WO2019156395A1 - Dispositif d'extraction d'adn à base de séparation de fluide latéral - Google Patents

Dispositif d'extraction d'adn à base de séparation de fluide latéral Download PDF

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WO2019156395A1
WO2019156395A1 PCT/KR2019/000980 KR2019000980W WO2019156395A1 WO 2019156395 A1 WO2019156395 A1 WO 2019156395A1 KR 2019000980 W KR2019000980 W KR 2019000980W WO 2019156395 A1 WO2019156395 A1 WO 2019156395A1
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pad
dna
sample
extraction device
dna extraction
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PCT/KR2019/000980
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English (en)
Korean (ko)
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김민곤
석영웅
안희섭
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광주과학기술원
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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
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5023—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures with a sample being transported to, and subsequently stored in an absorbent for analysis
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • 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
    • G01N1/34—Purifying; Cleaning
    • 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
    • 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
    • G01N1/4044—Concentrating samples by chemical techniques; Digestion; Chemical decomposition
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0631—Purification arrangements, e.g. solid phase extraction [SPE]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/069—Absorbents; Gels to retain a fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0825—Test strips

Definitions

  • the present invention is directed to a lateral flow separation based DNA extraction device.
  • Non-Patent Document 1 Niemz, A., T.M. Ferguson, and D.S. Boyle, Point-of-care nucleic acid testing for infectious diseases. Trends Biotechnol, 2011. 29 (5): p. 240-50].
  • Non-Patent Document 2 [2] Thatcher, S.A., DNA / RNA preparation for molecular detection. Clin Chem, 2015. 61 (1): p. 89-99].
  • Silica matrices can effectively extract DNA from various samples such as blood, urine, tissue, saliva, etc.
  • Non-Patent Document 3 [3] Handbook, B.D.P., Qiagen. Gmbh, Germany, June, 2005].
  • the present inventors have made a number of studies and efforts to develop a DNA extraction device that solves the above-mentioned problems. As a result, based on the lateral flow separation method, the present invention was completed by experimentally confirming that DNA extraction from various kinds of samples can be efficiently performed within a short time.
  • an object of the present invention is to provide a DNA extraction device based on lateral flow separation with high DNA extraction efficiency.
  • a sample pad (sample pad) is accommodated to the sample to be extracted DNA;
  • a loading pad containing a washing buffer carrying DNA emanated from a sample contained in the sample pad;
  • a wicking pad absorbing the wash buffer developed by the binding pad and positioned downstream of the binding pad.
  • the DNA extraction device includes a structure in which the loading pad, the sample pad, the binding pad, and the wicking pad are sequentially spaced apart from each other.
  • the loading pad, the sample pad, the binding pad and the wicking pad each consist of a porous membrane and have an absorbency to the wash buffer.
  • the loading pad, the sample pad, the binding pad, and the wicking pad have different absorption powers to the wash buffer.
  • said DNA extraction device further comprises a support on which said loading pad, said sample pad, said binding pad, and said wicking pad rest on a surface.
  • the support is made of a material that does not absorb the wash buffer and the DNA.
  • the DNA extraction device further includes a sealing member provided to surround the binding pad and including a hole for dense the DNA concentrated in the binding pad.
  • the DNA extraction device further comprises a case in which the loading pad, the sample pad, the binding pad and the wicking pad are accommodated.
  • the case may include a buffer injection hole formed at a position corresponding to the loading pad to inject the washing buffer into the loading pad; A sample injection hole formed at a position corresponding to the sample pad to inject the sample into the sample pad; And a DNA extraction port formed at a position corresponding to the binding pad to extract DNA concentrated in the binding pad.
  • said sample pad comprises a lysis buffer for extracting DNA from said sample.
  • Method for extracting DNA according to another embodiment of the present invention, a method for extracting DNA from a sample using a DNA extraction device according to an embodiment of the present invention, the method for extracting the DNA, the Injecting a sample into the sample pad to accommodate the sample in the sample pad; Injecting the wash buffer into the loading pad to convey DNA extracted from the sample contained in the sample pad to the binding pad; Injecting the wash buffer into the loading pad, and then standing for a predetermined time to condense DNA released from the sample onto the binding pad; And extracting DNA concentrated in the binding pad.
