WO2016126141A1 - Dna extraction disc apparatus and dna extraction method using same - Google Patents

Abstract

The present invention provides: a DNA extraction disc apparatus, wherein a temperature sensitive material is positioned in a cell disruption chamber to directly measure the temperature of a sample in the chamber and thus to control the temperature in the cell disruption chamber through the feedback control of the on/off of a heater, and thus the DNA extraction disc apparatus chemically performs a cell disruption procedure on the sample to purify and extract intracellular DNA or amplify the extracted DNA; and a DNA extraction method using the same.

Description

DNA extraction disk device and DNA extraction method using same

The present invention relates to a DNA extraction disk device using a temperature sensitive material and a DNA extraction method using the same. More specifically, the present invention is to break down the cells (cells) and enzymes to extract DNA required for various nucleic acid analysis. The present invention relates to a DNA extraction disk apparatus for heating and controlling a sample at an active temperature of and a DNA extraction method using the same.

Molecular diagnostics using nucleic acids have become a major issue in the field of food hygiene and forensic science because of their high accuracy, reproducibility, and rapidity. However, in spite of these advantages, many additional measurement facilities have to be equipped. Recently, many studies have been conducted to implement them in the form of Lab on a Disc. The biggest obstacle to implementing molecular diagnostics using nucleic acids on a Lab on a Disc is the integration of cell lysis processes to extract DNA.

Recently, several integrated technologies for cell destruction have been developed to improve efficiency and economics, and a device for such a cell destruction process is disclosed in US Patent Document US 7157049 (Invention: Optical bio-discs and fluidic circuits for analysis of cells and methods relating). it is shown in FIG.

However, existing cell-on-disruption methods use physical cell destruction, which requires an external device and is less efficient than nucleic acid cell destruction. The disadvantage is that it is very difficult. Nevertheless, the existing Lab on a Disc-based methods chose physical cell destruction methods because they are much easier to implement than chemical cell destruction methods that require precise temperature control. The advantage of the chemical cell destruction method is that the efficiency of cell destruction can be maximized by activating the enzyme by temperature control.

Therefore, there is an urgent need to develop a technology that can apply chemical cell destruction methods to Lab on a Disc.

In order to apply the chemical cell destruction method to the Lab on a Disc, the following two conditions must be satisfied.

First, in Lab on a Disc, chambers necessary for various functions are integrated together on the Disc. Only the chamber part responsible for cell destruction function during cell destruction must be selectively temperature controlled. Otherwise, during heating for cell destruction, sites unrelated to cell destruction may be heated or affected simultaneously, causing problems in overall performance.

Second, in order to activate proteinaseK, an enzyme added during chemical cell destruction, temperature control of 60 degrees is essential. In the case of Lab on a Disc, a series of bioprocesses are performed by centrifugal force by rotational force, so Measurements should be made non-contact.

In order to satisfy these two conditions, the present invention provides a cell destruction chamber for storing an enzyme and at the same time storing an enzyme to help the cell destruction of the sample; A heating device for activating the enzyme; Temperature sensitive material disposed within the cell disruption chamber; And a camera for measuring the temperature of the sample in the cell destruction chamber by measuring the transparency, liquidity, or fluorescence of the temperature sensitive material, thereby feeding back the heating device so that the enzyme is activated at an optimal temperature. Feedback) Provides a DNA extraction disk device that can separate the DNA contained in the cell by controlling and a DNA extraction method using the same.

The present invention has been made to solve the problems of the prior art, an object of the present invention is to place a temperature sensitive material in the cell destruction chamber to directly measure the temperature of the sample in the cell destruction chamber and thereby to feed back the heating device. By controlling the temperature in the cell destruction chamber by the activation temperature of the enzyme by controlling, the cell extraction process is performed chemically to provide a DNA extraction disk device for separating the DNA in the cell and a DNA extraction method using the same.

Another object of the present invention, further comprising a silica membrane (silica membrane) for binding to the DNA in the disk to remove the debris (debri) contained in the cells destroyed in the cell destruction chamber by centrifugal force by the disk rotation To provide a DNA extraction disk device for obtaining the DNA and the DNA extraction method using the same,

Specifically, the present invention provides a DNA extraction disk device including a cell destruction step of destroying bacterial cells contained in a sample by a chemical method, a washing step of removing debris, and a DNA extraction step, and a DNA extraction method thereof.

Hereinafter, in the present invention, DNA is a reverse transcription from deoxyribonucleic acid, RNA, which is obtained from bacteria, viruses, plants, animal body parts (hair, flesh, etc.) or secretions thereof (acupuncture, urine, etc.), blood or food samples. It is characterized in that it is any one selected from cDNA (Complementary DNA) samples obtained by reverse transcription)

Bacteria in the sample were taken from food according to Korean Food Standards Codex or FDA food code of the United States, and injected into 225 ml of liquid medium suitable for target pathogens for homogenization. 18-24 hours after enrichment culture, using a colony of separated cultures in a selective agar medium suitable for the target pathogen;

Centrifugation to porous filters using pellets of bacteria obtained by centrifugation after carrying out enrichment culture for the target pathogen, or homogenized by stomacher from food. preferably through a filter).

Hereinafter, in the present invention, "DNA extraction disk" is used interchangeably with "disk".

DNA extraction disc device according to the invention,

DNA extraction discs; A drive controller for rotating and driving the DNA extraction disk; A heating device for adjusting the temperature of the cell disruption chamber of the DNA extraction disk; And an optical sensor for measuring the temperature of the cell destruction chamber,

The DNA extraction disk is a sample injection port for injecting a sample; And an enzyme for chemically destroying the cells contained in the sample while storing the injected sample, a heating film for absorbing the heat generated from the heating device or transferring the absorbed heat, and a temperature. And a cell disruption chamber consisting of a temperature sensitive chamber that stores a temperature sensitive material.

In the present invention, the temperature sensitive material is preferably a paper coated or dyed with a phase change material, a low-melting alloy, or a temperature sensitive fluorescence material.

The paper uses a porous membrane or art paper and art paper is preferred in the present invention.

The phase change material may be a paraffin-based, non-paraffin-based, inorganic-based divorce-based, metal-based, etc. among the materials that change from the solid phase to the liquid phase. Polyacrylic pigments based on latex (MMA) or acrylonitrile, diaminostilbene dyes, fluorescein, thioflavin, eosin, rhodamine Rhodamine B and the like, and in the present invention, polyacrylic pigments based on acrylonitrile are preferred.

