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WO2018169307A1 - Procédé de concentration microbienne au moyen de diatomite et procédé d'extraction d'acides nucléiques - Google Patents

Procédé de concentration microbienne au moyen de diatomite et procédé d'extraction d'acides nucléiques Download PDF

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WO2018169307A1
WO2018169307A1 PCT/KR2018/002994 KR2018002994W WO2018169307A1 WO 2018169307 A1 WO2018169307 A1 WO 2018169307A1 KR 2018002994 W KR2018002994 W KR 2018002994W WO 2018169307 A1 WO2018169307 A1 WO 2018169307A1
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Prior art keywords
nucleic acid
dimethyl
composition
diatomaceous earth
silane compound
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PCT/KR2018/002994
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English (en)
Korean (ko)
Inventor
신용
조비
Original Assignee
울산대학교 산학협력단
재단법인 아산사회복지재단
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Priority claimed from KR1020180028131A external-priority patent/KR102136694B1/ko
Application filed by 울산대학교 산학협력단, 재단법인 아산사회복지재단 filed Critical 울산대학교 산학협력단
Priority to EP18767296.9A priority Critical patent/EP3597773A4/fr
Priority to US16/492,606 priority patent/US20210079374A1/en
Publication of WO2018169307A1 publication Critical patent/WO2018169307A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B33/00—Silicon; Compounds thereof
    • C01B33/113—Silicon oxides; Hydrates thereof
    • C01B33/12—Silica; Hydrates thereof, e.g. lepidoic silicic acid
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples

Definitions

  • the present invention relates to a method for enriching microorganisms and a method for extracting nucleic acids using diatomaceous earth, in particular diatomaceous earth modified with a silane compound.
  • Diatoms are unicellular algae that are distributed in various sizes from 2 ⁇ m to 2 mm and are representative phytoplankton commonly found on the earth. Diatoms account for 20-25% of the total primary production of land and 40% of marine biomass production, and have played an important role in ecosystems as marine producers for millions of years.
  • microalgae have high photosynthetic efficiency relative to cell mass and are mainly used for the production of lipids, biofuels, and bioavailable resources through culture.
  • diatomaceous earth is a natural siliceous material which is a main component of diatomaceous cell membrane, and is known as bio-silica because it is calcined at 450 ° C. and composed of silica-rich silica on the surface.
  • the size of the bio-silica is about 10-20 ⁇ m.
  • An object of the present invention is a composition for concentrating microorganisms comprising diatomaceous earth modified with a silane compound as an active ingredient, a method and kit for concentrating microorganisms using the same;
  • a nucleic acid extracting composition comprising diatomaceous earth modified with a silane compound as an active ingredient, a nucleic acid extracting method and kit using the same;
  • it provides a composition for microbial enrichment and nucleic acid extraction, including a diatomaceous earth modified with a silane compound as an active ingredient, a method and kit for extracting nucleic acid from the concentrated microorganism at the same time as the microorganism concentration using the same.
  • the present invention provides a composition for concentrating microorganisms comprising diatomaceous earth modified with a silane compound as an active ingredient.
  • the present invention provides a method for concentrating microorganisms comprising contacting a sample containing microorganisms with diatomaceous earth modified with the silane compound.
  • the present invention also provides a microbial enrichment kit comprising the composition.
  • the present invention also provides a nucleic acid extracting composition comprising diatomaceous earth modified with a silane compound as an active ingredient.
  • the present invention comprises the steps of (1) reacting by adding a diatomaceous earth modified with a silane compound to the nucleic acid sample to be extracted; And (2) provides a nucleic acid extraction method comprising the step of eluting the nucleic acid from the reaction.
  • the present invention comprises the steps of (1) adding and mixing the diatomaceous earth modified with the silane compound to the nucleic acid sample to be extracted; (2) dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS) and dimethyl 3,3'-dithiobis Reacting by adding one or more selected from the group consisting of dimethyl 3,3'-dithiobispropionimidate (DTBP); And (3) provides a nucleic acid extraction method comprising the step of eluting the nucleic acid from the reaction.