  • the present invention has the following excellent effects.
  • the lateral flow separation based DNA extraction device has very high DNA extraction efficiency. That is, according to the lateral flow separation based DNA extraction device according to an embodiment of the present invention, only a very short time (about 3 minutes) is required to extract DNA from various kinds of samples.
  • the lateral flow separation-based DNA extraction device does not require additional processing such as centrifugation, multiple washing processes, and thus can be usefully applied to the field of DNA extraction in a particularly limited environment. .
  • the lateral flow separation-based DNA extraction device can extract DNA from a variety of samples, there is an effect that can be pursued low cost, rapidity, and simplicity.
  • the lateral flow separation-based DNA extraction device has a relatively simple structure, can be easily and quickly produced, there is an effect that can be easily mass production and commercialization.
  • FIG. 1A-1C illustrate a DNA extraction device according to one embodiment of the invention.
  • 2A to 7D are diagrams showing experimental results on the performance of the DNA extraction device according to an embodiment of the present invention.
  • FIG. 1A-1C illustrate a DNA extraction device according to one embodiment of the invention.
  • the DNA extraction device 1 includes a sample pad 110, a loading pad 120, a binding pad 130, a wicking pad 140, and a transfer pad 150. , A support 160, a sealing member 170, and a case 180.
  • the sample pad 110 is made of a material that can contain and contain a sample for DNA extraction.
  • the sample means a biological substance to be subjected to DNA extraction.
  • the sample includes cell culture fluid, saliva, blood, serum, artificial bile, milk, and the like, and may be derived from any biological source, such as a physiological fluid.
  • the sample pad 110 is treated to have alkalinity.
  • the sample pad 110 includes a lysis buffer for destroying target cells in the sample to release DNA from the target cells.
  • the loading pad 120 includes a washing buffer which carries DNA extracted from the sample contained in the sample pad 110. Due to the difference in liquid absorbency between the sample pad 110, the loading pad 120, the binding pad 130, and the wicking pad 140, which will be described later, the washing buffer injected into the loading pad 120 and accommodated in the loading pad 120 is provided. Is deployed downstream of the DNA extraction device. That is, the washing buffer developed from the loading pad 120 passes through the sample pad 110, the binding pad 130, and the wicking pad 140 sequentially, thereby binding the DNA leaked from the sample contained in the sample pad 110. The pad 130 is carried.
  • the binding pad 130 (binding pad) is concentrated in the DNA carried by the wash buffer.
  • the binding pad 130 is made of a material that is biocompatible so that DNA can be concentrated.
  • the DNA extraction device includes a sealing member 170 surrounding the binding pad 130.
  • the sealing member 170 is provided to cover the binding pad 130 as a whole on the surface of the binding pad 130.
  • holes are formed on the sealing member 170 to dense the DNA concentrated in the binding pad 130.
  • DNA concentrated in the binding pad 130 is densely packed into holes formed on the sealing member 170.
  • the DNA extraction device according to the present embodiment includes a sealing member 170 that dense DNA, thereby maximizing DNA extraction efficiency.
  • the wicking pad 140 absorbs the wash buffer deployed to the binding pad 130.
  • the wicking pad 140 is located downstream of the binding pad 130.
  • the wash buffer is absorbed into the wicking pad 140 from the sample pad 110 to the binding pad 130.
  • Each of the loading pad 120, the sample pad 110, the binding pad 130 and the wicking pad 140 is made of a porous membrane.
  • the porous materials that make up the porous membrane include fibrous paper, microporous membranes of cellulosic materials, cellulose, cellulose derivatives such as cellulose acetate, porous such as nitrocellulose, glass fibers, cartons, nylon Gels and the like.
  • each of the loading pad 120, the sample pad 110, the binding pad 130, and the wicking pad 140 are liquid absorbent. Specifically, each of the loading pad 120, the sample pad 110, the binding pad 130, and the wicking pad 140 has an absorbency to the washing buffer injected into the loading pad 120 described above.