The low temperature melting alloy may be a kind of bismuth (Bismuth) alloy and has a characteristic that the melting temperature varies depending on the blending ratio. Representative low-temperature melting alloys include Wood's metal, Field's metal, Cerrolow 136 and Cerrolow 117. The composition of wood metal is bismuth 50%, lead 26.7%, indium 8.5% and cadmium 10%. It is melted at 70 ℃. The composition of the field metal is composed of bismuth 32.5%, tin 16.5%, indium 51%, and melts at 62 ℃. Cerrolow 136 alloy is composed of bismuth 29%, lead 18%, tin 12%, indium 21%, and melts at 58 ℃. The Cerrolow 117 alloy is composed of bismuth 44.7%, lead 22.6%, tin 8.3%, indium 19.1%, cadmium 5.3% and melts at 47.2 ° C. Fields metal is preferred in the present invention.

The temperature sensitive material is preferably arranged in an array in the cell disruption chamber.

In the present invention, the heating device is preferably a laser heating device or an induction heating device.

The heating film of the present invention is preferably a metal plate, it is further preferred to be heated by the laser heating device further comprises a black coating surface by black paint. The black paint absorbs heat from the laser heater so that heat is transferred through the cell destruction chamber by the metal plate to heat the sample in the cell destruction chamber.

 The black paint is preferably a heat resistant paint which is not melted by the laser heater.

The metal plate is preferably in the range of 0.01 to 0.1 mm aluminum (alumnium) to magnetic metal material.

The induction heating device generates a magnetic force line in the coil when an alternating current is sent to the coil, and the magnetic force line causes a eddy current to the metal plate placed on the coil by Faraday's law of electromagnetic induction. It generates and heats a metal plate.

Another aspect of the invention the heating film is preferably a black film

The black film absorbs laser light from the laser heater to heat the sample in the DNA destruction chamber.

The black film is preferably a heat resistant film which is not melted by a laser heating device.

The heat resistant film is preferably an aramid film, a polyethylene terephthalate (PET) film, a polyimide film.

In another aspect of the present invention, it is preferable that the heating film is a black coating layer formed on one surface of the cell destruction chamber by black paint.

Still another object of the present invention is a silica membrane for binding to DNA from the cells destroyed in the cell destruction chamber; A washing chamber storing a washing solution for washing the silica membrane; A waste chamber for storing debris from the cells destroyed in the cell destruction chamber or for storing impurities from the washing of the silica membrane; An extraction buffer chamber configured to wash DNA bound to the silica membrane by the washing solution, and to store an extraction buffer for extracting DNA bound onto the silica membrane; A DNA chamber for collecting DNA extracted from the silica membrane or for amplifying the collected DNA; And a channel for connecting the chambers to provide a passage through which the fluid can flow, and a valve for controlling the flow of the fluid.

The fluid is moved along the flow path by the centrifugal force due to the disk rotation.

The silica membrane is washed using the washing solution to remove impurities from the silica membrane, and finally, the extraction buffer is introduced into the silica membrane to obtain pure purified DNA when the extraction process is performed. Lose. The purified DNA is moved to the DNA chamber to perform a DNA amplification process as needed.

In the present invention, the sample is preferably mixed with a buffer for enhancing the cell destruction mechanism and the silica membrane adsorption force of DNA, and injected into the cell destruction chamber.

 The buffer aids cell destruction by combining EDTA, glucose, lysozyme, SDS, and the like, which are used for cell destruction, and contains guanidine hydrochloride, Tris-HCL, and the like to bind DNA on the silica membrane.

The buffer may be divided into a buffer for cell destruction and a buffer for DNA silica membrane adsorption. In this case, the former is called a lysis buffer and the latter is called a binding buffer, but when the two functions are combined, it is usually called a binding buffer, and if necessary depending on the characteristics of the binding buffer. Isopropanol can be added to proceed with bacterial destruction. Isopropanol is responsible for separating cellular proteins and DNA. In the present invention, isopropanol is preferably mixed with a binding buffer and injected into a cell destruction chamber to proceed with cell destruction.

Another aspect of the invention, the enzyme RnaseA can be added to the sample. Enzyme RnaseA induces the hydrolysis of RNA and removes false positive factors caused by RNA when analyzing DNA extraction results, helping to confirm pure DNA extraction results.

In another aspect of the invention, the silica membrane may be used in place of a positively charged material, such as a porous plastic or bead having a positive charge.

DNA is negatively charged due to the 5'-terminal phosphate group as a whole.

Due to the potential difference between the positively charged material and the negative charge of the DNA, the DNA is attracted to each other and the DNA is adsorbed onto the surface of the positively charged material. The larger the potential difference, the greater the electrostatic interaction, resulting in a larger adsorption efficiency.

Therefore, the surface of the positively charged material (postive charge) is preferably introduced to increase the DNA adsorption efficiency by introducing an amine group having a large positive charge value.

Another object of the present invention is characterized in that the DNA chamber of the DNA extraction disk stores the enzyme and buffer solution required for the DNA amplification process and performs DNA amplification.

For the DNA amplification process of the DNA extraction disk, the DNA chamber stores a buffer solution containing various enzymes such as primers, including dNTP, and a separate polymer for storage of polymerase. It is preferred to have a laase chamber.

In the present invention, the DNA amplification process is performed by repeatedly performing a thermo cycle of a polymer chain reaction (PCR),

It is characterized by being made by isothermal amplification.

The PCR is denaturation (~ 95 ^o C), annealing (~ 50 ^o C), and extension (~ 72) ^{o This is} achieved by running a thermal cycle in the DNA chamber that periodically repeats the three temperatures consisting of C), but the isothermal amplification is performed for about 90 minutes at one specific temperature (eg 60 ^o C). By heating.

In the present invention, the DNA extraction disk device is characterized in that it further comprises a stirrer (stirrer) for mixing the sample of the cell disruption chamber.

The stirrer includes a stirring magnet in the cell disruption chamber for sample homogenization and circulation in the cell disruption chamber; And a stirring motor having a small permanent magnet attached to the stirring magnet to perform the stirring operation by exerting an attractive force and a reaction force.

The stirrer stores enzymes that assist cell destruction, such as proteinaseK, in a dry powder form or in a buffer solution in a cell destruction chamber, injects a sample into the cell destruction chamber and mixes and homogenizes the enzyme well during the cell destruction process. Be sure to

In the present invention, the DNA extraction disk device is characterized in that it further comprises a fluorescent sensor or turbidity sensor (turbidity meter) for quantitatively analyzing the DNA amplification product (product).

The driving control unit includes a turn table for placing the DNA extraction disk; And a motor for rotating the DNA extraction disk on the turn table.

For the DNA extraction disk of the present invention, the disk diameter of the disk is preferably 120 mm, 80 mm, 60 mm or 32 mm, and the disc diameter is preferably 1.2 mm to 10 mm.

In the present invention, the optical sensor is a photodiode, a camera, a photodiode array, a spectrometer, a charge-coupled device (CCD), a complementary metal-oxide-semiconductor (CMOS) image sensor, a laser It is preferred that any one of the laser power meters is used.