  • DMA dimethyl adipimidate
  • DMP dimethyl pimelimidate
  • DMS dimethyl suberimidate
  • DTBP dimethyl 3,3'-dithiobis Reacting by adding one or more selected from the group consisting of dimethyl 3,3'-dithiobispropionimidate
  • DTBP dimethyl 3,3'-dithiobispropionimidate
  • the present invention also provides a nucleic acid extraction kit comprising the composition.
  • the present invention also provides a composition for microbial enrichment and nucleic acid extraction comprising diatomaceous earth modified with a silane compound as an active ingredient.
  • the present invention comprises the steps of (1) contacting the sample containing the microorganism to the diatomaceous earth modified with the silane compound to concentrate the microorganism; (2) dissolving the concentrated microorganism; And (3) eluting the nucleic acid from the lysate, and providing a method for extracting the nucleic acid from the concentrated microorganism at the same time as the microorganism is concentrated.
  • the present invention comprises the steps of (1) contacting the sample containing the microorganism to the diatomaceous earth modified with the silane compound to concentrate the microorganism; (2) dissolving the concentrated microorganism; (3) dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS) and dimethyl 3,3'-diti in the lysate Reacting by adding any one or more selected from the group consisting of dimethyl 3,3'-dithiobispropionimidate (DTBP); And (4) provides a method for extracting nucleic acid from the concentrated microorganism simultaneously with the concentration of the microorganism comprising eluting the nucleic acid from the reaction.
  • DMA dimethyl adipimidate
  • DMP dimethyl pimelimidate
  • DMS dimethyl suberimidate
  • DTBP dimethyl 3,3'-diti in the lysate Reacting by adding any one or more selected from the group consisting of dimethyl 3,3'-d
  • the present invention also provides a microbial enrichment and nucleic acid extraction kit comprising the composition.
  • the present invention also provides the use of diatomaceous earth modified with a silane compound for microbial enrichment.
  • the present invention also provides the use of diatomaceous earth modified with silane compounds for nucleic acid extraction.
  • the present invention also provides the use of diatomaceous earth modified with silane compounds for microbial enrichment and nucleic acid extraction.
  • the present invention relates to a method for concentrating microorganisms and nucleic acid extraction method using diatomaceous earth, conventional methods for detecting microorganisms do not use the entire solution in more than 1 ml of the patient sample, extracting nucleic acids using only a part of them Detection has been performed using this. 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. Therefore, the present inventors have developed a technology capable of concentrating microorganisms about 4-8 times using existing diatomaceous earth, and extracting nucleic acids directly using dimethyl adipimidate (DMA). Developed methods. By enabling microbial enrichment and nucleic acid extraction techniques in one tube, it is expected to significantly reduce external contamination, cost, time and complexity.
  • DMA dimethyl adipimidate
  • Figure 2 shows the results of experimental validation in the pathogen concentration process.
  • D diatomaceous earth
  • DA diatomaceous earth modified with APTES.
  • Figure 3 is the result of optimizing the pathogen concentration experimental conditions by changing the modified diatomaceous earth concentration.
  • Figure 7 shows the results of experimental validation in the DNA extraction process.
  • D diatomaceous earth
  • DA diatomaceous earth modified with APTES.
  • D diatomaceous earth
  • DA diatomaceous earth modified with APTES.
  • RNA extraction experiment 12 is a result of optimizing RNA extraction experiment conditions by varying the concentration of diatomaceous earth (DA) modified with APTES.
  • DA diatomaceous earth
  • Figure 13 is the result of optimizing the RNA extraction experimental conditions by varying the reaction time.
  • FIG. 16 shows experimental results of applying an amine-functionalized diatomaceous earth-based, dimethyl suberimidate assisted system (ADD system) based on RNA extraction from pathogen Brucella sample.
  • ADD system dimethyl suberimidate assisted system
  • the present invention provides a composition for concentrating microorganisms comprising diatomaceous earth modified with a silane compound as an active ingredient.