  • the liquid absorbing power of the loading pad 120, the sample pad 110, the binding pad 130, and the wicking pad 140 are different from each other. More specifically, the liquid absorption of the loading pad 120 is the lowest, the liquid absorption of the wicking pad 140 is the largest. That is, the liquid absorbing power increases as the loading pad 120, the sample pad 110, the binding pad 130, and the wicking pad 140 go.
  • the DNA extraction device includes a structure in which the loading pad 120, the sample pad 110, the binding pad 130, and the wicking pad 140 are sequentially spaced apart from each other. That is, the DNA extraction device according to the present embodiment includes a structure in which a plurality of pads are arranged in the order of the liquid absorbing force. That is, the DNA extraction device according to the present embodiment is provided with a loading pad 120 having the lowest liquid absorbing force on the upstream side, and a pad having a large liquid absorbing force toward the downstream side, and a wicking pad ( 140) is provided. Because of this structure, the wash buffer injected into the loading pad 120 may naturally develop toward the wicking pad 140.
  • the transfer pad 150 connects the loading pad 120, the sample pad 110, the binding pad 130, and the wicking pad 140 to allow the washing buffer to move. That is, the transfer pad 150 is provided between the loading pad 120 and the sample pad 110, between the sample pad 110 and the binding pad 130, and between the binding pad 130 and the wicking pad 140, respectively. .
  • the support 160 supports the loading pad 120, the sample pad 110, the binding pad 130, the wicking pad 140, and the transfer pad 150.
  • the support 160 is a material capable of supporting the loading pad 120, the sample pad 110, the binding pad 130, the wicking pad 140, and the transfer pad 150. It may be formed of any material so long as it is a material having liquid impermeability (liquid impermeability).
  • the loading pad 120, the sample pad 110, the binding pad 130, and the wicking pad 140 are sequentially provided on the support 160.
  • a transfer pad 150 is provided between the loading pad 120 and the sample pad 110, between the sample pad 110 and the binding pad 130, and between the binding pad 130 and the wicking pad 140. .
  • the case 180 accommodates the loading pad 120, the sample pad 110, the binding pad 130, the wicking pad 140, the transfer pad 150, and the support 160.
  • the case 180 is composed of an upper case 180 'and a lower case 180 ". On the surface of the lower case 180", a loading pad 120, a sample pad 110, a binding pad 130, and a wicking pad are provided. 140, the transfer pad 150 and the support 160 are placed. A buffer injection hole, a sample injection hole, and a DNA extraction hole are formed in the upper case 180 'coupled to the lower case 180 ".
  • the buffer injection hole is formed at a position corresponding to the loading pad 120.
  • the user may inject the washing buffer into the loading pad 120 through the buffer inlet.
  • the sample injection hole is formed at a position corresponding to the sample pad 110.
  • the user may inject a sample into the sample pad 110 through the sample inlet.
  • the DNA extraction port is formed at a position corresponding to the binding pad 130. More specifically, the DNA extraction port is provided to be aligned in line with the holes provided in the sealing member 170 surrounding the binding pad 130. The user may extract DNA concentrated in the binding pad 130 through the DNA extraction port.
  • Glass pads were purchased from Millipore (Billerica, MA, USA). Asymmetric PES membranes are available from Pall Co. (Port Washington, NY, USA). ELISA sealing tape was purchased from Excel Scientific (Victroville, CA, USA). Oligonucleotide primers were purchased from Genotech (Daejeon, South Korea). Saliva and whole blood from a single donor were purchased from innovative research (Novi, Michigan, USA).
  • S. aureus (ATCC 23235) was incubated in a Tryptic Soy Broth (Hach, Co., USA) for 18 hours at 200 rpm shaking incubator at a temperature of 37 °C. Cultured cells were diluted 10-fold in fresh Tryptic Soy Broth medium and then placed on on Tryptic Soy Agar (Hach) plates incubated at 37 ° C. for 18 hours. After overnight incubation, each colony point was calculated once, after which the number of S. aureus cultures was recorded. S.aureus cultures with recorded numbers were used for the experiments after dilution with PBS buffer.