The optical sensor may measure the temperature of the sample by measuring the amount of fluorescence from a temperature sensitive fluorescence dye installed in the cell destruction chamber or by measuring the fluidity or transparency of the temperature sensitive material installed in the cell destruction chamber. The optical sensor is also preferably used as a fluorescence sensor or turbidity meter for quantitatively analyzing the DNA amplification product.

Since the emission intensity of the fluorescent dye varies with the sample temperature in the cell destruction chamber, the temperature-sensitive fluorescent dye may measure the temperature of the sample by measuring the emission intensity of fluorescence according to the excitation of the light emitting diode with the optical sensor. Can be measured.

In the case of fluorescence analysis of the DNA amplification product, the amount of DNA can be quantitatively measured by measuring the emission intensity of fluorescence according to excitation of the light emitting diode with the optical sensor.

When turbidity analysis of the DNA amplification product (product), the amount of DNA can be quantitatively measured by measuring the turbidity according to excitation of the light emitting diode with the optical sensor.

Preferably, the disk is made of a silicon wafer, polypropylene, polyacrylate, polyvinyl alcohol, polyethylene, polymethyl methacrylate (PMMA), cyclic olefin polymer (COC) and polycarbonate It may be formed of one or more selected from the group. However, plastics are preferred for economic reasons and for ease of processing.

The disk is preferably composed of an upper substrate, an intermediate substrate and a lower substrate, which are preferably bonded by an adhesive.

In this case, the disk is formed by laminating and bonding the upper substrate, the intermediate substrate and the lower substrate, and includes a double-sided adhesive tape for bonding each substrate.

The double-sided adhesive tape is surface treated with a special adhesive (gluing agent) on both sides of the release paper such as paper, vinyl, polyester film, polyethylene film, and other synthetic materials, and high sealing according to the conditions required It is possible to select and use a pressure-sensitive adhesive material which has characteristics such as sealing), buffering, vibration damping, impact resistance, heat resistance, adsorption, and adhesion.

In another aspect of the present invention, it is preferred that the double-sided adhesive tape does not use a release paper or backing, and that an adhesive (gluing agent) itself forms the double-sided adhesive tape.

The double-sided adhesive tape is preferably in the form of an adhesive (gluing agent) without double-coating the adhesive on a release paper or using a release paper, the adhesive is hot melt (silicone, silicone, rubber, modified silicone, acrylic) , Materials such as polyamide, polyamide, polyolefin, teflon, polyester, epoxy, UV curable adhesive, UV adhesive, thermoplastic resin, and the like may be used.

Still another object of the present invention is a DNA extraction disk apparatus according to the present invention, comprising: injecting a sample into a cell destruction chamber through a sample inlet; Heating the cell destruction chamber by a heating device; A sample stirring step of rocking a stirring magnet during the heating step; A temperature measuring step of measuring the temperature of the cell destruction chamber by reading a change in temperature sensitive material (transparency, flowability, fluorescence amount or volume change) through an optical sensor; Controlling the on-off of the heating device according to the temperature value obtained in the temperature measuring step to maintain the activation temperature of the enzyme; Binding to the silica membrane while the DNA destroyed by the heating step passes through the silica membrane under centrifugal force due to disk rotation; A washing step of washing the DNA-bound silica membrane using a washing solution to send impurities to a waste chamber; And after the washing step, the DNA is separated from the silica membrane using an extraction buffer to extract DNA bound to the silica membrane, and then the extracted DNA is sent to a DNA chamber. The present invention provides a DNA extraction method using an extraction disk device.

In the present invention, the DNA extraction method using the DNA extraction disk device is a DNA amplification step for increasing the number of DNA obtained in the extraction step; And a product analysis step of identifying an amplification product by turbidity and fluorescence measurement.

 In the present invention, the DNA extraction method using the DNA extraction disk device is characterized in that it further comprises a disk authentication step of verifying whether the disc is genuine or reused over the Internet by embedding the RF IC on the disk. The disc of the present invention should not be reused for single use. The ID information of the disk is sent to the server and stored in each step of the disk authentication, so that the disk having the same ID information has been used in the past by checking the history information on the server when the disk is used, thereby reusing the disk. You can automatically check whether or not.

In the present invention, the DNA extraction method using the DNA extraction disk device is characterized in that it further comprises a result output step of displaying the result obtained in the output analysis step on the display unit, or providing to the outside through the Internet network. .

In the present invention, the DNA extraction method using the DNA extraction disk device, after the results sending step, receiving the hygiene score from the public office (for example, the Ministry of Health, Welfare, Education, Food and Drug Administration) through the Internet network remotely billboard It characterized in that it further comprises a hygiene score disclosure step to display.

The hygiene score is a score indicating how clean the establishment is. Therefore, guests will choose the restaurant based on the sanitary scores of the billboards posted on the outside.

The hygiene score is preferably determined by the presence or absence of detection of target pathogens from food and the frequency of inspections of the establishment.

In addition, in the present invention, the mobile phone user can receive not only the location information of the restaurant, but also the sanitary score information of the restaurant when searching for a restaurant, which will further contribute to the restaurant user's choice of restaurant and the hygiene of the business.

As described above, the present invention relates to a DNA extraction disk apparatus using a temperature sensitive material and a DNA extraction method using the same. More particularly, the present invention relates to a cell destruction process, a DNA purification and extraction process, DNA, which are essential for a DNA analysis apparatus. Provided are a DNA extraction disk apparatus for performing DNA analysis more easily and efficiently by integrating an amplification process on one disk, and a DNA extraction method using the same.

1 shows that the cell disruption chamber is engraved on a disc,

2 shows a side view and a perspective view thereof in various embodiments of the cell disruption chamber 29,

3 is another embodiment of a temperature sensitive chamber of a cell disruption chamber,

4 (a) and 4 (b) is an embodiment for performing the stirring operation by exerting attractive force and repulsive force on the stirring magnet in the sample stirring chamber for sample homogenization and circulation in the cell destruction chamber,

5 is an embodiment of a DNA extraction disk apparatus including a drive control unit for driving a DNA extraction disk,

6, 7, 8, and 9 are various embodiments of a DNA extraction disk incorporating a valve, a cell disruption process, a DNA purification process, a DNA extraction process, and a DNA amplification process,

10 is a cross-sectional view showing an embodiment in which the valve is opened by a capes channel as another embodiment of the valve,

11 is an embodiment of the valve.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1 shows that the cell disruption chamber 29 is engraved on the disk 100. Feed that may be used in the present invention is injected into the cell disruption chamber 29 through a sample inlet 28. The cell destruction chamber 29 includes a heating film 30 covering the cell destruction chamber 29; A sample stirring chamber 32b; Sample stirring auxiliary chambers 32a and 32c; And a temperature sensitive chamber 16 filled with a temperature sensitive material 24.