  • 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),
  • the microorganism may be any negatively charged microorganism such as bacteria, viruses or cells, and more preferably Brucella ovis or para influenza virus (PIV3) described in the embodiment of the present invention.
  • PIV3 para influenza virus
  • the concentration of the microorganism may be 10 0 cfu / ml to 10 7 cfu / ml, but is not limited thereto.
  • the present invention also provides a method for concentrating microorganisms comprising contacting a sample containing microorganisms with diatomaceous earth modified with a silane compound.
  • 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 most suitable for (3-aminopropyl) triethoxysilane (APTES) described in the embodiment of the present invention.
  • APTES (3-aminopropyl) triethoxysilane
  • 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 microorganism may be any negatively charged microorganism such as bacteria, viruses or cells, and more preferably Brucella ovis or para influenza virus (PIV3) described in the embodiment of the present invention.
  • PIV3 para influenza virus
  • the concentration of the microorganism may be 10 0 cfu / ml to 10 7 cfu / ml, but is not limited thereto.
  • the step of contacting the microorganism to the diatomaceous earth modified with the silane compound may be contacted for 20 to 60 minutes, but is not limited thereto.
  • 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 also provides a nucleic acid extracting composition comprising diatomaceous earth modified with a silane compound as an active ingredient.
  • 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 most suitable for (3-aminopropyl) triethoxysilane (APTES) described in the embodiment of the present invention.
  • APTES (3-aminopropyl) triethoxysilane
  • the composition is dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS) and dimethyl 3,3'-dity It may further include any one or more selected from the group consisting of dimethyl propionimate (Dimethyl 3,3'-dithiobispropionimidate; DTBP).
  • DMA dimethyl adipimidate
  • DMP dimethyl pimelimidate
  • DMS dimethyl suberimidate
  • DTBP dimethyl 3,3'-dity
  • the nucleic acid may be DNA or RNA, but is not limited thereto.
  • the present invention comprises the steps of (1) reacting by adding a diatomaceous earth modified with a silane compound to the nucleic acid sample to be extracted; And (2) provides a nucleic acid extraction method comprising the step of eluting the nucleic acid from the reaction.
  • the present invention comprises the steps of (1) adding and mixing the diatomaceous earth modified with the silane compound to the nucleic acid sample to be extracted; (2) dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS) and dimethyl 3,3'-dithiobis Reacting by adding one or more selected from the group consisting of dimethyl 3,3'-dithiobispropionimidate (DTBP); And (3) provides a nucleic acid extraction method comprising the step of eluting the nucleic acid from the reaction.
  • DMA dimethyl adipimidate
  • DMP dimethyl pimelimidate
  • DMS dimethyl suberimidate
  • DTBP dimethyl 3,3'-dithiobis Reacting by adding one or more selected from the group consisting of dimethyl 3,3'-dithiobispropionimidate
  • DTBP dimethyl 3,3'-dithiobispropionimidate
  • 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 most suitable for (3-aminopropyl) triethoxysilane (APTES) described in the embodiment of the present invention.
  • APTES (3-aminopropyl) triethoxysilane
  • 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 also provides a composition for microbial enrichment and nucleic acid extraction comprising diatomaceous earth modified with a silane compound as an active ingredient.
  • 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 most suitable for (3-aminopropyl) triethoxysilane (APTES) described in the embodiment of the present invention.
  • APTES (3-aminopropyl) triethoxysilane
  • the microorganism may be any negatively charged microorganism such as bacteria, viruses or cells, and more preferably Brucella ovis or para influenza virus (PIV3) described in the embodiment of the present invention.
  • PIV3 para influenza virus
  • the concentration of the microorganism may be 10 0 cfu / ml to 10 7 cfu / ml, but is not limited thereto.
  • the composition is dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS) and dimethyl 3,3'-dity It may further comprise the step of reacting by adding any one or more selected from the group consisting of dimethyl propionimate (Dimethyl 3,3'-dithiobispropionimidate; DTBP).