  • Genomic DNA was extracted from freshly cultured S. aureus cells by the QIAamp DNA Mini kit (Qiagen, Valencia, CA). 20 ⁇ L of samples containing S. aureus cells were added to the Qiagen kit lysis buffer and then processed by wash buffer. The extracted DNA was separated by 50 ⁇ L of ultrapure water, and the concentration of the separated DNA was measured by UV-vis spectrometer or CFX96 real-time PCR instrument (Bio-rad Laboratories, Hercules, CA).
  • DNA extracted from S. aureus cells was fluorescently labeled by the ARES Alexa Fluor 647 DNA labeling kit (Life Technologies, Carlsbad, Calif.). Nick translation was used for the initial representation of the amine functionality of the template genomic DNA.
  • DNA solution treated by alcohol precipitation was purified by QIAquick PCR purification kit (Qiagen). Final concentrations of fluorescence labeled DNA were determined by qPRC.
  • Primers for quantitative real-time PCR were designed to target the Staphylococcus aureus 165 ri bosomal RNA gene from the NCBI database.
  • the DNA sequence of the forward primer was 5'- GCACATCTTGACGGTACCTAATC-3 'and the DNA sequence of the reverse primer was 5'- CGCGCTTTACGCCCAATAA-3'.
  • PCR mixtures used dNTPs, Taq polymerase and reaction buffer.
  • a 20 ⁇ L reaction sample was mixed with 100 nM forward and reverse primers, 1 ⁇ commercial qPCR mixture, and 2 ⁇ L template DNA.
  • the absorbance at 260 nm of the genomic DNA obtained from the Qiagen extraction kit was measured by UV-vis spectrometer from which the absolute concentration of DNA was calculated.
  • Concentrated DNA was serially diluted 10 fold from 50 ng to 0.05 ng, real time PCR curves were obtained for each solution (FIG. 6A), and S. aureus DNA was quantified.
  • Real-time PCR was performed by the CFX96 real-time PCR instrument. Quantification of the analyzed DNA was obtained by the relationship between the amount of DNA and the Cq value. (FIG. 6B)
  • the DNA extraction device is a transfer pad 150 (loading pad), loading pad 120 (loading pad), sample pad 110 (sample pad), binding pad ( 130 and a wicking pad 140.
  • the transfer pad 150 is vivid plasma separation-GF (Pall), which is one type of asymmetric PES membrane. Glass pad GF / C grade (Whatman) was used as the loading pad 120, conjugate pad (Pall) was used as the sample pad 110, and glass pad GF / F grade (Whatman) was used as the binding pad 130. Was used. Wicking pad 140 was provided downstream to induce the flow of fluid in the DNA extraction device.
  • a transfer pad 150 cut to an appropriate length was attached on a backing card 160.
  • Loading pads 120 (10 mm), sample pads 110 (5 mm) and wicking pads 140 (15 mm) cut to uniform length were attached to the remaining portions of the support 160.
  • the backing card was cut into strips of uniform 5 mm length. Each strip was then placed in a printed case 180 for DNA extraction testing.
  • the binding pad 130 (5 mm x 5 mm) was attached by an ELISA sealing tape (sealing member 170) including a hole having a diameter of 1.5 mm.
  • 20 ⁇ L of Lysis buffer 200 mM NaOH with 1% SDS
  • the case 180 was designed by Solidwork software and manufactured by 3D printing.
  • the printed case 180 includes an upper case 180 'and a lower case 180 ".
  • the three holes included in the upper cover are the buffer inlet, the sample inlet, and the DNA outlet, respectively. 120, sample pad 110 and binding pad 130. All solutions for DNA extraction in the DNA extraction device were injected through the inlets, and the final DNA solution was extracted through the DNA outlet by a pipette.
  • DNA extraction by DNA extraction device consists of three steps: (1) sample (2) buffer (3) extraction. A 20 ⁇ L volume sample solution containing S. aureus cells was injected through the sample inlet and loaded into the sample pad 110 on the DNA extraction device. Then, 75 ⁇ L of wash buffer (15% (v / v) isopropyl alcohol) was injected immediately after sample solution injection through the buffer inlet. After 3 minutes of washing buffer injection, 2 ⁇ L of D.W was injected and extracted three times through the DNA outlet. Finally, the extracted 2 ⁇ L DNA solution was analyzed by real time PCR.