Reference numeral 44 is a heating device for heating the heating film 30 by a non-contact method, preferably a laser heating device or an induction heating device.

In the present invention, the laser heating device 44 is preferably composed of one or more laser modules.

In one embodiment of the present invention, the heating film 30 is preferably a metal plate, and the metal plate has a black coating surface 30a coated on one side by black paint and heated by the laser heating device 44. Is preferred. The black coating surface 30a absorbs the light generated from the laser heater 44 to transfer heat to the metal plate 30 to heat the sample in the cell destruction chamber 29.

In another embodiment of the present invention, the heating film 30 is preferably a magnetic metal plate,

When the induction heating device 44 sends an alternating current to a coil (not shown), a magnetic force line is generated in the coil, and the magnetic force line faces the coil by Faraday's law of electromagnetic induction. By generating an eddy current in the magnetic metal plate 30 placed in position, the magnetic metal plate 30 is heated to heat the sample in the cell destruction chamber 29.

In the present invention, the cell disruption chamber 29 is an enzyme that breaks down proteins, and it is preferable to have an enzyme proteinaseK in the form of a powder in the chamber 29 for activating destruction of the cell membrane.

In the present invention, the temperature sensitive chamber 16 is preferably configured in which a chamber for storing a plurality of temperature sensitive materials 24 is arranged in an array form.

1 shows one embodiment in which six chambers for storing temperature sensitive material 24 are arranged in an array.

Reference numeral 170 denotes a disk gap.

When the temperature sensitive chamber 16 is arranged in an array, it is possible to determine whether the cell destruction chamber 29 is uniformly heated from the plurality of temperature sensitive materials 24. In addition, the temperature sensitive material 24 has a hysteresis phenomenon showing a characteristic difference between a temperature rise and a fall, and this hysteresis phenomenon is proportional to the volume of the temperature sensitive material 24, so that the temperature By dividing the sensitive material 24 into small amounts and arranging them in an array, there is an advantage in that the hysteresis phenomenon can be minimized.

In one embodiment of the present invention, the temperature sensitive material 24 is preferably a phase change material, preferably having a melting point of 55 ~ 65 ℃.

In this case, when the internal temperature of the cell destruction chamber 29 reaches 55-65 ° C., which is the temperature of the activity of the enzyme proteinaseK, cell destruction is most active and phase change in the temperature sensitive chamber 16 is achieved. The phases change from solid to liquid, turning them from opaque white to transparent. At this time, by observing the transparency of the phase change material 24 by the optical sensor 46 installed below the temperature sensitive chamber 16, the temperature in the cell destruction chamber 29 can be measured in a non-contact manner.

That is, if the color of the phase change material 24 is opaque white, the heating device 44 is turned on to heat the cell destruction chamber 29. When the heating device 44 heats the cell destruction chamber 29, the temperature of the cell destruction chamber 29 is increased to reach the same temperature as the melting point of the phase change material 24. Is activated and the color of the phase change material 24 becomes transparent. In this case, the heating device 44 is turned off or the driving voltage of the heating device 44 is adjusted to a low temperature of the cell destruction chamber 29 to 55-65 ° C., which is an appropriate temperature for activating the enzyme proteinaseK. Keep it.

In another embodiment of the invention, the temperature sensitive material 24 is preferably a low-melting alloy, preferably having a melting point of 55-65 ° C.

In this case, when the internal temperature of the cell destruction chamber 29 reaches 55-65 ° C., which is the active temperature of the enzyme proteinaseK, cell destruction is most active and low-temperature fusion alloy in the temperature sensitive chamber 16 low-melting alloys change from solid to liquid. At this time, the temperature in the cell destruction chamber 29 can be measured in a non-contact manner by observing the liquidity of the low-temperature fusion alloy 24 by the optical sensor 46 installed below the temperature sensitive chamber 16. have.

That is, if the low-temperature fusion alloy 24 is in the solid phase, the heating device 44 is turned on to heat the cell destruction chamber 29. When the heating device 44 heats the cell destruction chamber 29, the temperature of the cell destruction chamber 29 is increased to reach the same temperature as the melting point of the low temperature fusion alloy 24. Is activated and the low-temperature melting alloy 24 melts into a liquid state. In this case, the heating device 44 is turned off or the driving voltage is adjusted low to maintain the temperature of the cell destruction chamber 29 at 55-65 ° C., which is an appropriate temperature for activating the enzyme proteinaseK.

In another embodiment of the present invention, the temperature sensitive material 24 is preferably paper coated or dyed with a temperature sensitive fluorescence material.

In this case, the emitted fluorescence intensity from the paper 24 changes with the rise and fall of the internal temperature of the cell destruction chamber 29. At this time, by observing the fluorescence intensity emitted from the paper 24 by the optical sensor 46 installed below the temperature sensitive chamber 16, the temperature in the cell destruction chamber 29 can be measured in a non-contact manner. . The temperature sensitive fluorescent material 24 is preferably excited by a light emitting diode 48 having a wavelength of 465 nm and the optical sensor 46 has light passing through a wavelength between 550 nm and 625 nm. It is preferred to have a filter (not shown) in front of the optical sensor 46. The optical filter selectively passes a wavelength band belonging to the fluorescence emitted from the temperature sensitive fluorescent material 24.

If the fluorescence intensity emitted from the paper 24 is lower than the lower limit temperature (eg, 55 ° C.) at which the enzyme proteinaseK is activated, the heating device 44 is turned on to turn on the cell destruction chamber. (29) is heated. In addition, as the heating device 44 heats the cell destruction chamber 29, the temperature of the cell destruction chamber 29 is increased, so that the fluorescence intensity emitted from the paper 24 is the upper limit temperature at which the enzyme proteinaseK is activated. If the temperature is higher than 65 (eg, 65), the heating device 44 may be turned off or the driving voltage may be adjusted to lower the internal temperature of the cell destruction chamber 29 to reduce the activity of the enzyme proteinaseK. It is kept at 55-65 degreeC which is the temperature which enables.

In addition, the sample stirring chamber 32B is provided with a stirring magnet 31 moving by the magnetic force of the permanent magnet 47a, so that the stirring magnet 31 is rotated as the permanent magnet 47a rotates or moves. By rocking, the sample in the cell destruction chamber 29 is stirred.

The sample stirring auxiliary chambers 32a and 32c not only provide a passage through which the sample circulates during the stirring operation of the sample, but also provide a structure to prevent the stirring magnet 31 from being separated from the sample stirring chamber 32b.

In the present invention, the low-temperature melting alloy 24 is further provided with a magnetic bead (mangitc bead) to measure the fluidity of the low-temperature melting alloy 24 according to the movement of the permanent magnet 47a by the optical sensor 46 Is preferred.

