 변성 단계; 95℃에서 45초, 59℃에서 45초(Actin), 및 72℃에서 45초를 1사이클로하여 총 45 사이클; 및 72℃, 10분 동안 최종 연장 단계로 이루어져 있다. 5 내지 10 ㎕의 DNA를 1 × PCR 완충액(Qiagen), 2.5 mM 염화마그네슘(MgCl2), 0.25 mM 데옥시뉴클레오티드 트리포스페이트(deoxynucleotide triphosphate), 25 pmol의 각 프라이머, 및 Taq DNA 중합효소 1 유닛을 포함하는 총 부피 25 ㎕에서 증폭하였다.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. 5-10 μl of DNA was added to 1 × PCR buffer (Qiagen), 2.5 mM magnesium chloride (MgCl 2 ), 0.25 mM deoxynucleotide triphosphate, 25 pmol of each primer, and 1 unit of Taq DNA polymerase. Amplified at a total volume of 25 μl.
실시간 PCR(real time-PCR) 분석을 위해, 다음 단계가 LightCycler 2.0(로슈 다이어그노스틱스)에서 제공된 방법을 하기와 같이 변형하였다. 5 내지 10 ㎕의 DNA를 4 ㎕의 LightCycler FastStart DNA Master 믹스, 25 pmol의 각 프라이머, 1 × PCR 완충액 2 ㎕(큐아젠, 힐덴, 독일), 2.5 mM 염화마그네슘(MgCl2), 0.25 mM 데옥시뉴클레오티드 트리포스페이트(deoxynucleotide triphosphate), 및 증류수를 포함하는 총 부피 20 ㎕에서 증폭하였다. 95℃에서 10분 동안 처음 예비 처리한 후 95℃에서 10초, 59℃에서 30초(Actin), 및 72℃에서 10초를 1사이클로하여 총 50 사이클을 수행하고, 30초 동안 40℃에서 냉각 단계를 거쳤다. SYBR 녹색 신호를 가진 증폭된 생성물은 LightCycler 2.0 (Roche Diagnostics)에서 수행하였다.For real time-PCR analysis, the following steps were modified to the method provided in LightCycler 2.0 (Roche Diagnostics) as follows. 5-10 μl of DNA is mixed with 4 μl of LightCycler FastStart DNA Master, 25 pmol of each primer, 2 μl of 1 × PCR buffer (Qiagen, Hilden, Germany), 2.5 mM magnesium chloride (MgCl 2 ), 0.25 mM deoxy Amplification was performed at a total volume of 20 μl containing nucleotide triphosphate, and distilled water. After the first pretreatment at 95 ° C. for 10 minutes, a total of 50 cycles were performed with 10 cycles at 95 ° C., 30 seconds at 59 ° C. (Actin), and 10 seconds at 72 ° C., followed by cooling at 40 ° C. for 30 seconds. I went through the steps. Amplified product with SYBR green signal was performed in LightCycler 2.0 (Roche Diagnostics).
그리고, 도 4 및 도 5의 End-point PCR(도 4(a) 및 도 5(a))과 실시간 PCR(도 4(b) 및 도 5(b))을 참조하면, DTBP를 이용하여 추출한 DNA의 End-point PCR 결과, Qiagen 제품에 비해 HCT116는 약 1.3배, MCF는 약 2.6배 높은 양의 DNA가 추출된 것을 확인할 수 있었고, 실시간 PCR 결과는 Qiagen 제품과 크게 차이가 나지 않는 것을 확인할 수 있었다. 4 and 5, the end-point PCR (FIGS. 4A and 5A) and real-time PCR (FIGS. 4B and 5B) are extracted using DTBP. As a result of 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.
<< 실시예Example 4>  4> DTBPDTBP 분석을 이용한 박테리아 세포로부터의 DNA 추출 DNA Extraction from Bacterial Cells Using Assays
박테리아 세포에서 DTBP 분석 성능을 확인하기 위해, DTBP 분석을 이용하여 추출된 DNA를 사용하여 PCR 기반 DNA 증폭을 실시하였다. 모든 프라이머는 대장균 (Escherichia coli), 마이코박테리움 압세수스(Mycobacterium abscessus), 마이코박테리움 고르도니에(Mycobacterium gordonae) 및 살모넬라 균주(Salmonella Strains) (살모넬라 티피뮤리움(Salmonella Typhimurium), 살모넬라 뉴폴트(Salmonella Newport), 및 살모넬라 세인트폴(Salmonella Saintpaul))의 상용 프라이머를 사용하였다. 본 실시예에서 사용한 해당 균주의 상용 프라이머는 미국 IDT(https://www.idtdna.com)에서 구입하여 사용하였다.In order to confirm the performance of DTBP analysis in bacterial cells, 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).