  • cell culture fluid human saliva, human whole blood, human serum, artificial sputum, milk, and cotton wool were prepared. Then, 1 ⁇ L volume of S. aureus cells were injected into prepared cell culture medium having a volume of 20-50 ⁇ L, human saliva, human whole blood, human serum and the like, and tested by a DNA extraction device.
  • the DNA extraction device As described above, the DNA extraction device according to one embodiment of the present invention is shown in FIG. 1.
  • the procedure and example of sample processing for DNA extraction in a DNA extraction device is shown in FIG. 1A.
  • DNA extraction via a DNA extraction device is possible only with a simple procedure of (1) sample injection, (2) buffer injection, and (3) DNA extraction. And this process can be performed in 3 minutes.
  • Various kinds of samples can be filtered by the DNA extraction device. Lateral flow separation based on the porous structure of the DNA extraction device can be widely applied to complex samples without additional pretreatment, regardless of the viscosity, concentration and condition of the sample containing the bacterial cells.
  • the lysis buffer of the sample pad 110 is mixed with the sample solution to destroy the target cells to release the DNA.
  • the wash buffer is injected directly into the loading pad 120 to transport and concentrate the released DNA to the binding pad 130.
  • DNA purified and concentrated in the binding pad 130 is eluted by a micropipette.
  • the structure of the DNA extraction device and the designed case 180 are shown in FIG. 1B.
  • the positions of the three holes on the designed case 180 correspond to the positions of the loading pad 120, the sample pad 110, and the binding pad 130.
  • the shape of the hole, strip contour and pressure of the case 180 prevent overflow and at the same time increase DNA extraction efficiency and improve fluid flow reproducibility within the porous structure.
  • the fully assembled DNA extraction device is shown in FIG. 1C.
  • the size of the actual DNA extraction device was compared with the coin.
  • the DNA extraction device has a compact design and a size that is handheld and usable, making it well suited for nucleic acid testing in limited environments.
  • the DNA extraction device is designed such that various porous materials are appropriately placed such that genomic DNA concentration is induced on the binding pad 130 depending on the relative affinity difference of the DNA molecules.
  • the loading pad 120, the sample pad 110, the binding pad 130, and the wicking pad 140 are arranged according to their respective characteristics, as shown in FIG. 2A.
  • the binding affinity of various porous materials could be estimated by fluorescence labeled genomic DNA.
  • 2B and 2C are diagrams showing fluorescence images obtained by loading fluorescence-labeled genomic DNA and processing by washing buffer and analysis results of the test substance.
  • the binding affinity of each material for DNA molecules can be compared from the remaining amount of DNA related to the fluorescence intensity after washing. Affinity with fluorescently labeled genomic DNA was higher in glass filter 1 than in other materials, which means that glass filter 1 can concentrate DNA with binding pad 130 in the DNA extraction device.
  • the conjugate pad can be effectively used as the sample pad 110, because the binding affinity of the conjugate pad is very low compared to other materials. Most of the DNA molecules in the sample solution did not remain in the sample pad 110 during the operation of the DNA extraction device.
  • 2D and 2E show the fluorescence intensity of the binding pad 130 during the operation of the DNA extraction device. 2D and 2E, it can be seen that the fluorescence intensity of the binding pad 130 is saturated after 3 minutes, which means that 3 minutes is enough time to concentrate the DNA on the binding pad 130. it means.
  • fluorescence labeled genomic DNA was used to optimize the analysis component of DNA extraction.
  • the fluorescence intensity of the fluorescently labeled DNA provides information about the distribution of DNA molecules in the flow stream from which optimal conditions for various parameters of DNA extraction could be found.
  • the volume of the wash buffer and the ashes of each portion of the DNA extraction device were optimized from the comparison of the fluorescence signal at the binding pad 130. In all optimization tests, the most effective analytical elements were first tested by fluorescently labeled DNA, and then optimized conditions were identified through quantitative real-time PCR analysis.
  • lysis buffer was directly related to DNA release and showed different results in the fluorescence test.
  • various types of lysis buffers have been developed, such as enzyme lysis, alkali lysis and detergents.