For example, when the internal temperature of the cell destruction chamber 29 reaches 55-65 ° C., the temperature of the activity of the enzyme proteinaseK, the low-temperature melting alloy in the temperature sensitive chamber 16 changes from solid to liquid. Therefore, since the magnetic beads move as the permanent magnet 47a moves, the low-temperature fusion alloy 24 may have fluidity and may be observed by the optical sensor 46. When the low melting alloy 24 has high fluidity, the heating device is turned off, while when the low melting alloy 24 has lost its fluidity, the heating device 44 is turned on or By lowering the driving power to maintain a constant internal temperature of the cell destruction chamber 29 to 55 ~ 65 ℃.

2 shows a side view and a perspective view of various embodiments of the cell disruption chamber 29.

2 (a) and 2 (c) show a case in which the temperature sensitive material 24 is arranged in an array in the temperature sensitive chamber 16, and FIG. 2 (b) shows a temperature on the upper surface of the temperature sensitive chamber 16. FIG. The case where the sensitive material 24 is arrange | positioned is shown.

The temperature sensitive material 24 of FIG. 2 (b) is preferably a phase change material, preferably having a melting point of 55-65 ° C.

In this case, when the internal temperature of the cell destruction chamber 29 reaches 55-65 ° C., which is the temperature of the activity of the enzyme proteinaseK, cell destruction is most active, and an image of the upper surface of the temperature sensitive chamber 16 is formed. The change materials 24 undergo a phase change from solid to liquid to change from opaque white to transparent. At this time, by observing the transparency of the phase change material 24 by the optical sensor 46 installed below the temperature sensitive chamber 16, the temperature in the cell destruction chamber 29 can be measured in a non-contact manner.

In the embodiment of FIG. 2A, the heating film 30 is a metal plate, and the metal plate has a black coating surface 30a coated on one side by black paint and is heated by the heating device 44. Is preferred.

2 (b) and 2 (c), the heating film 30 is a case where one side of the upper substrate 100a of the disc is implemented by a black coating layer 30 coated with black paint. It is preferred to be heated by the heating device 44.

3 is another embodiment of the temperature sensitive chamber 16 of the cell destruction chamber 29 or the DNA chamber 50, in which the chamber is configured in the form of a thermometer in the temperature sensitive chamber 16 to form the temperature sensitive material 24. It shows an embodiment of storing the.

In the present embodiment, the temperature sensitive material 24 is preferably a material that changes in volume with temperature change, and a phase change material or a low-melting alloy is preferable. As the temperature rises in the cell destruction chamber 29 by the heating device 44, the temperature sensitive material 24 changes from solid to liquid phase and thus expands in volume. Reference numeral 25a denotes a temperature sensitive channel providing a movement path of the temperature sensitive material 24 according to the volume expansion of the temperature sensitive material 24 due to the temperature rise, and reference numeral 25b denotes a scale indicating the degree of volume expansion.

The scale 25b is read by the optical sensor 46 to determine whether the internal temperature of the cell destruction chamber 29 is in the range of 55 to 65 ° C., which is the active temperature of the enzyme proteinaseK, and accordingly the heating Device 44 is feedback controlled.

Alternatively, the scale 25b is read by the optical sensor 46 to determine the internal temperature of the DNA chamber 50, whereby the heating device 44 is feedback controlled.

4 (a) and 4 (b) exert an attractive force and reaction force against the stirring magnet 31 in the sample stirring chamber 32b for homogenizing and circulating the sample in the cell destruction chamber 29, thereby stirring operation. As an embodiment for carrying out, Figure 4 (a) is provided with a permanent magnet 47a on the slider 211 to perform the stirring operation, the stirring in accordance with the repetition of the forward and backward movement of the slider 211 With respect to the magnet 31, an embodiment for exerting an attractive force and a reaction force to perform a stirring operation on the sample in the cell destruction chamber 29 is shown.

The slider 211 is controlled to move in a radial direction according to the rotation of the slider motor 109 by worm gear connecting portions 109a and 109b connected to the slide motor 109 axis.

With respect to the sample stirring chamber 32b, the slider 211 is moved back and forth to make the stirring magnet 31 swing.

The slider 211 is slidably moved using the slide arms 108a and 108b as guides. The slide arms 108a and 108b are fastened to the body of the drive control unit via screws 110a, 110b, 110c and 110d. Reference numeral 113 is a turn table for placing the disk.

Another aspect of this embodiment is to observe the transparency of the phase change material on the slider 211, to observe the liquidity (liquidity) of the low-temperature melting alloy, to observe the fluorescence intensity emitted from the paper, or the temperature By monitoring the volume change of the sensitive material it is characterized in that it is equipped with an optical sensor 46 capable of measuring the temperature in the cell destruction chamber 29 in a non-contact manner.

Another aspect of this embodiment is characterized in that the valve opening and closing means 49 for controlling the opening and closing of the valve on the disc is mounted on the slider 211. Space addressing of the valve is possible by adjusting the disk rotation angle and the radial distance of the slider 211.

In the present invention, the valve opening and closing means 49 is preferably a laser diode, and first performs space addressing on the valve to be opened, and then turns on the laser diode 49 to turn on the valve. Melting and opening are more preferred.

4 (b) shows a permanent magnet 47a attached to a shaft 300a of the stirring motor 300, so that the stirring magnet 31 in the chamber 32B for sample stirring during the rotation of the stirring motor 300, Exercising the attractive force and the reaction force is an embodiment for performing the stirring operation.

5 shows an embodiment of the DNA extraction disk apparatus 200 including a drive control unit for driving the DNA extraction disk 100.

The driving control unit includes a turn table 113 for placing the DNA extraction disk; A brushless motor 102 for rotating the DNA extraction disk 100 on the turn table 113; And a central control unit 101. In this embodiment, a brushless motor suitable for low noise and high speed rotation is preferred as the disk rotation motor.

Reference numeral 211 denotes a slider which allows the movement in the radial direction, and the radial movement of the slider 211 is controlled by the step motor 109.

Reference numeral 322 is a sanitary score billboard for the central control unit 101 to remotely receive a hygiene score from a public office through an internet connection to display a sanitary score of a business. The hygiene score is preferably displayed on the hygiene score billboard 322 via a wireless connection.

 Reference numeral 350 denotes a body that supports the DNA extraction disk apparatus 200. A circuit board 140 is jointly fastened to the body 350 at the bottom of the DNA extraction disk device 200, and the central control unit 101, the storage device 112, and the USB and the Internet are connected to the circuit board 140. An input / output device 111 providing a connection is arranged and designed on the circuit board 140. The central controller 101 controls the brushless motor 102 to rotate or stop the disk 100, the heating device 44, the optical sensor 46, the light emitting diode 48, In addition to controlling the valve opening and closing means 49, the stirring motor 300, and the hygiene score billboard 322, the optical sensor 46 and the valve opening and closing designed and arranged on the slider 211 by the control of the slide motor 109. The movement of the means 49 and the permanent magnet 47a is controlled.