  • DMA dimethyl adipimidate
  • DMP dimethyl pimelimidate
  • DMS dimethyl suberimidate
  • DTBP dimethyl 3,3'-dity
  • the nucleic acid may be DNA or RNA, but is not limited thereto.
  • the present invention comprises the steps of (1) contacting the sample containing the microorganism to the diatomaceous earth modified with the silane compound to concentrate the microorganism; (2) dissolving the concentrated microorganism; And (3) eluting the nucleic acid from the lysate, and providing a method for extracting the nucleic acid from the concentrated microorganism at the same time as the microorganism is concentrated.
  • the present invention comprises the steps of (1) contacting the sample containing the microorganism to the diatomaceous earth modified with the silane compound to concentrate the microorganism; (2) dissolving the concentrated microorganism; (3) dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS) and dimethyl 3,3'-diti in the lysate Reacting by adding any one or more selected from the group consisting of dimethyl 3,3'-dithiobispropionimidate (DTBP); And (4) provides a method for extracting nucleic acid from the concentrated microorganism simultaneously with the concentration of the microorganism comprising eluting the nucleic acid from the reaction.
  • DMA dimethyl adipimidate
  • DMP dimethyl pimelimidate
  • DMS dimethyl suberimidate
  • DTBP dimethyl 3,3'-diti in the lysate Reacting by adding any one or more selected from the group consisting of dimethyl 3,3'-d
  • 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 most suitable for (3-aminopropyl) triethoxysilane (APTES) described in the embodiment of the present invention.
  • APTES (3-aminopropyl) triethoxysilane
  • 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 microorganism may be any negatively charged microorganism such as bacteria, viruses or cells, and more preferably Brucella ovis or para influenza virus (PIV3) described in the embodiment of the present invention.
  • PIV3 para influenza virus
  • the concentration of the microorganism may be 10 0 cfu / ml to 10 7 cfu / ml, but is not limited thereto.
  • 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 lysis buffer or a protease for lysing the cells in the sample to release the nucleic acid from inside the cell, and for adjusting the pH required for effective microbial concentration or nucleic acid extraction. Buffers and the like may be additionally included.
  • the kit may be in the form of a well-plate, a tube, a column or a microfluidic chip, but the kit may be in the form of any commercially available form. It is possible.
  • the well-plate may be manufactured without limitation, such as 96 wells, 384 wells.
  • the kit may be a single composition or the individual components of the composition for microbial enrichment or nucleic acid extraction may be stored separately for mixing before use.
  • the components of the composition for microbial enrichment or nucleic acid extraction in the kit of the present invention may be packaged separately, or may be packaged in one or more mixtures in any combination of components.
  • Each of the individual components or one or more mixtures may be placed in different containers and may comprise the entire kit in a single container.
  • the present invention also provides the use of diatomaceous earth modified with a silane compound for microbial enrichment.
  • the present invention also provides the use of diatomaceous earth modified with silane compounds for nucleic acid extraction.
  • the present invention also provides the use of diatomaceous earth modified with silane compounds for microbial enrichment and nucleic acid extraction.
  • Brucella ovis was used as the test pathogen.
  • 1 ml of 10 6 cfu / ml Brucella solution was used.
  • HCT-116 cancer cells were used as test samples.
  • 1 ml of 10 5 cells / ml cell solution was used.
  • 100 ⁇ l elution buffer was used for final DNA extraction.
  • DNA was extracted using diatomaceous earth (D) and diatomaceous earth (DA) modified with APTES, and dimethyl adipimidate (DMA) was added.
  • D diatomaceous earth
  • DA diatomaceous earth
  • DMA dimethyl adipimidate
  • the sample is allowed to react at room temperature until the sample becomes clear (within 1 minute).
  • the reaction is carried out at 56 ° C. for 20 minutes to bind DNA to the modified diatomaceous earth (DA).
  • DA modified diatomaceous earth
  • the mixture is centrifuged for 1 minute using a microcentrifuge. Carefully remove the supernatant.
  • the mixture is centrifuged for 1 minute. Using a pipette, transfer the supernatant to a new microtube.