  • enzyme lysis alkali lysis and detergents.
  • an alkali dissolution method that is capable of rapid alkali decomposition, completely denatures a protein, and reacts under complex conditions with a simple composition has been applied to the DNA extraction device of the present invention.
  • the suitable composition of the alkali dissolution buffer in the dry state was tested from the porous structure of the sample pad 110.
  • FIG. 7B shows comparative data showing that the dissolution effect is dependent on the additive detergent of alkaline dissolution buffer.
  • DNA elution methods were compared with each other via the concentration of DNA. This was to find a suitable method for inducing fluid flow without further processing in the DNA extraction process by the DNA extraction device according to the present invention.
  • Various structures or traditional methods have been tested.
  • the hole in the sealing tape covering the binding pad 130 induced an effective flow of the spilled DNA, resulting in an increase in the DNA concentration of the binding pad 130.
  • Figure 3a is a diagram showing the results of DNA extraction from S.aureus cells of 10 1 to 10 6 CFU in PBS.
  • the DNA extraction device showed DNA extraction efficiencies comparable to the control (results from the Qiagen kit) at various concentration ranges. At S.aureus cell numbers below 10 4 CFU, the DNA concentration of the DNA extraction device was higher than the control.
  • the DNA extraction device according to the present invention can be used particularly in the field of rapid diagnosis for low concentration samples, as it can be seen that high efficiency is shown for a sample including a low number of cells.
  • DNA extraction devices could be used to effectively purify samples with varying volumes.
  • FIG. 3B 5.5 ⁇ 10 2 CFU of S.aureus cells in PBS solution with a volume of 5 ⁇ L-100 ⁇ L were processed, and similar concentrations of DNA were obtained.
  • the DNA extraction device showed an accurate and reliable DNA extraction effect.
  • the DNA extraction device showed higher DNA extraction efficiency in this volume range compared to the control.
  • the DNA extraction device performed DNA extraction as expected from the PBS buffer.
  • the standard deviation was increased in complex samples such as saliva and milk. Contaminants contained in complex samples affected DNA extraction but were not an important factor affecting lateral flow separation in the DNA extraction device.
  • the DNA extraction device showed higher efficiency than the Qiagen kit because the contact area of the sample pad 110 included in the DNA extraction device is higher than that of the silica column included in the Qiagen kit.
  • artificial bile also showed high DNA concentrations, since viscous samples were effectively filtered by lateral flow separation by the porous structure of the DNA extraction device.
  • the DNA extraction device was somewhat less efficient than the Qiagen kit.
  • the DNA extraction device has the advantage of being able to extract DNA from complex samples easily and quickly while having a simple structure.
  • Porous membranes can be used as materials for DNA storage and transportation.
  • the DNA extraction device of the present invention based on porous membranes can also be used for storage, storage and transport of DNA molecules.
  • FIG. 5 shows that the DNA extraction device can be effectively used for storage, storage and transport of DNA molecules. Referring to Figure 5, it can be seen that DNA can be effectively extracted from S.aureus cells stored for 8 weeks in atmospheric conditions. These results show that DNA extraction devices can be a useful alternative to original samples requiring pretreatment and DNA extraction.
  • the DNA extraction device of the present invention can be effectively used for DNA extraction of complex samples based on porous materials.
  • the DNA extraction device of the present invention was able to effectively extract the DNA of the gram-positive bacteria S. aureus, it was confirmed that the effective DNA extraction can be performed for various types of samples.
  • the DNA extraction device of the present invention has the effect of extracting DNA from various types of complex samples with simple manipulation.

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Abstract

La présente invention concerne un dispositif d'extraction d'ADN à base de séparation de fluide latéral. Un dispositif d'extraction d'ADN selon un mode de réalisation de la présente invention peut comprendre : un plot d'échantillon pour recevoir un échantillon à partir duquel l'ADN doit être extrait; un plot de chargement pour recevoir un tampon de lavage portant l'ADN libéré de l'échantillon reçu par le plot d'échantillon; un plot de liaison pour enrichir l'ADN transporté par le tampon de lavage; et un plot à effet de mèche, situé en aval du plot de liaison, pour absorber le tampon de lavage déployé dans le plot de liaison.