In addition, the central controller 101 controls the display unit 320 and the button input unit 321 to provide a user interface to the DNA extraction disk device 200 to the user.

In the present invention, a unique ID of the disc 100 to the central control unit 101, preferably via the wireless RF IC 188 on the disc at the time of loading of the disc to the turntable 113. The central control apparatus 101 recognizes that the DNA extraction disk 100 is loaded by transmitting the information wirelessly.

Reference numeral 104 is a crimping means of the disk 100 loaded in the disk cavity 170 is compressed by a magnetic attraction force with the turn table 113 is preferably designed to allow vertical movement and idling. Reference numeral 108 denotes an RF power supply for supplying power to the RF IC 188 by electromagnetic induction.

6, 7, 8, and 9 illustrate various embodiments of a DNA extraction disk 100 incorporating a cell disruption process, a DNA purification process, a DNA extraction process, and a DNA amplification process.

The DNA extraction disk 100 illustrated in FIGS. 6, 7, 8, and 9 includes a sample inlet 28 for injecting a sample; A cell destruction chamber (29) comprising an enzyme and a temperature sensitive chamber (24) for chemically destroying cells contained in the sample while storing the injected sample; The DNA and debris generated from the cells destroyed in the cell destruction chamber 29 are captured by the centrifugal force during the rotation of the disk 100 while the remaining debris is passed through as it is. Silica membrane 51; A membrane chamber 51a on which the silica membrane 51 is fixed; Washing solution chamber 1 (58A) and washing solution storing washing solution 1 (washing solution 1) and washing solution 2 (washing solution 2) for washing the silica membrane (51) capturing the DNA, respectively Chamber 2 58B; Waste chamber for storing debris passing through the silica membrane 51 or for storing impurities generated during the purification and washing of the silica membrane 51 by the cleaning solution 1 and the cleaning solution 2. 52; An extraction solution chamber 59 storing an extraction buffer for extracting DNA bound to the silica membrane 51 washed by the washing solution 1 and the washing solution 2; And a DNA chamber 50 for storing DNA extracted from the silica membrane 51 by the extraction buffer or for amplifying the stored DNA.

Another aspect of the invention is to further comprise a porous plastic filter (not shown) in the membrane chamber (51a), it is preferred to mechanically support the silica membrane (51). The porous plastic filter can prevent the silica membrane 51 from deforming mechanically by centrifugal force during the rotation of the disk 100.

The disc 100 has valves 56A, 56B, 56C, 56D at the outlet of the cell disruption chamber 29, the wash solution chamber 1 58A, the wash solution chamber 2 58B, and the extract solution chamber 59, respectively. In this case, the liquids stored in these chambers are moved along the channel 57 by the centrifugal force generated during the rotation of the disk 100 when the valve is opened.

The valves 56A, 56B, 56C, 56D, 71b are all initially closed and the valve 71a is opened from the beginning, so that the cell breaking process in the cell destruction chamber 29 is completed by the valve opening and closing means 49 The DNA is captured on the silica membrane 51 while the DNA and the debris generated in the cell destruction process are moved along the channel 57 by the centrifugal force according to the rotation of the disk 100. The remaining debris is passed through and stored in the residue chamber 52.

Thereafter, the valve 56B is opened, and then, by centrifugal force due to the rotation of the disk 100, the washing solution 1 stored in the washing solution chamber 1 58A moves along the channel 57 to capture the DNA. Clean the silica membrane 51. At this time, impurities generated during the cleaning process are stored in the residue chamber 52.

Thereafter, the valve 56C is opened, and then, by centrifugal force due to the rotation of the disk 100, the washing solution 2 stored in the washing solution chamber 2 58B moves along the channel 57 to capture the DNA. Clean the silica membrane 51. At this time, the impurities generated during the cleaning process are stored in the waste chamber 52.

Thereafter, the valve 71a is closed by the valve opening and closing means 49, and the valve 71b and the valve 56D are opened.

Then, by centrifugal force due to the rotation of the disk 100, the extraction buffer stored in the extraction solution chamber 59 moves along the channel 57 to bind the DNA bound on the silica membrane 51. Extracted and moved to the DNA chamber 50.

6 and 7, reference numerals 71a and 71b denote valves. The valve 71b is initially closed while the valve 71a is open from the beginning so that debris passed as it is without binding to the silica membrane 51 by centrifugal force during the rotation of the disc 100 is left. In addition to being transferred to the chamber 52 and stored therein, impurities generated during the washing of the silica membrane 51 by the cleaning solution 1 and the cleaning solution 2 are transferred to the waste chamber 52 and stored.

Thereafter, valve 71b and valve 56D are opened while valve 71a is closed.

In this case, when the disk 100 is rotated, the extraction buffer stored in the extraction solution chamber 59 passes through the silica membrane 51 to extract DNA and transfer the DNA to the DNA chamber 50.

In FIG. 8 and FIG. 9, reference numeral 77 denotes a Coriolis channel, which is a channel using the Coriolis effect, which is a physical law, and is composed of channels 77a and 77b.

The channel 77b and the channel 77a are connected to the outlet of the membrane chamber 51a and branched in opposite directions to form an arch. The channel 77b is connected to the waste chamber 52 and the channel 77a is the DNA. Connected to the chamber 50 and the liquid collected in the membrane chamber 51a when the disk rotates counterclockwise moves to the dreg chamber 52 via the channel 77b, while the disk rotates clockwise. The liquid collected in the membrane chamber 51a is transferred to the DNA chamber 50 via the channel 77a.

Therefore, the debris passed as it is without binding to the silica membrane 51 is transferred to the dreg chamber 52 by centrifugal force during the rotation of the disc 100, or the washing solution 1 and the washing solution 2 When the impurities generated during the cleaning of the silica membrane 51 are transferred to the waste chamber 52, the disk 100 is rotated counterclockwise.

In addition, when the extraction buffer stored in the extraction solution chamber 59 passes through the silica membrane 51 to extract DNA and transfer the DNA to the DNA chamber 50, the disk 100 is rotated clockwise.

The closing of the valves 56A, 56B, 56C, 56D, 71b is preferably to close the hole of the valve by the black membrane.

The black membrane is black vinyl, black polyester film, black paint coated PVDF, black hotmelt, black polyethylene film, black polypropylene, black PVC vinyl (polyvinyl chloride) , Black PET (Polyethylene Terephthalate, Poly-Ethylene-Terephthalate) film and other black synthetic materials are preferred, and the valve opening and closing means is preferably a laser diode.