최적화 반응을 위해, 100 mM 트리스-염산 (pH 8.0)을 함유하는 용출 완충액, 10mM의 EDTA, 1%의 SDS, 10%의 트리톤 X-100 및 리소자임 20 mg/㎖을 DTBP(50 mg/㎖)와 혼합하였다. 본 발명의 DTBP 방법의 유효성을 검증하기 위해 일반적인 PCR을 수행하였다. 대장균(E. coli) XL1 블루 균주(한국미생물자원센터로부터 구입; https://kctc.kribb.re.kr)를 50 ㎍/㎖의 테트라사이클린(tetracycline)과 Luria-Bertani(LB) 배지에 접종하고, 쉐이킹 상태(shaking condition)로 37℃에서 하루 동안 배양하였고, 103∼107의 콜로니 형성 단위(colony forming unit; CFU)의 시료를 시험을 위해 사용하였다. DTBP 분석 및 큐아젠 키트 분석에서 사용하기 위해 배양시킨 대장균(Escherichia coli), 마이코박테리움 압세수스(Mycobacterium abscessus), 마이코박테리움 고르도니에(Mycobacterium gordonae) 및 살모넬라 균주(Salmonella Strains)(살모넬라 티피뮤리움(Salmonella Typhimurium), 살모넬라 뉴폴트(Salmonella Newport), 살모넬라 세인트폴(Salmonella Saintpaul)) 및 브루셀라 오비스(Brucella Ovis)에서 박테리아 DNA를 추출하였다. 본 실시예에서 사용한 균주는 한국미생물자원센터(https://kctc.kribb.re.kr)로부터 구입하여 사용하였다.For the optimization reaction, 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) was inoculated into 50 μg / ml of tetratracycline and Luria-Bertani (LB) medium. and it was cultured for one day at 37 ℃ with shaking conditions (shaking condition), 10 3 ~10 7 colony forming units; the sample (CFU colony forming unit) was used for testing. Escherichia 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).
박테리아 유전자의 유전학적 분석을 위해, DTBP 분석 및 큐아젠 키트 분석에서 추출된 DNA 2 ㎕를 1 × PCR 완충액(큐아젠, 힐덴, 독일), 2.5 mM 염화마그네슘(MgCl2), 0.25 mM 데옥시뉴클레오티드 트리포스페이트(deoxynucleotide triphosphate), 각 프라이머 25 pmol, 및 Taq DNA 중합효소 1 유닛을 포함하는 총 부피 25 ㎕를 사용하여 15분 동안 95℃에서 증폭하였다; 95℃에서 30초, 60℃에서 30초(마이코박테리움 압세수스(Mycobacterium abscessus), 마이코박테리움 고르도니에(Mycobacterium gordonae), 살모넬라 균주(Salmonella Strains) 및 브루셀라 오비스(Brucella Ovis)), 및 72℃에서 30초를 1 사이클로하여 총 45 사이클; 및 72℃, 7분 동안 최종 연장 단계. PCR 증폭물들은 PCR 생성물을 에티듐 브로마이드(EtBr)(시그마-알드리치)를 함유한 2% 아가로즈 젤 상에서 분리하는 젤 전기영동법으로 가시화되었다. 상기 젤은 Gel Doc System(Bio-Rad)을 사용하여 가시화되었다. DNA 농도 및 순도의 측정은 UV 분광분석기(Perkin-Elmer)로 수행되었다. For genetic analysis of bacterial genes, 2 μl of DNA extracted from DTBP assay and Qiagen kit assay was added to 1 × PCR buffer (Qiagen, Hilden, Germany), 2.5 mM magnesium chloride (MgCl 2 ), Amplification at 95 ° C. for 15 minutes using a total volume of 25 μl comprising 0.25 mM deoxynucleotide triphosphate, 25 pmol of each primer, and 1 unit of Taq DNA polymerase; 30 seconds at 95 ° C, 30 seconds at 60 ° C (Mycobacterium abscessus, Mycobacterium gordonae, Salmonella strains and Brucella Ovis) A total of 45 cycles of 30 seconds at 72 ° C. in one cycle; And 72 ° C., final extension step for 7 minutes. 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).