PCT/KR2019/000980 2018-02-09 2019-01-23 Dispositif d'extraction d'adn à base de séparation de fluide latéral WO2019156395A1 (fr)

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KR1020180016031A KR102037411B1 (ko) 2018-02-09 2018-02-09 측방 유동 분리 기반의 dna 추출 디바이스
KR10-2018-0016031 2018-02-09

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KR102510536B1 (ko) * 2020-09-18 2023-03-15 광주과학기술원 증폭/검출 키트, 이를 이용한 광열 pcr 증폭 방법 및 미생물 검출 방법
KR102597405B1 (ko) * 2020-12-04 2023-11-07 (주)오상헬스케어 현장진단형 핵산추출장치
KR102284766B1 (ko) * 2020-12-16 2021-08-02 주식회사 에이아이더뉴트리진 고농도의 dna 추출이 가능한 측방 분리 추출 기반의 dna 추출 키트
KR102370566B1 (ko) 2021-07-21 2022-03-07 주식회사 에이아이더뉴트리진 원스텝 형광 다중 핵산진단 방법
KR102370580B1 (ko) 2021-07-21 2022-03-07 주식회사 에이아이더뉴트리진 원스텝 다중 핵산 진단이 가능한 유체흐름조절 오프너 기반 페이퍼 칩 구조물
KR102370572B1 (ko) 2021-07-21 2022-03-07 주식회사 에이아이더뉴트리진 원스텝 다중 핵산 진단이 가능한 유체흐름조절 오프너 기반 페이퍼 칩 비색진단법
KR102370553B1 (ko) 2021-07-21 2022-03-07 주식회사 에이아이더뉴트리진 원스텝 다중 핵산 비색 검출이 가능한 측면유동 페이퍼 칩 검출방법
KR102370561B1 (ko) 2021-07-21 2022-03-07 주식회사 에이아이더뉴트리진 원스텝 다중 핵산 비색 검출이 가능한 페이퍼 칩 검출방법
KR102392570B1 (ko) 2021-11-30 2022-04-29 주식회사 에이아이더뉴트리진 임질 진단용 조성물 및 다중 등온증폭 프라이머 세트, 그리고 이를 이용한 신속성, 정확성 및 휴대성이 향상된 키트와 진단키트를 통한 육안진단방법
KR102392573B1 (ko) 2021-11-30 2022-04-29 주식회사 에이아이더뉴트리진 클라미디아, 임질을 포함한 다중 성 매개 감염병 진단용 프라이머 세트 및 이를 이용한 동시다중 분자진단 방법과 랩온페이퍼 기반 진단 키트
KR102393392B1 (ko) 2021-11-30 2022-05-02 주식회사 에이아이더뉴트리진 매독 진단용 조성물 및 다중 등온증폭 프라이머 세트, 그리고 이를 이용한 신속성, 정확성 및 휴대성이 향상된 키트와 진단키트를 통한 육안진단방법
KR102468964B1 (ko) 2021-11-30 2022-11-22 주식회사 에이아이더뉴트리진 뎅기, 지카 및 치쿤구니야를 포함한 다중 모기 매개 감염병 진단용 프라이머 세트와 이를 이용한 동시다중 분자진단 방법과 랩온페이퍼 기반 진단 키트
KR102392567B1 (ko) 2021-11-30 2022-04-29 주식회사 에이아이더뉴트리진 클라미디아 진단용 조성물 및 다중 등온증폭 프라이머 세트, 그리고 이를 이용한 신속성, 정확성 및 휴대성이 향상된 키트와 진단키트를 통한 육안진단방법
KR102437064B1 (ko) 2022-02-28 2022-08-26 주식회사 에이아이더뉴트리진 측방 유동성의 일체형 시스템에 기반으로 정제 및 검출이 동시에 이루어지는핵산 검출용 랩-온-페이퍼 플랫폼
KR102447967B1 (ko) 2022-02-28 2022-09-27 주식회사 에이아이더뉴트리진 히팅 시스템을 포함하는 랩온페이퍼 플랫폼

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