The black membrane has a high absorbance and is easily heated and melted by irradiation of a laser beam by a laser diode to open the valve.

The DNA extraction disk 100 illustrated in FIG. 7 is an embodiment in which two pairs of the DNA extraction disk 100 illustrated in FIG. 6 are arranged in a symmetrical structure, and the disk is rotated when the disk 100 is rotated because of the symmetrical structure. The overall weight balance of (100) provides the advantage of minimizing vibration and noise during high-speed rotation of the disk (100).

9 is an embodiment in which the DNA extraction disk 100 illustrated in FIG. 9 is arranged in a symmetrical structure facing two pairs of the DNA extraction disk 100 illustrated in FIG. 8, when the disk 100 is rotated due to the symmetrical structure. The overall weight balance of the disk 100 provides an advantage of minimizing vibration and noise during high-speed rotation of the disk 100.

FIG. 10 is a side view and a plan view of another embodiment in which the valve 71b is opened by a capes channel 72 as another embodiment of the valve 71b. Fig. 10 (a) shows the case where valve 71b is closed by black vinyl 85, while Fig. 10 (b) shows the case where valve 71b is open.

In this embodiment, the disk 100 is preferably made of an upper substrate 100a, an intermediate substrate 100b and a lower substrate 100c, which are preferably bonded by an adhesive. For convenience, the top view of FIG. 10 is drawn for the intermediate substrate 100b and the lower substrate 100c, except for the upper substrate 100a.

In the case of Fig. 10 (a), the closing of the valve 71b is closed by black vinyl 85 disposed between the upper substrate 100a and the intermediate substrate 100b, and the liquid in the membrane chamber 51a is filled with the DNA chamber ( Cannot go to 50).

In the case of FIG. 10B, the valve 71b is opened by forming a capillary channel 72 on the black vinyl 85 while melting the black vinyl 85 by the valve opening and closing means 49. In this case, the liquid in the membrane chamber 51a may move to the DNA chamber 50 by the centrifugal force due to the rotation of the disk 100.

In the present invention, the capillary channel (72) extends from the outlet of the membrane chamber (51a) to the hole (71b) of the valve by black vinyl (85) to form a closing channel (closing channel); The valve 71b is formed by turning on a laser diode 49 to melt the black vinyl 85 from the outlet of the membrane chamber 51a to the hole 71b of the valve, thereby forming an opening channel. It characterized in that the opening.

In the crossing of the Red Sea of Moses, just as Moses points to the Red Sea, laser diode 49 breaks the black vinyl 85 into a channel on black vinyl 85. Thus, in the present invention, the channel 72 is named as a capillary channel.

The black vinyl is black polyester film, black hotmelt, black paint coated PVDF, black polyethylene film, black PP, black paint, black PVC vinyl ), Black PET (Polyethylene Terephthalate, Poly-Ethylene-Terephthalate) film and other black synthetic materials are preferred.

11 is an embodiment of the valve 71a. FIG. 11 (a) shows the case where the valve 71a is opened and FIG. 11 (b) shows the case where the valve 71a is closed. The valve 71a is initially opened as shown in FIG. 11A, so that the liquid collected in the membrane chamber 51a by centrifugal force can freely move to the dreg chamber 52.

In this embodiment, the disk 100 is preferably made of an upper substrate 100a, an intermediate substrate 100b and a lower substrate 100c, which are preferably bonded by an adhesive. For convenience, the top view of FIG. 11 is drawn for the intermediate substrate 100b and the lower substrate 100c, except for the upper substrate 100a.

Reference numeral 78b denotes a PCM chamber which stores a phase change material 78, and when heated by the laser diode 49, the phase change material 78 changes from solid to liquid.

Reference numeral 79 denotes a black coating surface made of black paint, which is present on the bottom surface of the phase change material 78 so that when the laser diode 49 is turned on, the light emitted from the laser diode 49 is highly efficient. Can absorb. Accordingly, the black coating surface 79 is heated, and the phase change material 78 melts by this heat, and the phase change material 78 flows into the channel, thereby closing the valve 71a as shown in FIG. 11 (b). . After the phase change material 78 enters the channel, the laser diode is turned off, causing the phase change material 78 to turn into a solid state so that the valve 71a remains closed.

Claims (22)

1. A DNA extraction disk including a sample inlet for injecting a sample, a heating film for absorbing heat to transfer heat to the sample, and a cell destruction chamber including a temperature sensitive chamber in which a temperature sensitive material is stored;

   A valve for controlling fluid flow in the DNA extraction disk;

   A drive control unit including a turn table on which the DNA extraction disk is mounted, a disk rotation motor for rotating the turn table, and a central control unit for driving and controlling the disk rotation motor;

   A heating device including a laser heating device or an induction heating device for heating the heating film;

   A stirring magnet accommodated in the cell destruction chamber, a stirring motor having a permanent magnet attached thereto to provide stirring movement to the stirring magnet, or a slider having a permanent magnet moving along the radial direction of the DNA extraction disk; A stirrer for stirring the sample in the cell disruption chamber; and

   And an optical sensor for measuring transparency, liquidity, or fluorescence of the temperature sensitive material to measure the temperature of the cell destruction chamber.

   DNA extraction disk device.
2. The method of claim 1,

   On the slider is mounted valve opening and closing means for controlling the opening and closing of the optical sensor or the valve,

   DNA extraction disk device.
3. The method of claim 1,

   The temperature sensitive material is provided with a temperature sensitive fluorescent material,

   The DNA extraction disk apparatus further includes a light emitting diode for irradiating light to excite the temperature sensitive fluorescent substance, and an optical filter for passing a wavelength band belonging to the fluorescence emitted from the temperature sensitive fluorescent substance in front of the optical sensor. ,

   DNA extraction disk device.
4. The method of claim 1,

   A silica membrane which captures DNA from the cells destroyed in the cell destruction chamber and passes the residues through;

    A membrane chamber having the silica membrane mounted therein and connected to the cell destruction chamber through a first flow path;

   At least one cleaning solution chamber storing a cleaning solution for cleaning the silica membrane and connected to the membrane chamber through the first flow path;

   An extraction solution chamber storing an extraction buffer for extracting DNA attached to the silica membrane washed by the washing solution and connected to the membrane chamber through the first flow path;

   A residue chamber for storing the residue passing through the silica membrane and connected to the membrane chamber via a second flow path; And

   And a DNA chamber configured to store DNA extracted from the silica membrane by the extraction buffer, or to be connected to the membrane chamber through a third flow path for performing an amplification process on the stored DNA.