DTBP를 이용하여 브루셀라 오비스(Brucella Ovis)로부터 추출된 DNA로 PCR 분석을 수행한 결과, Qiagen 제품과 크게 차이가 나지 않는 것을 확인할 수 있었다(도 6). As a result of PCR analysis with DNA extracted from Brucella Ovis using DTBP, it was confirmed that there is no significant difference from Qiagen products (FIG. 6).
<< 실시예Example 5>  5> DTBPDTBP 분석을 이용한 인간  Human with analysis 체액로부터의From body fluids DNA 추출 DNA extraction
인간의 체액으로 DTBP 분석 능력을 검증하기 위해, 체액 시료 200 ㎕(전혈 및 소변)을 상기 박막 장치에 유입하여 DNA를 추출하였다. 먼저, 앞서 제작된 박막 장치 내로 프로테이나제 K와 DTBP를 포함한 용출 완충액과 체액 시료를 각각 유입한 후, 마이크로 채널 챔버로 이동시켜 체액 시료 내 DNA와 DTBP 간의 복합체를 형성시킨 후, 실시예 2와 동일한 방법으로 DNA를 추출하였다. 이때, 용출 완충액과 체액 시료는 시린지 펌프(KD Scientific, MA)를 사용하여 2개의 다른 입구에 각각 1.5 ㎖/hr의 유속으로 10분간 유입시켰으며, DNA의 추출 및 정제를 위하여, 카트리지를 20분 동안 56℃에서 배양하였다. 그리고, 시린지 펌프에 의한 PBS 완충액 유입을 위한 입구의 유속은 4 ㎖/hr으로 10분간 증가시켰다. 마지막으로, 추출된 DNA를 100 ㎕의 용출 완충액으로 용출하였다. 또한, 비교를 위해 QIAmp DNA 미니 키트(힐덴, 독일)를 사용한 게놈성 DNA 추출에 200 ㎕의 전혈 또는 소변을 사용하였다. 모든 추출된 DNA는 UV 분광 광도계(Perkin-Elmer)에 의해 DNA 농도 및 순도가 결정되었다. In order to verify the DTBP analysis ability with human body fluids, 200 μl of body fluid samples (whole blood and urine) were introduced into the thin film device to extract DNA. First, 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. At this time, 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. Incubated at 56 ° C. In addition, the flow rate of the inlet for inflow of PBS buffer by the syringe pump was increased to 4 ml / hr for 10 minutes. Finally, the extracted DNA was eluted with 100 μl of elution buffer. In addition, 200 μl of whole blood or urine was used for genomic DNA extraction using the QIAmp DNA mini kit (Hilden, Germany) for comparison. All extracted DNA was determined for DNA concentration and purity by UV spectrophotometer (Perkin-Elmer).
<< 실시예Example 6>  6> DTBPDTBP 분석을 이용한 RNA 추출 RNA extraction using analysis
실시예 2와 동일한 방법으로 DTBP 분석을 이용하여 RNA를 추출하였고, 도 7(a)와 같이 외부에서 cDNA 합성 후 18S rRNA의 증폭을 통해 RNA 추출이 잘 되었음을 확인하였다.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.
그리고, 도 7(b)와 같이 DTBP 분석을 통해 RNA 추출과 cDNA 합성을 하나의 박막 장치 상에서 진행할 수 있었고, 추후 18S rRNA의 증폭을 통해 이러한 방법이 잘 개발된 것을 확인하였다.In addition, 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.
<< 실시예Example 7>  7> DTBP를DTBP 이용한 병원균(대장균) 농축 및 핵산 추출 Concentrate Pathogens (E. Coli) and Extract Nucleic Acids
앞서 제작한 박막 장치를 65℃에서 1시간 동안 3-아미노프로필(디에톡시)메틸실란(APDMS)로 표면 처리하였다. 병원균을 농축시키기 위해서, DTBP(100 mg/㎖)와 함께 병원균 시료를 1∼2 ㎖ 주입하였으며 100 ㎕/min의 유속으로 상온에서 농축시켰다. 농축된 시료는 100 ㎕에 현탁하였다. 본 실시예에서는 병원균 시료로 대장균(E. coli)을 사용하였다. 한편, Qiagen DNA Kit으로 DNA를 추출하여 비교하였다.The previously produced thin film device was surface treated with 3-aminopropyl (diethoxy) methylsilane (APDMS) at 65 ° C. for 1 hour. In order to concentrate the pathogen, 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 µl / min. The concentrated sample was suspended in 100 μl. In this example, E. coli was used as a pathogen sample. Meanwhile, DNA was extracted and compared with the Qiagen DNA Kit.