  • HCT-116 cell concentration was varied from 10 0 cells / ml to 10 7 cells / ml. As shown in FIG. 10, Q-PCR results showed that DNA extraction was effective even at low cell concentrations.
  • HCT-116 cancer cells were used as test samples.
  • 1 ml of 10 5 cells / ml cell solution was used.
  • 100 ⁇ l elution buffer was used for final RNA extraction.
  • RNA extraction ii. 40 ⁇ l of DA was used for RNA extraction.
  • HCT-116 cell concentration was varied from 10 1 cells / ml to 10 6 cells / ml.
  • Q-PCR results showed that RNA extraction was effective even at low cell concentrations.
  • DA diatomaceous earth
  • Example 6> for diagnosis Amine Modified Diatomaceous earth based systems (amine-functionalized, diatomaceous earth-based, dimethyl suberimidate assisted system; Application of ADD system
  • the ADD system of the present invention was able to successfully extract RNA from biological samples. Compared with traditional analytical methods, the optimized protocol of the ADD system is expected to be useful for nucleic acid-based diagnostics.
  • Human brucellosis an endemic disease in many countries and regions, is a worldwide common infectious disease.
  • using the ADD system was able to detect even at very low Brucella concentration of 10 0 CFU / mL, which was confirmed that the detection sensitivity is 100 times more improved than the existing kit.
  • NTC no-template control
  • the ADD system of the present invention further confirmed clinical utility using RNA extracted from 10 5 CFU / mL Brucella samples derived from urine. Since Brucella is a transfection pathogen, we included the same concentration of Brucella in human urine and sheep urine samples. Although the overall Ct value of Brucella in urine was slower than PBS, it was confirmed that the RNA extracted using the ADD system was faster than the RNA extracted using the existing kit (FIG. 16c).
  • RNA samples extracted by the ADD system showed faster Ct values compared to existing kits.
  • we tested a relatively large volume of samples using the ADD system increasing the detection limit by loading more samples.
  • using the ADD system was able to extract more RNA from a large volume (1 mL) sample compared to using the other system (Fig.
  • the Ct value for the 1 mL sample derived RNA was about 2 cycles faster than the 200 ⁇ L sample, clearly indicating that the total amount of extracted RNA increased when the sample volume was large.
  • the protocol was optimized for 200 ⁇ L samples, the ADD system could also be used for RNA extraction of large volume samples. Therefore, the ADD system proposed in the present invention can be usefully used for RNA extraction and pathogen detection based on nucleic acid amplification by RT-qPCR.

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Abstract

La présente invention concerne un procédé de concentration microbienne au moyen de diatomite et un procédé d'extraction d'acides nucléiques. Une technique a été mise au point, qui permet d'atteindre environ de 4 à 8 fois la concentration microbienne au moyen de diatomite lorsqu'elle est comparée à l'état de la technique, et un procédé a été mis au point, qui permet d'extraire directement des acides nucléiques de micro-organismes concentrés au moyen de diméthyl adipimidate (DMA) ou analogue. Il est prévu que les techniques de concentration microbienne et d'extraction d'acides nucléiques admissibles dans un seul tube réduisent de manière significative la contamination provenant de l'extérieur, les coûts, le temps, la complexité et autres.
PCT/KR2018/002994 2017-03-15 2018-03-14 Procédé de concentration microbienne au moyen de diatomite et procédé d'extraction d'acides nucléiques WO2018169307A1 (fr)

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Application Number Priority Date Filing Date Title
EP18767296.9A EP3597773A4 (fr) 2017-03-15 2018-03-14 Procédé de concentration microbienne au moyen de diatomite et procédé d'extraction d'acides nucléiques
US16/492,606 US20210079374A1 (en) 2017-03-15 2018-03-14 Microbial concentration method using diatomite and nucleic acid extraction method

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KR10-2017-0032581 2017-03-15
KR20170032581 2017-03-15
KR1020180028131A KR102136694B1 (ko) 2017-03-15 2018-03-09 규조토를 이용한 미생물 농축 방법 및 핵산 추출 방법
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