   DNA extraction disk device.
5. The method of claim 4, wherein

   An outlet of the cell destruction chamber, an outlet of the cleaning solution chamber, and an outlet of the extraction solution chamber are provided with a first valve, a second valve, and a third valve, respectively, and the third flow path is provided with a fifth valve,

   DNA extraction disk device.
6. The method of claim 1,

   The valve comprises a black synthetic material, the DNA extraction disk device further comprises valve opening and closing means for melting the black synthetic material by a laser diode to open the valve,

   DNA extraction disk device.
7. The method of claim 5, wherein

   The fifth valve is provided on the third flow path connecting the membrane chamber and the DNA chamber and is closed by a black synthetic material extending from the outlet of the membrane chamber to the hole of the fifth valve. and a capillary channel that opens the fifth valve by forming an opening channel by melting the black synthetic material from the outlet of the membrane chamber to the hole of the fifth valve by a laser diode. further comprising a channel),

   DNA extraction disk device.
8. The method of claim 1,

   The valve,

   A PCM chamber for storing a black coated surface and a phase change material disposed in contact with the black coated surface,

   When the black coating surface is heated by a laser diode, the flow path is closed by the liquefied phase change material.

   DNA extraction disk device.
9. The method of claim 1,

   Characterized in that it further comprises a hygiene score billboard for displaying the hygiene score by remotely feedback the hygiene score from the public office through the Internet connection by the central control unit,

   DNA extraction disk device.
10. A DNA extraction disk including a sample injection port into which a sample is injected and a cell destruction chamber including a temperature sensitive chamber in which a temperature sensitive material is stored;

    A valve for controlling fluid flow in the DNA extraction disk;

    A drive controller for driving and controlling the rotation of the DNA extraction disk;

    A heating device for heating the cell destruction chamber; And

    An optical sensor for measuring the temperature of the cell destruction chamber;

    The temperature of the cell destruction chamber is measured by detecting the change of the temperature sensitive material heated by the heating device by the optical sensor,

    DNA extraction disk device.
11. The method of claim 10,

    The temperature sensitive material is provided as a phase change material or a temperature sensitive fluorescent material,

    The temperature of the cell destruction chamber is measured by detecting a change in transparency, liquidity or fluorescence of the temperature sensitive material by the optical sensor.

    DNA extraction disk device.
12. (a) injecting a sample into a cell disruption chamber of a DNA extraction disk;

    (b) the temperature of the cell destruction chamber by heating the cell destruction chamber by means of a heating device and detecting a change in transparency, liquidity or fluorescence of the temperature sensitive material included in the cell destruction chamber through an optical sensor; Measuring; And

    (c) controlling the heating device such that based on the temperature of the cell destruction chamber being measured, the temperature in the cell destruction chamber is maintained within a temperature range that destroys the cells of the sample;

    DNA extraction method.
13. The method of claim 12,

    (d) rotating the DNA extraction disc after opening the valve of the cell disruption chamber so that the sample of cell destruction completes through the silica membrane and moves toward the membrane chamber;

    (e) rotating the DNA extraction disc after opening the valve of the wash chamber such that the wash solution stored in the wash chamber passes through the silica membrane to the membrane chamber;

    (f) rotating the DNA extraction disc after opening the valve of the extraction solution chamber so that the extraction buffer stored in the extraction solution chamber passes through the silica membrane to the membrane chamber;

    (g) amplifying the DNA extracted from the silica membrane and contained in the DNA chamber;

    (h) calculating the amount of amplified DNA by measuring the turbidity or fluorescence of the DNA chamber; And

     (i) displaying the result obtained in the step of calculating the amount of the amplified DNA on a display unit or sending the result to the outside through an internet network; Further comprising,

    DNA extraction method.
14. The method of claim 13,

    (j) a disk authentication step of transmitting ID information on the disk to a server through the Internet to check whether the disk is genuine or reuse the disk from the server;

    DNA extraction method.
15. The method of claim 13,

    (k) a sanitary score disclosure step of displaying the sanitary score on a sanitary score billboard by receiving a feedback of the sanitary score remotely from a government office (for example, the Ministry of Health, Welfare, Education, and Food and Drug Administration) through the Internet network; Further comprising,

    DNA extraction method.
16. Circular disk body; And

    And at least one cell disruption chamber and a DNA chamber provided in the circular disk body.

    The cell destruction chamber,

    A sample inlet for injecting a sample into the cell destruction chamber;

    A heating film that absorbs heat provided by an external heating device and transfers the heat to the chamber; And

    A temperature sensitive chamber in which a temperature sensitive material is stored;

    DNA extraction discs.
17. The method of claim 16,

    In the chamber,

    Enzymes for chemically destroying or amplifying the cells contained in the sample or

    Further provided with a stirring magnet for stirring the sample,

    DNA extraction discs.
18. The method of claim 16,

    The temperature sensitive material is provided with a phase change material, low temperature melting alloy, temperature sensitive fluorescent material, or paper coated or dyed with temperature sensitive fluorescent material,

    DNA extraction discs.
19.  The method of claim 16,

    A temperature sensitive channel extending from said temperature sensitive chamber, said temperature sensitive channel providing a passage through which said temperature sensitive material inflated with temperature increase enters said temperature sensitive chamber; And

    And a scale provided in the temperature sensitive channel for sensing the temperature of the chamber by identifying a degree of volume expansion of the temperature sensitive material.

    DNA extraction discs.
20. The method of claim 16,

    A silica membrane which captures DNA from the cells destroyed in the cell destruction chamber and passes the residues through; And a membrane chamber having the silica membrane mounted therein and connected to the cell destruction chamber through a first flow path.

    At least one cleaning solution chamber storing a cleaning solution for cleaning the silica membrane and connected to the membrane chamber through the first flow path;

    An extraction solution chamber storing an extraction buffer for extracting DNA attached to the silica membrane washed by the washing solution and connected to the membrane chamber through the first flow path;

    A residue chamber for storing the residue passing through the silica membrane and connected to the membrane chamber via a second flow path; And

    A DNA chamber connected to the membrane chamber via a third flow path for storing DNA extracted from the silica membrane by the extraction buffer or performing an amplification process on the stored DNA; And

    An outlet of the cell destruction chamber, an outlet of the washing solution chamber, and an outlet of the extraction solution chamber are each provided with a first valve, a second valve, and a third valve,

    DNA extraction discs.
21. The method of claim 20,

    The second and third flow passages are connected together to the outlet of the membrane chamber and branched in opposite directions to form an arch, so that the second flow passage is connected to the waste chamber and the third flow passage is connected to the DNA chamber. And the liquid collected in the membrane chamber when the disk rotates counterclockwise moves to the ground chamber via the second flow path, while the liquid collected in the membrane chamber when the disk rotates clockwise moves the third flow path. A Coriolis channel that travels to the DNA chamber via

    DNA extraction discs.
22. 22. A pair of DNA extracting disks according to any one of claims 16 to 21 is arranged and integrated in a symmetrical structure facing each other on the circular disk body.

    DNA extraction discs.

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