그 결과, DTBP로 농축을 진행하면, 기존 Qiagen 방법으로 농축 단계 없이 핵산 추출을 하는 경우보다 RT-PCR Ct 값이 절대값(absolute)에 근접하게 단축되는 것을 확인하였다(도 8).As a result, when the concentration was carried out by DTBP, it was confirmed that the RT-PCR Ct value was shortened closer to the absolute value (absolute) than the case of nucleic acid extraction without the concentration step by the existing Qiagen method (Fig. 8).
한편, 본 발명의 하나의 칩에서 병원균(대장균) 농축 및 핵산 추출을 동시에 진행했을 때, 다른 방법에 비해서 훨씬 Ct 값이 단축되고, 절대값(absolute)과 비슷하게 나오는 것을 확인하였다. 즉, 본 발명에 따른 방법이 1∼2 ㎖ 내에 있는 병원체를 효과적으로 농축할 수 있었으며, 동시에 핵산 추출 효율도 높은 것을 확인할 수 있었다(도 9). On the other hand, when one path of the present invention (E. coli) concentration and nucleic acid extraction is carried out at the same time, it was confirmed that the Ct value is much shorter than the other method, similar to the absolute value (absolute). That is, the method according to the present invention was able to effectively concentrate the pathogen in the 1-2 mL, and at the same time it was confirmed that the nucleic acid extraction efficiency is also high (Fig. 9).
<< 실시예Example 8>  8> DTBP를DTBP 이용한 병원균( Pathogens used PIV3PIV3 -RNA 바이러스) 농축 -RNA virus) enrichment
본 발명에 따른 방법의 병원균(바이러스) 검출 효율을 확인하기 위하여 PIV3-RNA 바이러스 감염이 의심되는 4개의 샘플에 대하여 기존에 널리 사용되는 방법인 Qiagen 키트와의 비교 실험을 수행하였다. In order to confirm the efficiency of detecting pathogens (viruses) of the method according to the present invention, a comparison experiment with a Qiagen kit, which is a widely used method, was performed on four samples suspected of PIV3-RNA virus infection.
즉, 각 샘플을 1 ㎖씩 취하여 Qiagen 키트로 추출하여 PIV3-RNA 바이러스 검출을 테스트한 경우(도 10의 1, 2, 3, 4 레인)와 본 발명에 따라 DTBP로 농축하여 추출 후 PIV3-RNA 바이러스 검출을 테스트한 경우(도 10의 1-1, 2-1, 3-1, 4-1 레인)를 비교하였다. In other words, 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.
그 결과, 1 ㎖의 시료에서 PIV3-RNA 바이러스를 DTBP 방식을 이용하여 농축했을 때, 1번 샘플의 경우, 기존 방법으로는 음성으로 진단되었으나, 본 발명에 따른 DTBP 농축 후에 양성으로 확인되었다. 2, 3번 샘플의 경우 확실한 음성이며, 4번 샘플은 기존에 양성이던 샘플의 밴드 세기(intensity)를 확연히 증가시킨 것을 확인할 수 있었다(도 10).As a result, when the PIV3-RNA virus was concentrated in a 1 ml sample using the DTBP method, the sample 1 was negatively diagnosed by the conventional method, but was positive after the DTBP concentration according to the present invention. In case of samples 2 and 3, the positive signal was confirmed, and sample 4 was able to confirm that the intensity of the band (intensity) of the previously positive sample was significantly increased (FIG. 10).
이상과 같이, 본 발명은 비록 한정된 실시예와 도면에 의해 설명되었으나, 본 발명은 이것에 의해 한정되지 않으며 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 본 발명의 기술 사상과 아래에 기재될 청구범위의 균등 범위 내에서 다양한 수정 및 변형이 가능함은 물론이다.As mentioned above, although this invention was demonstrated by the limited embodiment and drawing, this invention is not limited by this, The person of ordinary skill in the art to which this invention belongs, Of course, various modifications and variations are possible within the scope of equivalents of the claims to be described.

Claims (26)

  1. 디메틸 3,3'-디티오비스프로피온이미데이트(Dimethyl 3,3'-dithiobispropionimidate; DTBP)를 유효성분으로 포함하는 미생물 농축용 조성물.Dimethyl 3,3'-dithiobispropionimidate (Dimethyl 3,3'-dithiobispropionimidate; DTBP) as an active ingredient for a composition for concentrating microorganisms.
  2. 제1항에 있어서, 상기 미생물은 박테리아, 바이러스 또는 세포인 것을 특징으로 하는 미생물 농축용 조성물.The composition of claim 1, wherein the microorganism is a bacterium, a virus or a cell.
  3. 제1항 또는 제2항의 조성물을 포함하는 미생물 농축 키트.A microbial enrichment kit comprising the composition of claim 1.
  4. 대상물에 아민기를 도입하여 개질하는 단계(제1단계); 및Introducing and modifying an amine group into the object (first step); And
    상기 개질된 대상물 상에 미생물이 함유된 시료와 DTBP를 접촉시키는 단계(제2단계)를 포함하는 미생물 농축 방법.Contacting a sample containing microorganisms with DTBP on the modified object (second step).
  5. 제4항에 있어서, 상기 대상물은 박막장치, 자성 비드(magnetic bead), 링 공진기(ring resonator) 또는 나노입자(nanoparticle) 중 어느 하나인 것을 특징으로 하는 미생물 농축 방법.The method of claim 4, wherein the object is any one of a thin film device, a magnetic bead, a ring resonator, and a nanoparticle.
  6. 제4항에 있어서, 상기 대상물은 표면에 실란 화합물로 개질된 것을 특징으로 하는 미생물 농축 방법.The method of claim 4, wherein the object is a microorganism concentration method characterized in that the surface is modified with a silane compound.
  7. 제6항에 있어서, 상기 실란 화합물은 하기 화학식 1로 표시되는 화합물인 것을 특징으로 하는 미생물 농축 방법.The method of claim 6, wherein the silane compound is a microorganism concentration method, characterized in that the compound represented by the formula (1).
    [화학식 1][Formula 1]
    Figure PCTKR2017009254-appb-I000005
    Figure PCTKR2017009254-appb-I000005
    상기 식에서, R1 내지 R3는 각각 같거나 다를 수 있으며, C1 내지 C4의 알킬 또는 C1 내지 C4의 알콕시 중 어느 하나이고, R4는 아미노(C1 내지 C10)알킬, 3-(2-아미노(C1 내지 C4)알킬아미노)(C1 내지 C4)알킬 또는 3-[2-(2-아미노(C1 내지 C4)알킬아미노)(C1 내지 C4)알킬아미노](C1 내지 C4)알킬 중 어느 하나임.Wherein 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.
  8. 제7항에 있어서, 상기 실란 화합물은 (3-아미노프로필)트리에톡시실란((3-aminopropyl)triethoxysilane; APTES), (3-아미노프로필)트리메톡시실란((3-aminopropyl)trimethoxysilane), (1-아미노메틸)트리에톡시실란((1-aminomethyl)triethoxysilane), (2-아미노에틸)트리에톡시실란((2-aminoethyl)triethoxysilane), (4-아미노부틸)트리에톡시실란((4-aminobutyl)triethoxysilane), (5-아미노펜틸)트리에톡시실란((5-aminopentyl)triethoxysilane), (6-아미노헥실)트리에톡시실란((6-aminohexyl)triethoxysilane), 3-아미노프로필(디에톡시)메틸실란(3-aminopropyl(diethoxy)methylsilane; APDMS), N-[3-(트리메톡시실릴)프로필]에틸렌디아민(N-[3-(trimethoxysilyl)propyl]ethylenediamine), [3-(2-아미노에틸아미노)프로필]트리메톡시실란([3-(2-aminoethylamino)propyl]trimethoxysilane; AEAPTMS) 및 3-[(트리메톡시실릴)프로필]디에틸렌트리아민(3-[(trimethoxysilyl)propyl]diethylenetriamine; TMPTA)로 이루어진 군에서 선택된 어느 하나 이상인 것을 특징으로 하는 미생물 농축 방법.The method of claim 7, wherein the silane compound is (3-aminopropyl) triethoxysilane (APTES), (3-aminopropyl) trimethoxysilane ((3-aminopropyl) trimethoxysilane), (1-aminomethyl) triethoxysilane ((1-aminomethyl) triethoxysilane), (2-aminoethyl) triethoxysilane, (4-aminobutyl) triethoxysilane (( 4-aminobutyl) triethoxysilane), (5-aminopentyl) triethoxysilane, (6-aminohexyl) triethoxysilane, 3-aminopropyl Diethoxy) methylsilane (3-aminopropyl (diethoxy) methylsilane; APDMS), N- [3- (trimethoxysilyl) propyl] ethylenediamine (N- [3- (trimethoxysilyl) propyl] ethylenediamine), [3- ( 2-aminoethylamino) propyl] trimethoxysilane ([3- (2-aminoethylamino) propyl] trimethoxysilane; AEAPTMS) and 3-[(trimethoxysilyl) propyl] diethylenetriamine (3-[(trimethoxysilyl) propyl] diethylenetri amine; TMPTA) microbial enrichment method, characterized in that any one or more selected from the group consisting of.
  9. 제4항에 있어서, 상기 미생물이 함유된 시료는 미생물에 감염된 것으로 의심되는 객체의 분변, 소변, 눈물, 타액, 피부의 외부 분비물, 호흡관의 외부 분비물, 장관의 외부 분비물, 소화관의 외부 분비물, 혈장, 혈청, 혈액, 척수액, 림프액, 체액 및 조직으로 이루어진 그룹에서 선택된 어느 하나인 것을 특징으로 하는 미생물 농축 방법.The method of claim 4, wherein 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, A method for concentrating microorganisms, characterized in that any one selected from the group consisting of plasma, serum, blood, spinal fluid, lymph, body fluid and tissue.
  10. DTBP를 유효성분으로 포함하는 핵산 추출용 조성물.Nucleic acid extraction composition comprising DTBP as an active ingredient.
  11. 제10항에 있어서, 상기 핵산은 DNA 또는 RNA인 것을 특징으로 하는 핵산 추출용 조성물.The nucleic acid extracting composition of claim 10, wherein the nucleic acid is DNA or RNA.
  12. 제10항 또는 제11항의 조성물을 포함하는 핵산 추출 키트.A nucleic acid extraction kit comprising the composition of claim 10.
  13. 대상물에 아민기를 도입하여 개질하는 단계(제1단계);Introducing and modifying an amine group into the object (first step);
    상기 개질된 대상물 상에 핵산 시료와 DTBP를 주입하고 핵산과 상기 DTBP 간의 복합체를 형성시키는 단계(제2단계); 및Injecting a nucleic acid sample and DTBP onto the modified object and forming a complex between the nucleic acid and the DTBP (second step); And
    상기 복합체가 형성된 대상물에 용출 완충액을 처리하여 핵산을 추출하는 단계(제3단계)를 포함하는 핵산 추출방법.The nucleic acid extraction method comprising the step of extracting the nucleic acid by treating the object with the complex formed in the elution buffer (third step).
  14. 제13항에 있어서, 상기 대상물은 박막장치, 자성 비드(magnetic bead), 링 공진기(ring resonator) 또는 나노입자(nanoparticle) 중 어느 하나인 것을 특징으로 하는 핵산 추출방법.The nucleic acid extracting method of claim 13, wherein the object is any one of a thin film device, a magnetic bead, a ring resonator, and a nanoparticle.
  15. 제13항에 있어서, 상기 대상물은 표면에 실란 화합물로 개질된 것을 특징으로 하는 핵산 추출방법.The method of claim 13, wherein the object is modified with a silane compound on its surface.
  16. 제15항에 있어서, 상기 실란 화합물은 하기 화학식 1로 표시되는 화합물인 것을 특징으로 하는 핵산 추출방법.The method of claim 15, wherein the silane compound is a nucleic acid extraction method, characterized in that the compound represented by the formula (1).
    [화학식 1][Formula 1]
    Figure PCTKR2017009254-appb-I000006
    Figure PCTKR2017009254-appb-I000006
    상기 식에서, R1 내지 R3는 각각 같거나 다를 수 있으며, C1 내지 C4의 알킬 또는 C1 내지 C4의 알콕시 중 어느 하나이고, R4는 아미노(C1 내지 C10)알킬, 3-(2-아미노(C1 내지 C4)알킬아미노)(C1 내지 C4)알킬 또는 3-[2-(2-아미노(C1 내지 C4)알킬아미노)(C1 내지 C4)알킬아미노](C1 내지 C4)알킬 중 어느 하나임.Wherein 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.
  17. 제16항에 있어서, 상기 실란 화합물은 (3-아미노프로필)트리에톡시실란((3-aminopropyl)triethoxysilane; APTES), (3-아미노프로필)트리메톡시실란((3-aminopropyl)trimethoxysilane), (1-아미노메틸)트리에톡시실란((1-aminomethyl)triethoxysilane), (2-아미노에틸)트리에톡시실란((2-aminoethyl)triethoxysilane), (4-아미노부틸)트리에톡시실란((4-aminobutyl)triethoxysilane), (5-아미노펜틸)트리에톡시실란((5-aminopentyl)triethoxysilane), (6-아미노헥실)트리에톡시실란((6-aminohexyl)triethoxysilane), 3-아미노프로필(디에톡시)메틸실란(3-aminopropyl(diethoxy)methylsilane; APDMS), N-[3-(트리메톡시실릴)프로필]에틸렌디아민(N-[3-(trimethoxysilyl)propyl]ethylenediamine), [3-(2-아미노에틸아미노)프로필]트리메톡시실란([3-(2-aminoethylamino)propyl]trimethoxysilane; AEAPTMS) 및 3-[(트리메톡시실릴)프로필]디에틸렌트리아민(3-[(trimethoxysilyl)propyl]diethylenetriamine; TMPTA)로 이루어진 군에서 선택된 어느 하나 이상인 것을 특징으로 하는 핵산 추출 방법.The method of claim 16, wherein the silane compound is (3-aminopropyl) triethoxysilane (APTES), (3-aminopropyl) trimethoxysilane ((3-aminopropyl) trimethoxysilane), (1-aminomethyl) triethoxysilane ((1-aminomethyl) 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; APDMS), N- [3- (trimethoxysilyl) propyl] ethylenediamine (N- [3- (trimethoxysilyl) propyl] ethylenediamine), [3- ( 2-aminoethylamino) propyl] trimethoxysilane ([3- (2-aminoethylamino) propyl] trimethoxysilane; AEAPTMS) and 3-[(trimethoxysilyl) propyl] diethylenetriamine (3-[(trimethoxysilyl) propyl] diethylenetr nucleic acid extraction method, characterized in that any one or more selected from the group consisting of iamine;
  18. DTBP를 유효성분으로 포함하는 미생물 농축 및 핵산 추출용 조성물.Microbial enrichment and nucleic acid extraction composition comprising DTBP as an active ingredient.
  19. 제18항에 있어서, 상기 미생물은 박테리아, 바이러스 또는 세포인 것을 특징으로 하는 미생물 농축 및 핵산 추출용 조성물.The composition of claim 18, wherein the microorganism is a bacterium, a virus or a cell.
  20. 제18항에 있어서, 상기 핵산은 DNA 또는 RNA인 것을 특징으로 하는 미생물 농축 및 핵산 추출용 조성물.The composition of claim 18, wherein the nucleic acid is DNA or RNA.
  21. 제18항 내지 제20항 중 어느 한 항의 조성물을 포함하는 미생물 농축 및 핵산 추출 키트. 21. A microbial enrichment and nucleic acid extraction kit comprising the composition of any one of claims 18-20.
  22. 대상물에 아민기를 도입하여 개질하는 단계(제1단계); Introducing and modifying an amine group into the object (first step);
    상기 개질된 대상물 상에 미생물이 함유된 시료와 DTBP를 접촉시켜 미생물을 농축시키는 단계(제2단계);Concentrating the microorganism by contacting the sample containing the microorganism with DTBP on the modified object (second step);
    상기 농축된 미생물로부터 핵산을 분리하는 단계(제3단계); Separating nucleic acid from the concentrated microorganism (third step);
    상기 분리된 핵산과 상기 DTBP 간의 복합체를 형성시키는 단계(제4단계); 및Forming a complex between the separated nucleic acid and the DTBP (fourth step); And
    상기 복합체가 형성된 대상물에 용출 완충액을 처리하여 핵산을 추출하는 단계(제5단계)를 포함하는 미생물 농축과 동시에 상기 농축된 미생물로부터 핵산을 추출하는 방법.A method of extracting nucleic acid from the concentrated microorganism at the same time as the concentration of the microorganism comprising the step of extracting the nucleic acid by treating the object with the complex formed in the elution buffer (step 5).
  23. 제22항에 있어서, 상기 대상물은 표면에 실란 화합물로 개질된 것을 특징으로 하는 미생물 농축과 동시에 상기 농축된 미생물로부터 핵산을 추출하는 방법.23. The method of claim 22, wherein the object is modified with a silane compound on a surface thereof, and at the same time the nucleic acid is extracted from the concentrated microorganism.
  24. 제23항에 있어서, 상기 실란 화합물은 하기 화학식 1로 표시되는 화합물인 것을 특징으로 하는 미생물 농축과 동시에 상기 농축된 미생물로부터 핵산을 추출하는 방법.24. The method of claim 23, wherein the silane compound is a compound represented by the following Chemical Formula 1, wherein the nucleic acid is concentrated and the nucleic acid is extracted from the concentrated microorganism at the same time.
    [화학식 1][Formula 1]
    Figure PCTKR2017009254-appb-I000007
    Figure PCTKR2017009254-appb-I000007
    상기 식에서, R1 내지 R3는 각각 같거나 다를 수 있으며, C1 내지 C4의 알킬 또는 C1 내지 C4의 알콕시 중 어느 하나이고, R4는 아미노(C1 내지 C10)알킬, 3-(2-아미노(C1 내지 C4)알킬아미노)(C1 내지 C4)알킬 또는 3-[2-(2-아미노(C1 내지 C4)알킬아미노)(C1 내지 C4)알킬아미노](C1 내지 C4)알킬 중 어느 하나임.Wherein 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.
  25. 제24항에 있어서, 상기 실란 화합물은 (3-아미노프로필)트리에톡시실란((3-aminopropyl)triethoxysilane; APTES), (3-아미노프로필)트리메톡시실란((3-aminopropyl)trimethoxysilane), (1-아미노메틸)트리에톡시실란((1-aminomethyl)triethoxysilane), (2-아미노에틸)트리에톡시실란((2-aminoethyl)triethoxysilane), (4-아미노부틸)트리에톡시실란((4-aminobutyl)triethoxysilane), (5-아미노펜틸)트리에톡시실란((5-aminopentyl)triethoxysilane), (6-아미노헥실)트리에톡시실란((6-aminohexyl)triethoxysilane), 3-아미노프로필(디에톡시)메틸실란(3-aminopropyl(diethoxy)methylsilane; APDMS), N-[3-(트리메톡시실릴)프로필]에틸렌디아민(N-[3-(trimethoxysilyl)propyl]ethylenediamine), [3-(2-아미노에틸아미노)프로필]트리메톡시실란([3-(2-aminoethylamino)propyl]trimethoxysilane; AEAPTMS) 및 3-[(트리메톡시실릴)프로필]디에틸렌트리아민(3-[(trimethoxysilyl)propyl]diethylenetriamine; TMPTA)로 이루어진 군에서 선택된 어느 하나 이상인 것을 특징으로 하는 미생물 농축과 동시에 상기 농축된 미생물로부터 핵산을 추출하는 방법.The method of claim 24, wherein the silane compound is (3-aminopropyl) triethoxysilane (APTES), (3-aminopropyl) trimethoxysilane ((3-aminopropyl) trimethoxysilane), (1-aminomethyl) triethoxysilane ((1-aminomethyl) triethoxysilane), (2-aminoethyl) triethoxysilane, (4-aminobutyl) triethoxysilane (( 4-aminobutyl) triethoxysilane), (5-aminopentyl) triethoxysilane, (6-aminohexyl) triethoxysilane, 3-aminopropyl Diethoxy) methylsilane (3-aminopropyl (diethoxy) methylsilane; APDMS), N- [3- (trimethoxysilyl) propyl] ethylenediamine (N- [3- (trimethoxysilyl) propyl] ethylenediamine), [3- ( 2-aminoethylamino) propyl] trimethoxysilane ([3- (2-aminoethylamino) propyl] trimethoxysilane; AEAPTMS) and 3-[(trimethoxysilyl) propyl] diethylenetriamine (3-[(trimethoxysilyl) propyl] diethylenetr iamine; TMPTA) at the same time as the microorganisms characterized in that at least one selected from the group consisting of extracting nucleic acids from the concentrated microorganisms.
  26. 제22항에 있어서, 상기 미생물이 함유된 시료는 미생물에 감염된 것으로 의심되는 객체의 분변, 소변, 눈물, 타액, 피부의 외부 분비물, 호흡관의 외부 분비물, 장관의 외부 분비물, 소화관의 외부 분비물, 혈장, 혈청, 혈액, 척수액, 림프액, 체액 및 조직으로 이루어진 그룹에서 선택된 어느 하나인 것을 특징으로 하는 미생물 농축과 동시에 상기 농축된 미생물로부터 핵산을 추출하는 방법.The method of claim 22, wherein the sample containing the microorganism is fecal, urine, tear, saliva, external secretion of the skin, external secretion of the respiratory tract, external secretion of the intestinal tract, external secretion of the digestive tract, A method for extracting nucleic acid from the concentrated microorganism simultaneously with the microorganism, characterized in that any one selected from the group consisting of plasma, serum, blood, spinal fluid, lymph, body fluid and tissue.
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